SUMMARY Specific members of the intestinal microbiota dramatically affect inflammatory bowel disease (IBD) in mice. In humans, however, identifying bacteria that preferentially affect disease susceptibility and severity remains a major challenge. Here, we used flow cytometry-based bacterial cell sorting and 16S sequencing to characterize taxa-specific coating of the intestinal microbiota with immunoglobulin A (IgA−SEQ) and show that high IgA−coating uniquely identifies colitogenic intestinal bacteria in a mouse model of microbiota-driven colitis. We then used IgA−SEQ and extensive anaerobic culturing of fecal bacteria from IBD patients to create personalized disease-associated gut microbiota culture collections with pre-defined levels of IgA coating. Using these collections, we found that intestinal bacteria selected on the basis of high coating with IgA conferred dramatic susceptibility to colitis in germ-free mice. Thus, our studies suggest that IgA−coating identifies inflammatory commensals that preferentially drive intestinal disease. Targeted elimination of such bacteria may reduce, reverse, or even prevent disease development.

Crohn’s disease (CD), a form of inflammatory bowel disease, has a higher prevalence in Ashkenazi Jewish than in non-Jewish European populations. To define the role of non-synonymous mutations, we performed exome sequencing of Ashkenazi Jewish patients with CD, followed by array-based genotyping and association analysis in 2,066 CD cases and 3,633 healthy controls. We detected association signals in the LRRK2 gene that conferred CD risk (N2081D variant, P=9.5×10−10) or protection (N551K variant, tagging R1398H-associated haplotype, P=3.3×10−8). These variants affected CD age of onset, disease location, LRRK2 activity, and autophagy. Bayesian network analysis of CD patient intestinal tissue further implicated LRRK2 in CD pathogenesis. Analysis of the extended LRRK2 locus in 24,570 CD cases, patients with Parkinson’s disease (PD), and healthy controls revealed extensive pleiotropy, with similar genetic effects between CD and PD in both Ashkenazi Jewish and non-Jewish cohorts. The LRRK2 N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. Like the G2019S mutation, the N2081D variant is associated with increased kinase activity, whereas neither N551K nor R1398H on the protective haplotype altered kinase activity. R1398H, but not N551K, increased GTPase activity, thereby deactivating LRRK2. The presence of shared LRRK2 alleles in CD and PD provides refined insight into disease mechanisms and may have major implications for the treatment of these two seemingly unrelated diseases.

In eukaryotes, a key step in the initiation of translation is the binding of the eukaryotic initiation factor 4E (eIF4E) to the cap structure of the mRNA. Subsequent recruitment of several components, including the small ribosomal subunit, is thought to allow migration of initiation complexes and recognition of the initiation codon. Mitogens and cytokines stimulate the phosphorylation of eIF4E at Ser 209 , but the functional consequences of this modification have remained a major unresolved question. Using fluorescence spectroscopy and surface plasmon resonance techniques, we show that phosphorylation of eIF4E markedly reduces its affinity for capped RNA, primarily due to an increased rate of dissociation. Variant eIF4E proteins harboring negatively charged acidic residues at position 209 also showed decreased binding to capped RNA. Furthermore, a basic residue at position 159 was shown to be essential for cap binding. Although eIF4E-binding protein 1 greatly stabilized binding of phosphorylated eIF4E to capped RNA, in the presence of eIF4E-binding protein 1 the phosphorylated form still dissociated faster compared with nonphopshorylated eIF4E. The implications of our findings for the mechanism of translation initiation are discussed.In eukaryotic cells, all nucleus-encoded mRNAs possess a so-called "cap structure" at their 5Ј-end. This cap consists of a methylated 5Ј-5Ј bound guanosine triphosphate (m 7 GTP) moiety. The cap plays a key role in the translation of the mRNA by permitting recruitment of the eukaryotic initiation factors (eIFs) 1 required for the attachment of the ribosome and correct initiation of translation. The protein that interacts directly with the cap is eIF4E, which forms a complex with the scaffolding protein eIF4G, which in turn recruits other factors. These include eIF4A and eIF4B, which are involved in unwinding secondary structure in the 5Ј-untranslated region of the mRNA, and the poly(A)-binding protein, which by interacting with the 3Ј-tail of the mRNA circularizes it (1, 2). eIF4E also binds to small regulatory proteins termed eIF4E-binding proteins (4E-BPs). These compete with eIF4G for binding to overlapping sites on eIF4E and thus inhibit formation of initiation complexes (3, 4).It has long been known that eIF4E undergoes regulated phosphorylation, and the site has been identified as Ser 209 (5, 6). Phosphorylation of eIF4E is increased by mitogenic stimuli that activate translation (reviewed in Ref. 7) and by cytokines (8, 9). Recently, two kinases were identified that phosphorylate Ser 209 in eIF4E and are targets for the mitogen-activated extracellular signal-regulated kinase and stress/cytokine-activated p38 mitogen-activated protein kinase pathways (9 -13). These enzymes (Mnk1 and Mnk2) also associate with eIF4G in vivo (11,12,14). Despite these advances and the large number of studies on the changes of eIF4E phosphorylation in vivo, the key issue of the effect of phosphorylation on the function of eIF4E has received little attention and has remained a major question in the ...

ObjectivesThis study was aimed to assess the diversity of the meconium microbiome and determine if the bacterial community is affected by maternal diabetes status.MethodsThe first intestinal discharge (meconium) was collected from 23 newborns stratified by maternal diabetes status: 4 mothers had pre-gestational type 2 diabetes mellitus (DM) including one mother with dizygotic twins, 5 developed gestational diabetes mellitus (GDM) and 13 had no diabetes. The meconium microbiome was profiled using multi-barcode 16S rRNA sequencing followed by taxonomic assignment and diversity analysis.ResultsAll meconium samples were not sterile and contained diversified microbiota. Compared with adult feces, the meconium showed a lower species diversity, higher sample-to-sample variation, and enrichment of Proteobacteria and reduction of Bacteroidetes. Among the meconium samples, the taxonomy analyses suggested that the overall bacterial content significantly differed by maternal diabetes status, with the microbiome of the DM group showing higher alpha-diversity than that of no-diabetes or GDM groups. No global difference was found between babies delivered vaginally versus via Cesarean-section. Regression analysis showed that the most robust predictor for the meconium microbiota composition was the maternal diabetes status that preceded pregnancy. Specifically, Bacteroidetes (phyla) and Parabacteriodes (genus) were enriched in the meconium in the DM group compared to the no-diabetes group.ConclusionsOur study provides evidence that meconium contains diversified microbiota and is not affected by the mode of delivery. It also suggests that the meconium microbiome of infants born to mothers with DM is enriched for the same bacterial taxa as those reported in the fecal microbiome of adult DM patients.

22Coronavirus disease 2019 is caused by severe acute respiratory 23 syndrome coronavirus 2 (SARS-CoV-2). The spike protein that mediates 24 SARS-CoV-2 entry into host cells, is one of the major targets for vaccines and 25 therapeutics. Thus, insights into the sequence variations of S protein are key to 26 understanding the infection and antigenicity of SARS-CoV-2. Here, we observed a 27 dominant mutational variant at the 614 position of S protein (aspartate to glycine, 28 D614G mutation). Using pseudovirus-based assay, we found that S-D614 and 29 S-G614 protein pseudotyped viruses share a common receptor, human 30 angiotensin-converting enzyme 2 (ACE2), which could be blocked by recombinant 31 ACE2 with the fused Fc region of human IgG1. However, S-D614 and S-G614 32 protein demonstrated functional differences. First, S-G614 protein could be cleaved 33 by serine protease elastase-2 more efficiently. Second, S-G614 pseudovirus 34 infected 293T-ACE2 cells significantly more efficiently than the S-D614 pseudovirus, 35 Moreover, 93% (38/41) sera from convalescent COVID-19 patients could neutralize 36 both S-D614 and S-G614 pseudotyped viruses with comparable efficiencies, but 37 about 7% (3/41) convalescent sera showed decreased neutralizing activity against 38 S-G614 pseudovirus. These findings have important implications for SARS-CoV-2 39 transmission and immune interventions.40 42 43 44 SARS-CoV-2 is a novel coronavirus reported in 2019 that caused the recent 45 outbreak of coronavirus disease-2019 (COVID-19) 1 . By June 17, 2020, the World 46 Health Organization (WHO) reported that 8.06 million people worldwide had been 47 infected with SARS-CoV-2, and 440,290 individuals died of COVID-19. This 48 pandemic had a significant adverse impact on international social and economic 49 activities. The RNA genome of SARS-CoV-2 was rapidly sequenced to facilitate 50 diagnostic testing, molecular epidemiologic source tracking, and development of 51 vaccines and therapeutic strategies 2 . Coronaviruses are enveloped, 52 positive-stranded RNA viruses that contain the largest known RNA genomes to 53 date. The mutation rate for RNA viruses is extremely high, which may contribute to 54 its transmission and virulence. The only significant variation in the SARS-CoV-2 55 spike (S) protein is a non-synonymous D614G (Aspartate (D) to Glycine (G)) 56 mutation 3 . Primary data showed that S-G614 is a more pathogenic strain of 57 SARS-CoV-2 with high transmission efficiency 3 , however whether D614G 58 conversion in S protein affect the viral entry and infectivity in cell model is still 59 unclear. 60 61The S protein of coronavirus, the major determinant of host and tissue tropism, is a 62 major target for vaccines, neutralizing antibodies, and viral entry inhibitors 4,5 . 63Similar to SARS-CoV, the cellular receptor of SARS-CoV-2 is 64 angiotensin-converting enzyme 2 (ACE2); however, the SARS-CoV-2 S protein has 65 a 10-to 20-fold higher affinity for ACE2 than the corresponding S protein of 66 SARS-CoV 6,7 . Coronaviruses use two d...