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Background Levofloxacin(LVX) is used in treatment against bacterial infections but also affect the intestinal microbiota. Calcium(Ca) can form complex with LVX and prevent its absorption. Taking them at least 2 hours apart does not affect LVX peak plasma concentration(Cmax). Since, our in vitro study showed that Ca can increase LVX minimal inhibitory concentration (MIC) in some bacteria, we conducted a randomized control trial(RCT) to study whether oral CaCO3 can lower fecal LVX concentration and preserve gut microbiota diversity in healthy volunteers. Methods We conducted a RCT involving 20 healthy volunteers. All of them received a 5-day course of once-daily 500 mg LVX oral tablet at 8:00 AM. They were randomly assigned to treatment (taking 1,000 mg CaCO3 oral tablet twice daily at 12:00 PM and 6:00 PM) and control group (no CaCO3). The primary outcome was fecal LVX concentration by MIC and high performance liquid chromatography (HPLC) on day 2 and 5 after taking LVX. The secondary outcomes were fecal microbiota diversity by Shannon index (H) by 16s rDNA analysis, LVX Cmax by HPLC on day 1 and 5, and drug adverse events(AEs) in 4 weeks period. Figure 1.Demonstrates study methods. Results Each 10 volunteers were randomly assigned to treatment and control group. Mean fecal LVX concentration was higher in treatment than control group, 100.50 vs 53.21 µg/ml by MIC at day 5 (95% confidence interval [CI] 4.912, 89.73; p = 0.0242). There was no difference in mean fecal LVX concentrations by HPLC between treatment and control at day 2 and day 5. There was no difference in Cmax LVX at day 1 and day5 in treatment and control. No difference of fecal H, but treatment group had significantly declined in H(p = 0.0019). No serious AE, mild AEs were reported (3 in treatment and 5 in control groups), including nausea and diarrhea. Primary outcome Secondary outcome Conclusion In this study, CaCO3 is significantly related to higher fecal LVX level by MIC but does not significantly affect the LVX Cmax. However, rather than protecting gut microbiota from LVX, CaCO3 may lower gut microbiota diversity in the presence of LVX. Therefore, co-prescription of LVX and CaCO3 should be cautioned even without the concern about the absorption like when LVX is administered intravenously or when both drugs in oral forms are taken at different times. Shannon index diversity Figure 1 showed the Shannon diversity index. The mean diversity index of treatment group at day 0 was a bit higher than control group without statistical significance (p = 0.923). On day 2 and 5 of levofloxacin treatment, mean diversity index was higher in control group than in treatment group, also without statistical significance (p = 0.426 on day 2 and 0.237 on day 5). On day 14, mean diversity index was higher in treatment group. On day 28, mean diversity index was returned close to baseline at day 0. Figure 3. Taxonomic profile Details of microbiota composition in different taxa were demonstrated in Figure 2, different color represented different taxa compositions at genus level. Disclosures All Authors: No reported disclosures.

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types., Graphical abstract, D’Antonio et al. characterize associations between GWAS signals for COVID-19 disease and eQTLs in 69 human tissues to identify causal variants and their underlying molecular mechanisms. They show that diverse symptoms and disease severity of COVID-19 are associated with variants affecting gene expression in a wide variety of tissues.

Sindbis virus (SINV) is an alphavirus that causes age-dependent encephalomyelitis in mice. Within 7–8 days after infection infectious virus is cleared from neurons through the antiviral effects of antibody and interferon-gamma (IFNγ), but RNA persists. To better understand changes in viral RNA associated with immune-mediated clearance we developed recombinant strains of SINV that have genomic and subgenomic viral RNAs tagged with the Broccoli RNA aptamer that binds and activates a conditional fluorophore for live cell imaging of RNA. Treatment of SINV-Broccoli-infected cells with antibody to the SINV E2 glycoprotein had cell type-specific effects. In BHK cells, antibody increased levels of intracellular viral RNA and changed the primary location of genomic RNA from the perinuclear region to the plasma membrane without improving cell viability. In undifferentiated and differentiated AP7 (dAP7) neuronal cells, antibody treatment decreased levels of viral RNA. Occasional dAP7 cells escaped antibody-mediated clearance by not expressing cell surface E2 or binding antibody to the plasma membrane. IFNγ decreased viral RNA levels only in dAP7 cells and synergized with antibody for RNA clearance and improved cell survival. Therefore, analysis of aptamer-tagged SINV RNAs identified cell type- and neuronal maturation-dependent responses to immune mediators of virus clearance.

Sindbis virus (SINV) can infect neurons and cause encephalomyelitis in mice. Nonstructural proteins are translated from genomic RNA and structural proteins from subgenomic RNA. While visualization of viral proteins in living cells is well developed, imaging of viral RNAs has been challenging. RNA aptamers that bind and activate conditional fluorophores provide a tool for RNA visualization. We incorporated cassettes of two F30-scaffolded dimers of the Broccoli aptamer into a SINV cDNA clone using sites in nsP3 (genomic RNA), the 3′UTR (genomic and subgenomic RNAs) and after a second subgenomic promoter resulting in 4–28 Broccoli copies. After addition of the cell-permeable 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI-1T) conditional fluorophore and laser excitation, infected cells emitted green fluorescence that correlated with Broccoli copy numbers. All recombinant viruses replicated well in BHK and undifferentiated neural cells but viruses with 14 or more Broccoli copies were attenuated in differentiated neurons and mice. The signal survived fixation and allowed visualization of viral RNAs in differentiated neurons and mouse brain, as well as BHK cells. Subgenomic RNA was diffusely distributed in the cytoplasm with genomic RNA also in perinuclear vesicle-like structures near envelope glycoproteins or mitochondria. Broccoli aptamer-tagging provides a valuable tool for live cell imaging of viral RNA.

Sindbis virus (SINV) infection of neurons in the brain and spinal cord in mice provides a model system for investigating recovery from encephalomyelitis and antibody-mediated clearance of virus from the central nervous system (CNS). To determine the roles of IgM and IgG in recovery, we compared the responses of immunoglobulin-deficient activation-induced adenosine deaminase-deficient (AID −/− ), secretory IgM-deficient (sIgM −/− ), and AID −/− sIgM −/− double-knockout (DKO) mice with those of wild-type (WT) C57BL/6 mice for disease, clearance of infectious virus and viral RNA from brain and spinal cord, antibody responses, and B cell infiltration into the CNS. Because AID is essential for immunoglobulin class switch recombination and somatic hypermutation, AID −/− mice produce only germ line IgM, while sIgM −/− mice secrete IgG but no IgM and DKO mice produce no secreted immunoglobulin. After intracerebral infection with the TE strain of SINV, most mice recovered. Development of neurologic disease occurred slightly later in sIgM −/− mice, but disease severity, weight loss, and survival were similar between the groups. AID −/− mice produced high levels of SINV-specific IgM, while sIgM −/− mice produced no IgM and high levels of IgG2a compared to WT mice. All mice cleared infectious virus from the spinal cord, but DKO mice failed to clear infectious virus from brain and had higher levels of viral RNA in the CNS late after infection. The numbers of infected cells and the amount of cell death in brain were comparable. We conclude that antibody is required and that either germ line IgM or IgG is sufficient for clearance of virus from the CNS. IMPORTANCE Mosquito-borne alphaviruses that infect neurons can cause fatal encephalomyelitis. Recovery requires a mechanism for the immune system to clear virus from infected neurons without harming the infected cells. Antiviral antibody has previously been shown to be a noncytolytic means for alphavirus clearance. Antibody-secreting cells enter the nervous system after infection and produce antiviral IgM before IgG. Clinical studies of human viral encephalomyelitis suggest that prompt production of IgM is associated with recovery, but it was not known whether IgM is effective for clearance. Our studies used mice deficient in production of IgM, IgG, or both to characterize the antibody necessary for alphavirus clearance. All mice developed similar signs of neurologic disease and recovered from infection. Antibody was necessary for virus clearance from the brain, and either early germ line IgM or IgG was sufficient. These studies support the clinical observation that prompt production of antiviral antibody is a determinant of outcome.

Viral RNA studies often rely on in situ hybridization and reverse transcriptase-PCR to provide snapshots of RNA dynamics in infected cells. To facilitate analysis of cellular RNAs, aptamers Spinach and Spinach2 that bind and activate the conditional fluorophore 3, 5-difluoro-4-hydroxybenzylidene imidazolinon have been developed. To determine the feasibility of applying this technology to viral RNA, we have used cDNA clones of the TE strain of Sindbis virus (SINV) to construct multiple viruses containing one or two copies of tRNA-scaffolded Spinach2 after a second subgenomic promoter, TEds-1Sp and TEds-2Sp within the 3′UTR, TE-1UTRSp, or after a second subgenomic promoter and in the 3′UTR, TEds-1Sp+1 UTRSp. TEds-1Sp+1 UTRSp gave the brightest signal and replicated well in cell culture, while TEds-2Sp was the dimmest and replicated poorly. Selection of baby hamster kidney cells infected with TEds-1Sp+1 UTRSp for improved signal intensity identified a virus with a stronger signal and point mutations in the tRNA scaffold. Imaging of SINV in BHK cells showed RNA to be concentrated in filopodia that contacted and transferred RNA to adjacent cells. The effect of treatment with anti-E2 antibody, which effects non-cytolytic clearance of SINV from neurons, on viral RNA was cell-type-dependent. In antibody-treated BHK cells, intracellular viral RNA increased and spread of infection continued. In undifferentiated and differentiated AP7 neuronal cells antibody treatment induced viral RNA clearance. Both viruses with two inserted aptamers were prone to deletion. These studies form the basis for further development of aptamer-labelled viral RNAs that will facilitate functional studies on the dynamics of infection and clearance.

Biologically relevant, three-dimensional extracellular matrix is an essential component of in vitro vasculogenesis models. WI-38 fibroblasts assemble a 3D matrix that induces endothelial tubulogenesis, but this model is challenged by fibroblast senescence and the inability to distinguish endothelial cell-derived matrix from matrix made by WI-38 fibroblasts. Matrices produced by hTERT-immortalized WI-38 recapitulated those produced by wild type fibroblasts. ECM fibrils were heavily populated by tenascin-C, fibronectin, and type VI collagen. Nearly half of the total type I collagen, but only a small fraction of the type IV collagen, were incorporated into ECM. Stable hTERT-WI-38 transfectants expressing TagRFP-fibronectin incorporated TagRFP into ~90% of the fibronectin in 3D matrices. TagRFP-fibronectin colocalized with tenascin-C and with type I collagen in a pattern that was similar to that seen in matrices from wild type WI-38. Human Umbilical Vein Endothelial Cells (HUVEC) formed 3D adhesions and tubes on WI38-hTERT-TagRFP-FN-derived matrices, and the TagRFP-fibronectin component of this new 3D human fibroblast matrix model facilitated the demonstration of concentrated membrane type 1 metalloprotease and new HUVEC FN and collagen type IV fibrils during EC tubulogenesis. These findings indicate that WI-38-hTERT- and WI-38-hTERT-TagRFP-FN-derived matrices provide platforms for the definition of new matrix assembly and remodeling events during vasculogenesis.

We describe a case of microsporidial myositis in a healthy man from Thailand. The small subunit rRNA sequence of this microsporidium is novel and has a close phylogenetic relationship with Endoreticulatus, a genus of lepidopteran microsporidia. Myositis could be caused by more genera of microsporidia than previously known.

Sindbis virus (SINV) infection of neurons results in nonfatal viral encephalomyelitis and provides a model system for understanding recovery from virus infection of the central nervous system (CNS). Infection is followed by clearance of infectious virus, a gradual decrease in viral RNA, and then long-term maintenance of low levels of viral RNA. Antibody to the E2 glycoprotein is important for virus clearance, and B cells enter the CNS along with CD4 + and CD8 + T cells during the early clearance phase. Antibody-secreting cells (ASCs) are present in the CNS and become enriched for SINV-specific ASCs. We have evaluated the factors within the CNS that facilitate continued local antibody production after infection. Expression of CXCL9, CXCL10, CCL1, CCL2, and CCL5 chemokine mRNAs increased early, and infiltrating B cells expressed CXCR3, CXCR5, and CCR7. The mRNAs for IL-10 and IL-21, cytokines important for B cell proliferation and differentiation, rose rapidly and remained elevated long after clearance of infectious virus. Active proliferation of B cells, as indicated by Ki-67 expression, continued for months. Bromodeoxyuridine (BrdU) labeling of proliferating cells showed that ASCs produced in the draining cervical lymph nodes during the early germinal center response were preferentially retained in the CNS. Sustained increase in B-cell-activating factor (BAFF) mRNA in the CNS and BAFF receptor expression by B cells coincided with the long-term maintenance of SINV-specific ASCs in the brain. We conclude that multiple changes in the brain microenvironment facilitate B-cell entry and support proliferation and differentiation and long-term survival of antiviral ASCs during recovery from alphaviral encephalomyelitis.

In February, 2006, a 45-year-old woman went to her local hospital with diff use abdominal pain. When the hospital realised that she might have a serious illness, she was brought by ambulance to our hospital, for insurance reasons. By the time she arrived, the pain had been present for 15 h. Her aortic valve had been replaced in 1999, but she had had no abdominal problems until mid-2005, when she developed a tendency to constipation. She had therefore taken 1–2 tablets of a senna-based laxative, once or twice a week. She had seen no need to tell her doctors about the laxative. For 3 weeks preceding her admission she had taken several tablets of laxative a day, and passed loose, watery stools up to three times a day. The day before the pain started, she had taken many tablets, after passing no stool for 2 days. She had then passed three watery stools, followed by three bloody stools— some blood was fresh and red, and some old and black. She had no other medical history of note; notably, she had no psychiatric disorder. She was on no drugs other than warfarin, which she had taken ever since her operation. She took her warfarin regularly, and her international normalised ratio (INR) had been in the therapeutic range for years. Her diet, including her consumption of green vegetables, varied little. She took no herbal medications. 25 days before admission, her INR had been 2·3. She was hypotensive and tachycardic. Her abdomen was distended, with widespread tenderness and guarding. Rectal examination revealed fresh blood. Blood tests showed a haemoglobin concentration of only 84 g/L; the platelet count was normal, at 164×109/L, but the INR was 11·9; the activated partial thromboplastin time was 92·4 s (control time 29·3 s). The concentrations of bilirubin and aspartate aminotransferase were slightly high, at 33·5 μmol/L and 48 U/L, respectively, but the results of liver function tests were otherwise normal. CT of the abdomen showed extensive haematoma in the pelvic, paracolic, and subhepatic regions. We admitted the patient for several days, during which we gave her vitamin K, fresh frozen plasma, and packed red cells. She subsequently resumed taking warfarin, and stopped taking laxatives in excess; her INR rapidly returned to the target range, where it has remained ever since. When last seen, in November, 2007, she was well. Vitamin K is an essential cofactor for γ-carboxylation, the post-translational modifi cation of coagulation factors II, VII, IX, and X (fi gure). Although vitamin K is also a cofactor for the γ-carboxylation of protein C and protein S, lack of vitamin K reduces coagulation. Green leafy vegetables have especially high con centrations of vitamin K, but the vitamin K in vegetable oils and soya may be more bioavailable. Many patients on warfarin are aware—as, indeed, was our patient—that their dietary consumption of vitamin K should remain fairly constant. However, not all are aware that diarrhoea can reduce absorption of vitamin K, and increase the risk of bleeding. Although some physicians recommend halving the dose of warfarin when the patient has diarrhoea, many doctors seem unaware of the increased risk of bleeding—and many textbooks, formularies, and patient-information leafl ets fail to mention the increased risk, or do so only in passing. Cases like ours will continue to occur, until doctors and patients are better informed.