The use of circulating cell-free (cf) DNA to monitor cancer progression and response to therapy has significant potential but there is only limited data on whether this technique can detect the presence of low frequency subclones that may ultimately confer therapy resistance. In this study, we sought to evaluate whether whole-exome sequencing of cfDNA can accurately profile the mutation landscape of metastatic melanoma. We used whole-exome sequencing (WES) to identify variants in matched tumor-derived genomic (g) DNA and plasma-derived cfDNA isolated from a cohort of 10 metastatic cutaneous melanoma patients. WES parameters such as sequencing coverage and total sequencing reads were comparable between gDNA and cfDNA. There was significant concordance between gDNA and cfDNA based on the total number of variants identified and the degree of overlap in variants which was independent of the site of tumor biopsy. The mutant allele frequency of common single nucleotide variants was lower in cfDNA reflecting lower read depth and dilution of circulating tumor DNA in the circulation by other cfDNA species. In addition to known melanoma driver mutations, several other melanoma-associated mutations were found to be concordant between matched gDNA and cfDNA. This study highlights that WES of cfDNA can capture clinically-relevant mutations present in melanoma metastases, but does not appear to provide any additional unique information on tumor heterogeneity. Targeted deep sequencing may be required to detect low frequency genomic aberrations known for predicting therapy resistance.

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders globally, and leads to an excessive loss of dopaminergic neurons in the substantia nigra of the brain. Circulating cell-free DNA (ccf-DNA) are double-stranded DNA fragments of different sizes and origins that are released into the serum and cerebrospinal fluid (CSF) due to cell death (i.e., necrosis and apoptosis) or are actively released by viable cells via exocytosis and NETosis. Methods: Using droplet digital polymerase chain reaction (ddPCR) we comprehensively analyzed and distinguished ccfDNA and ccf mtDNA in the serum and CSF of PD and control patients. Results: The quantitative analysis of serum ccf-DNA in PD patients demonstrated significant increase of ccf mtDNA and ccfDNA compared to healthy control patients and significantly higher copy of ccf mtDNA when compared to ccfDNA. Next, the serum ccf mtDNA levels significantly increased in male PD patients compared to healthy male controls. Furthermore, CSF ccf mtDNA in PD patients increased significantly compared to ccfDNA, and ccf mtDNA decreased in PD patients compared to healthy controls. These decreases were not statistically significant, but were in agreement with previous data. In-terestingly, ccf mtDNA increased in healthy control patients in both serum and CSF as compared to ccfDNA. Limitations: the small sample size of serum and CSF were the main limitations of this study. Conclusion: To the best of our knowledge, this is the first comprehensive study on serum and CSF of PD patients using ddPCR and indicating the distribution of the copy number of ccf mtDNA, as well as ccfDNA. If validated, we suggest that ccf mtDNA has greater than ccfDNA potential to lead the development of novel treatments for PD patients.

Recent advancements in molecular diagnostics have highlighted the potential of circulating cell-free DNA (ccfDNA) as a biomarker in various diseases. This study aims to explore the role of ccfDNA in the early diagnosis, prognosis, and therapeutic monitoring of diseases, with a focus on oncology. We conducted a comprehensive review and analysis of current literature, combined with experimental approaches involving advanced genomic sequencing and bioinformatics techniques. Our research systematically characterizes the molecular composition of ccfDNA, including its origins, size distribution, and methylation patterns, across different disease states. Furthermore, we investigate the dynamics of ccfDNA release and clearance in the bloodstream, elucidating mechanisms that could influence its diagnostic accuracy. In the context of cancer, we analyze the correlation between tumor-derived ccfDNA and disease progression, response to therapy, and overall survival. Our findings indicate that ccfDNA holds promise as a non-invasive biomarker for early disease detection, real-time monitoring of treatment efficacy, and potentially, predicting relapse. This study contributes to the growing field of liquid biopsies and opens new avenues for personalized medicine, emphasizing the need for standardized protocols in ccfDNA analysis to maximize its clinical utility. The implications of our findings extend beyond oncology, suggesting potential applications of ccfDNA in other pathological conditions.

Circulating tumor cells (CTCs) are cells released from the primary tumor and intravasate into the blood or lymphatic vessels, where they are transported to distant sites and act as seeds that initiate cancer metastases. Recent advances in CTC research have shown their relevance as prognostic markers for early and metastatic disease detection, predictive biomarkers for relapse, and response to medical intervention or therapy. The rapidly evolving landscape of CTC biology has opened new avenues for understanding cancer progression, metastasis, and treatment response. Additionally, translating these findings into clinical applications holds promise for improving cancer diagnostics, prognosis, and personalized therapeutic strategies. This review discusses the significance of CTCs in cancer research and their associated challenges. We explore recent developments in the detection and characterization of CTCs and their implications in cancer research and clinical practice.

Circulating cell-free DNA (cfDNA) refers to small fragments of DNA molecules released after programmed cell death and necrosis in several body fluids such as blood, saliva, urine, and cerebrospinal fluid. The discovery of cfDNA has revolutionized the field of non-invasive diagnostics in the oncologic field, in prenatal testing, and in organ transplantation. Despite the potential of cfDNA and the solid results published in recent literature, several challenges remain, represented by low abundance, need for highly sensitive assays and analytical issues. In this review, the main technical advances in cfDNA analysis are presented and discussed, with a comprehensive examination of the current available methodologies applied in each field. Considering the potential advantages of cfDNA, this biomarker is increasing its consensus among clinicians, as it consents to monitor patients’ conditions in an easy and non-invasive way, offering a more personalized care. Nevertheless, cfDNA analysis is still considered a research marker to be further validated, and very few centers are implementing its analysis in the real-life assistance. As technical improvements are enhancing the performances of cfDNA analysis, its application will transversally improve patients’ quality of life.

Early diagnosis and treatment have always been highly desired in the fight against cancer, and detection of circulating tumor DNA (ctDNA) has recently been touted as highly promising for early cancer screening. Consequently, the detection of ctDNA in liquid biopsy gains much attention in the field of tumor diagnosis and treatment, which has also attracted research interest from the industry. However, traditional gene detection technology is difficult to achieve low cost, real-time and portable measurement of ctDNA. Electroanalytical biosensors have many unique advantages such as high sensitivity, high specificity, low cost and good portability. Therefore, this review aims to discuss the latest development of biosensors for minimal-invasive, rapid, and real-time ctDNA detection. Various ctDNA sensors are reviewed with respect to their choices of receptor probes, detection strategies and figures of merit. Aiming at the portable, real-time and non-destructive characteristics of biosensors, we analyze their development in the Internet of Things, point-of-care testing, big data and big health.

Effective biomarkers are essential to the early diagnosis of non-small cell lung cancer (NSCLC). Herein, a retrospective study of 49 newly diagnosed and recurrent NSCLC patients, 31 patients with benign pulmonary disease and 24 healthy volunteers was conducted, to evaluate the diagnostic value of circulating rare cells for NSCLC. The expression of circulating tumor cells (CTCs) and circulating tumor-derived endothelial cells (CTECs) in peripheral blood were measured by subtraction enrichment-immunostaining-fluorescence in situ hybridization (SE-iFISH). The level of CTCs (P＜0.001) and CTECs (P＜0.001) was significantly higher in NSCLC group than that in benign pulmonary disease group. The proportion of small CTCs (P＜0.001) and CTECs (P＜0.0001) significantly increased from benign lung disease individuals to NSCLC patients. The AUC of ROC curves of total CTCs and CTECs were 0.815 (95%CI: 0.722~0.907), 0.739 (95%CI: 0.618~0.860), respectively. The cut-off values for discriminating NSCLC with benign lung disease patients were total CTCs 11.5 units/6ml and total CTECs 10.5 units/6ml, with sensitivity and specificity being 67.3% and 83.9%, 77.6% and 77.4%, respectively. When CTCs and CTECs were combined, predictive value significantly increased to 82.6% as measured by the area under the curve. Small CTCs and triploid CTCs had high positive predictive value (PPV) and positive likelihood ratio (LR+) of the diagnosis of NSCLC in early stage. CTCs and CTECs can not only be used as new biomarkers for the diagnosis of NSCLC, but can also improve diagnostic performance of the early stage NSCLC. Moreover, the combined examination of CTCs and CTECs is be superior to the single.

Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and high-risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. Particularly, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, where many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed association with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders.

Introduction: Circulating tumor DNA (ctDNA) is newly diagnosed tumor DNA that can easily represent a tumor’s genetic and epigenetic change. Pheochromocytomas (PCCs) and Paragangliomas (PGLs) are rare tumors of adrenal gland tissue that have the potential to be detected by ctDNA. We aimed to study the potential of the methylation status of RDBP, SDHB, and SDHC genes in ctDNA of PCCs/PGLs patients as a diagnostic biomarker. Materials and Methods: Clinical data, fresh frozen tissue, the blood of 12 PCCs/PGL patients, and the blood of 12 age/sex-matched normal patients were collected. The methylation status of RDBP, SDHB, and SDHC was compared between cases and controls by MS-HRM analysis. Results: Amongst six promoter regions of RDBP, SDHB, and SDHC, promoter methylation quantification of SDHCa and RDBPb was significantly different between PCCs/PGLs and controls. SDHCa was methylated in 49.93% of PCCs/PGLs cases vs. 8.33 % of control samples, p-value: 0.026, area under curve AUC=0.757, and RDBPb in 74.9% of PCCs/PGLs cases vs. 25.0% of control samples, p-value: 0.032, AUC=0.750. Conclusions: This study suggests the ctDNA potential for a less invasive source of tumor epigenetic modification in PCCs/PGLs malignancies. The SDHCa and RDBPb hypermethylation warrant further exploration as diagnostic tools for PCCs/PGLs.

Currently, there are few effective methods to monitor patients with gynecologic malignancies, including ovarian and endometrial cancers. The ‘tumor fraction’ of circulating tumor DNA may offer a minimally-invasive method to evaluate tumor aggressiveness via simple blood draw. To date, there is limited data on utility of ctDNA TFx interrogation in gynecologic malignancies, although there is evidence that TFx may be low or undetectable in malignancies primarily confined to the peritoneal cavity. The objective was a descriptive assessment of ctDNA TFx in a cohort of patients with ovarian and endometrial cancer. The association of TFx to continuous and categorical baseline clinicopathologic factors and progression-free survival was assessed. In a large cohort comprised of 210 plasma samples from 78 patients with biopsy-proven ovarian and endometrial cancer, plasma samples collected between 4/2018 and 4/2020 were subjected to shallow whole genome sequencing with determination of TFx. Overall, we demonstrate that TFx was low for most samples with mean TFx for ovarian cancer 5.5% and endometrial cancer 2.4%, yet can be quantified above lower limit of detection in many patients particularly when evaluating serial samples. There was no significant association between TFx level and clinical (primary vs recurrent, best response) or pathologic (e.g. histology, grade) features. Our data demonstrates low TFx levels in ovarian and endometrial cancers likely reflect that tumor types are ‘low ctDNA shed’, suggesting that higher sensitivity assessments may be required for clinical utility.

Pediatric brain tumors are the leading cause of cancer-related death in children. Diagnosis cur-rently relies on surgical biopsy to obtain tumor tissue for histological analysis. There is a great need for less invasive approaches to molecularly profile tumors and monitor therapeutic response. Liquid biopsy analysis of circulating tumor biomarkers in biofluids like cerebrospinal fluid (CSF) and blood offers a promising minimally invasive strategy. This review discusses recent advances in developing liquid biopsy assays using next-generation sequencing, digital PCR, and emerging techniques to detect circulating tumor DNA (ctDNA), cells (CTCs), and exosomes in pediatric brain tumors. The ability to use these circulating biomarkers for non-invasive diagnosis, thera-peutic targeting, tracking response to therapy, and understanding resistance mechanisms is ex-amined. Technical and clinical validation challenges to translate liquid biopsies into routine clin-ical practice are also discussed.

There is no routine approach to identify patients with gynecologic malignancies at high risk for chemotherapy-resistance. Circulating tumor DNA (ctDNA) tumor fraction (TFx) represents a minimally-invasive approach to tumor profiling, with as yet limited data on utility in gynecologic malignancies. The objective was to investigate the use of ctDNA TFx in a cohort of patients with ovarian and endometrial cancer. Plasma samples from patients with biopsy-proven ovarian or endometrial cancer collected between 4/2018 and 4/2020 were subjected to shallow whole genome sequencing with determination of TFx via ichorCNA package. The association of TFx to continuous and categorical baseline clinicopathologic factors and progression-free survival was assessed. 210 plasma samples from 78 patients with gynecologic cancers were analyzed. Mean TFx for ovarian cancer was 5.5% and endometrial cancer 2.4% and there was no significant difference in TFx among histology either for endometrial or ovarian cancers. Grade was associated with significant difference in ‘sentinel’ TFx among ovarian cancers but not endometrial cancers. ctDNA TFx dynamics over time demonstrated rapid clearance of ctDNA in most patients with ovarian cancer, while endometrial cancer had consistently low TFx. ctDNA TFx is feasible in ovarian and endometrial cancers and may be a valuable important tool for prognostication.

Separation and interpretation of rebellious Circulating Tumor Cells (CTCs) originating from the primary tumor or cancer tissue plays a significant role in diagnostics, cancer progression analyses, suitable medicine exploration, and treatment proficiency examination. Cancer metastasis occurs when CTCs spread throughout the body and invade healthy tissues, leading to new tumors in that area. Although a dramatic rate of deaths begins from spreading CTCs around the body, valuable measures have been made to control their development. However, the first step is separating these harmful cells from the bloodstream and investigating their features. Having examined the characteristics of CTCs as cancer’s main strength, researchers can introduce complementary treatments that can affect cancerous cells without damaging the healthy cells. Therefore, according to their unique characteristics, numerous techniques have been established for continuous and fast separation and sorting of CTCs. Nevertheless, few separators enjoy the efficient performance and appropriate accuracy and can be produced in mass numbers due to the available fabrication equipment. Microfabrication advancements enable separators to combine the advantages of active and passive methods in a small-scale platform for probing individual cells and separation purposes. Reduction in reagents, sample volume, analysis time, and less harmfulness to patients are some of the motivations that encourage researchers to employ microfluidic instruments for CTCs separation from other blood cells over the last two decades. However, microfabrication limitations mean effective separators, and the diagnostic option they provide, are not readily available. Addressing these limitations requires optimizing the design and fabrication of separators such that they are reduced in their size and fabrication cost, while also maintaining high-throughput separating capability. The emergence of the Lab-On-a-Chip (LOC) and then Lab-On-a-CD (LOCD) technologies, having more inherent benefits than conventional microfluidic devices, has created new opportunities and become increasingly widespread in recent years. Evidence suggests that employing single methodologies or integrating approaches without sufficient understanding of potential outcomes is unlikely to result in successful diagnostic results. This paper contributes an extensive review of several separation systems, including fundamental theories and experimental details, and describes detailed operating principles and device performance.

Background: Cell-free DNA (cfDNA) analysis has become a promising tool for diagnosis, prognosis and monitoring of lymphoma cases. Until now, research in this area has mainly focused in aggressive lymphomas with scanty information from other lymphoma subtypes. Methods: We selected 256 patients diagnosed with lymphomas including a large variety of B-cell and T-cell non-Hodgkin and Hodgkin lymphomas and quantified cfDNA from plasma at the time of diagnosis. We further selected 49 large B cell lymphomas (LBCL) and analyzed cfDNA levels at diagnosis (pre-therapy) and after therapy. In addition, we performed NGS in cfDNA and tissue in this cohort of LBCL. Results: Lymphoma patients showed a statistically significant higher cfDNA concentration than healthy controls (mean 53.0 ng/mL vs 5.6 ng/mL, p <0.001). The cfDNA concentration correlated with lymphoma subtype, lactate dehydrogenase, International Prognostic Index (IPI) score, Ann Arbor (AA) and B-symptoms. In 49 LBCL cases, the cfDNA concentration decreased after therapy in cases who achieved complete response (CR), and increased in non-responders. The median cfDNA at diagnosis of patients who achieved CR and later relapsed was higher (81.5 ng/mL) compared with levels of those who did not (38.6 ng/mL). A concordance of 84% was observed between NGS results in tumor and cfDNA samples. Higher VAF in cfDNA correlated with advanced stage and bulky disease. Conclusions: cfDNA analysis can be easily performed in almost all lymphoma cases. The cfDNA concentration correlated with the characteristics of aggressiveness of the lymphomas and in LBCL with the response achieved after therapy. These results support the utility of cfDNA analysis as a complementary tool in the management of lymphoma patients.

Blood-based circulating cell free DNA(cfDNA) detection offers a non-invasive and easily accessible way for early cancer detection. Despite the extensive utility of cfDNA, there are still many challenges to develop the clinical biomarkers. For example, cfDNA with genetic alterations often compose a small portion of the DNA circulating in plasma, which can be confounded by cfDNA contributed by normal cells. Therefore, filtering out the potential false-positive cfDNA mutations from health population will be important for the cancer-based biomarkers. Additionally, many low-frequency genetic alterations are easily overlooked in small amount of cfDNA-based cancer test. We hypothesize that, the combination of diverse types of cancer studies on cfDNA can provide us a new insight to identify low-frequency genetic variant across cancer types for early clinical detection of cancers. By building a standardized computational pipeline for 1358 cfDNA samples across seven cancer types, we prioritize 129 shard genetic variants in the major cancer types. Further functional analysis of the 129 variants found that they are mainly enriched in ribosome pathways such as cotranslational protein targeting to membrane, some of which are tumor suppressor, oncogene and related to cancer initiation. In summary, our integrative analysis revealed the important roles of ribosome proteins as the common biomarkers in early cancer diagnosis.

Cell-free DNA (cfDNA) analysis is a pivotal tool in non-invasive diagnostics, including cancer monitoring and prenatal testing. However, the preanalytical phase, particularly the choice of anticoagulant, significantly impacts cfDNA integrity and yield. This study aims to compare cfDNA yield, stability, and DNase activity across plasma-citrate, plasma-heparin, plasma-EDTA, and serum, to identify the most suitable specimen for cfDNA analysis among the tested anticoagulants. Blood samples from 15 healthy volunteers were collected in four types of tubes (citrate, heparin, EDTA, and serum). cfDNA was extracted and quantified using qPCR, and endogenous DNase activity was assessed through hydrolysis probe assays. Samples were incubated at 37°C for 24 hours to evaluate cfDNA degradation rates. Statistical analyses were performed using repeated measures ANOVA and Sidak's multiple comparison tests. Baseline cfDNA levels were highest in serum, followed by heparin-plasma, while EDTA and citrate-plasma exhibited the lowest levels. DNase activity was fully inhibited in EDTA-plasma, partially inhibited in citrate-plasma, and active in both heparin-plasma and serum. cfDNA degradation was most pronounced in heparin-plasma (85.3%) and serum (55.6%), compared to minimal degradation in EDTA (8%) and citrate-plasma (13.3%). Among the tested anticoagulants, EDTA-plasma proved to be the most suitable specimen for cfDNA analysis due to its low DNase activity and minimal gDNA contamination. Citrate-plasma, with intermediate DNase inhibition, could be an alternative if the citrate concentration were increased. Heparin-plasma and serum showed significant cfDNA degradation, rendering them unsuitable for cfDNA analysis. These findings highlight the importance of anticoagulant selection in preserving cfDNA integrity for diagnostic applications.

Biomarker directed selection of targeted anti-neoplastic agents such as immune check-point inhibitors, small molecule inhibitors and monoclonal antibodies form an important aspect of cancer treatment. Immunohistochemistry (IHC) analysis of the tumor tissue is the method of choice to evaluate the presence of these biomarkers. However, a significant barrier to biomarker testing on tissue is the availability of an adequate amount of tissue and need for repetitive sampling due to tumor evolution. Also, tumor tissue testing is not immune to inter- and intra-tumor heterogeneity. We describe the analytical and clinical validation of a Circulating Tumor Cell (CTC) assay to accurately assess the presence of PD-L1 22C3 and PD-L1 28.8, ER, PR and HER2, from patients with solid tumors to guide the choice of suitable targeted therapies. Analytically, the test has high sensitivity, specificity, linearity and precision. Based on a blinded case control study, the clinical sensitivity and specificity for PD-L1 (22C3 and 28.8) was determined to be 90% and 100% respectively. The clinical sensitivity and specificity was 83% and 89% for ER; 80% and 94% for PR; 63% and 89% for HER2 (by ICC); and 100% and 92% for HER2 (by FISH), respectively. The performance characteristics of the test support its suitability and adaptability for routine clinical use.

Circulating tumor DNA (ctDNA), the tumor-derived cell-free DNA fragments in the bloodstream carrying tumor-specific genetic and epigenetic alterations, represents an emerging novel tool for minimal residual disease (MRD) assessment in patients with resected colorectal cancer (CRC). For many decades, precise risk-stratification following curative-intent colorectal surgery has remained an enduring challenge. The current risk stratification strategy relies on clinicopathologic characteristics of the tumors that lacks precision and results in over-and undertreatment in a significant proportion of patients. Consequently, a biomarker that can reliably identify patients harboring MRD would be of critical importance in refining patient selection for adjuvant therapy. Several prospective cohort studies have provided compelling data suggesting that ctDNA could be a robust biomarker for MRD that outperforms all existing clinicopathologic criteria. Numerous clinical trials are currently underway to validate the ctDNA-guided MRD assessment and adjuvant treatment strategies. Once validated, the ctDNA technology will likely transform the adjuvant therapy paradigm of colorectal cancer, supporting ctDNA-guided treatment escalation and de-escalation. The current article presents a comprehensive overview of the published studies supporting the utility of ctDNA for MRD assessment in patients with CRC. We also discuss ongoing ctDNA-guided adjuvant clinical trials that will likely shape future adjuvant therapy strategies for patients with CRC.

Background: Liquid biopsies are revolutionary tools to detect tumor-specific genetic alterations in body fluids, and cell-free DNA (cfDNA) can be used for molecular diagnosis in cancer patients. In brain tumors cerebrospinal fluid (CSF) cfDNA might be more informative than plasma cfDNA. Here, we assess the use of CSF cfDNA in pediatric embryonal brain tumors (EBT) for molecular diagnosis. Methods: CSF cfDNA of pediatric patients with medulloblastoma (n=18), ATRT (n=3), ETMR (n=1), CNS NB FOXR2 (n=2) and pediatric EBT NOS (n=1) (mean cfDNA concentration 48 ng/mL; range 4-442 ng/mL) and matched tumor genomic DNA were sequenced by WES, and/or a targeted sequencing approach, to determine single nucleotide variations (SNVs)/copy number alterations (CNA). A specific capture covering transcription start sites (TSS) of genes of interest was used for nucleosome footprinting in CSF cfDNA. Results: 15/25 CSF cfDNA samples yielded informative results, with informative CNA and SNVs in 11 and 15 cases, respectively. For cases with paired tumor and CSF cfDNA WES (n=15), a mean of 83 (range 1-160) shared SNVs was observed, including SNVs in classical medulloblastoma genes such as SMO and KMT2D. Interestingly, tumor-specific SNVs (mean 18; range 1-62) or CSF-specific SNVs (mean 5; range 0-25) were observed suggesting clonal heterogeneity. The TSS panel resulted in differential coverage profiles across all 112 studied genes in 7 cases, indicating distinct promoter accessibility. Conclusion: CSF cfDNA sequencing yielded informative results in 60% of cases (15/25), with WES feasible in 83% (15/18). These results pave the way for implementation of these novel approaches for molecular diagnosis and minimal residual disease monitoring.

Patients with heart failure (HF) with improved LVEF (HFimpEF) demonstrate better clinical outcomes when compared with individuals without restoration of cardiac function. Identification of predictors for HFimpEF may play a crucial role in individual management of HF with reduced EF (HFrEF). Cell-free nuclear (cf-nDNA) DNA are released from damaged cells and contribute to adverse cardiac remodeling, cardiac dysfunction and inflammation. The purpose of the study was to elucidate whether cf-nDNAs are associated with HFimpEF. It has been prescreened 1416 patients with HF using local database. Between October 2021 and August 2022 we included 452 patients chronic HFrEF after prescription of optimal guideline-based therapy and identified HFimpEF in 177 individuals from them according to the criteria of European Society of Cardiology. Measurements of circulating biomarkers were performed at baseline and in 6 months. Detection of cf-nDNA was executed with real time quantitative PCR (qPCR) using NADH dehydrogenase, ND2 and beta-2-microglobulin. We found that HFimpEF was associated with a significant decrease in the levels of cf-nDNA when compared with the patients from persistent HFrEF cohort. Presence of ischemia-induced cardiomyopathy (odds ration [OR] = 0.75; confidence interval [CI] = 0.62 – 0.88; p = 0.044), type 2 diabetes mellitus (OR = 0.77; CI = 0.71 – 0.82; p = 0.042) and digoxin administration (OR = 0.85; CI = 0.72-0.97; p = 0.042) were negative factors for HFimpEF, whereas NT-proBNP ≤1940 pmol/mL (OR = 1.42; 95% CI = 1.19-1.98, p = 0.001), relative decrease in NT-proBNP levels (&gt;35% vs. ≤35%) from baseline (OR = 1.52; 95% CI = 1.38-0.69, p = 0.001) and cf-nDNA ≤7.5 μmol/L (OR = 1.56; 95% CI = 1.07-2.94, p = 0.001) were positive predictors for HFimpEF. Multivariate logistic regression adjusted to ischemia-induced cardiomyopathy, IV HF NYHA class, and digoxin use yielded that NT-proBNP ≤1940 pmol/mL (OR = 1.43; 95% CI = 1.21-1.88, p = 0.001) and cf-nDNA ≤7.5 μmol/L (OR = 1.64; 95% CI = 1.19-2.15, p = 0.001) independently predicted HFimpEF. In conclusions: we established that the levels of cf-nDNA≤7.5 μmol/L independently predicted HFimpEF and improved discriminative ability of as single measured NT-proBNP as well as relative decrease in NT-proBNP levels ≤35% from baseline.

(1) Background: donor-derived circulating free DNA (dd-cfDNA) is an innovative biomarker with great potential for the early identification and prevention of graft damage; (2) Methods: Prospective study to analyze dd-cfDNA plasma levels in 30 kidney transplant patients during their post-transplant follow-up (15 days, 3, 6 and 9 months), to determine if the result could be of interest in the identification of possible adverse events, specially allograft rejection. In addition, the aim is also to verify the obtained data on sensitivity, specificity, NPV and PPV; (3) Results: Plasma levels of dd-cfDNA could be considered a novel biomarker of graft rejection after first term post-transplant up to several months before its clinical presentation, mainly in TCMR or ABMR. In this study, it appears to have no positive predictive value for borderline rejection or TCMR IA. Our results show a low NPV of the technique because other pathologies or biological post-transplant complications can also affect dd-cfDNA levels and they must be considered in the interpretation of the result.; (4) Conclusions: dd cfDNA could allow, together with other additional biomarkers in the context of a future score, avoid invasive biopsies in most cases, mainly in the case of suspected TCMR or ABMR.

Gastric cancer is the 5th most common disease in the world and the 4th most common cause of death. It is diagnosed through esophagogastroduodenoscopy with biopsy, however there are limitations in finding lesions in the early stages. Recently, research has been actively conducted to use liquid biopsy to diagnose various cancers, including gastric cancer. Various substances derived from cancer are reflected in the blood. By analyzing these substances, it was expected that not only the presence or absence of cancer but also the type of cancer can be diagnosed. However, the amount of these substances is extremely small, and even these have various variables depending on the characteristics of the individual or the characteristics of the cancer. To overcome these, we collected methylated DNA fragments using MeDIP and compared them with normal plasma to characterize gastric cancer tissue or patient’s plasma. And we attempted to diagnose gastric cancer using the characteristics of cancer reflected in the blood through the cancer tissue and patient’s plasma. As a result, we confirmed that the consistency of common methylated fragments between tissue and plasma was approximately 41.2%, and found the possibility of diagnosing and characterizing cancer using the characteristics of the fragments through SFR and 5'end-motif analysis.

Tumor growth, progression, and therapy resistance are crucial factors in the prognosis of cancer. Properties of three-dimensional tumor-like organoids (tumoroids) more closely resemble in vivo tumors compared to two-dimensionally cultured cells and are therefore effectively used for assays and drug screening. We here established a repurposed drug for novel anticancer research and therapeutics using a tumoroid-based screening system. We screened 6 pharmacologically active compounds by using an original tumoroid-based multiplex phenotypic screening system with matrix metalloproteinase 9 (MMP9) promoter-driven fluorescence reporter for the evaluation of both tumoroid formation and progression. The effects of one of the hit compounds were examined on tumor formation and progression in vitro and in vivo. Antiparkinson drug benztropine was the most effective compound uncovered by the screen. Benztropine significantly inhibited in vitro tumoroid formation, cancer cell survival, and MMP9 promoter activity. Benztropine also reduced the activity of oncogenic signaling transducers and trans-activators for MMP9, including STAT3, NF-κB, and β-catenin, and properties of cancer stem cells / cancer-initiating cells. Benztropine and GBR-12935 directly targeted the dopamine transporter DAT/SLC6A3, whose genetic alterations such as amplification were correlated with poor prognosis for cancer patients. Benztropine also inhibited tumor growth, circulating tumor cell (CTC) number, and rate of metastasis in a tumor allograft model in mice. In conclusion, we propose the repurposing of benztropine for anticancer research and therapeutics that can suppress tumor progression, CTC, and metastasis of aggressive cancers by reducing key pro-tumorigenic factors.

Background: The AR-V7 splice variant is a cause of castration-resistant prostate cancer (CRPC). However, testing for the presence of AR-V7 by RT-PCR shows AR-V7 positivity in healthy individuals. We hypothesized that the positivity reflected contamination by hematopoietic cells. We tried a novel CTC enrichment instrument using Celsee® to clear hematopoietic cells. Methods: We tested whole blood or Celsee-enriched samples for AR-V7 by RT-PCR and included samples from 41 CRPC patients undergoing sequential therapy. We evaluated the associations between AR-V7 status and clinical factors. We evaluated factors affecting AR-V7 positivity. Results: AR-V7 positivity was lower in Celsee-enriched than in whole blood specimens. AR-V7 and clinical factors did not predict the therapy effectiveness. We found no significant differences in the effectiveness of Enz/Abi upon AR-V7 evaluation. All AR-V7 positive patients had resistance to Enz/Abi. DTX, CBZ, and Radium223 treatment showed no significant difference in the treatment effectiveness, regardless of AR-V7 presence. AR-V7 was more frequently positive than EOD2 in cases with bone metastases. Conclusion: Celsee® CTC enrichment suppresses the AR-V7 false positivity. All AR-V7 positive patients presented resistance to Enz/Abi. DTX, CBZ, and Radium223 were effective and remained treatment options. AR-V7 positivity should progressively appear in patients with advanced bone metastases.

Education outside the classroom (EOtC) can be beneficial for students. The relationship between biological stress markers and sedentary behavior (SB) plus physical activity (PA) is insufficiently evaluated in school settings. This exploratory study aims to evaluate the association between students’ cortisol, plus circulating cell-free deoxyribonucleic acid (cfDNA) levels, and their SB, light PA (LPA) and moderate-to-vigorous PA (MVPA) during outdoor and indoor classes in different seasons. We assessed data from an education outside the classroom (EOtC) program (n = 48; intervention group [IG], n = 37; control group [CG], n = 11). We sampled data on 3 school-days in three seasons (fall, spring, and summer) in normal teaching indoors (CG) and outdoor lessons (IG) in the forest. SB and PA were evaluated by accelerometry, and cortisol and cfDNA levels by saliva samples. The compositional data analysis approach analyzed SB and PA. Fitted Bayesian hierarchical linear models evaluated the association between cortisol and cfDNA, and SB/LPA/MVPA. A steady decline of cortisol in the outdoor setting is associated with relatively high levels of LPA. SB and MVPA tended to exhibit a similar effect in the indoor setting. CfDNA is positively associated with a relatively high amount of SB in the IG, the same association is likely for LPA and MVPA in both groups. LPA seems to support a healthy cortisol decrease in children during outdoor lessons. The relevance of SB/PA as a composition in relation to students stress response in school should be emphasized. This study facilitates the formulation of straightforward and directed hypotheses for further research.

Background: Current strategies in circulating tumor cell (CTC) isolation in pancreatic cancer heavily rely on the EpCAM and cytokeratin cell status. EpCAM is generally not considered as a good marker given its transitory change during Epithelial to Mesenchymal Transition (EMT) or reverse EMT. There is a need to identify other surface markers to capture the complete repertoire of PDAC CTCs. The primary objective of the study is to characterize alternate surface bi-omarkers to EpCAM on CTCs that express low or negligible levels of surface EpCAM in pancre-atic cancer patients. Methods: Flow cytometry and surface mass spectrometry were used to iden-tify proteins expressed on the surface of PDAC CTCs in culture. CTCs were grown under condi-tions of attachment and in co-culture with naïve neutrophils. Putative biomarkers were then validated in GEMMs and patient samples. Results: Surface proteomic profiling of CTCs identi-fied several novel protein biomarkers. ALCAM was identified as a novel robust marker in GEMM models and in patient samples. Conclusions: We identified several novel surface bi-omarkers on CTCs expressed under differing conditions of culture. ALCAM was validated and identified as a novel alternate surface marker on EpCAMlow CTCs.

Inflammatory bowel disease (IBD), including Crohn’s Disease (CD) and Ulcerative Colitis (UC) are chronic multifactorial disorders which affect the gastrointestinal tract with variable extent. Despite extensive research, their etiology and exact pathogenesis are still unknown. Cell-free DNAs (cfDNAs) are defined as any DNA fragments which are free from origin cell and able to circulate into bloodstream with or without microvescicles. CfDNAs are now being increasingly studied in different human diseases, like cancer or inflammatory diseases. However, to date it is unclear how IBD etiology is linked to cfDNAs in plasma. Extrachromosomal circular DNA (eccDNA) are non-plasmidic, nuclear, circular and closed DNA molecules found in all eukaryotes tested. CfDNAs appear to play an important role in autoimmune diseases, inflammatory processes, and cancer; recently, interest has also grown in IBD, and their role in the pathogenesis of IBD has been suggested. We now suggest that eccDNAs also plays a role in IBD. In this review we have comprehensively collected available knowledge in literature regarding cfDNA, eccDNA, and structures involving them such as neutrophil extracellular traps and exosomes, and their role in IBD. Finally, we focused on old and novel potential molecular therapies and drug delivery systems, such as nanoparticles, for IBD treatment.

Dysbiosis, as reflected by the altered composition of the microbiome favoring pathogenic species, is a major contributor to colorectal cancer (CRC) development, which could lead to bacterial translocation into the bloodstream. This study aimed to evaluate the presence of circulated bacterial DNA (cbDNA) in CRC patients, with or without surgical removal of the primary tumor. In total, 100 participants enrolled in this prospective clinicolaboratory study, equally divided into four groups; healthy controls (Group 1), patients with non-metastatic CRC with surgical removal of the primary tumor (Group 2), patients with metastatic CRC with surgically excised (Group 3) or non-excised (Group 4) primary tumor. DNA extracted from peripheral blood was analyzed using PCR with specific primers targeting 16S rRNA, Escherichia coli (E. coli), and Fusobacterium nucleatum (F. nucleatum). Only the detection of F. nucleatum in the blood was significantly higher in Group 4 compared to Group 1 (p < 0.001), Group 2 (p = 0.023) and Group 3 (p = 0.023). The association of cbDNA with other clinical parameters or co-morbidities was also evaluated. These results highlighted the importance of bacterial translocation in CRC patients and the role of F. nucleatum as an intratumoral oncomicrobe associated with metastatic CRC.

Detection methods have been developed to prevent transmission of zoonotic or xenozoonotic porcine viruses after transplantation of pig organs or cells to the recipient (xenotransplantation). Eleven xenotransplantation-relevant viruses including porcine cytomegalovirus, a porcine roseolovirus (PCMV/PRV), porcine lymphotropic herpesviruses -1, -2, -3 (PLHV-1, 2, 3), porcine parvovirus (PPV), porcine circovirus 2, 3, 4 (PCV2, 3, 4), hepatitis E virus genotype 3 (HEV3), porcine endogenous retrovirus-C (PERV-C) and recombinant PERV-A/C have been selected. In the past, several pig breeds, minipigs and genetically modified pigs generated for xenotransplantation had been analyzed using these methods. Here, spleen, liver and blood samples from 10 German slaughterhouse pigs were screened using both, PCR-based and immunological assays. Five viruses: PCMV/PRV, PLHV-1, PLHV-3 and PERV-C were found in all animals, and PCV3 in one animal. Some animals were latently infected with PCMV/PRV as only virus-specific antibodies were detected. Others were also PCR positive in spleen and/or liver, indicative of an ongoing infection. These results provide important information on the viruses that infect German slaughterhouse pigs and together with results of previous studies revealed that the methods and test strategies efficiently work under field conditions.

Notwithstanding the fact that there is some improvement for an earlier detection of patients with lung cancer, the majority of them still present with a late-stage disease at the time of diagnosis. Next to the most frequently used factors affecting the prognosis of lung cancer patients (stage, performance and age) the recent application of biomarkers obtained by liquid profiling gained more acceptance. In our study we aimed to answer these questions: i) is the quantification of free-circulating methylated PTGER4 and SHOX2 plasma DNA an useful method for the therapy monitoring and is this also possible for patients treated with different therapy regimens?, and ii) is this approach possible when blood drawing tubes are used which allow for a delayed processing of blood samples? Baseline values for mPTGER4 and mSHOX2 do not allow for a clear discrimination between different response groups. In contrast the combination of the methylation values for both genes show a clear difference between responders vs non-responders at the time of re-staging. Additionally, blood drawing into tubes stabilizing the sample give researchers more flexibility.

The global incidence of cutaneous squamous cell carcinoma (cSCC), a prevalent and aggressive skin cancer, has risen significantly, posing a substantial public health challenge. This study investigates the tumor microenvironment (TME) of cSCC by focusing on the spatial distribution patterns of immune and vascular markers (CD31, CD20, CD4, and CD8) using fractal dimension (FD) analysis. Our analysis encompassed 141 cases, including 100 invasive cSCC and 41 specimens with pre-invasive lesions exclusively, and the rest being peripheral pre-invasive lesions from the invasive cSCC class. The FD values for each marker were computed and compared between pre-invasive and invasive lesion classes. The results revealed significant differences in FD values between the two classes for CD20 and CD31 markers, suggesting distinct alterations in B cell distribution and angiogenic activity during cSCC progression. However, CD4 and CD8 markers did not exhibit significant changes individually. Still, the CD4/CD8 ratio showed a significant difference, indicating a potential shift in the immune landscape as lesions progress from pre-invasive to invasive stages. These findings underscore the complexity and heterogeneity of the TME in cSCC and highlight the potential of FD analysis as a quantitative tool for characterizing tumor progression. Further research is needed to elucidate the implications of these differences in the clinical management of cSCC.

The study delves into the Zaalklapspruit valley-bottom wetland in South Africa, a site designed through ecological engineering and currently impacted by acid mine drainage due to an upstream defunct coal mine. The research conducted in 2022, aimed to uncover the dynamics of contaminant dispersal within such a wetland setting, focusing on the origins, routes, and ultimate recipients of various metals and sulfur compounds. Analysis revealed that the bottom sediment is rich in organic material, with pH levels observed between 6.05 and 6.59, and exhibited notably low oxidation-reduction potentials, reaching down to −219.67 mV at sample point S3. Notably, Manganese displayed the highest adsorption rates, whereas Iron was predominantly absorbed by the roots of Typha capensis and the algae Klebsormidium acidophilum. The wetland's macrophyte rhizospheres hosted a rich array of microbiota, including families like Helicobacteraceae and Hydrogenophilaceae, which are crucial in metal and sulfur processing. This investigation sheds light on the intricate biogeochemical processes taking place involving the wetland's sediment, macrophyte root systems, periphyton, and microbial populations, underscoring the potential of ecologically engineered wetlands in the remediation of waters affected by acid mine drainage.

Nose mask is a fomite and its usage was mandatory during the COVID-19 outbreak. Its effectiveness has been established; however, as a fomite, it can harbor opportunistic respiratory pathogens such as Streptococcus spp., which could complicate COVID-19 infections. This study profiled Streptococcus species resident on nose masks obtained from consenting participants. Phenotypic assays and 16S rRNA sequencing were used to identify Streptococcus spp. resident on the interior and exterior of 100 nose masks analyzed. Antibiotic resistance (AMR) was determined with disc diffusion and broth microdilution assays. AMR and virulence makers were profiled with primer-specific PCR amplification. 23% of the analyzed samples had Streptococcus isolates. The isolates were highly resistant to the antimicrobials, including conventional, last-resort antibiotics, disinfectants and antiseptics. All the isolates are multidrug resistant with 80-100% levels of AMR to the 16 antibiotics tested with a Multiple-Antibiotic Resistance index greater than 0.2, indicative of potential pathogens. The isolates harbored tetB, ermB, pbp2B, pbp2b1 (resistance) and lytA, lytA1, ply1, cbpA, lytA, pavA (virulence) markers, which might constitute a serious public health risk. Overall, the study emphasizes the need to assess mask usage and the risks associated with opportunistic pathogens that are resident on frequently used nose masks in the COVID-19 era.

This study focused on the precise detection and concordant analysis of circulating tumor cells (CTCs) to verify the usefulness of CTCs as promising clinical markers. CTCs are traditionally achieved using immune-fluorescence and morphological techniques. By combining two prostate cancer-specific genes (PSA and PSMA) and two epithelial-specific genes (EpCAM and KRT19), the CTC detection efficiency can be enhanced. The detection rates of localized prostate cancer (LPC), locally advanced prostate cancer (LAPC), metastatic hormone-sensitive prostate cancer (mHSPC), and metastatic castration-resistive prostate cancer (mCRPC) were 63.04 % (29/46), 70.59% (34/51), 94.34% (50/53), and 90% (126/140), respectively. Subsequent correlation analysis between the four genes, baseline PSA, and CTC count revealed that only the CTC count correlated with baseline PSA in the localized stages of prostate cancer, whereas all four genes were correlated with baseline PSA and CTC count in the metastatic stages of prostate cancer. The precise detection of CTCs using both numerical and transcriptomic analyses can improve the accuracy of biomarker investigations. This study provides valuable insights into the limitations of serum PSA testing and offers a promising alternative to diagnose and manage prostate cancer via the CTC number and specific genes for prostate cancer.

Liquid biopsy has been developed to diagnose and monitor cancer. Despite the recent advances in investigating early cancer detection and diagnosis strategies, there is still a lack of improvements in capturing (CTCs). To archive this, we developed an efficient CTC detection system by integrating gold nanoparticles and a microfluidic platform, which can achieve CTC capture within 120 min. The microdevice was functionalized by immobilizing antibodies specific to the target cells. The method was tested with spiked tumor cells in whole human blood with three pancreatic cancer cell lines and compared with purified cancer cells suspended in a phosphate buffer solution. Our results captured pancreatic cancer cells spiked in blood samples with efficiency (90±3.2)% and were used to isolate CTCs from patients with metastatic pancreatic cancer. This methodology will enable the production of functionalized, patterned surfaces that interact with tumor cells, enhancing the selective capture of CTCs from biological assays.

Acute and chronic graft versus host disease (a/cGVHD) are major complications of allogeneic stem cell transplantation and a frequent cause of non relapse-mortality. The treatment of a/cGVHD consists of steroid administration at full dose. In the case of aGVHD, around 60 % of patients with grade III/IV fails to respond (steroid-resistant, SR), requiring a second line therapy. Extracorporeal photopheresis (ECP) represents a good option for second line treatment, leading to multiorgan response in several patients. Here, we have evaluated the performances of a cytofluorimetric quality control of the first ECP procedure in 29 patients with severe SR aGVHD who underwent ECP. The quality control represents a check control of in vitro cell apoptosis/death induction in collected and irradiated CD3+ cells by ECP. Our analysis shows that in more than 70 % quality controls a relevant apoptotosis/death of CD3+ cells was observed after cell irradiation, as compared to non-irradiated controls. Albeit observed in a small patients’ series, the occurrence of relevant in vitro effect after ECP relates to less procedures and/or to a better survival on ECP. Age, disease, gender, aGVHD grade and time from transplant to the first ECP had not impact on ECP efficacy in this patients’ series.

Shigella and Salmonella have been implicated in neonatal sepsis, food poisoning, and bacteremia. Their increasing resistance to antibiotics has undermined treatment options, constituting a public health crisis. Also, the scarcity of new antibiotics in the development pipeline compounds multidrug-resistance (MDR). Hence, there is a need to potentiate antibiotics to mitigate resistance. This study leveraged riboflavin to re-sensitize fluoroquinolones and carbapenems against MDR Shigella and Salmonella spp. Disk diffusion, microbroth-dilution, chequerboard, and time-kill assays were used to determine resistance profiles and riboflavin-antibiotic activity. Biofilm-formation and bacterial viability (after riboflavin-antibiotic challenge) was determined with crystal-violet and Macrophage infection assays, respectively. Strains exhibited &gt;80% resistance to fluoroquinolones and carbapenems, with 160–320 µg MIC ranges and 0.5–2 FIC, indicating no synergy. At 40–80 µg riboflavin, there was two-fold reduction in bacterial survival, with meropenem-riboflavin exhibiting significant reduction and FIC of 0.075 indicating high synergistic activity. Biofilm-formation was altered (from strong to weak phenotype) with 40 µg riboflavin-antibiotic treatment. There was a 40–70% reduction in bacterial survival in THP-1 cells treated with meropenem-riboflavin after 3 hours, and complete clearance between 24-48 hpi. Riboflavin potentiated fluoroquinolones and meropenem, leading to modulation of the host immune response for the clearance of the strains.

Sepsis is a life-threatening syndrome triggered by infection and accompanied by high mortality, with antimicrobial resistances (AMRs) further escalating clinical challenges. Rapid and reliable detection of causative pathogens and AMRs are key factors for fast and appropriate treatment, in order to improve outcome in septic patients. However, current sepsis diagnostics based on blood culture is limited by low sensitivity and specificity while current molecular approaches fail to enter clinical routine. Therefore, we developed a Suppression PCR-based selective enrichment sequencing approach (SUPSETS), providing a molecular method combining multiplex suppression PCR with Nanopore sequencing to identify most common sepsis-causative pathogens and AMRs using plasma cell-free DNA. Applying only 1 mL of plasma, we target eight pathogens across three kingdoms and ten AMRs in a proof-of-concept study. SUPSETS was successfully tested on first ten clinical samples and revealed comparable results to clinical metagenomics while clearly outperforming blood culture. Several clinically relevant AMRs could be detected additionally. Furthermore, SUPSETS provided first pathogen and AMR-specific sequencing reads within minutes of starting sequencing, thereby potentially decreasing time to results to 11 - 13 hours and suggesting diagnostic potential in sepsis.

Keywords: Cell-free DNA, sepsis, oxidative stress, immunoredox modulation, cecal ligation and puncture, toll-like receptor

Background and Objectives: Age-related macular degeneration (AMD) is one of the major causes of central vision loss in the older age group and its dry form accounts for the majority of cases. Although several causes and mechanisms for the development and progression of AMD have previously been identified, the pathogenesis of this complex disease is still not entirely understood. As the inflammation and involvement of the immune system are strongly suggested to have a central role to promote the degenerative process and to stimulate the onset of complications, we aimed to analyze the prevalence of serum anti-retinal (ARA) and anti-endothelial cell antibodies (AECA) in patients with dry AMD and to determine their relationship with the clinical features of the disease, notably area of geographic atrophy (GA). Materials and Methods: We enrolled 41 patients with dry AMD and 50 sex- and age-matched healthy controls without AMD. The presence of ARA was determined by indirect immunofluorescence using monkey retina as an antigen substrate, while the presence of AECA was determined using cultivated human umbilical vein endothelial cells and primate skeletal muscle. Results: ARA were detected 36 (87.8%) AMD patients (titers range from 1:20 to 1:320) and in 16 (39.0%) (titers range from 1:10 to 1:40) of controls (p=0.0000). Twenty of the 41 patients (48.8%) were positive for AECA, while in a control group AECA were present only in 5 sera (10.0%). The titers of AECA in AMD patients ranged from 1:100 to 1:1000 and in the control group the AECA titers were 1:100 (p=0.0001). There were no significant correlations between the presence of AECA and disease activity. Conclusions: This study provides evidence for the possible involvement of an autoimmune process against retina and retinal vessel antigens in the pathogenesis of geographic atrophy associated with dry AMD.

As non-operative management (NOM) of esophageal and rectal cancer is becoming more prevalent, blood-biomarkers such as circulating tumor DNA (ctDNA) may provide clinical information in addition to endoscopy and imaging to aid in treatment decisions following chemotherapy and radiation therapy. In this feasibility study, we prospectively collected plasma samples from locally advanced esophageal (n=3) and rectal cancer (n=2) patients undergoing multimodal neoadjuvant therapy to assess the feasibility of serial ctDNA monitoring throughout neoadjuvant therapy. Using the DIDA-Seq error-correction method, we serially interrogated plasma cell-free DNA at 28-41 tumor-specific genomic loci throughout therapy and in surveillance with an average limit of detection of 0.016% mutant allele frequency. In both rectal cancer patients, ctDNA levels were persistently elevated following total neoadjuvant therapy with eventual detection of clinical recurrence prior to salvage surgery. Among the esophageal cancer patients, ctDNA levels closely correlated with tumor burden throughout and following neoadjuvant therapy, which was associated with a pathologic complete response in one patient. In this feasibility study, patient and tumor-specific ctDNA levels correlated with clinical outcomes throughout multi-modality therapy suggesting that serial monitoring of patient ctDNA has the potential to serve as a highly sensitive and specific biomarker to risk-stratify esophageal and rectal cancer patients eligible for NOM. Further prospective investigation is warranted.

This study presents the first genome assembly of the freshwater saprobe fungus Neonectria lugdunensis and a comprehensive phylogenomics analysis of the Nectriaceae family, examining genomic traits according to fungal lifestyles. The Nectriaceae family, one of the largest in Hypocreales, includes fungi with significant ecological roles and economic importance as plant pathogens, endophytes, and saprobes. The phylogenomics analysis identified 2684 single-copy orthologs, providing a robust evolutionary framework for the Nectriaceae family. We analyzed the genomic characteristics of 17 Nectriaceae genomes, focusing on their carbohydrate-active enzymes (CAZymes), biosynthetic gene clusters (BGCs), and adaptations to environmental temperatures. Our results highlight the adaptation mechanisms of N. lugdunensis, emphasizing its capabilities for plant-litter degradation and enzyme activity in varying temperatures. Comparative genomics of different Nectriaceae lifestyles revealed significant differences in genome size, gene content, repetitive elements, and secondary metabolite production. Endophytes exhibited larger genomes, more effector proteins, and BGCs, while plant pathogens had higher thermo-adapted protein counts, suggesting greater resilience to global warming. In contrast, the freshwater saprobe shows less adaptation to warmer temperatures and is important for conservation goals. This study underscores the importance of understanding fungal genomic adaptations to predict ecosystem impacts and conservation targets in the face of climate change.

Plant cells' ability to withstand abiotic stress is strongly linked to modifications in their mechanical characteristics. Nevertheless, the lack of a workable method for consistently tracking plant cell mechanical properties severely restricts our comprehension of the mechanical alterations in plant cells under stress. With Polyethylene Glycol (PEG6000), we created a drought-like environment, and we used the Double Resonator Piezoelectric Cytometry (DRPC) method to dynamically and non-invasively track changes in the stress (ΔS) created and viscoelasticity (storage modulus G' and loss modulus G") of protoplasts and suspension cells of rice during drought stress. The findings demonstrate that, rice suspension cells and protoplasts react mechanically differently to 5%–15% PEG6000 stress, implying distinct resistance mechanisms. However, neither of them can withstand 25% PEG6000 stress, they respond mechanically similarly to 25% PEG6000 stress. The results of DRPC are further corroborated by the morphological alterations of rice cells and protoplasts observed under an optical microscope. To sum up, the DRPC technique functions as a precise cellular mechanical sensor and offers novel research tools for the evaluation of plant cell adversity and differentiating between the mechanical reactions of cells and protoplasts under abiotic stress.

γδT have functions of innate and adaptive immunity, with the potential to induce durable responses while being well-tolerated, with limited adverse effects, making it attractive as a tool for immunotherapy. γδT faces challenges as a frontline tool in clinical oncology, with limited response rates due to difficulties in reaching tumor sites with consistent cytotoxic activity and strength. Modulated Electro-hyperthermia (mEHT) is a loco-regional treatment, whereby energy-transmission from an electromagnetic field selectively targets the plasma membrane of tumor cells, inducing apoptosis and activating immune cells. We hypothesized that mEHT could enhance therapeutic effects by drawing γδT to tumor cells, while also rendering tumor cells to be more susceptible to cytotoxic effects. In this study, NOD/SCID mice harboring subcutaneous human HepG2 tumors were treated with intravenous injections of γδT after mEHT treatment. This method increased infiltration of γδT into the tumor site, significantly inhibiting tumor growth as compared to monotherapy with either modality. These data suggest that γδT could mediate a potent anti-tumor effect when combined with mEHT, and provide a strong rationale for combining these modalities in clinical application for cancer treatment.

The efficient preparation of single-stranded DNA (ssDNA) rings, as a macromolecular construction approach with topological features, has aroused much interest due to their numerous applications in biotechnology and DNA nanotechnology. However, an extra splint is essential for enzymatic circularization, and by-products of multimers are usually present at high concentrations. Here, we proposed a simple and robust strategy of using permuted precursor (linear ssDNA) for circularization by forming an intramolecular dynamic nick using a part of the linear ssDNA substrate itself as the template. After the simulation of the secondary structure for desired circular ssDNA, the linear ssDNA substrate is designed to have its ends on the duplex part (≥5 bp). By using this permuted substrate with 5'-phosphate, the splint-free circularization is simply carried out by T4 DNA ligase. Very interestingly, formation of only several base pairs (2–4) flanking the nick is enough for ligation, although they form only instantaneously under ligation conditions. More significantly, the 5 bp intramolecular duplex part commonly exists in genomes or functional DNA, demonstrating the high generality of our approach. Our findings are helpful for understanding the mechanism of enzymatic DNA ligation from the viewpoint of substrate binding.

We have designed cell penetrating peptides that target the leucine zipper transcription factors ATF5, CE-BPB and CEBPD and that promote apoptotic death of a wide range of cancer cell types, but not normal cells, in vitro and in vivo. While such peptides have the potential for clinical application, their mechanisms of action are not fully understood. Here, we show that one such peptide, Dpep, compromises glucose uptake and glycolysis in a cell-context-dependent manner (in about 2/3 of cancer lines assessed). These actions are dependent on induction of tumor suppressor TXNIP (thioredoxin interacting protein) mRNA and protein. Knockdown studies show that TXNIP significantly contributes to apoptotic death in those cancer cells in which it is induced by Dpep. The metabolic actions of Dpep on glycolysis led us to explore combinations of Dpep with clinically approved drugs metformin and atovaquone that inhibit oxidative phosphorylation and that are in trials for cancer treatment. Dpep showed additive to synergistic activities in all lines tested. In summary, we find that Dpep induces TXNIP in a cell-context-dependent manner that in turn sup-presses glucose uptake and glycolysis and contributes to apoptotic death of a range of cancer cells.

We investigated the development of a simple and sensitive assay for the identification of minute amounts of animal and human biological material. The suggested system uses isothermal amplification of DNA in a rapid assay format, which returns the results in as little as 40 minutes from sampling. The assay used for the proof of concept employs the novel handheld SaLux19 device (Max-Planck Innovation prototype, Germany). This well-established infield detection system could also be utilized for forensic scenarios. The results presented here demonstrated that the assay is sufficiently specific and sensitive and can detect the presence of Sus scrofa nucleic acids.

Cell-culture meat is a novel concept to replace traditional meat produced from animals, and to satisfy rising food demands due to the world population growth. It is still a young field and requires to solve technical issues including cell sources and types, efficient culture for large scale production, taste and texture for customer satisfaction. The current review summarized traditional beef meat production and evaluation methods for customer satisfaction. In particular, serum biomarkers for beef meat evaluation are discussed, which can be applicated for cell-culture meat production.

Currently available DNA detection techniques frequently require compromises between simplicity, speed, accuracy, and cost. Here, we propose a simple, label-free and cost-effective DNA detection platform developed at screen printed carbon electrodes (SPCEs) modified with reduced graphene oxide (RGO) and gold nanoparticles (AuNPs). The preparation of the detection platform involved a two-step electrochemical procedure based on GO reduction onto SPCEs followed by the electrochemical reduction of HAuCl4 to facilitate the post-grafting reaction with AuNPs. The final sensor was fabricated by the simple physical adsorption of a single-stranded DNA (ssDNA) probe onto AuNPs-RGO/SPCE electrode. Each preparation step was confirmed by morphological and structural characterization using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy, respectively. Furthermore, the electrochemical properties of the modified electrodes have been investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results demonstrated that the introduction of AuNPs onto RGO/SPCEs led to an enhancement in surface conductivity, characteristic that favored an increased sensitivity in detection. The detection process relied on the change in the electrochemical signal induced by the binding of target DNA to the bioreceptor, and was monitored particularly by the change in the charge transfer resistance of a [Fe(CN)6]4−/3− redox couple added in the test solution.

Cell free systems are a widely used research tool in systems and synthetic biology and a promising platform for manufacturing of proteins and chemicals. In the past, cell free biology was primarily used to better understand fundamental biochemical processes. Notably, E. coli cell free extracts were used in the 1960s to decipher the sequencing of the genetic code. Since then, the transcription and translation capabilities of cell free systems have been repeatedly optimized to improve energy efficiency and protein yield. Today, cell free systems, in combination with the rise of synthetic biology, have taken on a new role as a promising technology for just in time manufacturing of therapeutically important biologics and high-value small molecules. They have also been implemented in an industrial scale for the production of antibodies and cytokines. In this review, we discuss the evolution of cell free systems, advancements in cell free protein synthesis, and cell free metabolic engineering, and conclude with discussing the importance and feasibility of mathematical modeling in cell free systems.

Reconstructing the Tree of Life remains a central goal in biology. Early methods, relying on small numbers of morphological or genetic characters, often yielded conflicting evolutionary histories, undermining confidence in the results. Investigations based on phylogenomics, which use hundreds to thousands of loci for phylogenetic inquiry, have provided a clearer picture of life's history, but certain branches remain problematic. To resolve difficult nodes on the Tree of Life, two recent studies tested the utility of synteny, the conserved collinearity of orthologous genetic loci in two or more organisms, for phylogenetics. Synteny exhibits compelling phylogenomic potential while also raising new challenges. We identify and discuss specific opportunities and challenges that bear on the value of synteny data and other rare genomic changes for phylogenomic studies. Synteny-based analyses of highly contiguous genome assemblies mark a new chapter in the phylogenomic era and the quest to reconstruct the Tree of Life.

We examine worldwide Property and Casualty and Life and Health insurance companies from 2004 until 2023, implementing panel regression models and mediation analysis to show that insurers raise their reserves when they face increasing costs for their potential environmental damages, ultimately reducing their profitability and underwriting capacity. Our findings extend to the insurance sector the previous evidence on banks, demonstrating that environmental damages could affect profits and solvency of financial intermediaries. These insights are important especially for insurance managers and regulators.

Iron oxide nanozymes are a form of nanomaterial with both superparamagnetic and enzyme-mimicking properties. Given the multifunctional nature of iron oxide nanozymes, it is attractive for creating iron oxide hybrid nanozymes through biomolecular modifications to imbue with auxillary properties. Such iron oxide hybrid nanozymes can be useful for rapid and cost-effective analysis of circulating tumor nucleic acids (ctNAs) in patient liquid biopsies during minimal residual disease (MRD) monitoring of cancer recurrence. Herein, the use of streptavidin-modified iron oxide hybrid nanozymes is reported for magnetic enrichment and bioelectrocatalytic sensing of three prostate cancer (PCa) ctRNA biomarkers with high detection specificity and sensitivity (10 copies) over an ultrabroad dynamic range (five orders of magnitude). Furthermore, the feasibility of ctRNA analysis for pre- and post-cancer treatment MRD monitoring is demonstrated using PCa urinary liquid biopsy samples.

We investigated the diagnostic capacity of selected circulating biomarkers (CBMs) for the early detection of bone metastasis (BMets) in patients with pancreatic neuroendocrine neoplasms (PanNENs). 115 patients with PanNENs and 40 controls were enrolled. We measured the serum levels of ferritin, cytokeratin 18 (CY18), CA19-9, CA125, AFP, CEA, and beta-2 microglobulin (B2M). 8 PanNENs patients developed BMets, and 107 remained BMets-free. We observed a significantly higher level of CA125 and CY18 in BM-PanNENs patients vs. non-BM-PanNENs patients (p = 0.01 and p = 0.04, respectively). CA125, CY18, and B2M area under receiver operator characteristic (AUROC) analyses differentiated BM-PanNENs from non-BM-PanNENs patients; CA125 area under the curve (AUC) 0.77, p < 0.01; CY18 AUC data were 0.72, p = 0.03, and B2M AUC 0.67, p = 0.02. Based on CBMs metrics in both subgroups, we reached a sensitivity/specificity for CA125 of 75/76%; for CY18 of 75/69%, for B2M of 100/50%, for CA125 and CY18 combination 93/90%, respectively. The useful CBMs for BM-PanNENs patients detection were CA125, CY18, and B2M. They seem to have the diagnostic capacity as a fair single biomarker and CA125&CY18 combination panel for the detection of BMets.

Tumors are complex tissues of malignant cells surrounded by a diverse cellular micro-environment with which they interact. The molecular and cellular underpinnings of cancer cells have been increasingly understood during the last few decades. Nonetheless, a great deal remains unknown regarding the ways in which tumour cells modify their surroundings and impact the organisation and makeup of cells. This review aims to concentrate on recent advancements in molecular characterization of tumor cells and its immunology, provides recent and general insight into cancer immunity and immunotherapy, and discusses the immunobiology of human adaptive and innate immunity to malignant growth of cancer cells, the mechanisms that support human immune response from beneath, an up-to-date scientific development to cancer immunotherapy, and how tumor and host responses can Encircle as a military tactic effective anti-cancer immunity.

Since their identification over twenty-five years ago, the plethora of cell penetrating peptides (CPP) and their applications has skyrocketed. These 5 to 30 amino acid long peptides have the unique property of breaching the cell membrane barrier while carrying cargoes larger then themselves into cells in an intact, functional form. CPPs can be conjugated to fluorophores, activatable probes, radioisotopes or contrast agents for imaging tissues, such as tumors. There is no singular mechanism for translocation of CPPs into a cell, and therefore, many CPPs are taken up by a multitude of cell types, creating the challenge of tumor specific translocation and hindering clinical effectiveness. Varying strategies have been developed to combat this issue and enhance their diagnostic potential by derivatizing CPPs for better targeting by constructing specific cell activated forms. These methods are currently being used to image integrin expressing tumors, breast cancer cells, human histiocytic lymphoma and protease secreting fibrosarcoma cells, to name a few. Additionally, identifying safe, effective therapeutics for malignant tumors has long been an active area of research. CPPs can circumvent many of the complications found in treating cancer with conventional therapeutics by targeted delivery of drugs into tumors, thereby decreasing off-target side effects, a feat not achievable by currently employed conventional chemotherapeutics. Myriad types of chemotherapeutics such as tyrosine kinase inhibitors, anti-tumor antibodies and nanoparticles can be functionally attached to these peptides leading to the possibility of delivering established and novel cancer therapeutics directly to tumor tissue. While much research is needed to overcome potential issues with these peptides, they offer a significant advancement over current mechanisms to treat cancer. In this review, we present a brief overview of the research leading to identification of CPPs with a comprehensive state of the art review on the role of these novel peptides in both cancer diagnostics as well as therapeutics.

Mesenchymal stem cells (MSCs) hold immense therapeutic potential due to their regenerative and immunomodulatory properties. However, to utilize this potential, it is crucial to optimize their in vitro cultivation conditions. Three-dimensional (3D) culture methods using cell-laden hydrogels aim to mimic the physiological microenvironment in vitro, thus preserving MSC biological functionalities. Cellulosic hydrogels are particularly promising due to their biocompatibility, sustainability, and tunability in terms of chemical, morphological, and mechanical properties. This study investigated the impact of (1) two physical crosslinking scenarios for hydrogels derived from anionic cellulose nanofibers (CNF) used to encapsulate adipose-derived MSCs (adMSCs) and (2) physiological culture conditions on the in vitro proliferation, differentiation, and extracellular vesicle (EV) production of these adMSCs. Our results revealed that additional Ca2+-mediated crosslinking, intended to complement self-assembly and gelation of aqueous to-CNF in the adMSC cultivation medium, adversely affected both the mechanical properties of the hydrogel spheres and the growth of the encapsulated cells. However, cultivation under dynamic and hypoxic conditions significantly improved the proliferation and differentiation of the encapsulated adMSCs. Furthermore, we demonstrated that adMSCs in CNF hydrogel spheres exhibited potential for scalable EV production with potent immunosuppressive capacities in a bioreactor system. These findings underscore the importance of physiological culture conditions and the suitability of cellulosic materials for enhancing the therapeutic potential of MSCs. Overall, our study provides valuable insights for optimizing the in vitro cultivation of MSCs for various applications, including tissue engineering, drug testing, and EV-based therapies.

Pulmonary hypertension (PH) is a progressive and potentially fatal complication of sickle cell disease (SCD) affecting 6-10% of adult SCD patients. Various mechanisms and theories have been evaluated to explain the pathophysiology of the disease. However, questions remain, particularly regarding the clinical heterogeneity of the disease in terms of symptoms, complications, and survival. Beyond the classical mechanisms that have been thoroughly investigated and include hemolysis, nitric oxide availability, endothelial disorders, thrombosis, and left heart failure, attention is focused nowadays on the potential role of the genes involved in such processes. Potential candidate genes are investigated through next-generation sequencing, with the TGF-β pathway being the initial target. This field of research may also provide novel targets for pharmacologic agents in the future as is already the case with idiopathic PH. The collection and processing of data and samples from multiple centers can yield safe results that will allow a better understanding of SCD-related PH as a part of the disease’s clinical spectrum. The following review attempts to capture the most recent findings of studies on gene polymorphisms that have been associated with PH in SCD patients.

Music has the innate potential to reach all parts of the brain, stimulates certain brain areas which are not achievable through other modalities. Music Therapy (MT) is being used for more than a century to treat individuals who needs personalized care. MT optimizes motor, speech and language responsibilities of the brain and improves cognitive performance. Pervasive developmentdisorder (PDD) is a multifaceted, neuro developmental disorder and autism spectrum disorder (ASD) comes under PDD, which is defined by deficiencies in three principal spheres: social connection with others, communicative and normal movement skills. The conventional imaging studies illustrate reduced brain area connectivity in people with ASD, involving selected parts of the brain cortex. People with ASD express much interest in musical activities which engages the brain network areas and improves communication and social skills.The main objective of this review is to analyze the potential role of MT in treating the neurological conditions, particularly ASD. Evidence based studies have reported the extensive therapeutic application of music on various part of the brain in a nonverbal child with autism through hearing or making music.Hence we hypothesized that MT intervention can improve the communication capacity in people with ASD, than customary neurorestoration therapy alone.

With its unique properties and potential applications, nanoparticle-based delivery platforms for vac-cines have gained significant attention in recent years. Nanoparticles have the advantages of enhancing immunogenicity, targeting delivery and improving stability, providing a new solution for drug and vaccine delivery. And in some clinical studies, a variety of nanoparticle delivery platforms have been gradually applied to a wide range of vaccine applications. Current research priorities are exploring various types of nanoparticles as vaccine delivery system to enhance vaccine stability and immunogen-icity. Lipid nanoparticles have shown promising potential in preclinical and clinical studies to efficiently deliver antigens to immune cells. Moreover, lipid nanoparticles (LNPs) and other nanoparticles as nu-cleic acids especially mRNA delivery system, have shown vast potential for vaccine development. In this review, we present various vaccine platforms with an emphasis on nanoparticles for mRNA nano-vaccine delivery vehicles. We introduce several novel nanoparticles delivery platforms for mRNA vac-cine such as lipid, polymer, protein-based nanoparticle and so on. In addition, we overview the an-ti-tumor immunity of nanovaccine against different tumors in cancer immunotherapy. Finally, we give an outlook future perspectives and remaining challenges for this promising technolo-gy-nanoparticle-based delivery platforms for vaccines.

The symmetry properties of an ornament contain information about its civilisation and its interactions with other cultural sources. Two-dimensional periodic ornaments can be strictly classified into a limited set of 17 mathematical symmetry groups, also known as wallpaper groups. The collection of ornaments thus classified for a civilisation is characteristic of the cultural group and serves as a fingerprint to identify that group. If the distribution of wallpaper groups is available for several societies, mathematical methods can be applied to determine similarities and differences between the art practices of these communities. This method allows a systematic approach to the general ornamental practices within a culture and their interactions in the form of similarity of fingerprints. We test the feasibility of the method on examples of medieval Armenians, Byzantium, Seljuks first in Persia and then in Anatolia and among Arabs in the Middle East. For this purpose we present the distribution of the planar ornaments and calculate the Euclidean distances in pairs. We tested to what extend geographical and religious factors could account for the observed similarity of ornamental groups between cultures. The results suggest an intensive interaction between the Seljuk Turks and Arab craftsmen who produced the ornaments. Therefore the cultural interactions are religiously motivated.

Due to the lack of a diagnostic tumor marker (TM), the diagnosis of pancreatic neuroendocrine neoplasia (PNEN) is usually delayed, and a large part of patients present with regional (RM) and distant metastases (DM). The project aimed to assess the serum TM levels in PNEN and their relationships with disease extent and grade. One hundred fifteen patients with PNEN and 40 healthy subjects (HS) were enrolled. Most of the PNEN were well-differentiated (93.04%) and non-functional (93.91%). Beta-2 microglobulin (BMG), carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA), cytokeratin 18 (CY18), and ferritin concentrations in PNEN patients were significantly higher than in HS (p &lt; 0.01). The highest area under the curve (AUC) ≥ 0.7 for differentiating PNEN patients from HS had CY18, CA19-9, and ferritin (p &lt; 0.001). PNEN disease was localized (LD) in 63 patients, 8 had RM, and 44 exhibited DM. Patients with metastatic PNEN (RM, DM) also showed significantly higher levels of CY18, CA125, and CEA than those without metastases (p &lt; 0.05). In conclusion, CY18, CA19-9, and ferritin were serum biomarkers that fair diagnosed PNEN disease. Elevated CY18, CA125, and CEA levels correlated with clinical stages (RM, DM) and elevated CY18, CA125 with high grade (G3) of disease.

The approval of monoclonal antibodies against programmed death-ligand 1 (PD-L1) and programmed cell death protein (PD1), has changed the landscape of cancer treatment. To date, many Immune Checkpoint Inhibitors (ICIs) have been approved by the FDA for the treatment of metastatic cancer as well as locally recurrent advanced cancer. However, immune-related adverse events (irAEs) of ICIs highlight the need for biomarker analysis with strong predictive value. Liquid biopsy is an important tool for clinical oncologists to monitor cancer patients and administer or change appropriate therapy. CTCs frequently express PD-L1 and this constitutes a clinically useful and non-invasive method to assess PD-L1 status in real time. This review summarizes all the latest findings about the clinical significance of CTC for the management of cancer patients during administration of immunotherapy and mainly focuses on the assessment of PD-L1 expression in CTCs.

Bladder cancer (BC) is one of the most lethal genitourinary malignancies owing to its propensity for recurrence and poor survival. The biochemical pathway, signal transducer and activator of transcription 3 (STAT3), has gained significance as a molecular pathway that promotes proliferation, invasion, and chemoresistance. In this study, we explored the targeting of STAT3 with TTI-101 and SH5-07 in bladder cancer (BCa) and elucidated the mechanisms in three-dimensional (3D) spheroid and tumoroid models. We optimized the growth of spheroids from human, rat, and mouse BCa cell lines (J82, NBT-II, and MB49, respectively), and tumoroids from BBN (N-butyl-N-(4-hydroxybutyl)-nitrosamine) induced rat bladder tumors. Cell viability was assessed using MTT and trypan blue assays. ATP production, ROS production, and calcium AM (CA)/EtBr staining were used to measure the spheroid and organoid inhibition and mitochondrial function. Western blot analysis was performed to evaluate the pharmacodynamic markers involved in cell proliferation, apoptosis, cancer stem cells (CSCs), and STAT3 signaling in BCa. We found that targeting STAT3 (using TTI-101 and SH5-07) significantly reduced the proliferation of BCa spheroids, and tumoroids, which was accompanied by decreased expression of pSTAT3, Cyclin D1, and PCNA. Our data also demonstrated that treatment with STAT3 inhibitors induced ROS production and cell death in BCa spheroids and organoids. STAT3 inhibition induced cell death was associated with the activation of caspase 3/7 and PARP cleavage. Moreover, TTI-101 and SH5-07 targets cancer stem cells by downregulating the expression of CD44 and CD133 in 3D models. This study provides the first evidence for the prevention of bladder cancer with small-molecule inhibitors TTI101 and SH5-07 via suppression of CSCs and STAT3 signaling.

Advanced chronic lymphocytic leukemia (CLL) is accompanied by increased circulating regula-tory T cells (Tregs) and increased susceptibility to severe infections, which were also shown to en-tail a striking induction of FOXP3 expression in Tregs. As homeostasis of the most suppressive CD45RA-FOXP3high activated Treg (aTreg) subset differs, it is critical to analyse homeostatic sig-naling in Treg subsets. Therefore, in this study, by using conventional and imaging flow cytome-try, we monitored STAT5 signaling/phosphorylation (pSTAT5) and investigated Treg subsets in relation to the Binet stage, the total tumor mass score (TTM) and disease course during follow-up of 37 patients with CLL. aTreg percentage was significantly increased among CD4+ T cells from patients with advanced disease and significantly correlated with the TTM. A subgroup of patients with higher aTreg percentages among CD4+FOXP3+ T cells at the start of therapy was character-ized by more frequent episodes of severe infections during follow-up, suggesting that aTreg frac-tion could represent a possible marker of severe disease course with infectious complications. Augmented homeostatic STAT5 signaling could support aTreg expansion, as higher pSTAT5 lev-els were significantly corelated with increased aTreg frequency among CD4+FOXP3+ T cells during follow-up of patients on therapy as well as following SARS-CoV-2 antigen-specific stimulation in vitro.

This study investigates the adoption of ecological innovations and management of occupational health hazards among Nigerian paddy rice farmers in response to field contamination and climate risks. Conducted across four major rice-producing states—Ekiti, Kwara, Edo, and Ogun—the research employed a semi-structured questionnaire developed from a comprehensive literature review and validated through peer review. Data were collected from 192 farmers through face-to-face interviews and guided dialogues, focusing on ecological innovations, vulnerability assessments, satisfaction with implemented methods, occupational health hazards, and perceptions of sustainable rice cultivation. Findings reveal that only 29.69% of farmers have adopted new measures to address contamination and climate risks, while 70.31% have not. Work-related injuries, such as cuts and abrasions, affected 28% of farmers, with minimal participation (3.1%) in safety training and support from agricultural or government agencies (1%). Correlation analysis demonstrated weak relationships among variables such as farmers' education, adoption of ecological innovations, satisfaction with results, and use of personal protective equipment (PPE). Importantly, farmers who adopted ecological innovations expressed significantly higher satisfaction (odds ratio = 11.613), as did those with prior exposure to pesticides and chemicals (odds ratio = 4.084) regarding safety practices. The study emphasizes the critical need for increased awareness, training, and support to promote sustainable farming practices and improve occupational health outcomes among paddy rice farmers in Nigeria.

The prospect of drinking water serving as a conduit for gut bacteria, artificially selected by disinfection strategies and lack of monitoring at the point of use, is concerning. Certain opportunistic pathogens, notably some nontuberculous mycobacteria (NTM), often exceed coliform bacteria levels in drinking water, posing safety risks. NTM and other microbiota resist chlorination and thrive in plumbing systems. When inhaled, opportunistic NTM can infect the lungs of immunocompromised or chronically ill patients, and the elderly, primarily postmenopausal women. When ingested with drinking water, NTM often survive stomach acidity, reach the intestines, migrate to other organs using immune cells as vehicles, potentially colonizing tumor tissue, including in breast cancer. The link between the microbiome and cancer is not new, yet the recognition of intratumoral microbiomes is a recent development. Breast cancer risk rises with age, and NTM infections emerged as a concern among breast cancer patients. In addition to studies hinting at a potential association between chronic NTM infections and lung cancer, NTM have also been detected in breast tumors at levels higher than normal adjacent tissue. Evaluating the risks of continued ingestion of contaminated drinking water is paramount, especially given the ability of various bacteria to migrate from the gut to breast tissue via entero-mammary pathways. This underscores a pressing need to revise water safety monitoring guidelines and delve into hormonal factors, which includes addressing the disproportionate impact of NTM infections and breast cancer on women and examining the potential health risks posed by the cryptic and unchecked microbiota from drinking water.

NSCLC is a very aggressive solid tumor, with a poor prognosis due to post-surgical recurrence. Analysis of the specific tumor and immune signatures of NSCLC samples is a critical step in prognostic evaluation and management decisions for patients after surgery. Routine histological assay has some limitations. Therefore, new diagnostic tools with the capability to quickly recognize NSCLC subtypes and correctly identify various markers are needed. We developed a technique for ex vivo expansion of cancer and immune cells from surgical tumor and lung tissue samples of patients with NSCLC (adenocarcinomas and squamous cell carcinomas) and their examination on ex vivo cell preparations and, parallelly, on histological sections after Romanovsky–Giemsa and immunofluorescent/immunochemical staining for cancer-specific and immune-related markers. As a result, PD-L1 expression was detected for some patients only by ex vivo analysis. Immune cell profiling in the tumor microenvironment revealed significant differences in the immunological landscapes between the patients’ tumors, with smokers’ macrophages with simultaneous expression of pro- and anti-inflammatory cytokines, neutrophils, and eosinophils being the dominant populations. The proposed ex vivo analysis may be used as an additional diagnostic tool for quick examining cancer and immune cells in whole tumor samples and avoiding false negatives in histological assays.

Cancer is a disease that encompasses multiple and different malignant conditions and is among the leading causes of death in the world. Therefore, the search for new pharmacotherapeutic options and potential candidates that can be used as treatments or adjuvants to control this disease is urgent. Natural products, especially those obtained from plants, have played an important role as a source of specialized metabolites with recognized pharmacological properties against cancer, therefore, they are an excellent alternative to be used. The objective of this research was to evaluate the action of the monoterpene isoespintanol (ISO) against the human tumor cell lines MDA-MB-231, A549, DU145, A2780, A2780-cis and the non-tumor line MRC-5. Experiments with MTT and fluorescence with propidium iodide (PI), DAPI and green plasma revealed the cytotoxicity of ISO against these cells; furthermore, morphological and chromogenic studies revealed the action of ISO on cell morphology and the inhibitory capacity on reproductive viability to form colonies in MDA-MB-231 cells. Likewise, 3D experiments validated the damage in these cells caused by this monoterpene. These results serve as a basis for progress in studies of the mechanisms of action of these compounds and the development of derivatives or synthetic analogues with a better antitumor profile.

Single-cell RNA sequencing (scRNA-seq) technique has enabled detailed analysis of gene expression at the single cell level, enhancing the understanding of subtle mechanisms that underly pathologies and drug resistance. To derive such biological meaning from sequencing data in oncology, some critical processing must be performed, including identification of the tumor cells by markers and algorithms that infer copy number variations (CNVs). We compared the performance of sciCNV, InferCNV, CopyKAT and SCEVAN tools that identify tumour cells by inferring CNVs from scRNA-seq data. Sequencing data from Pancreatic Ductal Adenocarcinoma (PDAC) patients, adjacent and healthy tissues were analysed, and the predicted tumor cells were compared to those identified by well-assessed PDAC markers. Results from InferCNV, CopyKAT and SCEVAN overlapped by less than 30% with InferCNV showing the highest sensitivity (0.72) and SCEVAN the highest specificity (0.75). We show that the predictions are highly dependent on the sample and the software used, and that they return so many false positives hence are of little use in verifying or filtering predictions made via tumor biomarkers. We highlight how critical this processing can be, warn against the blind use of these software and point out the great need for more reliable algorithms.

Background: Despite advances in the treatment of hepatocellular carcinoma (HCC) over the last few decades, treatment opportunities for patients with HCC remain limited. HCC is the most common form of liver cancer, accounting for approximately 90% of all cases worldwide. Moreover, apart from the current pharmacological interventions, hepatic resection and liver transplantation are the mainstay curative approaches for patients with HCC. Methods: This systematic review included phase I, II, III, and IV clinical trials (CTs) and randomized controlled trials (RCTs) on current treatments for patients with HCC in an Asian population (2013–2023). A total of 427 articles were screened, and 184 non-duplicate publications were identified. After screening titles and abstracts, 96 publications were excluded, and another 28 were excluded after full-text screening. The remaining 60 eligible RCTs/CTs were finally included. Results: Sixty clinical trials fulfilled our inclusion criteria and screened 36 drugs as monotherapy or combination therapy for HCC. Most studies used sorafenib alone or in combination with any of the treatment regimens. Lenvatinib or atezolizumab with bevacizumab was used for HCC after initial sorafenib treatment. Eighteen studies compared the efficacy of sorafenib with that of other drugs, including lenvatinib, cabozantinib, tepotinib, tigatuzumab, linifanib, erlotinib, resminostat, brivanib, tislelizumab, selumetinib, and refametinib. Conclusion: This study provides comprehensive insights into effective treatment interventions for HCC in Asian populations. The overall assessment indicated that sorafenib alone or in combination with atezolizumab and bevacizumab has been the first treatment choice in the past decade to achieve better outcomes in patients with HCC in an Asian population

In this comprehensive survey, we delve into the multifaceted role of palmitoylation across vari-ous cell death modalities in the oncological context. From its intricate correlations with tumor-igenesis, steered by the DHHC family, to the counter-process of depalmitoylation mediated by enzymes like PPT1. Innovations in detection methodologies have paralleled our growing under-standing, transitioning from rudimentary techniques to sophisticated modern methods. Central to our discourse are agents like GNS561 and DC661, promising heralds in palmitoylation-targeted cancer therapy. Collectively, this review accentuates palmitoylation's transformative potential in oncology, foreshadowing groundbreaking therapeutic strategies and deepening our molecular comprehension of cancer dynamics.

Mitochondria are dynamic organelles that can adjust and respond to different stimuli within a cell. This plastic ability allows them to effectively coordinate several cellular functions in cells and becomes particularly relevant in highly complex cells such as neurons. An imbalance in mitochondrial dynamics can disrupt mitochondrial function, leading to abnormal cellular function and ultimately to a range of diseases, including neurodegenerative disorders. Regulation of mRNA transport and local translation inside neurons is crucial for maintaining the proteome of distal mitochondria, which is vital for energy production and synaptic function. A significant portion of the axonal transcriptome is dedicated to mRNAs for mitochondrial proteins, emphasizing the importance of local translation in sustaining mitochondrial function in areas far from the cell body. In neurons, local translation and the regulation of mRNAs encoding mitochondrial-shaping proteins could be essential for synaptic plasticity and neuronal health. The dynamics of these mRNAs, including their transport and local translation, may influence the morphology and function of mitochondria, thereby affecting the overall energy status and responsiveness of synapses. Comprehending the mitochondria-related mRNAs regulation and local translation, as well as its influence on mitochondrial morphology near the synapses will help to better understand neuronal physiology and neurological diseases where mitochondrial dysfunction and impaired synaptic plasticity play a central role.

Background: The intrauterine and early extrauterine development represents a “window of opportunity” in the immunological development. The underlying mechanisms are still poorly understood. The aim of this study was to provide reference values B cell subpopulations in cord blood of term newborns, juveniles and in adults to find the spectrum of their physiological age-related variation. Methods: In this study, we used flow cytometry to evaluate human B lymphocytes and subpopulations in cord blood (n = 10), in peripheral blood from healthy juveniles aged 1 to 17 years (n=20) and from donors aged 24 to 62 years (n = 10). Results: Our findings showed increasing frequencies of IgM memory B cells, class-switched memory B cells, marginal zone B cells and plasmablasts, from cord blood to peripheral blood of juveniles and adults. In con-trast, the percentage of naïve B cells was higher in newborns than in juveniles and adults. The frequencies of were similar in cord blood and peripheral blood of adults. Interestingly, transitional B cells frequencies were similar in cord blood and adults but significantly lower in juveniles. Conclusions: The frequencies of circulating B cell subpopulation are subject to considerable changes during on-togeny, reflecting overlying effects of maturation and of the acquisition of an adaptive immune memory.

The DEAD-box RNA helicase DDX3X is a multifunctional protein involved in RNA metabolism and stress responses. In this study, we investigated the role of RG/RGG motifs in the dynamic process of liquid-liquid phase separation (LLPS) of DDX3X using cell-free assays and explored their potential link to cancer development through bioinformatic analysis. Our results demonstrate that the number, location, and composition of RG/RGG motifs significantly influence the ability of DDX3X to undergo phase separation and form self-aggregates. Mutational analysis revealed that the spacing between RG/RGG motifs and the number of glycine residues within each motif are critical factors in determining the extent of phase separation. Bioinformatic analysis of cancer genomic datasets uncovered a significant enrichment of DDX3X mutations in RG/RGG motifs across multiple cancer types, particularly in the N-terminal region of the protein. Furthermore, we found that DDX3X is co-expressed with the stress granule protein G3BP1 in several cancer types and can undergo co-phase separation with G3BP1 in a cell-free system, suggesting a potential functional interaction between these proteins in phase-separated structures. DDX3X and G3BP1 may interact through their RG/RGG domains and subsequently exert important cellular functions under stress situation. Collectively, our findings provide novel insights into the role of RG/RGG motifs in modulating DDX3X phase separation and their potential contribution to cancer pathogenesis.

Background: Emerging data indicate that radiotherapy can modulate antitumour immune responses. Regulatory T cells (Tregs) are a key immunosuppressive subpopulation of interest. We aimed to evaluate the impact of conventional fractionated radiotherapy on circulating Treg levels in melanoma patients and to determine the correlation between these changes and survival. Understanding radiotherapy–immunity interactions may facilitate personalized combination therapies. Methods: Peripheral blood samples from 32 melanoma patients were analysed to quantify Tregs before and 1-3 months after regional lymph node radiotherapy using flow cytometry. Tregs were characterized as CD4+/CD25+/CD127-. Total CD4+ T cells were also evaluated. Changes were compared to those in a control group of 30 tumor-free patients. Overall survival was correlated with T-cell subset dynamics induced by radiotherapy. Results: At baseline, the median percentage of Tregs was not significantly lower in melanoma patients than in controls (7.4% vs 8.32%, p=0.17). Radiotherapy markedly increased the median Treg count to 10.01% (p=0.0008), with a concomitant increase in total CD4+ cells (to 11.71%, p=0.001), indicating systemic expansion. Patients exhibiting coordinated increases in Treg and CD4+ T cells after treatment had a significantly improved prognosis compared with nonresponders (median OS 27.8 vs 13.1 months, p=0.001). Conclusions: Our study demonstrated the significant effects of standard fractionated radiotherapy on systemic Treg levels in melanoma patients. Radiation-induced changes in T-cell subset proportions hold prognostic value. Monitoring of circulating lymphocyte subsets could facilitate personalized combination therapies.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising chemotherapeutic agent because of its selective apoptotic action on cancer cells. However, resistance to TRAIL-induced apoptosis remains a challenge in many cancers. The gintonin-enriched Panax ginseng extract fraction (GEF) has diverse pharmacological benefits. We explored the combined efficacy of GEF and TRAIL in inducing apoptosis in human renal cell carcinoma (RCC) cells. The effect of GEF treatment on viability, clonogenic potential, wound healing, and TRAIL-induced apoptotic signaling of RCC cells was studied in vitro. Our investigation revealed that GEF pre-treatment sensitized RCC cells to TRAIL-induced apoptosis, as evidenced by DNA fragmentation and cell proliferation, colony formation, and migration inhibition. This sensitization was linked to the upregulation of death receptors 4 and 5 and alterations in apoptotic protein expression; notably, decreased expression of Mu-2-related death-inducing gene, a novel anti-apoptotic protein. Our findings underscore the necessity of caspase activation for GEF/TRAIL-induced apoptosis using the pan-caspase inhibitor Z-VAD-FMK. It demonstrates that GEF sensitizes human RCC cells to TRAIL-induced apoptosis by upregulating DR4/5 and modulating apoptotic protein expression. These findings suggest a promising strategy for overcoming TRAIL resistance in cancer therapy and highlight the potential of GEF as a valuable adjunct to TRAIL-based treatments.

Uterine Tumor Resembling Ovarian Sex Cord Stromal Tumor (UTROSCT) is a rare uterine mesenchymal neoplasm, which resembles ovarian sex cord tumors of the ovary. Although, in general, UTROSCT exhibits benign behavior with a favorable prognosis, this neoplasm is nonetheless classified as being of an uncertain ma-lignant potential, given its low rate of recurrence and the fact that it rarely pro-duces metastases (lymph nodes, epiploic appendix, omentum, small bowel, subcutaneous, lungs). Its histogenesis too is uncertain. Typically, UTROSCT oc-curs in peri-menopausal or menopausal women but can sometimes be observed in young women. Usually, this neoplasm can be found in the uterine corpus as a nodular intramural lesion, less frequently submucosal, subserosal or poly-poid/intracavitary. UTROSCT can cause abnormal bleeding, pelvic pain, enlarged uterus, mass sensation, but sometimes it is found purely by chance. This neo-plasm can be considered polyphenotypic on morphological, immunohistochem-ical, and genetic analyses. Generally, on microscopic examination, UTROSCT shows a predominant pattern of the cords, nests, and trabeculae typical of sex cord tumors of the ovary while, immunohistochemically, it is characterized by a co-expression of epithelial, smooth muscle, and sex cord markers. The aim of this review is to report clinical and pathological data and genetic alterations to estab-lish its impact on the prognosis and management of patients affected by this rare entity.

We present a numerical study in which large-scale bulk simulations of self-assembled DNA constructs have been carried out with a realistic coarse-grained model. The investigation aims at obtaining a precise, albeit numerically demanding, estimate of the free energy for such systems. We then, in turn, use these accurate results to validate a recently proposed theoretical approach that builds on a liquid-state theory, the Wertheim theory, to compute the phase diagram of all-DNA fluids. This hybrid theoretical/numerical approach, based on the lowest order virial expansion and a nearest-neighbor DNA model, can provide, in an undemanding way, a thermodynamic description of DNA associating fluids that is in semi-quantitative agreement with experiments. We show that the predictions of such scheme are as accurate as the ones obtained with more sophisticated methods. We also demonstrate the flexibility of the approach by incorporating non-trivial additional contributions that go beyond the nearest-neighbor model to compute the DNA hybridization free energy.

BRAF is one of the most frequently mutated oncogenes, with an overall frequency of about 50%. Targeting BRAF and its effector mitogen-activated protein kinase kinase 1/2 (MEK1/2) is now a key therapeutic strategy for BRAF-mutant tumors, and therapies based on dual BRAF/MEK inhibition showed significant efficacy in a broad spectrum of BRAF tumors. Nonetheless, BRAF/MEK inhibition therapy is not always effective for BRAF tumor suppression, and significant challenges remain to improve its clinical outcomes. First, certain BRAF tumors have an intrinsic ability to rapidly adapt to the presence of BRAF and MEK1/2 inhibitors by bypassing drug effects via rewired signaling, metabolic, and regulatory networks. Second, almost all tumors initially responsive to BRAF and MEK1/2 inhibitors eventually acquire therapy resistance via an additional genetic or epigenetic alteration(s). Overcoming these challenges requires identifying the molecular mechanism underlying tumor cell resistance to BRAF and MEK inhibitors and analyzing their specificity in different BRAF tumors. This review aims to update this information.

The anatomy of mature vertebrate hearts is marked by a looped design, which is roughly characterized by two components: (1), s-shaped (sigmoid) routing of the cardiac flow path(s), and (2,) bilaterally asymmetric (chiral) routing of the cardiac flow path(s). The looped heart design is regarded as a phylogenetically conserved feature among vertebrates and is thought to represent a significant determinant of the cardiac pumping function. It evolves during the embryonic period of development by a morphogenetic process called “cardiac looping”. During the past decades, remarkable progress has been made in the uncovering of the genetic, molecular, and biophysical factors contributing to cardiac looping. Thereby, a special focus was directed on the evolution of a bilaterally asymmetric (chiral) heart configuration, which is said to play a central role in the determination of the alignments and separations of the pulmonary and systemic flow paths in the multi-chambered hearts of tetrapods. In contrast to the recent progress made in uncovering of the biological driver of cardiac looping, our present knowledge about the functional consequences of cardiac looping is at an immature state. This article provides an overview and discussion of the currently available information about the looped design of vertebrate hearts and its functional implications.

Circulating tumor cells (CTCs) enumeration is a promising technique to predict cancer prognosis and treatment response. CTCs were evaluated in healthy subjects, cirrhotic controls and hepatocarcinoma (HCC) patients. CTCs were isolated using microfluidic system based on the expression of EpCAM, EGFR and three epithelial to mesenchymal transition (EMT) markers. Patients were stratified according to disease progression and exitus. Although counts of individual CTCs, clustered CTCs and α-fetoprotein (AFP) at basal level in patients with HCC were significantly increased compared with the values obtained in cirrhotic patients and control subjects, only individual CTCs (p=0.027), but not clustered CTCs (p=0.063) and AFP (p=0.072), were independent predictors of HCC development. The univariate regression model showed that basal levels of CTCs46 were related to high risk of HCC (Odds Ratio 3.467, p=0.011). The stratification of our cohort according to disease progression and death showed that basal individual CTCs 76 (Hazard Ratio 5.131, p=0.004) were related to disease progression, as well as the difference of clustered CTCs between 1-month and baseline levels 1.5 were related to death (Hazard Ratio 10.204, p=0.036). In conclusion, the preoperative and 1-month measurements of CTCs in blood constitute useful markers to predict the outcome of patients under TACE treatment.

JAK2 V617F is a somatic mutation associated with myeloproliferative neoplasm (MPN): polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In MPNs this mutation is associated with the germline GGCC (46/1) haplotype. Most studies consider the association of the JAK2 haplotypeGGCC_46/1 with some MPNs clinical parameters but no one analyzed the association between JAK2 haplotypeGGCC_46/1 and the onset of onco-drug resistance. Thus, we assessed for JAK2 46/1 haplotype correlation with therapy response in JAK2 V617F positive patients. Patients with MPN, selected by the Hematology Laboratory of “V. Fazzi” Hospital (LE), were analyzed with RLFP-PCR assay with rs10974944 SNP. Most of them had PV (63%) or PMF (61%). Among patients that developed onco-drug resistance, 58% had C/G genotype and only 11% had the G/G allele. No correlation between onco-drug resistance and JAK2 haplotype 46/1 variants emerged. Nevertheless, we observed that G/G allele seems to be related to MPNs evolution to myelofibrosis and to the development of onco-drug resistance clinical parameters (p-value= 0.002449; odd ratio of 3.701209). Thus, although other analyses are required, due to the narrow cardinality of sample, our findings suggest how the G/G allele could be useful for MPNs diagnosis and for the prediction of the disease outcome.

52 years have passed since President Nixon launched the "War on Cancer". The goals outlined by the President were not achieved because cancer treatment applied such as chemotherapy, radiotherapy, and targeted therapy have not fully met expectations. We suggest a new chemotherapeutic strategy: disrupting the communication between cancer cells and their microenvironment by chemical means. Immunological synapses that form between cancer cells and immune or other stromal cells provide an attractive target for this approach. Synapses form ligand-receptor clusters within interface of the interacting cells. Despite their differences, synapses share common properties: intercellular protein clusters; the proximity of these proteins; and their cooperative interaction making a synapse an unified functional unit. Synapses provide the limited space for the focused intercellular exchange of signaling molecules and particles. Therefore, destruction of synapses is expected to cause collapse of various tumor types. Additionally, the clustered arrangement of synapse components offers opportunities to increase treatment safety by reducing the concentration of cell impermeable agents used, to enhance its specificity applying cross-linking reagents, thus restricting modifications of surface-exposed molecules. By attaching a cleavable cell permeable toxic agent to a crosslinker should further enhance a killing potential in treating cancer. The proposed approach promises to be simple, universal, and less expensive than existing cancer therapy methods.

The purpose of this study was to elucidate patterns and mechanisms driving seasonal and inter-annual variations of carbon and nitrogen stable isotopes in the zooplankton crustacean commu-nity of Lake Maggiore (Italy), during the period 2009-2020. Different zooplankton taxa and groups revealed different ranges of d13C signatures, giving an insight into food sources and niche partition. In particular, cyclopoids had a restricted range with most negative d13C‰ values and increase in d13C fractionation with the establishment of water thermal vertical stratification, high-lighting the importance of vertical distribution as key factor for taxa coexistence in a vertically heterogenous environment. d13C of the zooplankton community and of Daphnia were positively related to water temperature (R2=0.58 p <0.0001 and R2=0.68 p <0.0001, respectively) and d13C Daphnia signature to chlorophyll-a (R2=0.32, p < 0.0001). Decomposition of time series data for zooplankton carbon and nitrogen signatures and environmental parameters identified increasing trends in water temperature, chlorophyll-a and water conductivity and decrease in nitrate, that matched changes in carbon isotopic signature trend in some zooplankton taxa (Bosmina, Daphnia and diaptomids). Overall, the observed patterns in zooplankton isotopic signatures were interpreted as integrations of climate warming effects in Lake Maggiore, affecting both food sources availability and environmental conditions.

Ovarian cancer is among the most common causes of female mortality. Despite improvements in diagnostic methods, non-specific symptoms and delayed gynecological exams can lead to late-stage ovarian tumor discovery. In this study, the effect of an anti-cancer compound, 3-amino-N-(3-chloro-2-methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide (Compound 1) was examined. Cytotoxicity, apoptosis, and metabolomic changes in ovarian cancer cell lines SK-OV-3 and OVCAR-3, as well as glycosphingolipid (GSL) expression, both on cancer stem cells (CSCs), marked as CD49f+ and non-CSCs (CD49f-) were explored. Treatment with Compound 1 reduced the percentage of CSCs compared to non-treated cells (P &lt; 0.001). The expression of eight GSLs on CSCs and non-CSCs was examined using flow cytometry. Both cell lines showed a change in the glycophenotype of the cells, either up or down of its expression, after the treatment. These findings raise the possibility of specifically targeting CSCs in ovarian cancer therapy. Additionally, treatment of Compound 1 resulted in a statistically significant increase in both early and late apoptosis (P&lt;0.001) suggesting a pivotal role in initiating programmed cell death by the apoptotic pathway. The metabolic analysis revealed that the treated cancer cells showed reduced metabolic activity compared to the control group (P &lt; 0.001)

Tumor immunotherapy represented by immune target blocking and chimeric antigen receptor cell therapy has developed rapidly. Among them, Natural Killer Cell (NK) is gradually becoming another preferred immunotherapy method after T cell immunotherapy because of its unique killing effect in innate immunity and adaptive immunity. Although NK cell therapy has achieved promising results in clinical trials, there are still some problems, such as low efficacy in solid tumors, insufficient penetration of NK cells and high cost of treatment. Nanomaterials are expected to be an excellent tool to improve the anti-tumor immune response of NK cells due to their advantages such as chemical specificity, biocompatibility and simple synthesis. Nanomaterials can enhance the cytotoxicity of NK cells, participate in the modification and noninvasive cell-tracking patterns of NK cells，and mediate their homing and infiltration, and thus significantly improve the efficiency of NK cell immunotherapy. In this review, we will briefly introduce the methods and mechanisms of tumor immunotherapy, emphasize the research progress of NK cell-based immunotherapy and imaging, and finally summarize the direction and research progress of nanomaterials in NK cell-based immunotherapy.

It has been more than three decades since the discovery of multifunctional factors, Non-POU Domain-Containing Octamer-Binding Protein, NonO and Splicing Factor, Proline-and Glutamine-Rich, SFPQ. Some of their functions, including their participations in transcriptional and posttranscriptional regulation as well as their contribution to paraspeckle subnuclear body organization have been well-documented. In this review, we focus on the roles of NonO and SFPQ in cell cycle with particular attention to their participation in nonhomologous end joining (NHEJ) and homologous recombination (HR) of DNA damage responses induced by irradiation. In these contexts, absence or malfunction of either or both lead to genome instability and ultimately to apoptosis, cellular senescence, and tumor development. This review also will shed light on NonO and SFPQ function in DNA/RNA/protein binding and their association with Ku heterodimer of the NHEJ pathway required for repair of DNA double-strand breaks (DSBs).

Two-dimensional silicon allotrodes– also called Sinicene– have recently experienced intensive scientific research interest due to their unique electrical, mechanical, and sensing characteristics. A novel silicene based nano-material has been enticed great amenities, partially because of its uniformity with graphene. Silicene is a highly sensitive for numerous sensors based on molecular sensing as pH sensor, gas sensor, ion sensor and biosensing are Deoxyribonucleic acid (DNA) nucleobase sensor, photonic sensor, cell-based biosensor, glucose sensor, and bioelectric nose sensor. Nowadays genetic research based on DNA hybridization, which is a vital tools for sensing material and it has various detection methods. Among of them, the detection method is frequency readout used to a label-free detection of DNA hybridization. In this paper we have compared the graphene and silicene quantum capacitance that has been proposed for a DNA hybridization detection method on wireless readout. These method shows, the strands of mismatched and complementary DNA have in different range of frequency to identify output efficiency. With respect to DNA concentration the output of silicene is almost sharply linear than graphene. In addition of field effect transistor, silicene opens a new opportunities due to its band gap whereas graphene indicates zero band gap. It can be stated that silicene is much more reliable as well as much stronger than multi-layered graphene.

Renal cell carcinoma (RCC) is the second most common cancer of the urinary system. The current therapeutic strategies are based on partial or total nephrectomy and/or targeted therapies based on immune checkpoint inhibitors to which patients are often refractory. Preventive and screening strategies are not existing and the few available biomarkers for RCC are characterized by the lack of sensitivity, outlining the need of novel noninvasive and sensitive biomarkers for an early diag-nosis and a better disease monitoring. Blood liquid biopsy (LB) is a non- or minimally invasive procedure for a more representative view of tumor heterogeneity than the tissue biopsy, poten-tially allowing real-time monitoring of cancer evolution. Growing interest is focused on the extra-cellular vesicles (EVs) secreted by either healthy or tumoral cells and recovered in a variety of bio-logical matrices, blood included. EVs are involved in cell-to-cell crosstalk transferring their mRNAs, miRNAs, and proteins content. In particular, transferred miRNAs may regulate the tu-morigenesis and proliferation also impacting on resistance to apoptosis, thus representing poten-tial useful biomarkers. Here we present the latest efforts in the identification of circulating miR-NAs in blood samples, focusing on the potential use of EV derived miRNAs as RCC diagnostic, prognostic markers.

Background: As cancer progresses, cells acquire traits that allow them to disperse and disseminate to distant locations in the body – a process known as metastasis. While in the vasculature, these cells are referred to as circulating tumour cells (CTCs) and can manifest either as single cells or clusters of cells (i.e., CTC clusters), with the latter being the most aggressive. The increased metastatic potential of CTC clusters is generally associated with cooperative group benefits in terms of survival, such as increased resistance to shear stress, anoikis, immune attacks and drugs. However, the adoption of a group phenotype poses a challenge when exiting the vasculature (extravasation) as the large size can hinder the passage through vessel walls. Despite their significant role in the metastatic process, the mechanisms through which CTC clusters extravasate remain largely unknown. Based on the observed in vivo association between CTC clusters and platelets, we hypothesized that cancer cells take advantage of the platelet-derived Transforming Growth Factor Beta 1 (TGF-β1) – a signalling factor that has been widely implicated in many aspects of cancer, to facilitate their own dissemination. To address this possibility, we evaluated the effect of exogenous TGF-β1 on an experimentally evolved non-small lung cancer cell line that we previously developed and used to investigate the biology of CTC clusters. Results: We found that exogenous TGF-β1 induced the dissociation of clusters into adherent single cells. Once adhered, cells released their own TGF-β1 and were able to individually migrate and invade in the absence of exogenous TGF-β1. Based on these findings we developed a model that involves a TGF-β1-mediated plastic switch between a cooperative phenotype and a single-celled stage that enables the extravasation of CTC clusters. Conclusions: This model allows for the possibility that therapies can be developed against TGF-β1 signalling components and/or TGF-β1 target genes to suppress the metastatic potential of CTC clusters. Considering the negative impact that metastasis has on cancer prognosis and the lack of therapies against this process, interfering with the ability of CTC clusters to switch between cooperative and individual behaviours could provide new strategies to improve patient survival.

Cholesterol homeostasis is pivotal for cellular function. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), also abbreviated as SOAT1, is an enzyme catalyzing the storage of excess cholesterol to cholesteryl esters. ACAT1 is an emerging target to treat diverse diseases including atherosclerosis, cancer, and neurodegenerative diseases. F12511 is a high affinity ACAT1 inhibitor. Previously, we developed a stealth liposome-based nanoparticle to encapsulate F12511 to enhance its delivery to the brain and showed its efficacy in treating a mouse model for Alzheimer’s disease (AD). In this study, we introduce F26, a close derivative of F12511 metabolite in rats. F26 was encapsulated in the same DSPE-PEG2000/phosphatidylcholine (PC) liposome-based nanoparticle system. We employed various in vitro and in vivo methodologies to assess F26's efficacy and toxicity compared to F12511. Results demonstrate that F26 is more effective and durable than F12511 in inhibiting ACAT1, in both mouse embryonic fibroblasts (MEFs) and in multiple mouse tissues including the brain tissues, without exhibiting any overt systemic or neurotoxic effects. This study demonstrates the superior pharmacokinetic and safety profile of F26 in wild-type mice, and suggests its therapeutic potential against various neurodegenerative diseases including AD.

Diffuse midline gliomas are among the deadliest human cancers and have had little progress in treatment in the last 50 years. Cell cultures of these tumors have been developed recently, but it is unknown the degree to which such cultures retain the characteristics of the source tumors. DNA methylation profiling offers a powerful tool to look at genome-wide epigenetic changes that are biologically meaningful and can help assess the similarity of cultured tumor cells to their in vivo progenitors. Paraffinized diagnostic tissue from three diffuse intrinsic pontine gliomas with H3 K27M mutations was compared with subsequent passages of neurosphere cell cultures from those tumors. Each cell line was passaged 3-4 times and analyzed with DNA methylation arrays and standard algorithms that provided comparison of diagnostic classification and cluster analysis. All samples tested maintained high classifier scores and clustered within the reference group of H3 K27M-mutant diffuse midline gliomas. There was a gain of 1q in all cell lines, with two cell lines initially manifesting the gain of 1q only during culture. In vitro cell cultures of H3 K27M-mutant gliomas maintain high degrees of similarity in DNA methylation profiles to their source tumor, confirming their fidelity even with some chromosomal changes.

Abstract Octreotide is the first synthetic peptide hormone, consisted of eight amino acids, that mimics the activity of somatostatin, a natural hormone in the body. During the past decades, advanced instrumentation and crystallographic software established X-ray Powder Diffraction (XRPD) as a valuable tool for extracting structural information of biological macromolecules. The latter was demonstrated by the successful structural determination of octreotide at a remarkably high d-spacing resolution (1.87 Å) (PDB code: 6vc1). This study focuses on the response of octreotide to different humidity levels and temperatures, with a particular focus on the stability of the polycrystalline sample. XRPD measurements were accomplished employing an Anton Paar MHC-trans humidity-temperature chamber installed within a laboratory X’Pert Pro diffractometer (Malvern Panalytical). The chamber is employed to control and maintain precise humidity and temperature levels of samples during XRPD data collection. Pawley analysis of the collected data sets revealed that the octreotide polycrystalline sample is remarkably stable and no structural transitions were observed. The compound retains its orthorhombic symmetry (space group: P212121, a = 18.57744(4) Å, b = 30.17338(6) Å, c = 39.70590(9) Å, d~ 2.35 Å). However, a characteristic structural evolution in terms of lattice parameters and volume of the unit-cell is reported mainly upon controlled relative humidity variation. In addition, an improvement of signal-to-noise ratio in the XRPD data under a cycle of dehydration/rehydration is reported. These results underline the importance of considering the impact of environmental factors, as humidity and temperature, in the context of structure-based drug design, thereby contributing to the development of more effective and stable pharmaceutical products.

Cell-free massive multiple input multiple output (MIMO) has the potential of providing joint services including joint initial access, efficient clustering of access points (APs) and pilot allocation to user equipments (UEs) over large coverage area with reduced interference. In cell-free massive MIMO, large coverage area corresponds to provision and maintenance of scalable quality of service requirements for infinitely large number of UEs. The research in cell free massive MIMO is mostly focused on time division duplex mode due to availability of channel reciprocity which aids in avoiding feedback overhead. However, frequency division duplex (FDD) protocol still dominates the current wireless standards and the provision of angle reciprocity aids in reducing this overhead. The challenge of providing a scalable cell-free massive MIMO system in FDD setting is also prevalent, since computational complexity regarding signal processing tasks such as channel estimation, precoding/combining and power allocation, becomes prohibitively high with increase in number of UEs. In this work, we consider an FDD based scalable cell-free network with angular reciprocity and dynamic cooperation clustering approach. We have proposed scalability for our FDD cell-free and perform comparative analysis with reference to channel estimation, power allocation and precoding/combining techniques. We present expressions for scalable spectral efficiency, angle based precoding/combining schemes and provide comparison of overhead between conventional and scalable angle based estimation as well as combining schemes. Simulations confirm that the proposed scalable cell-free network based on FDD scheme outperforms the conventional matched filtering scheme based on non-scalable precoding/combiming schemes. The angle based LP-MMSE in FDD cell-free network provides 14.3% improvement in spectral efficiency and 11.11% improvement in energy efficiency compared to non-scalable MF scheme.

In this study, the microbial strain was isolated from the humus soil to ferment Sophora flavescens and Eleutherococcus sessiliflorus extracts. The isolated microbial was identified as Bacillus genus by 16S rRNA sequence analysis. The fermented plant extracts exhibited the antifungal effects against four types of the plant pathogens such as P. carotorum, B. cinerea, C. fructicola Sau-3, and C. gloeosporioides according to incubation time. Especially, the fermented plant extracts showed the most active activities for Colletotrichum genus to inhibit mycelium growth. Metabolite changes in fermented S. flavescens and E. sessiliflorus extracts were confirmed through LC-Q-TOF/MS. Flavonoid and peptide derivatives were improved in fermented S. flavescens and E. sessiliflorus extracts compared to their unfermented one, respectively. This study suggested that the isolated Bacillus microbial fermentation could be a valuable tool in improving the bioactivity of S. flavescens and E. sessiliflorus extracts to have potential with more environmentally-friendly antifungal agents.

The stress-inducible heat shock protein 70 (Hsp70), which functions as a molecular chaperone and is frequently overexpressed in different cancer cell types, is presented on the cell surface of tumor cells and is actively released into the circulation in free and extracellular lipid vesicle-associated forms. Since the exact pathomechanism of endometriosis has not yet been elucidated but has been associated with the development of endometrial and ovarian cancer, we asked whether extracellular Hsp70 and circulating endometriotic cells (CECs) reflects the presence and development of endometriosis. Therefore, circulating levels of free and lipid microvesicle-associated Hsp70 were measured using the Hsp70-exo ELISA, and the presence of circulating CECs in the peripheral blood of patients with endometriosis was determined using membrane Hsp70 (mHsp70) and EpCAM monoclonal antibody (mAb)-based bead isolation approaches. Isolated CECs were further characterized by immunofluorescence using reagents directed against cytokeratin (epithelial), CD45 (leukocyte), CD105/CD44 (mesenchymal-stemness) and by comparative RNA analysis. Similar to the situation in patients with cancer, levels of circulating Hsp70 were elevated in the blood of patients with histologically proven endometriosis compared to a healthy control cohort, with the highest Hsp70 level in a patient with lymph node endometriosis. Moreover, CECs could be isolated using the cmHsp70.1 mAb- and to a lesser extent EpCAM mAb-based bead approaches in all patients with endometriosis. The longevity in cell culture and the expression of cytokeratin, CD105 and CD44, together with differentially expressed genes related to EMT reveals similarities of mHsp70 expressing CECs with circulating tumor cells (CTCs) and suggest a mesenchymal-stemness origin. These findings may support the involvement of mHsp70 positive, stem cell-like cells in the development of endometriotic lesions. In summary, elevated levels of Hsp70 in the blood and CECs could serve as liquid biopsy markers for endometriosis and help to elucidate the underlying pathomechanism of the disease.

A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices which have been classified as “possibly carcinogenic”, however their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (RE) may promote genomic instability, here we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of RE, such as long interspersed nuclear element1s (LINE-1), Alu short interspersed nuclear element and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines (HeLa, BE(2)C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE1 and ribosomal repeats in terms of both average profiles and organization of methylated and un-methylated CpG sites, in different ways in each of the three cell lines studied.

Radiation therapy (RT) is one of the main approaches of cancer therapy, including non-small cell lung cancer (NSCLC). Radiation-induced DNA double-stranded breaks (DSBs), single-stranded DNA breaks (SSBs) and oxidized nucleobases cause replication fork stalling, chromosomal instability or point mutagenesis and can lead to cell death. Translesion DNA synthesis (TLS) is one of the DNA damage tolerance (DDT) pathways promoting replication of damaged DNA. Here, we explored the effects of the knockout of primase-polymerase PRIMPOL (PRIMPOL −/−) and TLS polymerase POLI (POLI −/−) genes in two different monoclonal lines derived from human NSCLC A549 cells. Our study aimed to investigate the impact of their absence on the susceptibility of these cells to ionizing radiation (IR)-induced stress. Without IR, cells that do not have PRIMPOL or POLI experience a considerable rise in apoptosis by day 7 of continuous cultivation and a noteworthy decline in cell migration under spatial confinement when compared to the parental wild-type cells. Despite this, we have found that PRIMPOL −/− exhibited higher clonogenic survival compared to wild-type cell lines in response to IR stress. Both of these cell lines demonstrated remarkable resilience to radiation stress, surpassing the survival rate of the original A549 cells. POLI deficiency reduces cellular γH2AX foci accumulation, while the presence of highest number of γH2AX foci in PRIMPOL−/− cells was ATM-independent. We report for the first time that increased post-irradiation clonogenic survival of PRIMPOL −/− A549 cells might be associated with the increase of CD133+ but not CD44+ populations of cancer stem-like cells.

The spread of the infection caused by the new coronavirus SARS-CoV-2 (COVID-19) became pandemic on March 11, 2020. From the time of the first cases (in November 2019, Wuhan, China), to date, a large number of COVID-19 observations have been accumulated in different age groups of patients both in China and abroad. Published scientific data allows us to conclude that children suffer from COVID-19 much less often than adults and tolerate the disease in a milder form, often appear to be asymptomatic. There is currently no final answer why children are less susceptible to this virus; however, scientists are increasingly inclined to consider a complex effect of the immune response and components of the renin-angiotensin system (RAS), which according to recent studies affects not only the cardiovascular system, but is also responsible for the activation of inflammatory reactions. A hypothesis of genetic predisposition to the development of severe forms of COVID-19 has recently been made. We conducted a search for publications in the databases and showed current scientific ideas about COVID-19 pathogenesis and factors influencing the disease development in childhood. Childhood immunity may have several protective features against SARS-CoV-2: immaturity of particular elements of the innate immune response, constitutional lymphocytosis with a shift towards anti-inflammatory Th2-response, as well as "trained" immunity. The influence of renin-angiotensin system reactions in this review is shown from two perspectives: expression of ACE2 receptors and polymorphisms of certain genes of this system. It was established that ACE2 transmembrane protein is not only the entry point for the virus but also plays a regulatory role, turning the pro-inflammatory vasoconstrictor angiotensin II into anti-inflammatory angiotensin (1-7), which has vasodilating properties. Higher ACE2 content in children compared with adults helps maintain balance in the renin-angiotensin system and prevents the development of complications. It was also shown that the presence of certain genetic polymorphisms (AGTR1, AGTR2, ACE2, ACE) could determine the imbalance inside the RAS, leading to more pronounced reactions of alveolocytes, vascular endothelium and smooth muscle fibers in response to SARS-CoV-2 infection due to a shift towards vasoconstrictor, proliferative and profibrotic mechanisms.