NEDD4L downregulates autophagy and cell growth by modulating ULK1 and a glutamine transporter.

In mammals, autophagosome formation is initiated by ULK1 via the posttranslational modification of this protein. However, the precise role of ULK1 ubiquitination in modulating autophagy is unknown. Here, we show that NEDD4L, an E3 ubiquitin ligase, binds ULK1 in pancreatic cancer cells. ULK1 expression was stabilized in NEDD4L knockdown cells compared to that in control cells, suggesting that NEDD4L is involved in ULK1 ubiquitination and its subsequent degradation. Autophagy activity was enhanced in NEDD4L knockdown cells compared to control cells. NEDD4L-depleted cells exhibited an increase in the cellular oxygen consumption rate (OCR) and mitochondrial membrane potential, and maintained mitochondrial fusion status in response to metabolic stress. Enhanced OCR and mitochondrial fusion morphology in NEDD4L knockdown cells were repressed by siRNA targeting ULK1. In addition to ULK1, ASCT2, a glutamine transporter, was accumulated in NEDD4L-depleted cells; this is important for maintaining autophagy activation and mitochondrial metabolic function. Finally, the cellular growth and survival rate increased in NEDD4L knockdown cells compared to control cells. However, the genetic or pharmacological blockade of either ULK1 or ASCT2 in NEDD4L-depleted cells sensitized pancreatic cancer cells, particularly in response to nutrient deprivation. In a mouse xenograft model of pancreatic cancer, the use of autophagy inhibitors suppressed tumor growth more in NEDD4L-depleted cells than in tumors from control cells. NEDD4L and ULK1 levels were inversely correlated in two different pancreatic cancer mouse models-xenograft mouse and KPC mouse models. These results suggest that NEDD4L suppressed autophagy and mitochondrial metabolism by reducing cellular ULK1 or ASCT2 levels, and thus could repress the growth and survival of pancreatic cancer cells. Therefore, ubiquitin ligase-mediated autophagy plays a critical role in regulating mitochondrial metabolism, thereby contributing to the growth and survival of certain cancers with low NEDD4L levels.

Comparison of Swab Sampling Methods for Norovirus Recovery on Surfaces.

Human noroviruses (HuNoVs) can be easily transferred by the contacts of humans or fomites. Swab sampling methods are widely used for recovering HuNoVs from small surfaces of various fomites or hard-to-reach locations and swab sampling conditions are important for the accurate detection of HuNoVs, which have a low infectious dose and relatively long persistence under a range of environmental conditions. Therefore, to determine the suitable swab sampling method for recovering HuNoVs from various surfaces, we evaluated combinations of four swab materials (cotton, microdenier polyester [a type of microfiber], polyurethane foam, and rayon) and three elution buffer solutions (phosphate-buffered saline [PBS], PBS with 0.2% Tween-80, and 3% beef extract-50 mM glycine [pH 9.5]). First, we inoculated HuNoVs or murine noroviruses (MuNoVs), the surrogate of HuNoVs, onto test coupons (10 × 10 cm) consisting of three common surface materials (high-density polyethylene, stainless steel, and wood). Coupons were swabbed using a combination of each swab material and elution buffer, and the viral recovery was measured by real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) or plaque assay. By RT-qPCR, we confirmed that the cotton swab-PBS and microdenier polyester-PBS combinations had recovery efficiencies greater than 80% for viruses on plastic and stainless steel surfaces. The cotton swab-PBS combination had the highest recovery efficiency on all surface materials via the plaque assay. Therefore, a cotton or a microdenier polyester swab with PBS could be a useful method for sampling HuNoVs on various surfaces.

Merdimonas faecis gen. nov., sp. nov., isolated from human faeces.

A strictly anaerobic Gram-stain-positive, non-motile and non-spore-forming bacterial strain, BR31T, was isolated from a faecal sample of a healthy human. Bacterial colonies were ivory-coloured on GAM agar and composed of rod-shaped cells with rounded ends approximately 1.4-2.1×0.5-0.6 µm in size. According to comparative analysis based on 16S rRNA gene sequences, strain BR31T formed a distinct phylogenetic lineage that reflects a new genus within Clostridium cluster XIVa in the family Lachnospiraceae, with highest similarity to Eubacterium contortum DSM 3982T (94.6 %). The DNA G+C content was calculated to be 47.0 mol% from whole genome sequencing. Predicted genes associated with synthesis of teichuronic acid, polyamines, polar lipids and diaminopimelic acid were detected. The peptidoglycan contained meso-diaminopimelic acid and the main polar lipid detected was phosphatidylglycerol. The major metabolic end product of glucose was acetic acid, which was in agreement with those of most members of the family. However, the profile of major cellular fatty acids (C16 : 0, C14 : 0, summed feature 4 and C13 : 0) and overall enzyme activity demonstrated phenotypic differentiation of strain BR31T from other closely related genera. Thus, based on distinct phenotypic, phylogenetic, genotypic and chemotaxonomic characteristics, strain BR31T is considered to represent a novel species of a new genus, for which the name Merdimonas faecis gen. nov., sp. nov. is proposed. The type strain of Merdimonas faecis is BR31T (=KCTC 15482T=JCM 30748T).

Evaluation of Various Real-Time Reverse Transcription Quantitative PCR Assays for Norovirus Detection.

Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D's probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.

Sellimonas intestinalis gen. nov., sp. nov., isolated from human faeces.

A Gram-stain-positive and obligately anaerobic bacterial strain, BR72T, forming ivory yellow colonies was isolated from a faecal sample of a healthy Korean woman. 16S rRNA gene sequence analysis indicated that strain BR72T belongs to Clostridium cluster XIVa and represents a distinct phyletic line within the family Lachnospiraceae. The most closely related strains were Clostridium nexile DSM 1787T (94.1 % 16S rRNA gene sequence similarity), Coprococcus comes ATCC 27758T (93.5 %), Ruminococcus torques ATCC 27756T (93.5 %), Ruminococcus lactaris ATCC 29176T (93.5 %), Clostridium aerotolerans DSM 5434T (93.1 %) and Eubacterium fissicatena DSM 3598T (92.9 %). The DNA G+C content of strain BR72T based on its genome sequence was 45.3 mol%. The major cellular fatty acids were C16 : 0, C14 : 0, and iso-C17 : 1 I and/or anteiso-C17 : 1 B. Acetic acid was produced from glucose fermentation. Other physiological and biochemical comparisons allowed the phenotypic differentiation of strain BR72T from the members of the family Lachnospiraceae. Based on the phylogenetic and phenotypic findings, this strain is considered to represent a novel species of a new genus belonging to the family Lachnospiraceae and the name Sellimonas intestinalis gen. nov., sp. nov. is proposed. The type strain of Sellimonas intestinalis is BR72T ( = KCTC 15479T = JCM 30749T).