Search

Background and purpose A wide range of neuroradiological findings has been reported in patients with coronavirus disease 2019 (COVID-19), ranging from subcortical white matter changes to infarcts, haemorrhages and focal contrast media enhancement. These have been descriptively but inconsistently reported and correlations with clinical findings and biomarkers have been difficult to extract from the literature. The purpose of this study was to quantify the extents of neuroradiological findings in a cohort of patients with COVID-19 and neurological symptoms, and to investigate correlations with clinical findings, duration of intensive care and biomarkers in blood. Material and methods Patients with positive SARS-CoV-2 and at least one new-onset neurological symptom were included from April until July 2020. Nineteen patients were examined regarding clinical symptoms, biomarkers in blood and MRI of the brain. In order to quantify the MRI findings, a semi-quantitative neuroradiological severity scale was constructed a priori, and applied to the MR images by two specialists in neuroradiology. Results and conclusions The score from the severity scale correlated significantly with blood biomarkers of CNS injury (glial fibrillary acidic protein, total-tau, ubiquitin carboxyl-terminal hydrolase L1) and inflammation (C-reactive protein), Glasgow Coma Scale score, and the number of days spent in intensive care. The underlying radiological assessments had inter-rater agreements of 90.5%/86% (for assessments with 2/3 alternatives). Total intraclass correlation was 0.80. Previously reported neuroradiological findings in COVID-19 have been diverse and heterogenous. In this study, the extent of findings in MRI examination of the brain, quantified using a structured report, shows correlation with relevant biomarkers.

Background COVID-19 infection is known to cause a wide array of clinical chronic sequelae, but little is known regarding the long-term cardiac complications. We aim to report echocardiographic follow-up findings and describe the changes in left (LV) and right ventricular (RV) function that occur following acute infection. Methods Patients enrolled in the World Alliance Societies of Echocardiography-COVID study with acute COVID-19 infection were asked to return for a follow-up transthoracic echocardiogram. Overall, 198 returned at a mean of 129 days of follow-up, of which 153 had paired baseline and follow-up images that were analyzable, including LV volumes, ejection fraction (LVEF), and longitudinal strain (LVLS). Right-sided echocardiographic parameters included RV global longitudinal strain, RV free wall strain, and RV basal diameter. Paired echocardiographic parameters at baseline and follow-up were compared for the entire cohort and for subgroups based on the baseline LV and RV function. Results For the entire cohort, echocardiographic markers of LV and RV function at follow-up were not significantly different from baseline (all P > .05). Patients with hyperdynamic LVEF at baseline (>70%), had a significant reduction of LVEF at follow-up (74.3% ± 3.1% vs 64.4% ± 8.1%, P < .001), while patients with reduced LVEF at baseline (−20%) at baseline had significant improvement at follow-up (−15.2% ± 3.4% vs −17.4% ± 4.9%, P = .004). Patients with abnormal RV basal diameter (>4.5 cm) at baseline had significant improvement at follow-up (4.9 ± 0.7 cm vs 4.6 ± 0.6 cm, P = .019). Conclusions Overall, there were no significant changes over time in the LV and RV function of patients recovering from COVID-19 infection. However, differences were observed according to baseline LV and RV function, which may reflect recovery from the acute myocardial injury occurring in the acutely ill. Left ventricular and RV function tends to improve in those with impaired baseline function, while it tends to decrease in those with hyperdynamic LV or normal RV function.

Covalent inhibitors targeting the main protease (Mpro, or 3CLpro) of SARS-CoV-2 have shown promise in preclinical investigations. Herein, we report the discovery of two new series of molecules that irreversibly bind to SARS-CoV-2 Mpro. These acrylamide containing molecules were discovered using our covalent DNA-encoded library (DEL) screening platform. Following selection against SARS-CoV-2 Mpro, off-DNA compounds were synthesized and investigated to determine their inhibitory effects, the nature of their binding, and to generate preliminary structure-activity relationships. LC-MS analysis indicates a 1:1 (covalent) binding stoichiometry between our hit molecules and SARS-CoV-2 Mpro. Fluorescent staining assay for covalent binding in the presence of cell lysate suggests reasonable selectivity for SARS-CoV-2 Mpro. And lastly, inhibition of enzymatic activity was also observed against a panel of 3CLpro enzymes from different coronavirus strains, with IC50 values ranging from inactive to single digit micromolar. Our results indicate that DEL selection is a useful approach for identifying covalent inhibitors of cysteine proteases.

Background In the SARS-CoV-2/COVID-19 pandemic, we need to understand the impact of immunomodulatory medications on COVID-19 symptom severity in patients with inflammatory diseases, including the type 2/Th2 polarized skin disease, atopic dermatitis (AD). Objective Because it is believed that type 1/Th1 immunity controls viral infections and that there is a Th1/Th2 counter-regulation, we hypothesized that Th2 targeting with the IL-4Rα-antagonist, dupilumab, in patients with moderate-to-severe AD would rebalance the Th1/Th2 axis, potentially leading to attenuated COVID-19 symptoms. Methods A total of 1237 patients with moderate-to-severe AD in the Icahn School of Medicine at Mount Sinai Department of Dermatology were enrolled in a registry. Patients were screened for COVID-19-related symptoms and assigned a severity score (asymptomatic [0]-fatal [5]). Scores were compared among 3 treatment groups: dupilumab (n = 632), other systemic treatments (n = 107), and limited/no treatment (n = 498). Demographic and comorbid covariates were adjusted by multivariate generalized logistic regression models. Results The dupilumab-treated group showed reduced incidence and severity of COVID-19 symptoms versus other treatment groups. Dupilumab-treated patients were less likely to experience moderate-to-severe symptoms versus patients on other systemics (P = .01) and on limited/no treatment (P = .04), and less likely to experience any symptoms versus patients on other systemics (P = .01). This effect was seen in our entire cohort and in the subgroup of patients with verified COVID-19 or high-risk exposure. Conclusions Patients on dupilumab experienced less severe COVID-19 manifestations and lesser symptoms compared with patients on other systemics and on limited/no treatment. These results suggest that Th2 modulation with dupilumab may have a protective effect on anti-viral immune response in patients with AD.

Background & Aims Diarrhea is one of the most common illnesses and is often caused by bacterial infection. Recently, we have shown that human Na+/H+ exchanger NHE3 (hNHE3), but not non-human NHE3s, interacts with the E3 ubiquitin ligase Nedd4-2. We hypothesize that this property of hNHE3 contributes to the increased severity of diarrhea in humans. Methods We used humanized mice expressing hNHE3 in the intestine (hNHE3int) to compare the contribution of hNHE3 and mouse NHE3 to diarrhea induced by cholera toxin (CTX) and enteropathogenic Escherichia coli (EPEC). We measured Na+/H+ exchange activity and fluid absorption. The role of Nedd4-2 on hNHE3 activity and ubiquitination was determined by knockdown in Caco-2bbe cells. The effects of protein kinase A (PKA), the primary mediator of CTX-induced diarrhea, on Nedd4-2 and hNHE3 phosphorylation and their interaction were determined. Results The effects of CTX and EPEC were greater in hNHE3int mice than in control wild-type (WT) mice, resulting in greater inhibition of NHE3 activity and increased fluid accumulation in the intestine, the hallmark of diarrhea. Activation of PKA increased ubiquitination of hNHE3 and enhanced interaction of Nedd4-2 with hNHE3 via phosphorylation of Nedd4-2 at S342. S342A mutation mitigated the Nedd4-2–hNHE3 interaction and blocked PKA-induced inhibition of hNHE3. Unlike non-human NHE3s, inhibition of hNHE3 by PKA is independent of NHE3 phosphorylation, suggesting a distinct mechanism of hNHE3 regulation. Conclusions The effects of CTX and EPEC on hNHE3 are amplified, and the unique properties of hNHE3 may contribute to diarrheal symptoms occurring in humans., Graphical abstract

Background & Aims One of the features of ulcerative colitis (UC) is a defect in the protective mucus layer. This has been attributed to a reduced number of goblet cells (GCs). However, it is not known whether abnormal GC mucus secretion also contributes to the reduced mucus layer. Our aims were to investigate whether GC secretion was abnormal in UC and exists as a long-term effect of chronic inflammation. Methods Colonoids were established from intestinal stem cells of healthy subjects (HS) and patients with UC. Colonoids were maintained as undifferentiated (UD) or induced to differentiate (DF) and studied as three-dimensional or monolayers on Transwell filters. Total RNA was extracted for quantitative real-time polymerase chain reaction analysis. Carbachol and prostaglandin E2 mediated mucin stimulation was examined by MUC2 IF/confocal microscopy and transmission electron microscopy. Results Colonoids from UC patients can be propagated over many passages; however, they exhibit a reduced rate of growth and transepithelial electrical resistance compared with HS. Differentiated UC colonoid monolayers form a thin and non-continuous mucus layer. UC colonoids have increased expression of secretory lineage markers ATOH1 and SPDEF, along with MUC2 positive GCs, but failed to secrete mucin in response to the cholinergic agonist carbachol and prostaglandin E2, which caused increased secretion in HS. Exposure to tumor necrosis factor α (5 days) reduced the number of GCs, with a greater percentage decrease in UC colonoids compared with HS. Conclusions Chronic inflammation in UC causes long-term changes in GCs, leading to abnormal mucus secretion. This continued defect in GC mucus secretion may contribute to the recurrence in UC., Graphical abstract

Background & Aims Patient-derived tumor organoids recapitulate the characteristics of colorectal cancer (CRC) and provide an ideal platform for preclinical evaluation of personalized treatment options. We aimed to model the acquisition of chemotolerance during first-line combination chemotherapy in metastatic CRC organoids. Methods We performed next-generation sequencing to study the evolution of KRAS wild-type CRC organoids during adaptation to irinotecan-based chemotherapy combined with epidermal growth factor receptor (EGFR) inhibition. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 protein (Cas9)-editing showed the specific effect of KRASG12D acquisition in drug-tolerant organoids. Compound treatment strategies involving Aurora kinase A (AURKA) inhibition were assessed for their capability to induce apoptosis in a drug-persister background. Immunohistochemical detection of AURKA was performed on a patient-matched cohort of primary tumors and derived liver metastases. Results Adaptation to combination chemotherapy was accompanied by transcriptomic rather than gene mutational alterations in CRC organoids. Drug-tolerant cells evaded apoptosis and up-regulated MYC (c-myelocytomatosis oncogene product)/E2F1 (E2 family transcription factor 1) and/or interferon-α–related gene expression. Introduction of KRASG12D further increased the resilience of drug-persister CRC organoids against combination therapy. AURKA inhibition restored an apoptotic response in drug-tolerant KRAS–wild-type organoids. In dual epidermal growth factor receptor (EGFR)– pathway blockade-primed CRC organoids expressing KRASG12D, AURKA inhibition augmented apoptosis in cases that had acquired increased c-MYC protein levels during chemotolerance development. In patient-matched CRC cohorts, AURKA expression was increased in primary tumors and derived liver metastases. Conclusions Our study emphasizes the potential of patient-derived CRC organoids in modeling chemotherapy tolerance ex vivo. The applied therapeutic strategy of dual EGFR pathway blockade in combination with AURKA inhibition may prove effective for second-line treatment of chemotolerant CRC liver metastases with acquired KRAS mutation and increased AURKA/c-MYC expression., Graphical abstract

Background: Coronary artery disease (CAD) is one of the common genetic and clinical risk factors associated with cardiovascular and multifactorial disorder. ATP-binding cassette transporter A1 (ABCA1) gene plays an important role in lipid metabolism and in multiple studies associated with CAD. However, more studies are needed to identify the exact role of single nucleotide polymorphisms which may cause CAD. Objectives: The aim of this study is to investigate the genetic association of polymorphism g.1051G > A in the ABCA1 gene with CAD patients in the Saudi population. Methods: We included 315 confirmed CAD cases, and 205 non-CAD or control subjects in this case-control study. DNA isolation was carried out for all registered participants and the polymorphism g.1051G > A was genotyped with Polymerase Chain Reaction followed by Restriction Fragment Length Polymorphism analysis with EcoNI restriction enzyme. Results: Modifiable risk factors such as Body Mass Index, smoking and diabetes were strongly associated and non-modifiable risk factors such as hypertension (Systolic Blood Pressure and Diastolic Blood Pressure) and serum analysis such as Fasting Blood Glucose, Total cholesterol (TC), Triglyceride (TG) and LDL-c were significantly associated in CAD cases (p A polymorphism. Conclusion: The polymorphism g.1051G > A in the gene ABCA1 is closely associated with the existence of the CAD subjects. This polymorphism could also affect the serum levels of the lipid profile, suggesting a possible occurrence of CAD in the Saudi population.

Summary: Objectives: Patients with coronavirus disease 2019 (COVID-19) can present anorexia and weight loss due to their symptoms and eating disorder which can lead to immune system weakness and increase the duration of recovery time. We aim to assess the severity and duration of anorexia and weight loss within the infection and recovery period in these patients. Method: We retrospectively identified 233 COVID-19 patients (older than 18 years) were admitted to the Rasoul-e Akram Hospital, from August to December 2020. Their medical records were reviewed by researchers. Then, patients who had inclusion criteria were asked about duration and severity of anorexia, and also weight alternation during the infection and after the recovery period. Result: Analyzed data were collected from 233 COVID-19 patients showed the mean duration of anorexia was 7.08 ± 10.41 days with a significant loss of appetite (- 75.55 ± 88.09, P-value < 0.001) at the period of anorexia compare to appetite improvement. Also, results demonstrated patients, especially males and severe illness subjects, significantly lost weight (P-value

Background & Aims: Substitution of lysine for glutamic acid at residu 167 in Transmembrane 6 superfamily member 2 (TM6SF2) is associated with fatty liver disease and reduced plasma lipid levels. Tm6sf2-/- mice replicate the human phenotype but were not suitable for detailed mechanistic studies. As an alternative model, we generated Tm6sf2-/- rats to determine the subcellular location and function of TM6SF2. Methods: Two lines of Tm6sf2-/- rats were established using gene editing. Lipids from tissues and from newly secreted very low density lipoproteins (VLDLs) were quantified using enzymatic assays and mass spectrometry. Neutral lipids were visualized in tissue sections using Oil Red O staining. The rate of dietary triglyceride (TG) absorption and hepatic VLDL-TG secretion were compared in Tm6sf2-/- mice and in their wild-type littermates. The intracellular location of TM6SF2 was determined by cell fractionation. Finally, TM6SF2 was immunoprecipitated from liver and enterocytes to identify interacting proteins. Results: Tm6sf2-/- rats had a 6-fold higher mean hepatic TG content (56.1 ± 28.9 9 vs 9.8 ± 3.9 mg/g; P < .0001) and lower plasma cholesterol levels (99.0 ± 10.5 vs 110.6 ± 14.0 mg/dL; P = .0294) than their wild-type littermates. Rates of appearance of dietary and hepatic TG into blood were reduced significantly in Tm6sf2-/- rats (P < .001 and P < .01, respectively). Lipid content of newly secreted VLDLs isolated from perfused livers was reduced by 53% (TG) and 62% (cholesterol) (P = .005 and P = .01, respectively) in Tm6sf2-/- mice. TM6SF2 was present predominantly in the smooth endoplasmic reticulum and endoplasmic reticulum–Golgi intermediate compartments, but not in Golgi. Both apolipoprotein B-48 and acyl-CoA synthetase long chain family member 5 physically interacted with TM6SF2. Conclusions: TM6SF2 acts in the smooth endoplasmic reticulum to promote bulk lipidation of apolipoprotein B–containing lipoproteins, thus preventing fatty liver disease.

Graphical abstract, Introduction Neomenthol, a cyclic monoterpenoid, is a stereoisomer of menthol present in the essential oil of Mentha spp. It is used in food as a flavoring agent, in cosmetics and medicines because of its cooling effects. However, neomenthol has not been much explored for its anticancer potential. Additionally, targeting hyaluronidase, Cathepsin-D, and ODC by phytochemicals is amongst the efficient approach for cancer prevention and/or treatment. Objectives To investigate the molecular and cell target-based antiproliferative potential of neomenthol on human cancer (A431, PC-3, K562, A549, FaDu, MDA-MB-231, COLO-205, MCF-7, and WRL-68) and normal (HEK-293) cell lines. Methods The potency of neomenthol was evaluated on human cancer and normal cell line using SRB, NRU and MTT assays. The molecular target based study of neomenthol was carried out in cell-free and cell-based test systems. Further, the potency of neomenthol was confirmed by quantitative real-time PCR analysis and molecular docking studies. The in vivo anticancer potential of neomenthol was performed on mice EAC model and the toxicity examination was accomplished through in silico, ex vivo and in vivo approaches. Results Neomenthol exhibits a promising activity (IC50 17.3 ± 6.49 μM) against human epidermoid carcinoma (A431) cells by arresting the G2/M phase and increasing the number of sub-diploid cells. It significantly inhibits hyaluronidase activity (IC50 12.81 ± 0.01 μM) and affects the tubulin polymerization. The expression analysis and molecular docking studies support the in vitro molecular and cell target based results. Neomenthol prevents EAC tumor formation by 58.84% and inhibits hyaluronidase activity up to 10% at 75 mg/kg bw, i.p. dose. The oral dose of 1000 mg/kg bw was found safe in acute oral toxicity studies. Conclusion Neomenthol delayed the growth of skin carcinoma cells by inhibiting the tubulin polymerization and hyaluronidase activity, which are responsible for tumor growth, metastasis, and angiogenesis.

In this unprecedented crisis of severe acute respiratory syndrome coronavirus 2 and its associated coronavirus disease 2019 (COVID-19), polymerase chain reaction and then serological testing platforms have been massively developed to face the important screening demand. Polymerase chain reaction and serological testing platforms are not the only actors impacted by the crisis, transfusion services are facing important difficulties. A positive direct antiglobulin test is frequently observed for patients encountering COVID-19. Patients with severe symptoms may develop anaemia and become good candidates for blood transfusions. The interpretation of a positive direct antiglobulin test for patients recently transfused and suffering from COVID-19 is complex. The differentiation between COVID-19 induced antibodies and possible associated transfusion alloantibodies is therefore crucial. In this context, the elution technique incorporated in an appropriate decision-making process plays its full role. This intricate topic is presented through a case report followed by literature review and finally decision-making process for COVID-19 patients necessitating red blood cells administration.

Background Coronavirus disease 2019 (COVID-19) has spread worldwide and is also an important disease in Japan. Thus, the optimal treatment strategy for severe COVID-19 should be established urgently. The effects of combination treatment with baricitinib—a Janus kinase inhibitor, remdesivir, and dexamethasone (BRD) are unknown. Methods Patients who received combination therapy with BRD at the Japanese Red Cross Medical Center were enrolled in the study. All patients received baricitinib (≤14 d), remdesivir (≤10 d), and dexamethasone (≤10 d). The efficacy and adverse events were evaluated. Results In total, 44 patients with severe COVID-19 were enrolled in this study. The 28-d mortality rate was low at 2.3% (1/44 patients). The need for invasive mechanical ventilation was avoided in most patients (90%, 17/19 patients). Patients who received BRD therapy had a median hospitalization duration of 11 d, time to recovery of 9 d, duration of intensive care unit stay of 6 d, duration of invasive mechanical ventilation of 5 d, and duration of supplemental oxygen therapy of 5 d. Adverse events occurred in 15 patients (34%). Liver dysfunction, thrombosis, iliopsoas hematoma, renal dysfunction, ventilator-associated pneumonia, infective endocarditis, and herpes zoster occurred in 11%, 11%, 2%, 2%, 2%, 2%, and 2% of patients, respectively. Conclusions Combination therapy with BRD was effective in treating severe COVID-19, and the incidence rate of adverse events was low. The results of the present study are encouraging; however, further randomized clinical studies are needed.

Objectives SARS-CoV-2 rapid antigen tests (RAT) provide fast identification of infectious patients when RT-PCR results are not immediately available. We aimed to develop a prediction model for identification of false negative (FN) RAT results. Methods In this multicenter trial, patients with documented paired results of RAT and RT-PCR between October 1st 2020 and January 31st 2021 were retrospectively analyzed regarding clinical findings. Variables included demographics, laboratory values and specific symptoms. Three different models were evaluated using Bayesian logistic regression. Results The initial dataset contained 4,076 patients. Overall sensitivity and specificity of RAT was 62.3% and 97.6%. 2,997 cases with negative RAT results (FN: 120; true negative: 2,877; reference: RT-PCR) underwent further evaluation after removal of cases with missing data. The best-performing model for predicting FN RAT results containing 10 variables yielded an area under the curve of 0.971. Sensitivity, specificity, PPV and NPV for 0.09 as cut-off value (probability for FN RAT) were 0.85, 0.99, 0.7 and 0.99. Conclusion FN RAT results can be accurately identified through ten routinely available variables. Implementation of a prediction model in addition to RAT testing in clinical care can provide a decision guidance for initiating appropriate hygiene measures and therefore helps avoiding nosocomial infections., Graphical abstract Image, graphical abstract

Introduction The automated quantitative antigen test (QAT), which detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is suitable for mass screening. However, its diagnostic capability differentiated by time from onset and potential contribution to infectivity assessment have not been fully investigated. Methods A retrospective, observational study using nasopharyngeal swab specimens from coronavirus disease (COVID-19) inpatients was conducted using LumipulseⓇ SARS-CoV-2 antigen test. Diagnostic accuracy was examined for the early (up to 10 days after onset) and late (over 10 days after onset) stages. Time-course QAT changes and reverse‐transcription quantitative polymerase chain reaction tests results were displayed as locally estimated scatterplot smoothing curve, and receiver operating characteristic curve (ROC) analysis was used to determine the appropriate cutoff value for differentiating the early and late stages. Results We obtained 100 specimens from 68 COVID-19 patients, including 51 early-stage and 49 late-stage specimens. QAT sensitivity and specificity were 0.82 (0.72–0.90) and 0.95 (0.75–0.99) for all periods, 0.93 (0.82–0.98) and 1.00 (0.39–1.00) for the early stage, and 0.66 (0.48–0.82) and 0.93 (0.69–0.99) for the late stage, respectively. The ROC analysis indicated an ideal cutoff value of 6.93 pg/mL for distinguishing early-from late-stage specimens. The sensitivity, specificity, positive predictive value, and negative predictive value for predicting the late stage were 0.76 (0.61–0.87), 0.76 (0.63–0.87), 0.76 (0.61–0.87), and 0.76 (0.63–0.87). Conclusions QAT has favorable diagnostic accuracy in the early COVID-19 stages. In addition, an appropriate cutoff point can potentially facilitate rapid identification of noncontagious patients.

Although there have been several case reports and simulation models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission associated with air travel, there are limited data to guide testing strategy to minimize the risk of SARS-CoV-2 exposure and transmission onboard commercial aircraft. Among 9853 passengers with a negative SARS-CoV-2 polymerase chain reaction test performed within 72 hours of departure from December 2020 through May 2021, five (0.05%) passengers with active SARS-CoV-2 infection were identified with rapid antigen tests and confirmed with rapid molecular test performed before and after an international flight from the United States to Italy. This translates to a case detection rate of 1 per 1970 travelers during a time of high prevalence of active infection in the United States. A negative molecular test for SARS-CoV-2 within 72 hours of international airline departure results in a low probability of active infection identified on antigen testing during commercial airline flight.

A 33-year-old woman with newly diagnosed disseminated tuberculosis presented with acute heart failure and incidental findings of intracardiac thrombus, demonstrating possible tuberculous myocarditis. (Level of Difficulty: Intermediate.), Central Illustration

Coronavirus disease-2019 (COVID-19) is associated with systemic inflammation, endothelial activation, and multiorgan manifestations. Lipid-modulating agents may be useful in treating patients with COVID-19. These agents may inhibit viral entry by lipid raft disruption or ameliorate the inflammatory response and endothelial activation. In addition, dyslipidemia with lower high-density lipoprotein cholesterol and higher triglyceride levels portend worse outcomes in patients with COVID-19. Upon a systematic search, 40 randomized controlled trials (RCTs) with lipid-modulating agents were identified, including 17 statin trials, 14 omega-3 fatty acids RCTs, 3 fibrate RCTs, 5 niacin RCTs, and 1 dalcetrapib RCT for the management or prevention of COVID-19. From these 40 RCTs, only 2 have reported preliminary results, and most others are ongoing. This paper summarizes the ongoing or completed RCTs of lipid-modulating agents in COVID-19 and the implications of these trials for patient management., Central Illustration

Background & Aims Intestinal mucositis, a severe complication of antineoplastic therapeutics, is characterized by mucosal injury and inflammation in the small intestine. Therapies for the prevention and treatment of this disease are needed. We investigated whether 2’-fucosyllactose (2’-FL), an abundant oligosaccharide in human milk, protects intestinal integrity and ameliorates intestinal mucositis. Methods A mouse small intestinal epithelial (MSIE) cell line, mouse enteroid cultures, and human gastrointestinal tumor cell lines (AGS and HT29) were co-treated with the chemotherapy agent 5-fluorouracil (5-FU) and 2’-FL. Mice were injected intraperitoneally with 5-FU to induce intestinal mucositis. 2’-FL was administered in the drinking water to mice before (pretreatment) or concurrently with 5-FU injection. Body weight and pathologic changes were analyzed. Results 2’-FL alleviated 5-FU inhibition of cell growth in MSIE cells, but not in AGS and HT29 cells. The 5-FU–induced apoptosis in MSIE cells and enteroids was suppressed by 2’-FL. Compared with 5-FU treatment alone, 2’-FL pretreatment protected against body weight loss, and ameliorated inflammation scores, proinflammatory cytokine production, shortening of villi, epithelial cell apoptosis, goblet cell loss, and tight junctional complex disruption in the small intestine. 2’-FL concurrent treatment had less of an effect on intestinal mucositis than 2’-FL pretreatment. Interestingly, no effect of 2’-FL was observed on 5-FU–induced S-phase arrest in MSIE, AGS, and HT29 cells. Neither pretreatment nor concurrent treatment with 2’-FL affected 5-FU–induced inhibition of proliferation in MSIE cells. Conclusions This study shows a novel direct effect of 2’-FL in protecting small intestinal epithelial cells against apoptosis stimulated by 5-FU, which may contribute to prevention of 5-FU–induced intestinal mucositis., Graphical abstract

Multisystem inflammatory syndrome in children (MIS-C) can cause a myriad of cardiac manifestations, including coronary dilation and aneurysms; giant aneurysms are infrequent. We describe 3patients with giant coronary aneurysms associated with MIS-C, including the youngest case reported to date, treated with intravenous immunoglobulin, corticosteroids, and biologic agents. (Level of Difficulty: Intermediate.), Central Illustration

Spotted fever group rickettsiae (SFGR) represent gram-negative intracellular bacteria transmitted to human hosts through tick bites.1,2 Although Rocky Mountain spotted fever (RMSF) represents the prototypical rickettsiosis associated with SFGR, this group also includes Rickettsia parkeri. Among the SFGR, R parkeri may produce a distinct vesicular eruption preceded by an eschar. Thus, R parkeri rickettsiosis may clinically and histopathologically simulate rickettsialpox due to Rickettsia akari. Herein, we present a case of vesicular spotted fever due to R parkeri.

International audience; Background and aimsTo evaluate the prevalence and prognostic value of metabolic syndrome (MetS) in patients admitted for coronavirus disease 2019 (COVID-19).Methods and resultsIn this monocentric cohort retrospective study, we consecutively included all adult patients admitted to COVID-19 units between April 9 and May 29, 2020 and between February 1 and March 26, 2021. MetS was defined when at least three of the following components were met: android obesity, high HbA1c, hypertension, hypertriglyceridemia, and low HDL cholesterol. COVID-19 deterioration was defined as the need for nasal oxygen flow ≥6 L/min within 28 days after admission.We included 155 patients (55.5% men, mean age 61.7 years old, mean body mass index 29.8 kg/m2). Fifty-six patients (36.1%) had COVID-19 deterioration. MetS was present in 126 patients (81.3%) and was associated with COVID-19 deterioration (no-MetS vs MetS: 13.7% and 41.2%, respectively, p < 0.01). Logistic regression taking into account MetS, age, gender, ethnicity, period of inclusion, and Charlson Index showed that COVID-19 deterioration was 5.3 times more likely in MetS patients (95% confidence interval 1.3–20.2) than no-MetS patients.ConclusionsOver 81.3% of patients hospitalized in COVID-19 units had MetS. This syndrome appears to be an independent risk factor of COVID-19 deterioration.

Objective Neurosurgical patients are at a higher risk of having a severe course of coronavirus disease 2019 (COVID-19). The objective of this study was to determine morbidity, hospital course, and mortality of neurosurgical patients during the coronavirus disease 2019 (COVID-19) pandemic in a multicenter health care system. Methods A retrospective observational study was conducted to identify all hospitalized neurosurgical patients positive for COVID-19 from March 11, 2020 to November 2, 2020 at Mayo Clinic and the Mayo Clinic Health System. Results Eleven hospitalized neurosurgical patients (0.68%) were positive for COVID-19. Four patients (36.6%) were men and 7 (63.3%) were women. The mean age was 65.7 years (range, 35–81 years). All patients had comorbidities. The mean length of stay was 13.4 days (range, 4–30 days). Seven patients had a central nervous system malignancy (4 metastases, 1 meningioma, 1 glioblastoma, and 1 schwannoma). Three patients presented with cerebrovascular complications, comprising 2 spontaneous intraparenchymal hemorrhages and 1 ischemic large-vessel stroke. One patient presented with an unstable traumatic spinal burst fracture. Four patients underwent neurosurgical/neuroendovascular interventions. Discharge disposition was to home in 5 patients, rehabilitation facility in 3, and hospice in 3. Five patients had died at follow-up, 3 within 30 days from COVID-19 complications and 2 from progression of their metastatic cancer. Conclusions COVID-19 is rare among the inpatient neurosurgical population. In all cases, patients had multiple comorbidities. All symptomatic patients from the respiratory standpoint had complications during their hospitalization. Deaths of 3 patients who died within 30 days of hospitalization were all related to COVID-19 complications. Neurosurgical procedures were performed only if deemed emergent.

Genomic instability remains an enabling feature of cancer and promotes malignant transformation. Alterations of DNA damage response (DDR) pathways allow genomic instability, generate neoantigens, upregulate the expression of programmed death ligand 1 (PD-L1) and interact with signaling such as cyclic GMP–AMP synthase-stimulator of interferon genes (cGAS–STING) signaling. Here, we review the basic knowledge of DDR pathways, mechanisms of genomic instability induced by DDR alterations, impacts of DDR alterations on immune system, and the potential applications of DDR alterations as biomarkers and therapeutic targets in cancer immunotherapy., Graphical abstract This review summarizes basic knowledge of DNA damage response (DDR) pathways, mechanisms of genomic instability induced by DDR alterations, impacts of DDR alterations on immune system, and its potential applications as therapeutic targets in cancer immunotherapy.Image 1

The severe acute respiratory syndrome coronavirus 2 coronavirus disease 2019 (COVID-19) global pandemic has ushered in an era of hesitation in performing transplants in affected patients. This stems from the paucity of data regarding the testing modalities, long-term implications, and uncertain prognosis in this group of patients. Current guidance from the Centers for Disease Control recommends assessing symptoms rather than polymerase chain reaction (PCR) positivity. In light of these recommendations, we describe a case of an orthotopic liver transplant in a patient infected with COVID-19 with persistent PCR positivity for 40 days before retransplant. The patient's perioperative and postoperative course was uncomplicated. Our experience leads us to advocate for liver transplants in patients who are PCR positive for COVID-19 after careful individualized and multidisciplinary evaluation regarding their liver disease and COVID-19 symptomatology.

Salmonella mediated cancer therapy has achieved remarkable anti-tumor effects in experimental animal models, but the detailed mechanism remains unsolved. In this report, the active involvement of the host immune response in this process was confirmed by comparing the tumor-suppressive effects of Salmonella in immunocompetent and immunodeficient mice bearing melanoma allografts. Since flagella are key inducers of the host immune response during bacterial infection, flagella were genetically disrupted to analyse their involvement in Salmonella-mediated cancer therapy. The results showed that flagellum-deficient strains failed to induce significant anti-tumor effects, even when more bacteria were administered to offset the difference in invasion efficiency. Flagella mainly activate immune cells via Flagellin/Toll-like receptor 5 (TLR5) signalling pathway. Indeed, we showed that exogenous activation of TLR5 signalling by recombinant Flagellin and exogenous expression of TLR5 both enhanced the therapeutic efficacy of flagellum-deficient Salmonella against melanoma. Our study highlighted the therapeutic value of the interaction between Salmonella and the host immune response through Flagellin/TLR5 signalling pathway during Salmonella-mediated cancer therapy, thereby suggesting the potential application of TLR5 agonists in the cancer immune therapy., Graphical abstract Flagella play a critical role in Salmonella-mediated cancer therapy by activating host immune response through Flagellin/TLR5/NF-κB signalling pathway, indicating Flagellin's potential application in cancer immune therapy.Image 1

Background High scores in Medically Necessary, Time-sensitive (MeNTS) scoring system, used for elective surgical prioritization during Coronavirus Disease 2019 pandemic, are assumed to be associated with worse outcomes. We aimed to evaluate the MeNTS scoring system in patients undergoing elective surgery during restricted capacity of our institution with or without moderate or severe postoperative complications. Study Design In this prospective observational study, MeNTS scores of patients undergoing elective operations during May-June 2020 were calculated. Postoperative complication severity (classified as Group Clavien-Dindo, Medically Necessary Time-Sensitive (MeNTS) score was proposed to objectively prioritize elective surgery during COVID-19. Whether higher MeNTS score is associated with worse outcomes was not examined. Despite higher MeNTS score in patients with moderate and severe postoperative complication, it had low prediction capability for these complications.

Background & Aims To provide an adequate treatment strategy for chronic hepatitis B, it is essential to know which patients are expected to have a good prognosis and which patients do not require therapeutic intervention. Previously, we identified the substitution of isoleucine to leucine at amino acid 97 (I97L) in the hepatitis B core region as a key predictor among patients with stable hepatitis. In this study, we attempted to identify the point at which I97L affects the hepatitis B virus (HBV) life cycle and to elucidate the underlying mechanisms governing the stabilization of hepatitis. Methods To confirm the clinical features of I97L, we used a cohort of hepatitis B e antigen–negative patients with chronic hepatitis B infected with HBV-I97 wild-type (wt) or HBV-I97L. The effects of I97L on viral characteristics were evaluated by in vitro HBV production and infection systems with the HBV reporter virus and cell culture-generated HBV. Results The ratios of reduction in hepatitis B surface antigen and HBV DNA were higher in patients with HBV-I97L than in those with HBV-I97wt. HBV-I97L exhibited lower infectivity than HBV-I97wt in both infection systems with reporter HBV and cell culture-generated HBV. HBV-I97L virions exhibiting low infectivity primarily contained a single-stranded HBV genome. The lower efficiency of cccDNA synthesis was demonstrated after infection of HBV-I97L or transfection of the molecular clone of HBV-I97L. Conclusions The I97L substitution reduces the level of cccDNA through the generation of immature virions with single-stranded genomes. This I97L-associated low efficiency of cccDNA synthesis may be involved in the stabilization of hepatitis., Graphical abstract

Background & Aims Microbiota dysbiosis and mucosa-associated bacteria are involved in colorectal cancer progression. We hypothesize that an interaction between virulent pathobionts and epithelial defense promotes tumorigenesis. Methods Chemical-induced CRC mouse model was treated with antibiotics at various phases. Colonic tissues and fecal samples were collected in a time-serial mode and analyzed by gene microarray and 16S rRNA sequencing. Intraepithelial bacteria were isolated using a gentamicin resistance assay, and challenged in epithelial cultures. Results Our study showed that antibiotic treatment at midphase but not early or late phase reduced mouse tumor burden, suggesting a time-specific host–microbe interplay. A unique antimicrobial transcriptome profile showing an inverse relationship between autophagy and oxidative stress genes was correlated with a transient surge in microbial diversity and virulence emergence in mouse stool during cancer initiation. Gavage with fimA/fimH/htrA-expressing invasive Escherichia coli isolated from colonocytes increased tumor burden in recipient mice, whereas inoculation of bacteria deleted of htrA or triple genes did not. The invasive E.coli suppressed epithelial autophagy activity through reduction of microtubule-associated protein 1 light-chain 3 transcripts and caused dual oxidase 2–dependent free radical overproduction and tumor cell hyperproliferation. A novel alternating spheroid culture model was developed for sequential bacterial challenge to address the long-term changes in host–microbe interaction for chronic tumor growth. Epithelial cells with single bacterial encounter showed a reduction in transcript levels of autophagy genes while those sequentially challenged with invasive E.coli showed heightened autophagy gene expression to eliminate intracellular microbes, implicating that bacteria-dependent cell hyperproliferation could be terminated at late phases. Finally, the presence of bacterial htrA and altered antimicrobial gene expression were observed in human colorectal cancer specimens. Conclusions Invasive pathobionts contribute to cancer initiation during a key time frame by counterbalancing autophagy and oxidative stress in the colonic epithelium. Monitoring gut microbiota and antimicrobial patterns may help identify the window of opportunity for intervention with bacterium-targeted precision medicine., Graphical abstract

Recent infectious disease outbreaks, such as COVID-19 and Ebola, have highlighted the need for rapid and accurate diagnosis to initiate treatment and curb transmission. Successful diagnostic strategies critically depend on the efficiency of biological sampling and timely analysis. However, current diagnostic techniques are invasive/intrusive and present a severe bottleneck by requiring specialist equipment and trained personnel. Moreover, centralised test facilities are poorly accessible and the requirement to travel may increase disease transmission. Self-administrable, point-of-care (PoC) microneedle diagnostic devices could provide a viable solution to these problems. These miniature needle arrays can detect biomarkers in/from the skin in a minimally invasive manner to provide (near-) real-time diagnosis. Few microneedle devices have been developed specifically for infectious disease diagnosis, though similar technologies are well established in other fields and generally adaptable for infectious disease diagnosis. These include microneedles for biofluid extraction, microneedle sensors and analyte-capturing microneedles, or combinations thereof. Analyte sampling/detection from both blood and dermal interstitial fluid is possible. These technologies are in their early stages of development for infectious disease diagnostics, and there is a vast scope for further development. In this review, we discuss the utility and future outlook of these microneedle technologies in infectious disease diagnosis., Graphical abstract This review focused on the functionality of microneedle-based devices and their potential for medical diagnostics.Image 1

COVID-19, caused by SARS-CoV-2, is a highly infectious disease, and clinical laboratory detection has played important roles in its diagnosis and in evaluating progression of the disease. Nucleic acid amplification testing or gene sequencing can serve as pathogenic evidence of COVID-19 diagnosing for clinically suspected cases, and dynamic monitoring of specific antibodies (IgM, IgA, and IgG) is an effective complement for false-negative detection of SARS-CoV-2 nucleic acid. Antigen tests to identify SARS-CoV-2 are recommended in the first week of infection, which is associated with high viral loads. Additionally, many clinical laboratory indicators are abnormal as the disease evolves. For example, from moderate to severe and critical cases, leukocytes, neutrophils, and the neutrophil-lymphocyte ratio increase; conversely, lymphocytes decrease progressively but are over activated. LDH, AST, ALT, CK, high-sensitivity troponin I, and urea also increase progressively, and increased D-dimer is an indicator of severe disease and an independent risk factor for death. Severe infection leads to aggravation of inflammation. Inflammatory biomarkers and cytokines, such as CRP, SAA, ferritin, IL-6, and TNF-α, increase gradually. High-risk COVID-19 patients with severe disease, such as the elderly and those with underlying diseases (cardiovascular disease, diabetes, chronic respiratory disease, hypertension, obesity, and cancer), should be monitored dynamically, which will be helpful as an early warning of serious diseases.

BackgroundHealthcare workers (HCWs) who care for patients with the novel coronavirus infectious disease (COVID-19) are at an increased risk and fear contracting the infection themselves. HCWs are chronically exposed to very intense stress, both and physically and mentally. Hospitals must reduce both the physical and mental burden of HCWs on the front lines and ensure their safety. No prospective study has focused on the physical health complaints among HCWs engaged in the care of critically ill COVID-19 patients. This study aimed to investigate the occupational risk among HCWs of experiencing physical symptoms during the current COVID-19 pandemic.MethodsA twice-weekly questionnaire targeting HCWs who care for COVID-19 patients was performed at Osaka City University Hospital from April 30 to May 31, 2020 using a shareable Research Electronic Data Capture tool. The demographic characteristics of the participants, frequency of exposure to at-risk care, and physical complaints were evaluated.ResultsA total of 35 doctors, 88 nurses, and 35 technicians were engaged in the care of these critically ill COVID-19 patients. 76 HCWs participated in this study, of whom 24 (31.6%) were doctors, 43 (56.6%) were nurses, and 9 (11.8%) were technicians. The frequency of experiencing any physical symptom was 25.0% among HCWs. Exposure to at-risk care was significantly higher among nurses than among doctors (p < 0.001); likewise, the frequency of experiencing physical symptoms was higher among nurses than among doctors (p < 0.01). The multivariate analysis revealed that nurses (odds ratio 8.29; p = 0.01) might be independently at risk of experiencing physical symptoms.ConclusionsOur results indicate that occupational health care at hospitals must be allocated to HCWs who are highly exposed to at-risk care, particularly nurses engaged in the care of COVID-19 patients.

Nucleic acids duplex formation via hybridization is a crucial reaction in many processes and application across different disciplines. In life sciences the detection of mutations is an important application for which hybridization is used, e.g. in diagnostics via single-nucleotide variants (SNVs). This paper deals with the physicochemical aspects of hybridization-based detection of low-abundance mutations, which is challenging due to unavoidable competitive hybridization of high-abundant wild type sequence with the low-abundant variants. We apply two experimental methods based on theoretical hybridization models to show how sensing of DNA mutation can be significantly improved. This is implemented on two SNV biomarkers for which we first select a reference capture probe. This is a probe designed to match neither the wild type nor the SNV sequence, but to have an equal affinity to the wild-type as the SNV-matching probe. This allows the mutation-specific signal to be expressed as a ratiometric quantity, leading to increased assay robustness. Secondly, we selectively deplete the wild-type species by introducing an excess of wild-type-specific capture probes, and account for these depletion effects in the theoretical model. We demonstrate the detection of 0.05% mutant species in a wild-type background, which is an improvement of an order of magnitude in the limit of detection in comparison with the nodepletion case. This sensitivity is comparable with digital PCR results, showing performance suitable for e.g. clinical applications in liquid biopsy context. The principles of this work apply to a wide range of hybridizationbased DNA biosensing technologies, irrespective of the underlying transducer principle. This work was supported by the Research Foundation Flanders (FWO) and the Flemish Institute for Technological Research VITO nv (grant numbers 1S69320N, 1159719N ) and by the EU H2020 M3DLoC project (grant number 760662).

Objectives: To evaluate four sample treatments in a safe and straightforward procedure to detect SARS-CoV-2 in saliva. Methods: Four sample treatments were evaluated in a 3-step procedure to detect SARS-CoV-2 in saliva: 1) heating at 95 °C for 5 min for sample inactivation; 2) sample treatment; 3) analysis by reverse-transcription loop-mediated isothermal amplification (LAMP). Saliva samples used were from infected individuals or were spiked with known quantities of viral particles. Results: Three treatments had a limit of detection (LOD) of 500.000 viral particles per ml of saliva and could be used to detect individuals with potential to transmit the disease. The treatment of phosphate buffer, dithiothreitol, ethylenediaminetetraacetic acid and proteinase K, with an additional 95 °C heating step, yielded a lower LOD of 95; its sensitivity ranged from 100% in patients with nasopharyngeal swab reverse-transcriptase polymerase chain reaction cycle threshold values 30. Conclusions: This report highlights the importance of an adequate sample treatment for saliva to detect SARS-CoV-2 and describes a flexible procedure that can be adapted to point-of-care. Although its sensitivity when LAMP is used is lower than reverse-transcriptase polymerase chain reaction, this procedure can contribute to COVID-19 control by detecting individuals able to transmit the disease.

The outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has presented a global public health emergency. Although predominantly a pandemic of acute respiratory disease, corona virus infectious disease-19 (COVID-19) results in multi-organ damage that impairs cardiopulmonary (CP) function and reduces cardiorespiratory fitness. Superimposed on the CP consequences of COVID-19 is a marked reduction in physical activity that exacerbates CP disease (CPD) risk. CP exercise testing (CPET) is routinely used in clinical practice to diagnose CPD and assess prognosis; assess cardiovascular safety for rehabilitation; and delineate the physiological contributors to exercise intolerance and exertional fatigue. As such, CPET plays an important role in clinical assessments of convalescent COVID-19 patients as well as research aimed at understanding the long-term health effects of SARS-CoV-2 infection. However, due to the ventilatory expired gas analysis involved with CPET, the procedure is considered an aerosol generating procedure. Therefore, extra precautions should be taken by health care providers and exercise physiologists performing these tests. This paper provides recommendations for CPET testing during the COVID-19 pandemic. These recommendations include indications for CPET; pre-screening assessments; precautions required for testing; and suggested decontamination protocols. These safety recommendations are aimed at preventing SARS-CoV-2 transmission during CPET.

Background & Aims CD4+ T cells are regulated by activating and inhibitory cues, and dysregulation of these proper regulatory inputs predisposes these cells to aberrant inflammation and exacerbation of disease. We investigated the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of the CD4+ T-cell inflammatory response and exacerbation of the colitic phenotype. Methods We used Il10-/- spontaneous and CD4+CD45RBhi T-cell transfer models of colitis with PIR-B-deficient (Pirb-/-) mice. Flow cytometry, Western blot, and RNA sequencing analysis was performed on wild-type and Pirb-/- CD4+ T cells. In silico analyses were performed on RNA sequencing data set of ileal biopsy samples from pediatric CD and non–inflammatory bowel disease patients and sorted human memory CD4+ T cells. Results We identified PIR-B expression on memory CD4+ interleukin (IL)17a+ cells. We show that PIR-B regulates CD4+ T-helper 17 cell (Th17)-dependent chronic intestinal inflammatory responses and the development of colitis. Mechanistically, we show that the PIR-B– Src-homology region 2 domain-containing phosphatase-1/2 axis tempers mammalian target of rapamycin complex 1 signaling and mammalian target of rapamycin complex 1–dependent caspase-3/7 apoptosis, resulting in CD4+ IL17a+ cell survival. In silico analyses showed enrichment of transcriptional signatures for Th17 cells (RORC, RORA, and IL17A) and tissue resident memory (HOBIT, IL7R, and BLIMP1) networks in PIR-B+ murine CD4+ T cells and human CD4+ T cells that express the human homologue leukocyte immunoglobulin-like receptor subfamily B member 3 (LILRB3). High levels of LILRB3 expression were associated strongly with mucosal injury and a proinflammatory Th17 signature, and this signature was restricted to a treatment-naïve, severe pediatric CD population. Conclusions Our findings show an intrinsic role for PIR-B/LILRB3 in the regulation of CD4+ IL17a+ T-cell pathogenic memory responses., Graphical abstract

Background & Aims We used patient-derived organoids (PDOs) to study the epithelial-specific transcriptional and secretome signatures of the ileum during Crohn’s disease (CD) with varying phenotypes to screen for disease profiles and potential druggable targets. Methods RNA sequencing was performed on isolated intestinal crypts and 3-week-old PDOs derived from ileal biopsies of CD patients (n = 8 B1, inflammatory; n = 8 B2, stricturing disease) and non-inflammatory bowel disease (IBD) controls (n = 13). Differentially expressed (DE) genes were identified by comparing CD vs control, B1 vs B2, and inflamed vs non-inflamed. DE genes were used for computational screening to find candidate small molecules that could potentially reverse B1and B2 gene signatures. The secretome of a second cohort (n = 6 non-IBD controls, n = 7 CD, 5 non-inflamed, 2 inflamed) was tested by Luminex using cultured organoid conditioned medium. Results We found 90% similarity in both the identity and abundance of protein coding genes between PDOs and intestinal crypts (15,554 transcripts of 19,900 genes). DE analysis identified 814 genes among disease group (CD vs non-IBD control), 470 genes different between the CD phenotypes, and 5 false discovery rate correction significant genes between inflamed and non-inflamed CD. The PDOs showed both similarity and diversity in the levels and types of soluble cytokines and growth factors they released. Perturbagen analysis revealed potential candidate compounds to reverse B2 disease phenotype to B1 in PDOs. Conclusions PDOs are similar at the transcriptome level with the in vivo epithelium and retain disease-specific gene expression for which we have identified secretome products, druggable targets, and corresponding pharmacologic agents. Targeting the epithelium could reverse a stricturing phenotype and improve outcomes., Graphical abstract

Highlights • Next generation sequencing is a scalable solution to genotyping mutant mice. • Ratios of wild type and mutant sequence counts are used to call the genotype. • Hundreds of samples can be multiplexed into one sequencing experiment. • Amplification of high-homology genes can be easily filtered out during analysis., Genotyping of knockout alleles in mice is commonly performed by end-point PCR or gene-specific/universal cassette qPCR. Both have advantages and limitations in terms of assay design and interpretation of results. As an alternative method for high-throughput genotyping, we investigated next generation sequencing (NGS) of PCR amplicons, with a focus on CRISPR-mediated exon deletions where antibiotic selection markers are not present. By multiplexing the wild type and mutant-specific PCR reactions, the genotype can be called by the relative sequence counts of each product. The system is highly scalable and can be applied to a variety of different allele types, including those produced by the International Mouse Phenotyping Consortium and associated projects. One potential challenge with any assay design is locating unique areas of the genome, especially when working with gene families or regions of high homology. These can result in misleading or ambiguous genotypes for either qPCR or end-point assays. Here, we show that genotyping by NGS can negate these issues by simple, automated filtering of undesired sequences. Analysis and genotype calls can also be fully automated, using FASTQ or FASTA input files and an in-house Perl script and SQL database.

Multidrug resistance (MDR) mediated by ATP binding cassette subfamily B member 1 (ABCB1) is significantly hindering effective cancer chemotherapy. However, currently, no ABCB1-inhibitory drugs have been approved to treat MDR cancer clinically, mainly due to the inhibitor specificity, toxicity, and drug interactions. Here, we reported that three polyoxypregnanes (POPs) as the most abundant constituents of Marsdenia tenacissima (M. tenacissima) were novel ABCB1-modulatory pro-drugs, which underwent intestinal microbiota-mediated biotransformation in vivo to generate active metabolites. The metabolites at non-toxic concentrations restored chemosensitivity in ABCB1-overexpressing cancer cells via inhibiting ABCB1 efflux activity without changing ABCB1 protein expression, which were further identified as specific non-competitive inhibitors of ABCB1 showing multiple binding sites within ABCB1 drug cavity. These POPs did not exhibit ABCB1/drug metabolizing enzymes interplay, and their repeated administration generated predictable pharmacokinetic interaction with paclitaxel without obvious toxicity in vivo. We further showed that these POPs enhanced the accumulation of paclitaxel in tumors and overcame ABCB1-mediated chemoresistance. The results suggested that these POPs had the potential to be developed as safe, potent, and specific pro-drugs to reverse ABCB1-mediated MDR. Our work also provided scientific evidence for the use of M. tenacissima in combinational chemotherapy., Graphical abstract Type I polyoxypregnanes (POPs) as prodrugs can be biotransformed by gut microbiota to form active metabolites (type II POPs) that are specific, safe, and potent non-competitive inhibitors of ABCB1, showing unique mechanisms to reverse ABCB1-mediated cancer MDR.Image 1

Objective To report experience with fecal microbiota transplantation (FMT) for recurrent Clostridioides difficile infection (rCDI) and provide recommendations for management of rCDI and donor testing during the COVID-19 pandemic. Methods A retrospective study of patients with rCDI who underwent FMT from May 26, 2020, to September 30, 2020, with stool from well-screened donors with health and infectious screening and a newly implemented strategy for COVID-19 screening with every 2-week bookend testing with stool quarantine. Patients were followed up for development of rCDI and COVID-19. Results Of the 57 patients who underwent FMT for rCDI, 29 were tested for COVID-19 via nasopharyngeal polymerase chain reaction (PCR) and 22 via serology. All results were negative, except for 1 positive serology. Donor testing every 2 weeks for COVID-19 via serology and nasopharyngeal swab PCR was negative, except for 2 donors at 1 center who were excluded. Three patients had rCDI after FMT, and 1 underwent repeat FMT. One patient developed respiratory symptoms suggestive of COVID-19 and tested negative via nasopharyngeal PCR. Eleven patients who underwent COVID-19 testing for elective procedures or hospitalizations tested negative. No SARS-CoV-2 transmission was noted. Conclusions With appropriate donor screening, FMT can be performed safely for rCDI during the COVID-19 pandemic. Development of a validated stool assay for SARS-CoV-2 will simplify this process further.