Visualization of translation termination intermediates trapped by the Apidaecin 137 peptide during RF3-mediated recycling of RF1.

During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3. While high-resolution structures of the individual termination factors on the ribosome exist, direct structural insight into how RF3 mediates dissociation of the decoding RFs has been lacking. Here we have used the Apidaecin 137 peptide to trap RF1 together with RF3 on the ribosome and visualize an ensemble of termination intermediates using cryo-electron microscopy. Binding of RF3 to the ribosome induces small subunit (SSU) rotation and swivelling of the head, yielding intermediate states with shifted P-site tRNAs and RF1 conformations. RF3 does not directly eject RF1 from the ribosome, but rather induces full rotation of the SSU that indirectly dislodges RF1 from its binding site. SSU rotation is coupled to the accommodation of the GTPase domain of RF3 on the large subunit (LSU), thereby promoting GTP hydrolysis and dissociation of RF3 from the ribosome

Autoantibodies in Scurfy Mice and IPEX Patients Recognize Keratin 14

Scurfy mice have a deletion in the Foxp3 gene, resulting in a failure to generate Foxp3+ regulatory T cells, and they subsequently develop severe CD4+ T-cell-mediated autoimmune inflammation. Multiple organs are involved, but the skin is one of the main organs affected. During the course of disease, Scurfy mice develop autoantibodies; however, the targeted antigens are unknown. In this study, we show that Scurfy mice develop autoantibodies directed against skin antigens. Using western blot analysis, we found that Scurfy serum reacted with proteins in total skin lysate, as well as in a keratinocyte lysate. Most of the Scurfy sera tested identified a major band at 50kDa. Transfer of Scurfy CD4+ T cells into nu/nu mice yielded autoantibodies with similar reactivity. Further analysis using 2D western blots, followed by peptide mass fingerprinting, identified several keratins as targets. To confirm this observation, we chose one of the identified targets, keratin 14, and prepared recombinant proteins encompassing the N-terminal, middle, and C-terminal portions of the keratin 14 protein. Scurfy serum predominantly recognized the C-terminal fragment. Sera from patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, the human disease resulting from FOXP3 mutations, also recognized skin antigens, including keratin 14. Thus, the results of our study indicate that autoantibodies in Scurfy mice and patients with IPEX target keratins

Intimin, tir, and Shiga toxin 1 do not influence enteropathogenic responses to Shiga toxin-producing Escherichia coli in bovine ligated intestinal loops

Shiga toxin-producing Escherchia coli (STEC) comprises a group of attaching and effacing (A/E) enteric pathogens of animals and humans. Natural and experimental infection of calves with STEC may result in acute enteritis or subclinical infection, depending on serotype- and host-specific factors. To quantify intestinal secretory and inflammatory responses to STEC in the bovine intestine, serotypes that are associated with human disease (O103:H2 and O157:H7) were introduced into ligated mid-ileal loops in gnotobiotic and conventional calves, and fluid accumulation and recruitment of radiolabeled neutrophils were measured after 12 h. STEC serotype O103:H2, but not serotype O157:H7, elicited strong enteropathogenic responses. To determine if the inflammatory response to STEC O103:H2 in calves requires Shiga toxin 1 or intimate bacterial attachment to the intestinal epithelium, defined mutations were made in the stx1, eae, and tir genes. Our data indicate that some STEC induce intestinal inflammatory responses in calves by a mechanism that is independent of A/E-lesion formation, intimin, or Shiga toxin 1. This may have implications for strategies to reduce STEC carriage in cattle

The dolphin proline-rich antimicrobial peptide Tur1A inhibits protein synthesis by targeting the bacterial ribosome

Proline-rich antimicrobial peptides (PrAMPs) internalize into susceptible bacteria using specific transporters and interfere with protein synthesis and folding. To date, mammalian PrAMPs have so far only been identified in artiodactyls. Since cetaceans are co-phyletic with artiodactyls, we mined the genome of the bottlenose dolphin Tursiops truncates, leading to the identification of two PrAMPs, Tur1A and Tur1B. Tur1A, which is orthologous to the bovine PrAMP Bac7, is internalized into E. coli without damaging the membranes using the inner membrane transporters SbmA and YjiL/MdM. Furthermore, like Bac7, Tur1A also inhibits bacterial protein synthesis by binding to the ribosome and blocking the transition from the initiation to the elongation phase. By contrast, Tur1B is a poor inhibitor of protein synthesis and may utilize another mechanism of action. An X-ray structure of Tur1A bound within the ribosomal exit tunnel provides a basis to develop these peptides as novel antimicrobial agents

Gender Differences in Perceived Working Conditions of General Practitioners During the COVID-19 Pandemic-a Cross-Sectional Study.

BACKGROUND The ongoing COVID-19 pandemic has revealed gender-specific differences between general practitioners in adapting to the posed challenges. As primary care workforce is becoming increasingly female, in many countries, it is essential to take a closer look at gender-specific influences when the global health care system is confronted with a crisis. OBJECTIVE To explore gender-specific differences in the perceived working conditions and gender-specific differences in challenges facing GPs at the beginning of the COVID-19 pandemic in 2020. DESIGN Online survey in seven countries. PARTICIPANTS 2,602 GPs from seven countries (Austria, Australia, Switzerland, Germany, Hungary, Italy, Slovenia). Of the respondents, 44.4% (n = 1,155) were women. MAIN MEASURES Online survey. We focused on gender-specific differences in general practitioners' perceptions of working conditions at the beginning of the COVID-19 pandemic in 2020. KEY RESULTS Female GPs rated their skills and self-confidence significantly lower than male GPs (f: 7.1, 95%CI: 6.9-7.3 vs. m: 7.6, 95%CI 7.4-7.8; p < .001), and their perceived risk (concerned about becoming infected or infecting others) higher than men (f: 5.7, 95%CI: 5.4-6.0 vs. m: 5.1, 95%CI: 4.8-5.5; p = .011). Among female GPs, low self-confidence in the treatment of COVID-19 patients appear to be common. Results were similar in all of the participating countries. CONCLUSIONS Female and male GPs differed in terms of their self-confidence when dealing with COVID-19-related issues and their perceptions of the risks arising from the pandemic. To ensure optimal medical care, it is important that GPs realistically assess their own abilities and overall risk

Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyltRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation

Retinoic Acid and Rapamycin Differentially Affect and Synergistically Promote the Ex Vivo Expansion of Natural Human T Regulatory Cells

Natural T regulatory cells (Tregs) are challenging to expand ex vivo, and this has severely hindered in vivo evaluation of their therapeutic potential. All trans retinoic acid (ATRA) plays an important role in mediating immune homeostasis in vivo, and we investigated whether ATRA could be used to promote the ex vivo expansion of Tregs purified from adult human peripheral blood. We found that ATRA helped maintain FOXP3 expression during the expansion process, but this effect was transient and serum-dependent. Furthermore, natural Tregs treated with rapamycin, but not with ATRA, suppressed cytokine production in co-cultured effector T cells. This suppressive activity correlated with the ability of expanded Tregs to induce FOXP3 expression in non-Treg cell populations. Examination of CD45RA+ and CD45RA− Treg subsets revealed that ATRA failed to maintain suppressive activity in either population, but interestingly, Tregs expanded in the presence of both rapamycin and ATRA displayed more suppressive activity and had a more favorable epigenetic status of the FOXP3 gene than Tregs expanded in the presence of rapamycin only. We conclude that while the use of ATRA as a single agent to expand Tregs for human therapy is not warranted, its use in combination with rapamycin may have benefit

KLF13 sustains thymic memory-like CD8+ T cells in BALB/c mice by regulating IL-4–generating invariant natural killer T cells

Transcription factor KLF13 regulates the elevated numbers of iNKT cells in the BALB/c versus C57BL/6 thymus that results in production of sufficient levels of IL-4 to generate memory-like CD8+ T cells