Tissue Biopsies Of Patients Research Articles

Integrated DNA estimation in tissue biopsy and detection in liquid biopsy by HBV-targeted NGS assay.

Integrated HBV DNA (iDNA) plays a critical role in HBV pathogenesis, particularly in predicting treatment response and HCC. This study aimed to use an HBV hybridization-capture next-generation sequencing (HBV-NGS) assay to detect HBV-host junction sequences (HBV-JS) in a sensitive nonbiased manner to detect and estimate the iDNA fraction in tissue biopsies and HBV genetics by liquid biopsy. HBV DNA from plasmid monomers, HBV-HCC cell line (SNU398, Hep3B, and PLC/PRF/5), tissue biopsies of patients with serum HBV DNA <4 log IU/ml, and matched urine and plasma of HBV patients were assessed by HBV-NGS. Junction-specific qPCR (JS-qPCR) assays were developed to quantify abundant HBV-JS. We demonstrated high coverage uniformity, reproducibility across all HBV genotypes A-D, and 0.1% sensitivity for detecting iDNA by the HBV-NGS assay. The sequence and structures of iDNA molecules from SNU398 and Hep3B are reported. An iDNA estimation model was developed using six abundant HBV-JS sequences identified from tissue biopsies by HBV-NGS assay and validated using total DNA of SNU398 and Hep3B cells. Furthermore, the utility of the HBV-NGS assay for HBV genetic analysis in liquid biopsies was explored using matched plasma-urine samples from three patients with serum HBV DNA levels ranging from high to undetectable. HBV-JS was detected in all body fluids tested, regardless of viral load. These findings suggest that the iDNA fraction in tissue biopsies from patients with limited or undetectable serum HBV DNA can be estimated using a robust HBV-NGS assay, and a sensitive HBV genetics liquid biopsy can be obtained. This study highlights the potential of NGS-based methods to advance HBV management.

Cardiomyocyte-derived circulating extracellular vesicles allow a non-invasive liquid biopsy of myocardium in health and disease.

The ability to track disease without tissue biopsy in patients is a major goal in biology and medicine. Here, we identify and characterize cardiomyocyte-derived extracellular vesicles in circulation (EVs; "cardiovesicles") through comprehensive studies of induced pluripotent stem cell-derived cardiomyocytes, genetic mouse models, and state-of-the-art mass spectrometry and low-input transcriptomics. These studies identified two markers (POPDC2, CHRNE) enriched on cardiovesicles for biotinylated antibody-based immunocapture. Captured cardiovesicles were enriched in canonical cardiomyocyte transcripts/pathways with distinct profiles based on human disease type (heart failure, myocardial infarction). In paired myocardial tissue-plasma from patients, highly expressed genes in cardiovesicles were largely cardiac-enriched (vs. "bulk" EVs, which were more organ non-specific) with high expression in myocardial tissue by single nuclear RNA-seq, largely in cardiomyocytes. These results demonstrate the first "liquid" biopsy discovery platform to interrogate cardiomyocyte states noninvasively in model systems and in human disease, allowing non-invasive characterization of cardiomyocyte biology for discovery and therapeutic applications.

Distorted frequency and functionality of natural killer cells in pemphigus vulgaris: A potential therapeutic target

Pemphigus vulgaris (PV) is a rare autoimmune disorder where autoantibodies target the desmosomal proteins resulting in blistering of oral mucosa and skin. While the pathogenesis of PV is mainly mediated by the adaptive immune system, key players of innate immunity are also emerging. This study outlines the phenotypic as well as functional attributes of NK cells in PV. Through in-depth analysis using flow cytometry we identified an increase in the frequency of CD56+ CD3- NK cells and their subtypes in periphery. Along with this there is an increased frequency of IFNγ+ CD56bright CD16dim NK cells. mRNA expression of sorted NK cells for differentially expressed genes, particularly key transcription factors such as T-bet and EOMES, as well as surface receptors like NKG2D and KIR2D, and the cytokine IFNγ, displayed significant upregulation. A significant activation of NK cells was seen in the disease state. The levels of perforin and IFNγ were significantly elevated in the culture supernatants of patients. Additionally, a significantly higher cytotoxicity of NK cells in PV was observed. In lesioned tissues of PV, NK related markers were significantly increased. Lastly, we observed NK cells using confocal microscopy in the tissue biopsies of patients which showed significant infiltration of CD56+ CD3- NK cells at the lesional sites. This study aimed to shed light on the pivotal role of NK cells in the immunopathology of PV, offering a thorough understanding of their behaviour and changes in expression which might help in contributing to the development of novel therapeutics.

Utility of circulating tumor DNA (ctDNA) to inform treatment of patients with metastatic breast cancer.

1042 Background: Circulating tumor DNA (ctDNA) can overcome limitations of tissue biopsy (tbx) in patients with metastatic breast cancer (mBC). While tbx provides a snapshot of a tumor’s molecular profile from one site and time, ctDNA provides a more comprehensive landscape of the tumor genome. Serial liquid biopsies (lbx) can be easily obtained for monitoring clinical response and resistance over time. The Individualized Molecular Analyses Guide Efforts in Breast Cancer (IMAGE) II study evaluates tbx and serial lbx methods for tumor genomic profiling in mBC. Methods: IMAGE II (NCT02965755) is a prospective, multi-center trial to evaluate the clinical utility of lbx versus tbx. Eligible individuals had mBC of any subtype, with progression requiring therapy change. Tbx sequencing per standard of care was required unless biopsy was not technically feasible. Upon enrollment, we collected baseline plasma for tissue-agnostic comprehensive genomic profiling (CGP) by Foundation Medicine. Both lbx and tbx sequencing results were reviewed in real time by the Johns Hopkins Molecular Tumor Board to provide treatment recommendations. Clinical data was recorded. We collected serial lbx samples 1-2 weeks post initiation of next line of therapy, at first restaging, and at progression. Here we present the analysis of data collected at enrollment, and serial lbx results will be reported at a later date. Results: Total enrollment was 199 pts, of whom 194 were women (median age = 57 years, range 27-86 years). There were 140 HR+HER2-, 4 HR-HER2+, 20 HR+HER2+ and 35 triple negative (TNBC) cases. Metastases were found in bone only in 19 pts, or included lung in 99 pts, liver in 97 pts, and brain in 14 pts. Median prior lines of metastatic therapy was 2, with 44 pts (22.1%) having been on 4 or more, including antibody-drug conjugates (14 pts), immunotherapy (2), cytotoxic chemotherapy (74), endocrine (88), and targeted therapy (12). 9 pts were newly diagnosed with mBC at the time of enrollment. 110 pts (55%) had tbx sequencing. In lbx-based CGP results from 190 patients, the genes most frequently altered were TP53(90 patients), PIK3CA (63), ESR1 (54). Copy number amplifications were found in FGFR1 (21 patients) and FGFR3 (12), and copy number losses were found in PTEN in 3 patients. Of the 87 patients whose baseline lbx was profiled using FoundationOne Liquid CDx which allows calculation of ctDNA tumor fraction (TF), 69 (79.3%) pts had detectable ctDNA TF. 60 patients (69.0%) had ctDNA TF at least 1%. We will present the frequency of clinically actionable mutations detected via lbx vs tbx genomic profiling, accounting for sites of metastases, receptor subtype and prior therapy exposure, as well as the association between ctDNA TF and sites of disease. Conclusions: A high rate of ctDNA TF detection in pts with mBC highlights the value of lbx for CGP in this setting for detecting multiple alteration classes across mBC subtypes and metastatic sites. Clinical trial information: NCT02965755 .

Comparative analysis of genetic variants in pleural fluids and solid tissue biopsies of pleural mesothelioma patients: Implications for molecular heterogeneity assessment

ObjectivesThis study aims to determine whether the sequencing of DNA extracted from pleural fluids (PFs) of Pleural Mesothelioma (PM) patients accurately represents the genetic information obtained from the solid tissue counterpart biopsies with particular attention to the identification of single nucleotide variants (SNVs). Materials and methodsSingle pleural biopsy, PFs, and blood were collected from PM patients. DNA was extracted from these samples and then subjected to Whole-Exome Sequencing. ResultsA higher number of SNVs was identified in PFs than in solid tissue biopsies (STBs). Most SNVs were detected in PFs samples but not in STBs samples, while only a few SNVs were detected in STBs samples but not in PFs samples. ConclusionThe current findings support the notion that PFs might offer a more robust depiction of cancer's molecular diversity. Nonetheless, the current outcomes challenge the assertion that liquid biopsies can encompass the entirety of intra-patient variations. Indeed, a subset of potential cancer-driver SNVs was exclusively identified in STBs. However, relying solely on STBs would have precluded the detection of significant SNVs that were exclusively present in PFs. This implies that while PFs serve as a valuable complement to STBs, they do not supplant them.

Enhancing Diagnostic Capabilities for Occupational Lung Diseases Using LIBS Imaging on Biopsy Tissue.

Laser-induced breakdown spectroscopy (LIBS) imaging continues to gain strength as an influential bioanalytical technique, showing intriguing potential in the field of clinical analysis. This is because hyperspectral LIBS imaging allows for rapid, comprehensive elemental analysis, covering elements from major to trace levels consistently year after year. In this study, we estimated the potential of a multivariate spectral data treatment approach based on a so-called convex envelope method to detect exotic elements (whether they are minor or in trace amounts) in biopsy tissues of patients with occupational exposure-related diseases. More precisely, we have developed an approach called Interesting Features Finder (IFF), which initially allowed us to identify unexpected elements without any preconceptions, considering only the set of spectra contained in a LIBS hyperspectral data cube. This task is, in fact, almost impossible with conventional chemometric tools, as it entails identifying a few exotic spectra among several hundred thousand others. Once this detection was performed, a second approach based on correlation was used to locate their distribution in the biopsies. Through this unique data analysis pipeline to processing massive LIBS spectroscopic data, it was possible to detect and locate exotic elements such as tin and rhodium in a patient's tissue section, ultimately leading to a possible reclassification of their lung condition as an occupational disease. This review will thus demonstrate the potential of this new diagnostic tool based on LIBS imaging in addressing the shortcomings of approaches developed thus far. The proposed data processing approach naturally transcends this specific framework and can be leveraged across various domains of analytical chemistry, where the detection of rare events is concealed within extensive data sets.

Microbiome Dysbiosis Is Associated with Castration Resistance and Cancer Stemness in Metastatic Prostate Cancer.

Prostate cancer is the second leading cause of death in males in America, with advanced prostate cancers exhibiting a 5-year survival rate of only 32%. Castration resistance often develops during the course of treatment, but its pathogenesis is poorly understood. This study explores the human microbiome for its implications in castration resistance and metastasis in prostate cancer. RNA sequencing data were downloaded for the bone and soft tissue biopsies of patients with metastatic castration-resistant prostate cancer. These included both metastatic and adjacent normal biopsies. These sequences were mapped to bacterial sequences, yielding species-level counts. A vast majority of species were found to be significantly underabundant in the CRPC samples. Of these, numerous were found to correlate with the expression of known markers of castration resistance, including AR, PI3K, and AKT. Castration resistance-associated signaling pathways were also enriched with these species, including PI3K-AKT signaling and endocrine resistance. For their implications in cancer aggression and metastasis, cancer stem cell markers were further explored for a relation to these species. EGFR and SLC3A2 were widely downregulated, with a greater abundance of most species. Our results suggest that the microbiome is heavily associated with castration resistance and stemness in prostate cancer. By considering the microbiome's importance in these factors, we may better understand the highly aggressive and highly invasive nature of castration-resistant prostate cancer, allowing for the needed improvements in the treatment of this disease.

Digital methylation-specific PCR: New applications for liquid biopsy.

Epigenetic analysis is a fundamental part of understanding pathophysiological processes with potential applications in diagnosis, prognosis, and assessment of disease susceptibility. Epigenetic changes have been widely studied in chronic obstructive pulmonary disease (COPD), but currently, there is no molecular marker used to improve the treatment of patients. Furthermore, this progressive disease is a risk factor for the development of more severe COVID-19. Methylation-specific polymerase chain reaction (MSP-PCR) plays an important role in the analysis of DNA methylation profiles, and it is one of the most widely used techniques. In this context, the combination of MSP-PCR with emerging PCR technologies, such as digital PCR (dPCR), results in more accurate analyses of the DNA methylation profile of the genes under study. In this study, we propose the application of the MSP-dPCR technique to evaluate the methylation profile of the ADAM33 gene from saliva samples and lung tissue biopsies of patients with COPD and COVID-19. MSP-dPCR generated a measurable prediction of gene methylation rate, with the potential application of this combined technology for diagnostic and prognostic purposes. It has also proven to be a powerful tool for liquid biopsy applications.

Mycobacterium leprae is able to infect adipocytes, inducing lipolysis and modulating the immune response

Leprosy is a chronic infectious disease caused by the intracellular bacillus Mycobacterium leprae (M. leprae), which is known to infect skin macrophages and Schwann cells. Although adipose tissue is a recognized site of Mycobacterium tuberculosis infection, its role in the histopathology of leprosy was, until now, unknown. We analyzed the M. leprae capacity to infect and persist inside adipocytes, characterizing the induction of a lipolytic phenotype in adipocytes, as well as the effect of these infected cells on macrophage recruitment. We evaluated 3T3-L1-derived adipocytes, inguinal adipose tissue of SWR/J mice, and subcutaneous adipose tissue biopsies of leprosy patients. M. leprae was able to infect 3T3-L1-derived adipocytes in vitro, presenting a strong lipolytic profile after infection, followed by significant cholesterol efflux. This lipolytic phenotype was replicated in vivo by M. leprae injection into mice inguinal adipose tissue. Furthermore, M. leprae was detected inside crown-like structures in the subcutaneous adipose tissue of multibacillary patients. These data indicate that subcutaneous adipose tissue could be an important site of infection, and probably persistence, for M. leprae, being involved in the modulation of the innate immune control in leprosy via the release of cholesterol, MCP-1, and adiponectin.

Mycological profile of subcutaneous skin biopsy samples in suspected subcutaneous mycosis, Are we missing some fungi? A tertiary care hospital based study

Subcutaneous mycosis includes a heterogeneous group of fungal infections that develop at the site of transcutaneous trauma. A rise in surviving population of individuals with co-morbidities together with lapses in infection control practices especially in rural India has led to emergence of myriad species of fungi causing such infections in recent years. This poses a diagnostic challenge to the clinicians. We therefore felt a need to study the mycological profile of subcutaneous tissue biopsies suspected of fungal etiology in our hospital setup. This was a Retrospective cross-sectional observational study conducted for a period of 4 years from Jan 2015 to Jan 2019 in the Department of microbiology, Government medical College Srinagar. A total of 148 specimens of subcutaneous tissue biopsies of patients suspected of localized fungal Infection were processed by microscopy and culture for fungi. Among a total of 148 subcutaneous tissue biopsy samples 76 (51%) were positive for fungi on KOH and culture. Mycological profile revealed that majority of fungi were Dermatophytes (n=22; 29%) and Yeasts (n=21; 28%). Third most common causative fungi isolated were Chromoblastomycetes (n=8; 11%), Phaeohyphomycetes (n=8;11%) and Aspergilli (n=8;11 %) comprising, A.fumigatus (n=11) and A.terreus (n=03), followed by Hyalohypomycetes (n=4;5%), sporotrichosis (n=3;4%) and Zygomycetes (n=4 ;5.3%). : Dermatophytes and yeasts are emerging subcutaneous fungal infections. Active surveillance for all types of fungal infections should be done at all levels to address knowledge gaps that exist in etiology, pathogenesis and manifestations of fungal diseases of skin and subcutaneous tissue.

Unilateral Tonsillar Enlargement as Initial Presentation of Bilateral Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma.

Unilateral tonsillar enlargement is a common indication for tonsillectomy, but there are varying rates of malignancy among tonsils removed for asymmetry and a lack of clear guidelines for management within the literature. Lymphoma of the palatine tonsils is among the concerns leading to tonsillectomy, but chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) of the tonsil is rare. We report a case of primary CLL/SLL of the palatine tonsil in a 51-year-old gentleman who presented with tonsillar asymmetry and obstructive sleep apnea (OSA) but lacked signs and symptoms suspicious for malignancy, including lymphadenopathy and "B-symptoms." To our knowledge, only 7 cases of CLL/SLL of the palatine tonsil have been reported in the English literature, with the tonsil being the primary site of involvement in only 4 of those cases. Our unique case highlights the importance of thorough physical exam, family history, and tissue biopsy in patients presenting to the otolaryngologist with OSA and asymmetric tonsils.

Human intestinal organoids from Cronkhite-Canada syndrome patients reveal link between serotonin and proliferation.

Cronkhite-Canada Syndrome (CCS) is a rare, noninherited polyposis syndrome affecting 1 in every million individuals. Despite over 50 years of CCS cases, the etiopathogenesis and optimal treatment for CCS remains unknown due to the rarity of the disease and lack of model systems. To better understand the etiology of CCS, we generated human intestinal organoids (HIOs) from intestinal stem cells isolated from 2 patients. We discovered that CCS HIOs are highly proliferative and have increased numbers of enteroendocrine cells producing serotonin (also known as 5-hydroxytryptamine or 5HT). These features were also confirmed in patient tissue biopsies. Recombinant 5HT increased proliferation of non-CCS donor HIOs and inhibition of 5HT production in the CCS HIOs resulted in decreased proliferation, suggesting a link between local epithelial 5HT production and control of epithelial stem cell proliferation. This link was confirmed in genetically engineered HIOs with an increased number of enteroendocrine cells. This work provides a new mechanism to explain the pathogenesis of CCS and illustrates the important contribution of HIO cultures to understanding disease etiology and in the identification of novel therapies. Our work demonstrates the principle of using organoids for personalized medicine and sheds light on how intestinal hormones can play a role in intestinal epithelial proliferation.

Nerve transcriptomes in autoimmune and genetic demyelinating neuropathies: Pathogenic pathway assessment of nerve demyelination

The pathogenesis of autoimmune demyelinating neuropathies is poorly understood compared to inherited demyelinating forms. We performed whole transcriptome (RNA-Seq) using nerve biopsy tissues of patients with different autoimmune and inherited demyelinating neuropathies (CIDP n = 10, POEMS n = 18, DADS n = 3, CMT1 n = 3) versus healthy controls (n = 6). A limited number of differentially expressed genes compared to healthy controls were identified (POEMS = 125, DADS = 15, CMT = 14, CIDP = 5). Divergent pathogenic pathways including inflammatory, demyelinating and neurite regeneration such as with the triggering receptor expressed on myeloid cells (TREM1) part of the immunoglobulin superfamily and RhoGD1 are found. Shared and discordant pathogenic injury are discovered between autoimmune and inherited forms.

Multiplexed and Millimeter-Scale Fluorescence Nanoscopy of Cells and Tissue Sections via Prism-Illumination and Microfluidics-Enhanced DNA-PAINT.

Fluorescence nanoscopy has become increasingly powerful for biomedical research, but it has historically afforded a small field-of-view (FOV) of around 50 μm × 50 μm at once and more recently up to ∼200 μm × 200 μm. Efforts to further increase the FOV in fluorescence nanoscopy have thus far relied on the use of fabricated waveguide substrates, adding cost and sample constraints to the applications. Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT (PRIME-PAINT) for multiplexed fluorescence nanoscopy across millimeter-scale FOVs. Built upon the well-established prism-type total internal reflection microscopy, PRIME-PAINT achieves robust single-molecule localization with up to ∼520 μm × 520 μm single FOVs and 25-40 nm lateral resolutions. Through stitching, nanoscopic imaging over mm2 sample areas can be completed in as little as 40 min per target. An on-stage microfluidics chamber facilitates probe exchange for multiplexing and enhances image quality, particularly for formalin-fixed paraffin-embedded (FFPE) tissue sections. We demonstrate the utility of PRIME-PAINT by analyzing ∼106 caveolae structures in ∼1,000 cells and imaging entire pancreatic cancer lesions from patient tissue biopsies. By imaging from nanometers to millimeters with multiplexity and broad sample compatibility, PRIME-PAINT will be useful for building multiscale, Google-Earth-like views of biological systems.

Identifying infection in chronic wounds in a community setting: A systematic review of diagnostic test accuracy studies.

To determine the diagnostic accuracy of different methods currently available to identify infection in chronic wounds applicable to adult patients in a community setting. Systematic review of diagnostic test accuracy studies. Two authors independently completed screening, data extraction and quality and bias assessments (QUADAS2). Eligible studies compared a method (index test) for detecting infection (diagnosis of interest) with microscopy and culture of either deep tissue biopsy or wound swab (reference test) in adult patients with wounds of >4 weeks duration (participants). The results were synthesized narratively. We systematically searched CINAHL, Embase and Medline from 2011 to April 2022. Four studies were included, all recruiting from secondary care wound clinics. Two studies assessed the diagnostic accuracy of Moleculight i:X, a bacterial fluorescence imaging device against deep tissue biopsy culture. One study assessed the diagnostic accuracy of the elevation of various enzymes detected in wound fluid against wound swab microscopy of culture. One study assessed the diagnostic accuracy of bacterial protease activity against wound swab microscopy and culture. Sensitivities of these methods ranged from 50 to 75% and specificities from 47 to 100%. Only a small number of studies were included in this systematic review due to our strict inclusion criteria. We have not identified any methods for diagnosing infection in chronic wounds with either a sufficient quality of evidence to recommend their use in community settings at present. Further research is needed to develop and evaluate appropriate diagnostics for this purpose. This study highlights the paucity of research into wound diagnostics in a community setting and should prompt further research in this area. Accurate diagnostic tests have the potential to improve community-based wound care by optimizing antibiotic use and potentially improving healing time. PRISMA-DTA checklist. The PPI group for the NIHR Community Healthcare MIC were supportive of this topic of work.

Breast cancer prediction model based on clinical and biochemical characteristics: clinical data from patients with benign and malignant breast tumors from a single center in South China.

Breast cancer is the most prevalent cancer and is second leading cause of death from malignancy among women worldwide. In addition to tumor factors, the host characteristics of tumors have been paid more and more attention by the medical community. This study aimed to develop a breast cancer prediction model for the Chinese population using clinical and biochemical characteristics. This is a retrospective study. From 2012 to 2021, we selected 19,751 patients with breast diseases from the Guangdong Hospital of Traditional Chinese Medicine, which included 5660 patients with breast cancer and 14,091 patients with benign breast diseases-75% of patients were randomly assigned to the training group and 25% to the test group using a total of 34 clinical and biochemical characteristics. Significant clinical signs were investigated, and logistic regression with recursive feature elimination (RFE) model was used to develop a prediction model for distinguishing benign from malignant breast diseases. The prediction model's accuracy, precision, sensitivity, specificity, and area under the ROC curve (AUC) were calculated. Clinical statistics demonstrated that the prediction model comprised 19 clinical characteristics had statistical separability in both the training group and the test group, as well as good sensitivity and prediction. This model based on biochemical parameters demonstrates a significant predictive effect for breast cancer and may be useful as a reference for invasive tissue biopsy in patients undergoing BI-RADS 3 and 4A breast imaging.

Niclosamide Attenuates Inflammation-Associated Profibrotic Responses in Human Subepithelial Lung Myofibroblasts.

Niclosamide is a commonly used helminthicidic drug for the treatment of human parasitosis by helminths. Recently, efforts have been focusing on repurposing this drug for the treatment of other diseases, such as idiopathic pulmonary fibrosis. Subepithelial lung myofibroblasts (SELMs) isolated from tissue biopsies of patients undergoing surgery for lung cancer were stimulated with TNF-α (50 ng/mL), IL-1α (5 ng/mL), added alone or in combination, and TGF-β1 (5 ng/mL). After treatment with niclosamide at 30 nM and 100 nM concentrations, expression of collagen type I, collagen type III, and fibronectin was studied by total RNA isolation and qRT-PCR and protein collagen secretion with the use of Sircol collagen assay. The migration of SELMs was assessed by a wound-healing assay. Niclosamide had no effect on baseline SELM fibrotic factor expression. When stimulated with TGF-β1, IL-1α, and/or TNF-α, SELM expression of collagen type I, type III, and fibronectin were upregulated, as was the secretion of total collagen in the culture medium. Treatment with niclosamide attenuated the effects of cytokine stimulation leading to a notable decrease in the mRNA expression of collagen type I, type III, and fibronectin in a concentration-dependent manner. SELM collagen secretion was also reduced by niclosamide at 100 nM concentration when examined at the protein level. Migration of both TGF-β1 stimulated and unstimulated SELMs was also inhibited by niclosamide. In this study, we highlight the anti-fibrotic properties of niclosamide on SELMs under stimulation with pro-fibrotic and pro-inflammatory cytokines, thus proposing this compound as a possible new therapeutic agent against lung fibrosis.

The Relationship of Enterotoxigenic Bacteroides fragilis and Fusobacterium nucleatum Intestinal Colonization with Colorectal Cancer: A Case-Control Study Performed with Colon Biopsies

In recent years, it has been shown that some bacteria may be associated with colorectal cancer (CRC). In this study, it was aimed to investigate the role of Fusobacterium nucleatum and enterotoxigenic Bacteroides fragilis (ETBF) in the etiology of CRC by comparing the amounts of these bacteria in colon biopsy tissues of patients with CRC and healthy individuals. The amounts of F.nucleatum and ETBF were determined by quantitative polymerase chain reaction (qPCR) in colon biopsy samples taken from 35 CRC and 35 healthy individuals, and the results were compared in the patient and control groups. The detection rate and amounts of F.nucleatum were found to be statistically significantly higher in tissues of female patients with CRC compared to male patients (p= 0.003, p= 0.013, respectively). There was no statistically significant difference between the tissues of female and male patients with CRC in terms of detection rate and amount of ETBF (p= 0.521, p= 0.515, respectively). It was found that in the 50-74 age group, the amount of ETBF was statistically significantly higher in women and men with CRC compared to the controls (p= 0.005, p= 0.047, respectively), while the amount of F.nucleatum was statistically significantly higher in female patients compared to controls. However, no difference was found between male patients and controls (p= 0.009, p= 0.083). It was determined that the detection rate and amount of F.nucleatum in the tissues of patients with CRC, regardless of age and gender, were not statistically different from the controls (p= 0.473, p= 0.995, respectively), however, the detection rate of ETBF and the amount of ETBF were found to be statistically significantly higher (p= 0.002, p= 0.004, respectively). It has been determined that ETBF can play a role in the etiology of CRC in both men and women, and F.nucleatum only in women, in the age range of 50-74 years, when routine screenings for CRC are performed.

Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes

BackgroundStreptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist immuno-clearance, GAS adapt their genetic information and/or phenotype to the surrounding environment. Hyper-virulent streptococcal pyrogenic exotoxin B (SpeB) negative variants caused by covRS mutations are enriched during infection. A key driving force for this process is the bacterial Sda1 DNase.MethodsBacterial infiltration, immune cell influx, tissue necrosis and inflammation in patient´s biopsies were determined using immunohistochemistry. SpeB secretion and activity by GAS post infections or challenges with reactive agents were determined via Western blot or casein agar and proteolytic activity assays, respectively. Proteome of GAS single colonies and neutrophil secretome were profiled, using mass spectrometry.ResultsHere, we identify another strategy resulting in SpeB-negative variants, namely reversible abrogation of SpeB secretion triggered by neutrophil effector molecules. Analysis of NSTI patient tissue biopsies revealed that tissue inflammation, neutrophil influx, and degranulation positively correlate with increasing frequency of SpeB-negative GAS clones. Using single colony proteomics, we show that GAS isolated directly from tissue express but do not secrete SpeB. Once the tissue pressure is lifted, GAS regain SpeB secreting function. Neutrophils were identified as the main immune cells responsible for the observed phenotype. Subsequent analyses identified hydrogen peroxide and hypochlorous acid as reactive agents driving this phenotypic GAS adaptation to the tissue environment. SpeB-negative GAS show improved survival within neutrophils and induce increased degranulation.ConclusionsOur findings provide new information about GAS fitness and heterogeneity in the soft tissue milieu and provide new potential targets for therapeutic intervention in NSTIs.

Mechanism of cinobufacin anti-metastatic colorectal cancer based on integrated mining of proteomics and bioinformatics

In this study, the effector proteins of anti-metastatic colorectal cancer (CRC) were mined through proteomics based on the preliminary screening of cinobufacin anti-cancer genes using bioinformatics and the role of cinobufacin anti-metastatic CRC was explored. Proteomics was used to analyze the differential expression of proteins in biopsy tissues of patients with metastatic CRC and bioinformatics was adopted to screen for cinobufacin tumor suppressor genes. The effects of proteomics-mined effector protein CyclinD1 on cell proliferation and colony formation were evaluated by MTT assay and colony formation assay. The cinobufacin sensitivity of metastatic CRC cells were also assessed. After proteomic analysis, the common targets of “Cinobufacin-metastatic CRC” were screened out, among which the tumor-associated CyclinD1 was listed as its level was increased in biopsy tissues of patients with metastatic CRC. The preliminary screening of cinobufacin anti-cancer genes based on bioinformatics found that tumor suppressor gene P53 was closely related to metastatic CRC. CyclinD1 had a relation with P53 mRNA. Further studies showed that P53 was the direct target of CyclinD1 and CyclinD1 overexpression enhanced the sensitivity of metastatic CRC cells to cinobufacin. Using bioinformatics-based gene database analysis technology and proteomics mining technology, the differential expression of proteomics in the biopsy tissues of patients with metastatic CRC and the tumor suppressor genes regulated by cinobufacin were identified. CyclinD1 and P53 found by bioinformatics might be biomarkers for metastatic CRC prognosis, providing a new possible molecular target for anticancer therapy.