Unveiling the viral escape: Quantification of microfloc-bound viruses in precoagulation and membrane filtration.

The implementation of precoagulation before the physical removal process is expected to achieve a high virus removal rate. However, viruses may form small flocs and subsequently escape into the effluent during physical removal processes. This study evaluated how viruses in the microflocs could be quantified using conventional virus quantification methods (plaque assay and quantitative polymerase chain reaction (qPCR)) to reveal the risk of underestimating virus concentration. In this study, the microfloc dissolution phenomenon in phosphate buffer solution was employed as a floc dissolution test. Viruses in microflocs formed under the experimental conditions. assuming water treatments, were quantified before and after floc dissolution. The findings revealed that virus concentrations increased by 1.0-3.9 log plaque-forming units/mL according to the plaque assay and by 1.7-4.0 log copies/mL according to the qPCR. This increase occurred after the dissolution of microflocs that were prepared in the humic acid test water. In the case of treated wastewater, virus concentrations increased in all samples according to the plaque assay and in seven of eight samples according to the qPCR. Our results indicate the necessity of careful consideration of virus quantification after precoagulation and physical removal systems.

Correspondence of SARS-CoV-2 genomic sequences obtained from wastewater samples and COVID-19 patient at long-term care facilities.

Wastewater-based epidemiology (WBE) has been in the spotlight because of applicability of early detection of virus outbreak and new variants at the catchment area. However, there has been a notable absence of research directly confirming the association between SARS-CoV-2 in wastewater and patient specimens. In this study, we performed a quantitative and qualitative investigation with a genetic-level comparison of SARS-CoV-2 between COVID-19 patients and SARS-CoV-2 positive wastewater samples at long-term care facilities. Wastewater samples were collected via passive sampling from manholes, and SARS-CoV-2 load in wastewater was determined by qPCR. We performed correlation analysis between SARS-CoV-2 load and COVID-19 case number, which suggested that SARS-CoV-2 was detected from wastewater earlier than ascertainment of COVID-19 case. Six and six RNA samples from COVID-19 positive cases and wastewater, respectively, from two facilities were then applied for amplicon sequencing analysis. Mutation analysis revealed high sequence similarity of SARS-CoV-2 variants between wastewater and patient samples (>99 %). To the best of our knowledge, this is the first study demonstrating that WBE is also effective in predicting predominant SARS-CoV-2 variant at facility-level, which is helpful to develop early-warning system for outbreak occurrence with predominant variant.

Inactivation of coxsackievirus B5 by free chlorine under conditions relevant to drinking water treatment.

Chlorine disinfection is commonly applied to inactivate pathogenic viruses in drinking water treatment plants. However, the role of water quality in chlorine disinfection of viruses has not been investigated thoughtfully. In this study, we investigated the inactivation efficiency of coxsackievirus B5 (CVB5) by free chlorine using actual water samples collected from four full-scale drinking water treatment plants in Japan under strict turbidity management (less than 0.14 NTU) over a 12-month period. It was found that chlorine disinfection of CVB5 might not be affected by water quality. Japanese turbidity management might play an indirect role in controlling the efficiency of chlorine disinfection.

Development of a simple and low-cost method using Moringa seeds for efficient virus concentration in wastewater.

Effective virus concentration methods are essential for detecting pathogenic viruses in environmental waters and play a crucial role in wastewater-based epidemiology. However, the current methods are often expensive, complicated, and time-consuming, which limits their practical application. In this study, a simple and low-cost method was developed using the extract of Moringa oleifera (MO) seeds (MO method) to recover both enveloped and non-enveloped viruses, including pepper mild mottle virus (PMMoV), murine norovirus (MNV), Aichivirus (AiV), murine hepatitis virus (MHV), and influenza A virus subtype H1N1[H1N1] in wastewater. The optimal conditions for the MO method were determined to be a concentration of MO extract at the UV280 value of 0.308 cm-1 and an elution buffer (0.05 M KH2PO4, 1 M NaCl, 0.1 % Tween80 [v/v]) for recovering the tested viruses in wastewater. Compared to other commonly used virus concentration methods such as InnovaPrep, HA, PEG, and Centricon, the MO method was found to be more efficient and cost-effective in recovering the tested viruses. Moreover, the MO method was successfully applied to detect various types of viruses (PMMoV, AiV, norovirus of genotype II [NoV II], enterovirus [EV], influenza A virus [matrix gene] [IAV], and SARS-CoV-2) in raw wastewater. Thus, the developed MO method could offer a simple, low-cost, and efficient tool to concentrate viruses in wastewater.

Activity- and gene-based quantification of enteric viruses, F- specific RNA phage genogroups, pepper mild mottle virus, and Escherichia coli in surface water.

Polymerase chain reaction (PCR) is widely applied for the monitoring of pathogenic viruses in water environments. To date, several pretreatments to selectively detect genes from infectious viruses via PCR have been developed. This study was aimed to characterize and validate methods for quantifying active viruses and indicators and to evaluate the proportion of their active fractions in surface water (n = 42). Active E. coli and F-specific RNA phage (FRNAPH) genogroups were quantified using culture assays. In addition to these microbes, norovirus genogroups I (GI) and II, Aichi virus 1, and pepper mild mottle virus (PMMoV) were quantified by (reverse transcription)-quantitative PCR (RT-qPCR) with and without cis-dichlorodiammineplatinum (CDDP) treatment to exclude genes in inactive viruses. CDDP-RT-qPCR showed concentrations and detection frequencies comparable to or higher than culture assays. Consequently, although CDDP-RT-qPCR can suggest the presence of an inactive virus, it can also overestimate the activity of the virus in the environment. Differences between culture and CDDP-RT-qPCR and between CDDP-RT-qPCR and RT-qPCR varied among the viruses. CDDP-RT-qPCR showed a concentration comparable to the culture assay (within 1 log10 difference) in 93 % of positive samples for GI-FRNAPH but in <63 % of positive samples for GII- and GIII-FRNAPHs. GII-NoV was detected from 5 and 30 out of 42 samples via CDDP-RT-qPCR and RT-qPCR, respectively, and was suggested as inactivated by 2.0 log10 or higher in most of the samples. By contrast, concentrations of PMMoV determined by these two assays were not notably different. It is suggested that the operational conditions of wastewater treatment plants around the sites, rather than environmental stresses, affected the microbial inactivation. To better understand the infectivity of viruses in the environment, it is important to investigate them using sensitive detection methods at various sites, including the source of contamination.

Blood-based Proteomic Signatures Associated With MEN1-related Duodenopancreatic Neuroendocrine Tumor Progression.

PURPOSE: Patients with multiple endocrine neoplasia type 1 (MEN1) are predisposed to develop duodenopancreatic neuroendocrine tumors (dpNETs), and metastatic dpNET is the primary cause of disease-related mortality. Presently, there is a paucity of prognostic factors that can reliably identify patients with MEN1-related dpNETS who are at high risk of distant metastasis. In the current study, we aimed to establish novel circulating molecular protein signatures associated with disease progression. EXPERIMENTAL DESIGN: Mass spectrometry-based proteomic profiling was conducted on plasmas procured through an international collaboration between MD Anderson Cancer Center, the National Institutes of Health, and the University Medical Center Utrecht from a cohort of 56 patients with MEN1 [14 with distant metastasis dpNETs (cases) and 42 with either indolent dpNETs or no dpNETs (controls)]. Findings were compared to proteomic profiles generated from serially collected plasmas from a mouse model of Men1-pancreatic neuroendocrine tumors (Men1fl/flPdx1-CreTg) and control mice (Men1fl/fl). RESULTS: A total of 187 proteins were found to be elevated in MEN1 patients with distant metastasis compared to controls, including 9 proteins previously associated with pancreatic cancer and other neuronal proteins. Analyses of mouse plasmas revealed 196 proteins enriched for transcriptional targets of oncogenic MYCN, YAP1, POU5F1, and SMAD that were associated with disease progression in Men1fl/flPdx1-CreTg mice. Cross-species intersection revealed 19 proteins positively associated with disease progression in both human patients and in Men1fl/flPdx1-CreTg mice. CONCLUSIONS: Our integrated analyses identified novel circulating protein markers associated with disease progression in MEN1-related dpNET.

Proteogenomic landscape of gastric adenocarcinoma peritoneal metastases.

Advanced gastric adenocarcinoma (GAC) often leads to peritoneal carcinomatosis (PC) and is associated with very poor outcome. Here we report the comprehensive proteogenomic study of ascites derived cells from a prospective GAC cohort (n = 26 patients with peritoneal carcinomatosis, PC). A total of 16,449 proteins were detected from whole cell extracts (TCEs). Unsupervised hierarchical clustering resulted in three distinct groups that reflected extent of enrichment in tumor cells. Integrated analysis revealed enriched biological pathways and notably, some druggable targets (cancer-testis antigens, kinases, and receptors) that could be exploited to develop effective therapies and/or tumor stratifications. Systematic comparison of expression levels of proteins and mRNAs revealed special expression patterns of key therapeutics target notably high mRNA and low protein expression of HAVCR2 (TIM-3), and low mRNA but high protein expression of cancer-testis antigens CTAGE1 and CTNNA2. These results inform strategies to target GAC vulnerabilities.

Effects of surface hydrophobicity on the removal of F-specific RNA phages from reclaimed water by coagulation and ceramic membrane microfiltration.

Microfiltration (MF) has been widely adopted as an advanced treatment process to reduce suspended solids and turbidity in treated wastewater effluents designated for potable reuse. Although microfilter pores are much larger than viruses, the addition of a coagulant upstream of a microfilter system can achieve stable virus removal. Ceramic membranes have a narrow pore size distribution to achieve the high removal of contaminants. This study aims to evaluate virus log reduction using bench-scale coagulation and ceramic membrane MF. To investigate the effects of differences in net surface hydrophobicity, 18 sewage-derived F-specific RNA phages (FRNAPHs) were used for batch hydrophobicity and coagulation-MF tests. The capability of bench-scale coagulation and ceramic membrane MF under continuous automated long-term operation was tested to remove the lab reference strain MS2 and three selected FRNAPH isolates which varied by surface property. Median virus log reduction values (LRVs) exceeding 6.2 were obtained for all three isolates and MS2. Although coagulation and hydrophobicity were positively correlated, the virus isolate demonstrating the lowest level of hydrophobicity and coagulation (genogroup I) still exhibited a high LRV. Thus, coagulation and ceramic membrane MF systems may serve as viable options for virus removal during water reclamation and advanced treatment.

Kynureninase Upregulation Is a Prominent Feature of NFR2-Activated Cancers and Is Associated with Tumor Immunosuppression and Poor Prognosis.

The nuclear factor erythroid 2-related factor 2 (NRF2) pathway is frequently activated in various cancer types. Aberrant activation of NRF2 in cancer is attributed to gain-of-function mutations in the NRF2-encoding gene NFE2L2 or a loss of function of its suppressor, Kelch-like ECH-associated protein 1 (KEAP1). NRF2 activation exerts pro-tumoral effects in part by altering cancer cell metabolism. Previously, we reported a novel mechanism of NRF2 tumoral immune suppression through the selective upregulation of the tryptophan-metabolizing enzyme kynureninase (KYNU) in lung adenocarcinoma. In the current study, we explored the relevance of NRF2-mediated KYNU upregulation across multiple cancer types. Specifically, using a gene expression dataset for 9801 tumors representing 32 cancer types from The Cancer Genome Atlas (TCGA), we demonstrated that elevated KYNU parallels increased gene-based signatures of NRF2-activation and that elevated tumoral KYNU mRNA expression is strongly associated with an immunosuppressive tumor microenvironment, marked by high expression of gene-based signatures of Tregs as well as the immune checkpoint blockade-related genes CD274 (PDL-1), PDCD1 (PD-1), and CTLA4, regardless of the cancer type. Cox proportional hazard models further revealed that increased tumoral KYNU gene expression was prognostic for poor overall survival in several cancer types, including thymoma, acute myeloid leukemia, low-grade glioma, kidney renal papillary cell carcinoma, stomach adenocarcinoma, and pancreatic ductal adenocarcinoma (PDAC). Using PDAC as a model system, we confirmed that siRNA-mediated knockdown of NRF2 reduced KYNU mRNA expression, whereas activation of NFE2L2 (the coding gene for NRF2) through either small-molecule agonists or siRNA-mediated knockdown of KEAP1 upregulated KYNU in PDAC cells. Metabolomic analyses of the conditioned medium from PDAC cell lines revealed elevated levels of KYNU-derived anthranilate, confirming that KYNU was enzymatically functional. Collectively, our study highlights the activation of the NRF2-KYNU axis as a multi-cancer phenomenon and supports the relevance of tumoral KYNU as a marker of tumor immunosuppression and as a prognostic marker for poor overall survival.

Surfactant Treatment for Efficient Gene Detection of Enteric Viruses and Indicators in Surface Water Concentrated by Ultrafiltration.

The hollow fiber ultrafiltration (HFUF)-based microbial concentration method is widely applied for monitoring pathogenic viruses and microbial indicators in environmental water samples. However, the HFUF-based method can co-concentrate substances that interfere with downstream molecular processes-nucleic acid extraction, reverse transcription (RT), and PCR. These inhibitory substances are assumed to be hydrophobic and, therefore, expected to be excluded by a simple surfactant treatment before the silica membrane-based RNA extraction process. In this study, the efficacy and limitations of the sodium deoxycholate (SD) treatment were assessed by quantifying a process control and indigenous viruses using 42 surface water samples concentrated with HFUF. With some exceptions, which tended to be seen in samples with high turbidity (> 4.0 NTU), virus recovery by the ultrafiltration method was sufficiently high (> 10%). RNA extraction-RT-quantitative PCR (RT-qPCR) efficiency of the process control was insufficient (10%) for 30 of the 42 HFUF concentrates without any pretreatments, but it was markedly improved for 21 of the 30 inhibitory concentrates by the SD treatment. Detection rates of indigenous viruses were also improved and no substantial loss of viral RNA was observed. The SD treatment was particularly effective in mitigating RT-qPCR inhibition, although it was not effective in improving RNA extraction efficiency. The methodology is simple and easily applied. These findings indicate that SD treatment can be a good alternative to sample dilution, which is widely applied to mitigate the effect of RT-qPCR inhibition, and can be compatible with other countermeasures.

Mining the Immunopeptidome for Antigenic Peptides in Cancer.

Although harnessing the immune system for cancer therapy has shown success, response to immunotherapy has been limited. The immunopeptidome of cancer cells presents an opportunity to discover novel antigens for immunotherapy applications. These neoantigens bind to MHC class I and class II molecules. Remarkably, the immunopeptidome encompasses protein post-translation modifications (PTMs) that may not be evident from genome or transcriptome profiling. A case in point is citrullination, which has been demonstrated to induce a strong immune response. In this review, we cover how the immunopeptidome, with a special focus on PTMs, can be utilized to identify cancer-specific antigens for immunotherapeutic applications.

Novel UHRF1-MYC Axis in Acute Lymphoblastic Leukemia.

Ubiquitin-like, containing PHD and RING finger domain, (UHRF) family members are overexpressed putative oncogenes in several cancer types. We evaluated the protein abundance of UHRF family members in acute leukemia. A marked overexpression of UHRF1 protein was observed in ALL compared with AML. An analysis of human leukemia transcriptomic datasets revealed concordant overexpression of UHRF1 in B-Cell and T-Cell ALL compared with CLL, AML, and CML. In-vitro studies demonstrated reduced cell viability with siRNA-mediated knockdown of UHRF1 in both B-ALL and T-ALL, associated with reduced c-Myc protein expression. Mechanistic studies indicated that UHRF1 directly interacts with c-Myc, enabling ALL expansion via the CDK4/6-phosphoRb axis. Our findings highlight a previously unknown role of UHRF1 in regulating c-Myc protein expression and implicate UHRF1 as a potential therapeutic target in ALL.

A Comprehensive Search of Non-Canonical Proteins in Non-Small Cell Lung Cancer and Their Impact on the Immune Response.

There is substantial interest in mining neoantigens for cancer applications. Non-canonical proteins resulting from frameshift mutations have been identified as neoantigens in cancer. We investigated the landscape of non-canonical proteins in non-small cell lung cancer (NSCLC) and their induced immune response in the form of autoantibodies. A database of cryptoproteins was computationally constructed and comprised all alternate open reading frames (altORFs) and ORFs identified in pseudogenes, noncoding RNAs, and untranslated regions of mRNAs that did not align with known canonical proteins. Proteomic profiles of seventeen lung adenocarcinoma (LUAD) cell lines were searched to evaluate the occurrence of cryptoproteins. To assess the immunogenicity, immunoglobulin (Ig)-bound cryptoproteins in plasmas were profiled by mass spectrometry. The specimen set consisted of plasmas from 30 newly diagnosed NSCLC cases, pre-diagnostic plasmas from 51 NSCLC cases, and 102 control plasmas. An analysis of LUAD cell lines identified 420 cryptoproteins. Plasma Ig-bound analyses revealed 90 cryptoproteins uniquely found in cases and 14 cryptoproteins that had a fold-change >2 compared to controls. In pre-diagnostic samples, 17 Ig-bound cryptoproteins yielded an odds ratio ≥2. Eight Ig-bound cryptoproteins were elevated in both pre-diagnostic and newly diagnosed cases compared to controls. Cryptoproteins represent a class of neoantigens that induce an autoantibody response in NSCLC.

Genotype-dependent kinetics of enterovirus inactivation by free chlorine and ultraviolet (UV) irradiation.

Inactivation kinetics of enterovirus by disinfection is often studied using a single laboratory strain of a given genotype. Environmental variants of enterovirus are genetically distinct from the corresponding laboratory strain, yet it is poorly understood how these genetic differences affect inactivation. Here we evaluated the inactivation kinetics of nine coxsackievirus B3 (CVB3), ten coxsackievirus B4 (CVB4), and two echovirus 11 (E11) variants by free chlorine and ultraviolet irradiation (UV). The inactivation kinetics by free chlorine were genotype- (i.e., susceptibility: CVB5 < CVB3 ≈ CVB4 < E11) and genogroup-dependent and exhibited up to 15-fold difference among the tested viruses. In contrast, only minor (up to 1.3-fold) differences were observed in the UV inactivation kinetics. The differences in variability between the two disinfectants could be rationalized by their respective inactivation mechanisms: inactivation by UV mainly depends on the genomic size and composition, which was similar for all viruses tested, whereas free chlorine targets the viral capsid protein, which exhibited critical differences between genogroups and genotypes. Finally, we integrated the observed variability in inactivation rate constants into an expanded Chick-Watson model to estimate the overall inactivation of an enterovirus consortium. The results highlight that the distribution of inactivation rate constants and the abundance of each genotype are essential parameters to accurately predict the overall inactivation of an enterovirus population by free chlorine. We conclude that predictions based on inactivation data of a single variant or reference pathogen alone likely overestimate the true disinfection efficiency of free chlorine.

Mutational Activation of the NRF2 Pathway Upregulates Kynureninase Resulting in Tumor Immunosuppression and Poor Outcome in Lung Adenocarcinoma.

Activation of the NRF2 pathway through gain-of-function mutations or loss-of-function of its suppressor KEAP1 is a frequent finding in lung cancer. NRF2 activation has been reported to alter the tumor microenvironment. Here, we demonstrated that NRF2 alters tryptophan metabolism through the kynurenine pathway that is associated with a tumor-promoting, immune suppressed microenvironment. Specifically, proteomic profiles of 47 lung adenocarcinoma (LUAD) cell lines (11 KEAP1 mutant and 36 KEAP1 wild-type) revealed the tryptophan-kynurenine enzyme kynureninase (KYNU) as a top overexpressed protein associated with activated NRF2. The siRNA-mediated knockdown of NFE2L2, the gene encoding for NRF2, or activation of the NRF2 pathway through siRNA-mediated knockdown of KEAP1 or via chemical induction with the NRF2-activator CDDO-Me confirmed that NRF2 is a regulator of KYNU expression in LUAD. Metabolomic analyses confirmed KYNU to be enzymatically functional. Analysis of multiple independent gene expression datasets of LUAD, as well as a LUAD tumor microarray demonstrated that elevated KYNU was associated with immunosuppression, including potent induction of T-regulatory cells, increased levels of PD1 and PD-L1, and resulted in poorer survival. Our findings indicate a novel mechanism of NRF2 tumoral immunosuppression through upregulation of KYNU.

Removal of viruses from their cocktail solution by liquid-crystalline water-treatment membranes.

Liquid-crystalline (LC) water-treatment membranes obtained by in situ photopolymerization of ionic mesogenic monomers have been shown to efficiently remove viruses. In our previous works, bicontinuous cubic (Cubbi) and smectic (Sm) LC membranes prepared from ionic taper- and rod-shaped polymerizable mesogens, respectively, were used for this purpose. Here, we report the results of virus removal by columnar (Col) LC water-treatment membranes having ionic nanochannels obtained from ionic taper-shaped mesogens. These effects are compared with those obtained for Cubbi membranes. The effects of these Col and Cubbi LC ionic membranes on the removal of several viruses from their cocktail solution are also examined.

Capsid integrity detection of pathogenic viruses in waters: Recent progress and potential future applications.

Waterborne diseases caused by pathogenic human viruses are a major public health concern. To control the potential risk of viral infection through contaminated waters, a rapid, reliable tool to assess the infectivity of pathogenic viruses is required. Recently, an advanced approach (i.e., capsid integrity (RT-)qPCR) was developed to discriminate intact viruses (potentially infectious) from inactivated viruses. In this approach, samples were pretreated with capsid integrity reagents (e.g., monoazide dyes or metal compounds) before (RT -)qPCR. These reagents can only penetrate inactivated viruses with compromised capsids to bind to viral genomes and prevent their amplification, but they cannot enter viruses with intact capsids. Therefore, only viral genomes of intact viruses were amplified or detected by (RT-)qPCR after capsid integrity treatment. In this study, we reviewed recent progress in the development and application of capsid integrity (RT-)qPCR to assess the potential infectivity of viruses (including non-enveloped and enveloped viruses with different genome structures [RNA and DNA]) in water. The efficiency of capsid integrity (RT-)qPCR has been shown to depend on various factors, such as conditions of integrity reagent treatment, types of viruses, environmental matrices, and the capsid structure of viruses after disinfection treatments (e.g., UV, heat, and chlorine). For the application of capsid integrity (RT-)qPCR in real-world samples, the use of suitable virus concentration methods and process controls is important to control the efficiency of capsid integrity (RT-)qPCR. In addition, potential future applications of capsid integrity (RT-)qPCR for determining the mechanism of disinfection treatment on viral structure (e.g., capsid or genome) and a combination of capsid integrity treatment and next-generation sequencing (NGS) (capsid integrity NGS) for monitoring the community of intact pathogenic viruses in water are also discussed. This review provides essential information on the application of capsid integrity (RT-)qPCR as an efficient tool for monitoring the presence of pathogenic viruses with intact capsids in water.

Proteomic Profiling of the Tumor Microenvironment.

The tumor microenvironment forms a complex pro-tumorigenic milieu constituted by extracellular matrix, surrounding stroma, infiltrating cell populations, and signaling molecules. Proteomic studies have the potential to reveal how individual cell populations within the tumor tissue modulate the microenvironment through protein secretion and consequently alter their protein expression and localization to adapt to this milieu. As a result, proteomic approaches have uncovered how these dynamic components communicate and promote tumor development and progression. The characterization of these mechanisms is relevant for the identification of clinically targetable pathways and for the development of diagnostic tools. Here we describe a method based on the isolation of individual cell compartments and the chromatographic fractionation of intact proteins, followed by enzymatic digestion of individual fractions, and mass-spectrometry analysis, for the profiling of tumor microenvironment cell populations.