Five-year follow-up of 96 weeks peginterferon plus tenofovir disoproxil fumarate in hepatitis D.

BACKGROUND & AIMS: Until recently, pegylated interferon-alfa-2a (PEG-IFNa) therapy was the only treatment option for patients infected with hepatitis D virus (HDV). Treatment with PEG-IFNa with or without tenofovir disoproxil fumarate (TDF) for 96 weeks resulted in HDV RNA suppression in 44% of patients at the end of therapy but did not prevent short-term relapses within 24 weeks. The virological and clinical long-term effects after prolonged PEG-IFNa-based treatment of hepatitis D are unknown. METHODS: In the HIDIT-II study patients (including 40% with liver cirrhosis) received 180 µg PEG-IFNa weekly plus 300 mg TDF once daily (n = 59) or 180 µg PEG-IFNa weekly plus placebo (n = 61) for 96 weeks. Patients were followed until week 356 (5 years after end of therapy). RESULTS: Until the end of follow-up, 16 (13%) patients developed liver-related complications (PEG-IFNa + TDF, n = 5 vs PEG-IFNa + placebo, n = 11; p = .179). Achieving HDV suppression at week 96 was associated with decreased long-term risk for the development of hepatocellular carcinoma (p = .04) and hepatic decompensation (p = .009). Including complications irrespective of PEG-IFNa retreatment status, the number of patients developing serious complications was similar with (3/18) and without retreatment with PEG-IFNa (16/102, p > .999) but was associated with a higher chance of HDV-RNA suppression (p = .024, odds ratio 3.9 [1.3-12]). CONCLUSIONS: Liver-related clinical events were infrequent and occurred less frequently in patients with virological responses to PEG-IFNa treatment. PEG-IFNa treatment should be recommended to HDV-infected patients until alternative therapies become available. Retreatment with PEG-IFNa should be considered for patients with inadequate response to the first course of treatment. CLINICAL TRIAL REGISTRATION: NCT00932971.

Nematodes Degrade Extracellular Antibiotic Resistance Genes by Secreting DNase II Encoded by the nuc-1 Gene.

This study investigated the degradation performance and mechanism of extracellular antibiotic resistance genes (eARGs) by nematodes using batch degradation experiments, mutant strain validation, and phylogenetic tree construction. Caenorhabditis elegans, a representative nematode, effectively degraded approximately 99.999% of eARGs (tetM and kan) in 84 h and completely deactivated them within a few hours. Deoxyribonuclease (DNase) II encoded by nuc-1 in the excretory and secretory products of nematodes was the primary mechanism. A neighbor-joining phylogenetic tree indicated the widespread presence of homologs of the NUC-1 protein in other nematodes, such as Caenorhabditis remanei and Caenorhabditis brenneri, whose capabilities of degrading eARGs were then experimentally confirmed. C. elegans remained effective in degrading eARGs under the effects of natural organic matter (5, 10, and 20 mg/L, 5.26-6.22 log degradation), cation (2.0 mM Mg2+ and 2.5 mM Ca2+, 5.02-5.04 log degradation), temperature conditions (1, 20, and 30 °C, 1.21-5.26 log degradation), and in surface water and wastewater samples (4.78 and 3.23 log degradation, respectively). These findings highlight the pervasive but neglected role of nematodes in the natural decay of eARGs and provide novel approaches for antimicrobial resistance mitigation biotechnology by introducing nematodes to wastewater, sludge, and biosolids.

End-of-treatment HBcrAg and HBsAb levels identify durable functional cure after Peg-IFN-based therapy in patients with CHB.

BACKGROUND & AIMS: Functional cure can be sustained in a proportion of patients with chronic hepatitis B (CHB) who lose hepatitis B surface antigen (HBsAg) after pegylated interferon alpha (Peg-IFN-ɑ)-based treatment. In this study, we aimed to identify biomarkers associated with a durable functional cure and to dissect potential immunological mechanisms. METHODS: Of 257 nucleos(t)ide analogue-suppressed patients with CHB in the ANCHOR study, 80 patients randomly assigned to 96-week Peg-IFN-α-based therapy with 24-week off-treatment follow-up were included in this parallel study. Virologic and immunological biomarkers were examined dynamically. A response was defined as HBsAg loss or hepatitis B surface antibody (HBsAb) appearance at the end of treatment (EOT). Sustained response (SR) or durable functional cure was defined as sustained HBsAg loss with or without the appearance of HBsAb at the end of follow-up (EOF). RESULTS: Thirty-six (45.0%) out of 80 patients achieved a response at EOT; 58.3% (21/36) of responders maintained SR at EOF. Quantitative hepatitis B core-related antigen (qHBcrAg) and HBsAb at EOT were associated with SR, with AUROCs of 0.697 (0.512-0.882, p = 0.047) and 0.744 (0.573-0.915, p = 0.013), respectively. A combination of HBcrAg <4 log10U/ml and HBsAb >2 log10IU/L at EOT had a positive predictive value of 100% for SR with an AUROC of 0.822 (0.684-0.961, p = 0.001). These patients showed maintained proportions of HBV envelope-specific CD8+T and B cells, a markedly increased proportion of T follicular helper cells after Peg-IFN-ɑ discontinuation, and significantly higher proportions of HBV polymerase-specific CD8+T and CD86+CD19+B cells at EOF. CONCLUSIONS: Lower HBcrAg and higher HBsAb levels at EOT were associated with sustained cellular and humoral immune responses. They can be used to identify patients likely to achieve durable functional cure post Peg-IFN-based therapy. GOV IDENTIFIER: NCT02327416 LAY SUMMARY: Functional cure can be sustained in a proportion of patients with chronic hepatitis B after pegylated interferon alpha-based treatment. However, predicting who will achieve durable functional cure remains challenging. Herein, we show that low levels of hepatitis B core-related antigen and higher levels of hepatitis B surface antibodies at the end of treatment are linked to immunological responses and are associated with durable functional cure.

Meiotic viral attenuation through an ancestral apoptotic pathway.

The programmed release of apoptogenic proteins from mitochondria is a core event of apoptosis, although ancestral roles of this phenomenon are not known. In mammals, one such apoptogenic protein is Endonuclease G (EndoG), a conserved mitochondrial nuclease that fragments the DNA of dying cells. In this work, we show that budding yeast executes meiotically programmed mitochondrial release of an EndoG homolog, Nuc1, during sporulation. In contrast to EndoG's ostensible pro-death function during apoptosis, Nuc1 mitochondrial release is pro-survival, attenuating the cytosolic L-A and Killer double-stranded RNA mycoviruses and protecting meiotic progeny from the catastrophic consequences of their derepression. The protective viral attenuation role of this pathway illuminates a primordial role for mitochondrial release of EndoG, and perhaps of apoptosis itself.

Uniformity Correction of CMOS Image Sensor Modules for Machine Vision Cameras.

Flat-field correction (FFC) is commonly used in image signal processing (ISP) to improve the uniformity of image sensor pixels. Image sensor nonuniformity and lens system characteristics have been known to be temperature-dependent. Some machine vision applications, such as visual odometry and single-pixel airborne object tracking, are extremely sensitive to pixel-to-pixel sensitivity variations. Numerous cameras, especially in the fields of infrared imaging and staring cameras, use multiple calibration images to correct for nonuniformities. This paper characterizes the temperature and analog gain dependence of the dark signal nonuniformity (DSNU) and photoresponse nonuniformity (PRNU) of two contemporary global shutter CMOS image sensors for machine vision applications. An optimized hardware architecture is proposed to compensate for nonuniformities, with optional parametric lens shading correction (LSC). Three different performance configurations are outlined for different application areas, costs, and power requirements. For most commercial applications, the correction of LSC suffices. For both DSNU and PRNU, compensation with one or multiple calibration images, captured at different gain and temperature settings are considered. For more demanding applications, the effectiveness, external memory bandwidth, power consumption, implementation, and calibration complexity, as well as the camera manufacturability of different nonuniformity correction approaches were compared.

Optimization of Signal Peptide via Site-Directed Mutagenesis for Enhanced Secretion of Heterologous Proteins in Lactococcus lactis.

Secretion efficiency of heterologous proteins in the Generally Regarded As Safe (GRAS) Lactococcus lactis is often reported to be insufficiently low due to limitations such as poor targeting and translocation by the signal peptide or degradation by the host proteases. In this study, the secretion efficiency in the host was enhanced through the utilization of a heterologous signal peptide (SP) SPK1 of Pediococcus pentosaceus. The SPK1 was subjected to site-directed mutations targeting its tripartite N-, H-, and C-domains, and the effect on secretion efficiency as compared to the wild-type SPK1 and native lactococcal USP45 was determined on a reporter nuclease (NUC) of Staphylococcus aureus. A Fluorescence Resonance Energy Transfer (FRET) analysis indicated that four out of eight SPK1 variants successfully enhanced the secretion of NUC, with the best mutant, SPKM19, showing elevated secretion efficiency up to 88% (or by 1.4-fold) and an improved secretion activity yield of 0.292 ± 0.122 U/mL (or by 1.7-fold) compared to the wild-type SPK1. Modifications of the SPK1 at the cleavage site C-domain region had successfully augmented the secretion efficiency. Meanwhile, mutations in the H-domain region had resulted in a detrimental effect on the NUC secretion. The development of heterologous SPs with better efficacy than the USP45 has been demonstrated in this study for enhanced secretion of heterologous production and mucosal delivery applications in the lactococcal host.

A Systematic Study on the Optimal Nucleotide Analogue Concentration and Rate Limiting Nucleotide of the SARS-CoV-2 RNA-Dependent RNA Polymerase.

The current COVID-19 pandemic has highlighted the necessity of more efficient antiviral compounds. The antiviral efficacy of adenosine-based analogs, the main repurposed drugs for SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition, is mainly assessed through in vitro or cell-free polymerization assays, under arbitrary conditions that do not reflect the physiological environment. We show that SARS-CoV-2 RdRp inhibition efficiency of remdesivir and cordycepin, two common adenosine analogs, is influenced by endogenous adenosine level, and that the current clinically approved concentrations for COVID-19 treatment are suboptimal for effective RdRp inhibition. Furthermore, we identified GTP as the rate-limiting nucleotide of SARS-CoV-2 replication. Our results demonstrate that nucleotide sensitivity of the RdRp complex and competition of nucleoside analog drugs against endogenous concentrations of nucleotides are crucial elements to be considered when designing new SARS-CoV-2 antiviral compounds.

A Phase Ib Study of NUC-1031 in Combination with Cisplatin for the First-Line Treatment of Patients with Advanced Biliary Tract Cancer (ABC-08).

BACKGROUND: Cisplatin/gemcitabine is standard first-line treatment for patients with advanced biliary tract cancer (ABC). NUC-1031 (phosphoramidate transformation of gemcitabine) is designed to enhance efficacy by maximizing intratumoral active metabolites. METHODS: Patients with untreated ABC, Eastern Cooperative Oncology Group performance status 0-1 received NUC-1031 (625 or 725 mg/m2 ) and cisplatin (25 mg/m2 ) on days 1 and 8, every 21 days. Primary objectives were safety and maximum tolerated dose; secondary objectives were objective response rate (ORR), pharmacokinetics, progression-free survival (PFS), and overall survival (OS). RESULTS: Twenty-one patients (median age 61 years, n = 13 male; 17 cholangiocarcinoma, 2 ampullary, and 2 gallbladder cancer) received NUC-1031 625 mg/m2 (n = 8 and expansion n = 7; median six cycles) or 725 mg/m2 (n = 6; median 7.5 cycles). Treatment was well tolerated; most common treatment-emergent grade 3-4 adverse events occurring in more than one patient with 625 mg/m2 NUC-1031 were increased gamma-glutamyl transferase (GGT), 40%; alanine aminotransferase, 20%; bilirubin, 13%; neutropenia, 27%; decreased white cell count, 20%; thrombocytopenia, 13%; nausea, 13%; diarrhea, 13%; fatigue, 13%; and thrombus, 20% and with 725 mg/m2 , increased GGT, 67%, and fatigue, 33%. NUC-1031 725 mg/m2 was selected as the recommended dose with cisplatin in ABC. ORR was 33% (one complete response, six partial responses), DCR was 76%, median PFS was 7.2 months (95% confidence interval [CI], 4.3-10.1), and median OS was 9.6 months (95% CI, 6.7-13.1). The median estimates of area under the plasma concentration-time curve from time 0 to last measurable time and maximum concentration were highest for NUC-1031 (218-324 µg•h/mL and 309-889 µg/mL, respectively) and lowest for di-fluoro-deoxycytidine (0.47-1.56 µg•h/mL and 0.284-0.522 µg/mL, respectively). CONCLUSION: This is the first study reporting on the combination of NUC-1031 with cisplatin in ABC and demonstrated a favorable safety profile; 725 mg/m2 NUC-1031 in combination with cisplatin is undergoing phase III trial evaluation in ABC. (ClinicalTrials.gov ID: NCT02351765; EudraCT ID: 2015-000100-26). IMPLICATIONS FOR PRACTICE: The prognosis for patients with advanced biliary tract cancer (ABC) is approximately 1 year, and new treatment options are required. The cisplatin/gemcitabine combination is standard first-line treatment for patients with ABC. NUC-1031 is a phosphoramidate transformation of gemcitabine and is designed to enhance efficacy by maximizing intratumoral active metabolites. This phase Ib study (ABC-08) demonstrated a favorable safety profile of NUC-1031 in combination with cisplatin for the first-line treatment of patients with ABC, and 725 mg/m2 NUC-1031 was recommended in combination with cisplatin for phase III trial evaluation; the NuTide:121 global randomized study is currently enrolling.

Genome wide association study of agronomic and seed traits in a world collection of proso millet (Panicum miliaceum L.).

BACKGROUND: The climate crisis threatens sustainability of crop production worldwide. Crop diversification may enhance food security while reducing the negative impacts of climate change. Proso millet (Panicum milaceum L.) is a minor cereal crop which holds potential for diversification and adaptation to different environmental conditions. In this study, we assembled a world collection of proso millet consisting of 88 varieties and landraces to investigate its genomic and phenotypic diversity for seed traits, and to identify marker-trait associations (MTA). RESULTS: Sequencing of restriction-site associated DNA fragments yielded 494 million reads and 2,412 high quality single nucleotide polymorphisms (SNPs). SNPs were used to study the diversity in the collection and perform a genome wide association study (GWAS). A genotypic diversity analysis separated accessions originating in Western Europe, Eastern Asia and Americas from accessions sampled in Southern Asia, Western Asia, and Africa. A Bayesian structure analysis reported four cryptic genetic groups, showing that landraces accessions had a significant level of admixture and that most of the improved proso millet materials clustered separately from landraces. The collection was highly diverse for seed traits, with color varying from white to dark brown and width spanning from 1.8 to 2.6 mm. A GWAS study for seed morphology traits identified 10 MTAs. In addition, we identified three MTAs for agronomic traits that were previously measured on the collection. CONCLUSION: Using genomics and automated seed phenotyping, we elucidated phylogenetic relationships and seed diversity in a global millet collection. Overall, we identified 13 MTAs for key agronomic and seed traits indicating the presence of alleles with potential for application in proso breeding programs.

Upregulated-gene expression of pro-inflammatory cytokines, oxidative stress and apoptotic markers through inflammatory, oxidative and apoptosis mediated signaling pathways in Bovine Pneumonia.

Pneumonia is the acute inflammation of lung tissue and is multi-factorial in etiology. Staphylococcus aureus (S. aureus) is a harmful pathogen present as a normal flora of skin and nares of dairy cattle. In bovine pneumonia, S. aureus triggers to activates Toll-Like Receptors (TLRs), that further elicits the activation of the inflammation via NF-κB pathway, oxidative stress and apoptotic pathways. In the current study, pathogen-associated gene expression of the pro-inflammatory cytokines, oxidative stress and apoptotic markers in the lung tissue of cattle was explored in bovine pneumonia. Fifty lung samples collected from abattoir located in Wuhan city, Hubei, China. Histopathologically, thickening of alveolar wall, accumulation of inflammatory cells and neutrophils in perivascular space, hyperemia, hemorrhages and edema were observed in infected lungs as compared to non-infected lung samples. Furthermore, molecular identification and characterization were carried by amplification of S. aureus-specific nuc gene (270 base pairs) from the infected and non-infected lung samples to identify the S. aureus. Moreover, qPCR results displayed that relative mRNA levels of TLR2, TLR4, pro-inflammatory gene (IL-1ß, IL-6 and TNF-α) and apoptosis-associated genes (Bax, caspase-3 and caspase-9) were up-regulated except Bcl-2, which is antiapoptotic in nature, and oxidative stress related genes (Nrf2, NQO1, HO-1 and GCLC) which was down-regulated in infected pulmonary group. The relative protein expression of NF-κB, mitochondria-mediated apoptosis gene was up-regulated while Bcl-2 and Nrf2 pathway genes were downregulated in infected cattle lungs. Our findings revealed that genes expression levels of inflammatory mediators, oxidative stress and apoptosis were associated with host immunogenic regulatory mechanisms in the lung tissue during infection. Conclusively, the present study provides insights of active immune response via TLRs-mediated inflammatory, oxidative damage, and apoptotic paradox.

Quantitative HPLC-MS/MS determination of Nuc, the active metabolite of remdesivir, and its pharmacokinetics in rat.

Remdesivir is a nucleotide analog prodrug that has received much attention since the outbreak of the COVID-19 pandemic in December 2019. GS-441524 (Nuc) is the active metabolite of remdesivir and plays a pivotal role in the clinical treatment of COVID-19. Here, a robust HPLC-MS/MS method was developed to determine Nuc concentrations in rat plasma samples after a one-step protein precipitation process. Chromatographic separation was accomplished on Waters XBrige C18 column (50 × 2.1 mm, 3.5 µm) under gradient elution conditions. Multiple reaction monitoring transitions in electrospray positive ion mode were m/z 292.2 → 163.2 for Nuc and 237.1 → 194.1 for the internal standard (carbamazepine). The quantitative analysis method was fully validated in line with the United States Food and Drug Administration guidelines. The linearity, accuracy and precision, matrix effect, recovery, and stability results met the requirements of the guidelines. Uncertainty of measurement and incurred sample reanalysis were analyzed to further ensure the robustness and reproducibility of the method. This optimized method was successfully applied in a rat pharmacokinetics study of remdesivir (intravenously administration, 5 mg kg-1). The method can act as a basis for further pharmacokinetic and clinical efficacy investigations in patients with COVID-19. Graphical abstract.

Rapid Analysis for Staphylococcus aureus via Microchip Capillary Electrophoresis.

Staphylococcus aureus (S. aureus) is one of the most common pathogens for nosocomial and community infections, which is closely related to the occurrence of pyogenic and toxic diseases in human beings. In the current study, a lab-built microchip capillary electrophoresis (microchip CE) system was employed for the rapid determination of S. aureus, while a simple-to-use space domain internal standard (SDIS) method was carried out for the reliable quantitative analysis. The precision, accuracy, and reliability of SDIS were investigated in detail. Noted that these properties could be elevated in SDIS compared with traditional IS method. Remarkably, the PCR products of S. aureusnuc gene could be identified and quantitated within 80 s. The theoretical detection limit could achieve a value of 0.066 ng/µL, determined by the using SDIS method. The current work may provide a promising detection strategy for the high-speed and highly efficient analysis of pathogens in the fields of food safety and clinical diagnosis.

Hepatitis Flares Are Associated With Better Outcomes Than No Flare in Patients With Decompensated Cirrhosis and Chronic Hepatitis B Virus Infection.

BACKGROUND & AIMS: Little is known about the effects of baseline hepatitis flares (level of alanine aminotransferase ≥5-fold above the upper limit of normal) on the outcomes of patients with chronic hepatitis B virus (HBV) infection with decompensated cirrhosis treated with nucleos(t)ide analogues. We aimed to investigate these effects. METHODS: We performed a cohort study of 511 consecutive patients (78.1% men; 58.7% with flares at baseline) with chronic HBV infection and decompensated cirrhosis who were treated with nucleos(t)ide analogues as soon as decompensation was noted. Patients were enrolled from January 2002 to March 2018 at a tertiary care center in Taiwan and followed up for 16 years. RESULTS: Patients with hepatitis flares had higher mean baseline levels of HBV DNA (6.44 ± 1.52 vs 6.08 ± 1.46 log10 IU/mL; P = .003), hepatitis B surface antigen, and total bilirubin; prolonged prothrombin time; higher platelet counts (108.0 ± 42.9 vs 83.6 ± 44.7 103/µL; P < .001); and a higher proportion were infected with HBV genotype B, compared with patients without flares. Patients with flares had lower ratios of neutrophils to lymphocytes than patients with flares (6.14 ± 9.18 vs 9.12 ± 1.36; P = .019); were less likely than patients without flares to be positive for hepatitis B e antigen, ascites, esophageal varices, or splenomegaly; and a lower proportion died or underwent liver transplantation (46.5% vs 73.2% of patients without flares; P < .001), even though the patients without flares had similar short-term (<3 mo) outcomes. Factors associated independently with baseline flares were esophageal varices (odds ratio [OR], 0.165; 95% CI, 0.067-0.406), ascites (OR, 0.415; 95% CI, 0.178-0.969), levels of total bilirubin (OR, 1.158; 95% CI, 1.041-1.269), prolonged prothrombin time (OR, 1.095; 95% CI, 1.033-1.168), and higher platelet counts (OR, 1.009; 95% CI, 1.00-1.018). After we used propensity score matching to match patients with and without baseline flares, factors associated with the cumulative incidence of death or liver transplantation were flares (hazard ratio [HR], 0.491; 95% CI, 0.317-0.76), ratio of neutrophils to lymphocytes (HR, 1.278; 95% CI, 1.027-1.591), and prolonged prothrombin time (HR, 1.223; 95% CI, 1.052-1.423). CONCLUSIONS: In a 16-year study of patients with chronic HBV infection and decompensated cirrhosis treated with nucleos(t)ide analogues, a baseline flare of hepatitis was associated independently with better long-term (≥3 mo) outcomes than no flare.

NUC-1031/cisplatin versus gemcitabine/cisplatin in untreated locally advanced/metastatic biliary tract cancer (NuTide:121).

Gemcitabine/cisplatin is standard of care for first-line treatment of patients with advanced biliary tract cancer (aBTC); new treatments are needed. NUC-1031 is designed to overcome key cancer resistance mechanisms associated with gemcitabine. The tolerability/efficacy signal of NUC-1031/cisplatin in the Phase Ib ABC-08 study suggested that this combination may represent a more efficacious therapy than gemcitabine/cisplatin for patients with aBTC, leading to initiation of the global NuTide:121 study which will include 828 patients ≥18 years with untreated histologically/cytologically-confirmed aBTC (including cholangiocarcinoma, gallbladder or ampullary cancer); randomized (1:1) to NUC-1031 (725 mg/m2)/cisplatin (25 mg/m2) or gemcitabine (1000 mg/m2)/cisplatin (25 mg/m2), on days 1/8, Q21-days. Primary objectives are overall survival and objective response rate. Secondary objectives: progression-free survival, safety, pharmacokinetics, patient-reported quality of life and correlative studies. (Investigational new drug (IND) number: 139058, European Clinical Trials database: EudraCT Number 2019-001025-28, ClinicalTrials.gov identifier: NCT04163900).

A Median-Ratio Scene-Based Non-Uniformity Correction Method for Airborne Infrared Point Target Detection System.

Infrared detectors suffer from severe non-uniform noise which highly reduces image resolution and point target signal-to-noise ratio. This is the restriction for airborne point target detection systems in reaching the background limit. The existing methods are either not accurate enough, or too complex to be applied to engineering. To improve the precision and reduce the algorithm complexity of scene-based Non-Uniformity Correction (NUC) for an airborne point target detection system, a Median-Ratio Scene-based NUC (MRSBNUC) method is proposed. The method is based on the assumption that the median value of neighboring pixels is approximately constant. The NUC coefficients are calculated recursively by selecting the median ratio of adjacent pixels. Several experiments were designed and conducted. For both the clear sky scene and scene with clouds, the non-uniformity is effectively reduced. Furthermore, targets were detected in outfield experiments. For Target 1 48.36 km away and Target 2 50.53 km away, employing MRSBNUC the SNR of the target increased 2.09 and 1.73 times respectively compared to Two-Point NUC. It was concluded that the MRSBNUC method can reduce the non-uniformity of the detector effectively which leads to a longer detection distance and fewer false alarms of the airborne point target detection system.

FSH3 mediated cell death is dependent on NUC1 in Saccharomyces cerevisiae.

Family of Serine Hydrolases (FSH) members FSH1, FSH2 and FSH3 in Saccharomyces cerevisiae share conserved sequences with the human candidate tumor suppressor OVCA2. In this study, hydrogen peroxide (H2O2) exposure increased the expression of both mRNA and protein levels of FSH3 in wild-type (WT) yeast cells. The deletion of FSH3 improved the yeast growth rate under H2O2-induction as compared to WT control cells. The overexpression of FSH3 in WT yeast cells caused an apoptotic phenotype, including accumulation of reaction oxygen species, decreased cell viability and cell death. The double deletions fsh1Δ fsh2Δ, fsh1Δ fsh3Δ and fsh2Δ fsh3Δ displayed increased growth compared to WT cells. However, the overexpression of FSH3 effectively inhibited cell growth in all double deletions. Moreover, the overexpression of FSH3 in cells lacking NUC1 did not cause any growth defect in the presence or absence of H2O2. Our results suggest that FSH3 induced apoptosis of yeast in a NUC1 dependent manner.

An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease.

Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.

Robust HCV Genotype 3a Infectious Cell Culture System Permits Identification of Escape Variants With Resistance to Sofosbuvir.

BACKGROUND & AIMS: Direct-acting antivirals (DAAs) effectively eradicate chronic hepatitis C virus (HCV) infection, although HCV genotype 3a is less responsive to these drugs. We aimed to develop genotype 3a infectious cultures and study the effects of inhibitors of NS5A and NS5B and resistance to sofosbuvir-the only nucleotide analog approved for treatment of chronic HCV infection. METHODS: The developed HCV genotype 3a full-length genome (DBN3a), with a strain-DBN coding sequence, modified NS5B consensus sequence, pS52 untranslated regions, and coding mutations from a culture-efficient JFH1-based core-NS5A (DBN) recombinant, was transfected into Huh7.5 cells. The efficacy of selected DAAs was determined in dose-response assays, in which the number of HCV-infected cells was measured after incubation with different concentrations of the specific DAA. Long-term culture of infected Huh7.5 cells with increasing concentrations of sofosbuvir was used to promote selection of HCV-resistant variants. RESULTS: We engineered a DBN3a variant with 17 substitutions (DBN3acc) that had replication and propagation kinetics in Huh7.5 cells comparable with prototype J6/JFH1. The adaptive mutations also produced culture-efficient DBN-based recombinants with NS5B from HCV genotype 3a strains S52 and DH11. Compared with genotype 1a, genotype 3a was less sensitive to daclatasvir, ledipasvir, and elbasvir, but equally sensitive to ombitasvir, velpatasvir, beclabuvir, dasabuvir, MK-3682, and sofosbuvir. Exposure of Huh7.5 cells infected with DBN3a to sofosbuvir led to identification of an escape variant with substitutions in NS5B, including the resistance-associated substitution S282T. This variant showed increased infectivity of Huh7.5 cells, compared with DBN3a, and was genetically stable in cell cultures without sofosbuvir. Sofosbuvir, MK-3682, dasabuvir, or combinations of sofosbuvir and ledipasvir or sofosbuvir and velpatasvir had decreased efficacy against infection with the DBN3a sofosbuvir escape variant. CONCLUSIONS: We developed a system for highly efficient culture of HCV genotype 3a. Genotype 1a has a high genetic barrier to resistance for sofosbuvir, whereas resistance to this DAA can be induced in genotype 3a. We therefore isolated HCV genotype 3a variants with reduced sensitivity to sofosbuvir, with increased fitness and with cross-resistance to other NS5B inhibitors. These findings indicate that sofosbuvir escape variants could compromise the effectiveness of nucleotide analogs against HCV. GenBank accession numbers: KX280712-KX280716.

Three-Stage Decision-Making Model under Restricted Conditions for Emergency Response to Ships Not under Control.

A ship that is not under control (NUC) is a typical incident that poses serious problems when in confined waters close to shore. The emergency response to NUC ships is to select the best risk control options, which is a challenge in restricted conditions (e.g., time limitation, resource constraint, and information asymmetry), particularly in inland waterway transportation. To enable a quick and effective response, this article develops a three-stage decision-making framework for NUC ship handling. The core of this method is (1) to propose feasible options for each involved entity (e.g., maritime safety administration, NUC ship, and ships passing by) under resource constraint in the first stage, (2) to select the most feasible options by comparing the similarity of the new case and existing cases in the second stage, and (3) to make decisions considering the cooperation between the involved organizations by using a developed Bayesian network in the third stage. Consequently, this work provides a useful tool to achieve well-organized management of NUC ships.

Background Registration-Based Adaptive Noise Filtering of LWIR/MWIR Imaging Sensors for UAV Applications.

Unmanned aerial vehicles (UAVs) are equipped with optical systems including an infrared (IR) camera such as electro-optical IR (EO/IR), target acquisition and designation sights (TADS), or forward looking IR (FLIR). However, images obtained from IR cameras are subject to noise such as dead pixels, lines, and fixed pattern noise. Nonuniformity correction (NUC) is a widely employed method to reduce noise in IR images, but it has limitations in removing noise that occurs during operation. Methods have been proposed to overcome the limitations of the NUC method, such as two-point correction (TPC) and scene-based NUC (SBNUC). However, these methods still suffer from unfixed pattern noise. In this paper, a background registration-based adaptive noise filtering (BRANF) method is proposed to overcome the limitations of conventional methods. The proposed BRANF method utilizes background registration processing and robust principle component analysis (RPCA). In addition, image quality verification methods are proposed that can measure the noise filtering performance quantitatively without ground truth images. Experiments were performed for performance verification with middle wave infrared (MWIR) and long wave infrared (LWIR) images obtained from practical military optical systems. As a result, it is found that the image quality improvement rate of BRANF is 30% higher than that of conventional NUC.