Beyond Erectile Dysfunction: cGMP-Specific Phosphodiesterase 5 Inhibitors for Other Clinical Disorders.

Cyclic guanosine monophosphate (cGMP), an important intracellular second messenger, mediates cellular functional responses in all vital organs. Phosphodiesterase 5 (PDE5) is one of the 11 members of the cyclic nucleotide phosphodiesterase (PDE) family that specifically targets cGMP generated by nitric oxide-driven activation of the soluble guanylyl cyclase. PDE5 inhibitors, including sildenafil and tadalafil, are widely used for the treatment of erectile dysfunction, pulmonary arterial hypertension, and certain urological disorders. Preclinical studies have shown promising effects of PDE5 inhibitors in the treatment of myocardial infarction, cardiac hypertrophy, heart failure, cancer and anticancer-drug-associated cardiotoxicity, diabetes, Duchenne muscular dystrophy, Alzheimer's disease, and other aging-related conditions. Many clinical trials with PDE5 inhibitors have focused on the potential cardiovascular, anticancer, and neurological benefits. In this review, we provide an overview of the current state of knowledge on PDE5 inhibitors and their potential therapeutic indications for various clinical disorders beyond erectile dysfunction.

Rotavirus-mediated DGAT1 degradation: A pathophysiological mechanism of viral-induced malabsorptive diarrhea.

Gastroenteritis is among the leading causes of mortality globally in infants and young children, with rotavirus (RV) causing ~258 million episodes of diarrhea and ~128,000 deaths annually in infants and children. RV-induced mechanisms that result in diarrhea are not completely understood, but malabsorption is a contributing factor. RV alters cellular lipid metabolism by inducing lipid droplet (LD) formation as a platform for replication factories named viroplasms. A link between LD formation and gastroenteritis has not been identified. We found that diacylglycerol O-acyltransferase 1 (DGAT1), the terminal step in triacylglycerol synthesis required for LD biogenesis, is degraded in RV-infected cells by a proteasome-mediated mechanism. RV-infected DGAT1-silenced cells show earlier and increased numbers of LD-associated viroplasms per cell that translate into a fourfold-to-fivefold increase in viral yield (P < 0.05). Interestingly, DGAT1 deficiency in children is associated with diarrhea due to altered trafficking of key ion transporters to the apical brush border of enterocytes. Confocal microscopy and immunoblot analyses of RV-infected cells and DGAT1-/- human intestinal enteroids (HIEs) show a decrease in expression of nutrient transporters, ion transporters, tight junctional proteins, and cytoskeletal proteins. Increased phospho-eIF2α (eukaryotic initiation factor 2 alpha) in DGAT1-/- HIEs, and RV-infected cells, indicates a mechanism for malabsorptive diarrhea, namely inhibition of translation of cellular proteins critical for nutrient digestion and intestinal absorption. Our study elucidates a pathophysiological mechanism of RV-induced DGAT1 deficiency by protein degradation that mediates malabsorptive diarrhea, as well as a role for lipid metabolism, in the pathogenesis of gastroenteritis.

Grafting a Polymer Coating Layer onto Li1.2 Ni0.13 Co0.13 Mn0.54 O2 Cathode by Benzene Diazonium Salts to Facilitate the Cycling Performance and High-Voltage Stability.

Li-rich Mn-based cathodes have been regarded as promising cathodes for lithium-ion batteries because of their low cost of raw materials (compared with Ni-rich layer structure and LiCoO2 cathodes) and high energy density. However, for practical application, it needs to solve the great drawbacks of Li-rich Mn-based cathodes like capacity degradation and operating voltage decline. Herein, an effective method of surface modification by benzene diazonium salts to build a stable interface between the cathode materials and the electrolyte is proposed. The cathodes after modification exhibit excellent cycling performance (the retention of specific capacity is 84.2% after 350 cycles at the current density of 1 C), which is mainly attributed to the better stability of the structure and interface. This work provides a novel way to design the coating layer with benzene diazonium salts for enhancing the structural stability under high voltage condition during cycling.

Human Sapovirus Replication in Human Intestinal Enteroids.

Human sapoviruses (HuSaVs), like human noroviruses (HuNoV), belong to the Caliciviridae family and cause acute gastroenteritis in humans. Since their discovery in 1976, numerous attempts to grow HuSaVs in vitro were unsuccessful until 2020, when these viruses were reported to replicate in a duodenal cancer cell-derived line. Physiological cellular models allowing viral replication are essential to investigate HuSaV biology and replication mechanisms such as genetic susceptibility, restriction factors, and immune responses to infection. In this study, we demonstrate replication of two HuSaV strains in human intestinal enteroids (HIEs) known to support the replication of HuNoV and other human enteric viruses. HuSaVs replicated in differentiated HIEs originating from jejunum, duodenum and ileum, but not from the colon, and bile acids were required. Between 2h and 3 to 6 days postinfection, viral RNA levels increased up from 0.5 to 1.8 log10-fold. Importantly, HuSaVs were able to replicate in HIEs independent of their secretor status and histo-blood group antigen expression. The HIE model supports HuSaV replication and allows a better understanding of host-pathogen mechanisms such as cellular tropism and mechanisms of viral replication. IMPORTANCE Human sapoviruses (HuSaVs) are a frequent but overlooked cause of acute gastroenteritis, especially in children. Little is known about this pathogen, whose successful in vitro cultivation was reported only recently, in a cancer cell-derived line. Here, we assessed the replication of HuSaV in human intestinal enteroids (HIEs), which are nontransformed cultures originally derived from human intestinal stem cells that can be grown in vitro and are known to allow the replication of other enteric viruses. Successful infection of HIEs with two strains belonging to different genotypes of the virus allowed discovery that the tropism of these HuSaVs is restricted to the small intestine, does not occur in the colon, and replication requires bile acid but is independent of the expression of histo-blood group antigens. Thus, HIEs represent a physiologically relevant model to further investigate HuSaV biology and a suitable platform for the future development of vaccines and antivirals.

Miniaturized photoacoustic microscope for multi-segmental spinal cord imaging in freely moving mice.

Long-term and non-narcotic hemodynamic imaging is indispensable for observing factual physiological information of the spinal cord. Unfortunately, achieving label-free, high-resolution, and widefield spinal cord imaging for mice under freely moving conditions is challenging. In this study, we developed a miniaturized photoacoustic microscope along with a corresponding photoacoustic spinal window to realize high-resolution, multi-segmental hemodynamic imaging of the spinal cord for freely moving mice. The microscope has an outer size of 32 mm × 23 mm × 10 mm, a weight of 5.8 g, and a 4.4 µm lateral resolution within an effective field of view (FOV) of 2.6 mm × 1.8 mm. To eliminate the off-focus phenomena during spinal imaging, the microscope is equipped with a miniature motor to adapt the focal plane. Besides, the microscope is slidable along a customized rail on the window to expand the FOV. We evaluated the stability of the microscope and analyzed vascular images of the spinal cord under various physiological states. The results suggest that the microscope is capable of performing stable, multi-segmental spinal cord imaging in freely moving mice, offering new insights into spinal cord hemodynamics and neurovascular coupling research.

Photoacoustic expansion microscopy of melanosomes.

Optical resolution photoacoustic microscopy (OR-PAM) is a hybrid imaging method for visualizing organelles due to the high spatial resolution and abundant optical contrast. Usually, OR-PAM employs high numerical aperture (NA) objectives and high-frequency ultrasonic detectors to resolve three-dimensional (3D) microstructures of cells. Expansion microscopy (ExM) provides a nanoscale resolution by isotropically enlarging cells instead of utilizing ultrahigh NA objectives. In this Letter, we report the development of photoacoustic expansion microscopy (PA-ExM) that combines the advantages of OR-PAM and ExM for 3D organelle imaging using near-infrared light. We evaluate the performance of PA-ExM using label-free melanoma cells, where the image quality of melanosome distributions in expanded cells using a 40× objective is comparable to that of unexpanded cells using an oil-immersed 100× objective. The results suggest that PA-ExM possesses the great potential to study organelles.

Wearable photoacoustic watch for humans.

Longitudinal detection of hemodynamic changes based on wearable devices is imperative for monitoring human healthcare. Photoacoustic effect is extremely sensitive to variations in hemoglobin. Therefore, wearable photoacoustic devices are apt to monitor human healthcare via the observation of hemodynamics. However, the bulky system and difficulties in miniaturizing and optimizing the imaging interface restrict the development of wearable photoacoustic devices for human use. In this study, we developed a wearable photoacoustic watch with a fully integrated system in a backpack that has a size of 450 mm × 300 mm × 200 mm and an affordable weight of 7 kg for an adult to wear. The watch has a size of 43 mm × 30 mm × 24 mm, weighs 40 g, and features a lateral resolution of 8.7 µm, a field of view (FOV) of 3 mm in diameter, and a motorized adjustable focus for optimizing the imaging plane for different individuals. We recruited volunteers to wear the watch and the backpack and performed in vivo imaging of the vasculatures inside human wrists under the conditions of walking and human cuff occlusion to observe hemodynamic variations during different physiological states. The results suggest that the focus shifting capability of the watch makes it suitable for different individuals, and the compact and stable design of the entire system allows free movements of humans.

Non-telecentric photoacoustic microscopy for multi-scale imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) excels in precisely imaging a biological tissue based on absorption contrast. However, existing OR-PAMs are confined by fixed compromises between spatial resolution and field of view (FOV), preventing the integration of large FOV and local high-resolution within one system. Here, we present a non-telecentric OR-PAM (nTC-PAM) that empowers efficient adaptation of FOV and spatial resolution to match the multi-scale requirement of diverse biological imaging. Our method allows for a large-scale transformation in FOV and even surpassing the nominal FOV of the objective with minimal marginal degradation of the lateral resolution. We demonstrate the advantage of nTC-PAM through multi-scale imaging of the leaf phantom, mouse ear, and cortex. The results reveal that nTC-PAM can switch the FOV and spatial resolution to meet the requirements of different biological tissues, such as large-scale imaging of the whole cerebral cortex and high-resolution imaging of microvascular structures in local brain regions.

Cardiovascular adverse effects of antiviral therapies for COVID-19: Evidence and plausible mechanisms.

Antiviral therapeutics have made a critical contribution in mitigating the symptoms and clinical outcomes of the coronavirus disease of 2019 (COVID-19), in which a single-stranded RNA viral pathogen, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causes multi-organ injuries. Several antivirals were widely prescribed to treat COVID-19, either through the emergency use authorization (EUA) by the governmental regulatory agencies (i.e., remdesivir, paxlovid, molnupiravir, and the SARS-CoV-2-targeted monoclonal antibodies - tixagevimab and cilgavimab), as well as the repurposed use of the existing antiviral or antimalarial drugs (e.g., hydroxychloroquine, chloroquine, and ivermectin). Despite their efficacy in ameliorating COVID-19 symptoms, some adverse side-effects of the antivirals were also reported during the COVID-19 pandemic. Our current review has aimed to gather and extrapolate the recently published information concerning cardiovascular adverse effects caused by each of the antivirals. We also provide further discussion on the potential cellular mechanisms underlying the cardiovascular adverse effects of the selected antiviral drugs, which should be carefully considered when evaluating risk factors in managing patients with COVID-19 or similar infectious diseases. It is foreseeable that future antiviral drug development assisted with the newest artificial intelligence platform may improve the accuracy to predict the structures of biomolecules of antivirals and therefore to mitigate their associated cardiovascular adversities.

Silencing of ZNF610 suppresses cell proliferation and migration in lung adenocarcinoma.

Zinc finger proteins (ZNFs) play a significant role in the initiation and progression of tumors. Nevertheless, the specific contribution of ZNF610 to lung adenocarcinoma (LUAD) remains poorly understood. This study sought is to elucidate the role of ZNF610 in LUAD. Transcript data of LUAD were obtained from The Cancer Genome Atlas Program (TCGA) database and processed via R program. The expression of ZNF610 was assessed in various cell lines. To compare the proliferative capacity of cells with or without ZNF610 silencing, CCK8, cell colony formation assay, and Celigo label-free cell counting assay were employed. Furthermore, transwell migration and invasion assays were conducted to evaluate the migratory and invasive abilities of the cells. The expression levels of genes and proteins were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot techniques. In different LUAD cells, the expression level of ZNF610 was found to be significantly higher in LUAD cells compared to MRC-5 and BASE-2B cells. Moreover, the silencing of ZNF610 resulted in a decrease in cell proliferation and migration abilities. Additionally, the apoptosis rate of cells increased upon silencing ZNF610. Notably, the proportion of cells in the G0/G1 phase increased, while the proportion of cells in the S phase decreased following ZNF610 silencing. Finally, ß-catenin and snail were identified as downstream targets of ZNF610 in cells. Our findings suggest that silencing ZNF610 could inhibit LUAD cell proliferation and migration, possibly through the downregulation of ß-catenin and snail.

A diagnostic model for differentiating tuberculous spondylodiscitis from pyogenic spondylodiscitis based on pathogen-confirmed patients.

OBJECTIVE: This study aimed to distinguish tuberculous spondylodiscitis (TS) from pyogenic spondylodiscitis (PS) based on laboratory, magnetic resonance imaging (MRI) and computed tomography (CT) findings. Further, a novel diagnostic model for differential diagnosis was developed. METHODS: We obtained MRI, CT and laboratory data from TS and PS patients. Predictive models were built using binary logistic regression analysis. The receiver operating characteristic curve was analyzed. Both internal and external validation was performed. RESULTS: A total of 81 patients with PS (n = 46) or TS (n = 35) were enrolled. All patients had etiological evidence from the focal lesion. Disc signal or height preservation, skip lesion or multi segment (involved segments ≥ 3) involvement, paravertebral calcification, massive sequestra formation, subligamentous bone destruction, bone erosion with osteosclerotic margin, higher White Blood Cell Count (WBC) and positive result of tuberculosis infection T cell spot test (T-SPOT.TB) were more prevalent in the TS group. A diagnostic model was developed and included four predictors: WBC<7.265 * (10^9/L), skip lesion or involved segments ≥ 3, massive sequestra formation and subligamentous bone destruction. The model showed good sensitivity, specificity, and total accuracy (91.4%, 95.7%, and 93.8%, respectively); the area under the receiver operating characteristic curve (AUC) was 0.981, similar to the results of internal validation using bootstrap resampling (1000 replicates) and external validation set, indicating good clinical predictive ability. CONCLUSIONS: This study develop a good diagnostic model based on both CT and MRI, as well as laboratory findings, which may help clinicians distinguish between TS and PS.

Analysis of D-dimer levels for the detection of deep venous thrombosis for patients with spinal metastasis undergoing decompression with fixation.

BACKGROUND: Deep venous thrombosis (DVT) after spinal surgery has recently attracted increasing attention. Patients with spinal metastases who undergo decompression with fixation are at a high risk of developing DVT. D-dimer levels indicate the risk of DVT, and the purpose of our study was to investigate D-dimer levels as a predictor of DVT perioperatively. METHODS: We prospectively evaluated 100 patients with spinal metastases. D-dimer tests were performed twice: once before surgery and one day postoperatively. DVT was diagnosed by duplex ultrasonographic assessment of both lower extremities. Pulmonary embolisms (PEs) were diagnosed using multidetector computed tomography and pulmonary angiography. Perioperative serum D-dimer levels were compared between the DVT (+) and DVT (-) groups. The cutoff value of the D-dimer level was calculated using receiver operating characteristic analysis. RESULTS: Preoperative and postoperative DVT prevalences were 8.0% (8/100) and 6.6% (6/91), respectively, and none of the patients developed PE. Before surgery, there was no significant differences in D-dimer levels between the pre-DVT (+) and pre-DVT (-) groups. After surgery, the D-dimer level one-day postoperatively for the post-DVT (+) group (17.6 ± 11.8 mg/L) was significantly higher than that of the post-DVT (-) group (5.0 ± 4.7 mg/L). The cutoff value of the postoperative D-dimer level was 9.51(mg/L), and the sensitivity and specificity for the optimum threshold were 83.3% and 89.4%, respectively. CONCLUSIONS: Our findings suggest that preoperative D-dimer level may not be a predictor of DVT. Preoperative ultrasound examinations should be routinely performed in patients with spinal metastases. Postoperative D-dimer levels greater than 9.51(mg/L) are a predictive factor for the early diagnosis of DVT after spine surgery. TRIAL REGISTRATION: Our study was registered on Chinese Clinical Trial Registry (No.ChiCTR2000029737). Registered 11 February 2020 - Retrospectively registered, https://www.chictr.org.cn/index.aspx.

Antibiotic-Resistance Profiles and Genetic Diversity of Shigella Isolates in China: Implications for Control Strategies.

The urgent need for comprehensive and systematic analyses of Shigella as the key pathogen led us to meticulously explore the epidemiology and molecular attributes of Shigella isolates. Accordingly, we procured 24 isolates (10 from Xinjiang and 14 from Wuhan, China) and performed serotype identification and antimicrobial susceptibility testing. Resistance gene detection and homology analysis by polymerase chain reaction and pulsed-field gel electrophoresis (PFGE), respectively, were performed for genetic diversity analysis. All isolates were identified as Shigella flexneri, with 70% (35.4-91.9%) and 30% (8.1-64.6%) of the Xinjiang isolates and 85.7% (56.2-97.5%) and 14.3% (2/14, 2.5-43.9%) of the Wuhan isolates belonging to serotype 2a and serotype 2b, respectively. All isolates displayed resistance to at least two antibiotics and complete resistance to ampicillin. Multidrug resistance (MDR) was recorded in 70.8% (48.8-86.6%) of isolates, with Xinjiang isolates exhibiting relatively higher resistance to ampicillin-sulbactam, piperacillin, ceftriaxone, and aztreonam. Conversely, Wuhan isolates displayed higher MDR and resistance to tetracycline, ciprofloxacin, levofloxacin, and cefepime relative to Xinjiang isolates. Molecular scrutiny of antibiotic-resistance determinants revealed that blaTEM was the main mechanism of ampicillin resistance, blaCTX-M was the main gene for resistance to third- and fourth-generation cephalosporins, and tetB was the predominant gene associated with tetracycline resistance. Four Xinjiang and seven Wuhan isolates shared T1-clone types (>85%), and two Xinjiang and one Wuhan isolates were derived from the T6 clone with a high similarity of 87%. Six PFGE patterns (T1, T2, T5, T6-3, T8, and T10) of S. flexneri were associated with MDR. Thus, there is a critical need for robust surveillance and control strategies in managing Shigella infections, along with the development of targeted interventions and antimicrobial stewardship programs tailored to the distinct characteristics of Shigella isolates in different regions of China.

Small non-coding RNAome changes during human chondrocyte senescence as potential epigenetic targets in age-related osteoarthritis.

Chondrocyte senescence is a decisive component of age-related osteoarthritis, however, the function of small noncoding RNAs (sncRNAs) in chondrocyte senescence remains underexplored. Human hip joint cartilage chondrocytes were cultivated up to passage 4 to induce senescence. RNA samples were extracted and then analyzed using small RNA sequencing and qPCR. ß-galactosidase staining was used to detect the effect of sncRNA on chondrocyte aging. Results of small RNA sequencing showed that 279 miRNAs, 136 snoRNAs, 30 snRNAs, 102 piRNAs, and 5 rasiRNAs were differentially expressed in senescent chondrocytes. The differential expression of 150 sncRNAs was further validated by qPCR. Transfection of sncRNAs and ß-galactosidase staining were also performed to further revealed that hsa-miR-135b-5p, SNORA80B-201, and RNU5E-1-201 have the function to restrain chondrocyte senescence, while has-piR-019102 has the function to promote chondrocyte senescence. Our data suggest that sncRNAs have therapeutic potential as novel epigenetic targets in age-related osteoarthritis.

RETRACTED: Serebrovska et al. Intermittent Hypoxia-Hyperoxia Training Improves Cognitive Function and Decreases Circulating Biomarkers of Alzheimer's Disease in Patients with Mild Cognitive Impairment: A Pilot Study. Int. J. Mol. Sci. 2019, 20, 5405.

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Androgen-deprivation therapy with leuprolide increases abdominal adiposity without causing cardiac dysfunction in middle-aged male mice: effect of sildenafil.

Androgen-deprivation therapy (ADT) is the primary systemic therapy for treating advanced or metastatic prostate cancer (PCa), which has improved survival outcomes in patients with PCa. However, ADT may develop metabolic and cardiovascular adverse events that impact the quality of life and lifespan in PCa survivors. The present study was designed to establish a murine model of ADT with a gonadotropin-releasing hormone (GnRH) agonist leuprolide and to investigate its effects on metabolism and cardiac function. We also examined the potential cardioprotective role of sildenafil (inhibitor of phosphodiesterase 5) under chronic ADT. Middle-aged male C57BL/6J mice received a 12-wk subcutaneous infusion via osmotic minipumps containing either saline or 18 mg/4 wk leuprolide with or without 1.3 mg/4 wk sildenafil cotreatment. Compared with saline controls, leuprolide treatment significantly reduced prostate weight and serum testosterone levels, confirming chemical castration in these mice. The ADT-induced chemical castration was not affected by sildenafil. Leuprolide significantly increased the weight of abdominal fat after 12-wk treatment without a change in total body weight, and sildenafil did not block the proadipogenic effect of leuprolide. No signs of left ventricular systolic and diastolic dysfunction were observed throughout the leuprolide treatment period. Interestingly, leuprolide treatment significantly elevated serum levels of cardiac troponin I (cTn-I), a biomarker of cardiac injury, and sildenafil did not abolish this effect. We conclude that long-term ADT with leuprolide increases abdominal adiposity and cardiac injury biomarker without cardiac contractile dysfunction. Sildenafil did not prevent ADT-associated adverse changes.

Treating diabetes with combination of phosphodiesterase 5 inhibitors and hydroxychloroquine-a possible prevention strategy for COVID-19?

Type 2 diabetes (T2D) is one of the major risk factors for developing cardiovascular disease and the resultant devastating morbidity and mortality. The key features of T2D are hyperglycemia, hyperlipidemia, insulin resistance, and impaired insulin secretion. Patients with diabetes and myocardial infarction have worse prognosis than those without T2D. Moreover, obesity and T2D are recognized risk factors in developing severe form of COVID-19 with higher mortality rate. The current lines of drug therapy are insufficient to control T2D and its serious cardiovascular complications. Phosphodiesterase 5 (PDE5) is a cGMP specific enzyme, which is the target of erectile dysfunction drugs including sildenafil, vardenafil, and tadalafil. Cardioprotective effects of PDE5 inhibitors against ischemia/reperfusion (I/R) injury were reported in normal and diabetic animals. Hydroxychloroquine (HCQ) is a widely used antimalarial and anti-inflammatory drug and its hyperglycemia-controlling effect in diabetic patients is also under investigation. This review provides our perspective of a potential use of combination therapy of PDE5 inhibitor with HCQ to reduce cardiovascular risk factors and myocardial I/R injury in T2D. We previously observed that diabetic mice treated with tadalafil and HCQ had significantly reduced fasting blood glucose and lipid levels, increased plasma insulin and insulin-like growth factor-1 levels, and improved insulin sensitivity, along with smaller myocardial infarct size following I/R. The combination treatment activated Akt/mTOR cellular survival pathway, which was likely responsible for the salutary effects. Therefore, pretreatment with PDE5 inhibitor and HCQ may be a potentially useful therapy not only for controlling T2D but also reducing the rate and severity of COVID-19 infection in the vulnerable population of diabetics.

Chronic Exposure to Environmental Pollutant Ammonia Causes Damage to the Olfactory System and Behavioral Abnormalities in Mice.

Ammonia (NH3) is a major air pollutant. However, few studies have been extended beyond the histopathological changes in the olfactory mucosa to the impact of NH3 exposure on other parts of the olfactory system and olfactory functioning. Therefore, we assessed the effects of exogenous NH3 (either 20 ppm for the low exposure group or 200 ppm for the high exposure group) on the various parts of the olfactory system by histological observation, gene expression, immunochemistry, and chemical analyses. A total of 140 Institute of Cancer Research mice (4 weeks old), 70 females and 70 males (average body weight at the start: 21.5 ± 1.9 g), were used. The exposure lasted for 4 weeks, and the mice were exposed to the NH3 for 4 h per day. Our results showed that chronic exposure to NH3 damaged the olfactory system, with consequences for changing the foraging behavior and anxiety behavior. Our results also suggest that it is plausible that NH3 recruited T cells and activated microglia cells and astrocytes, leading to inflammation in the olfactory system. Increased release of proinflammatory cytokines (TNF-α, IL-1ß, IL-6, and interferon-γ) and reduced release of anti-inflammatory cytokines (IL-4 and IFN-beta) led to tissue damage and compromised the functions of the olfactory system.

Human norovirus exhibits strain-specific sensitivity to host interferon pathways in human intestinal enteroids.

Human noroviruses (HuNoVs) are the leading cause of viral gastroenteritis worldwide; yet currently, no vaccines or FDA-approved antiviral drugs are available to counter these pathogens. To understand HuNoV biology and the epithelial response to infection, we performed transcriptomic analyses, RT-qPCR, CRISPR-Cas9 modification of human intestinal enteroid (HIE) cultures, and functional studies with two virus strains (a pandemic GII.4 and a bile acid-dependent GII.3 strain). We identified a predominant type III interferon (IFN)-mediated innate response to HuNoV infection. Replication of both strains is sensitive to exogenous addition of IFNs, suggesting the potential of IFNs as therapeutics. To obtain insight into IFN pathway genes that play a role in the antiviral response to HuNoVs, we developed knockout (KO) HIE lines for IFN alpha and lambda receptors and the signaling molecules, MAVS, STAT1, and STAT2 An unexpected differential response of enhanced replication and virus spread was observed for GII.3, but not the globally dominant GII.4 HuNoV in STAT1-knockout HIEs compared to parental HIEs. These results indicate cellular IFN responses restrict GII.3 but not GII.4 replication. The strain-specific sensitivities of innate responses against HuNoV replication provide one explanation for why GII.4 infections are more widespread and highlight strain specificity as an important factor in HuNoV biology. Genetically modified HIEs for innate immune genes are useful tools for studying immune responses to viral or microbial pathogens.

Bile acids and ceramide overcome the entry restriction for GII.3 human norovirus replication in human intestinal enteroids.

Human noroviruses (HuNoVs) cause sporadic and epidemic outbreaks of gastroenteritis in all age groups worldwide. We previously reported that stem cell-derived human intestinal enteroid (HIE) cultures support replication of multiple HuNoV strains and that some strains (e.g., GII.3) replicate only in the presence of bile. Heat- and trypsin-treatment of bile did not reduce GII.3 replication, indicating a nonproteinaceous component in bile functions as an active factor. Here we show that bile acids (BAs) are critical for GII.3 replication and replication correlates with BA hydrophobicity. Using the highly effective BA, glycochenodeoxycholic acid (GCDCA), we show BAs act during the early stage of infection, BA-dependent replication in HIEs is not mediated by detergent effects or classic farnesoid X receptor or Takeda G protein-coupled receptor 5 signaling but involves another G protein-coupled receptor, sphingosine-1-phosphate receptor 2, and BA treatment of HIEs increases particle uptake. We also demonstrate that GCDCA induces multiple cellular responses that promote GII.3 replication in HIEs, including enhancement of 1) endosomal uptake, 2) endosomal acidification and subsequent activity of endosomal/lysosomal enzyme acid sphingomyelinase (ASM), and 3) ceramide levels on the apical membrane. Inhibitors of endosomal acidification or ASM reduce GII.3 infection and exogenous addition of ceramide alone permits infection. Furthermore, inhibition of lysosomal exocytosis of ASM, which is required for ceramide production at the apical surface, decreases GII.3 infection. Together, our results support a model where GII.3 exploits rapid BA-mediated cellular endolysosomal dynamic changes and cellular ceramide to enter and replicate in jejunal HIEs.