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Patients heterozygous for germline CBL loss-of-function (LOF) variants can develop myeloid malignancy, autoinflammation, or both, if some or all of their leukocytes become homozygous for these variants through somatic loss of heterozygosity (LOH) via uniparental isodisomy. We observed an upregulation of the inflammatory gene expression signature in whole blood from these patients, mimicking monogenic inborn errors underlying autoinflammation. Remarkably, these patients had constitutively activated monocytes that secreted 10 to 100 times more inflammatory cytokines than those of healthy individuals and CBL LOF heterozygotes without LOH. CBL-LOH hematopoietic stem and progenitor cells (HSPCs) outgrew the other cells, accounting for the persistence of peripheral monocytes homozygous for the CBL LOF variant. ERK pathway activation was required for the excessive production of cytokines by both resting and stimulated CBL-LOF monocytes, as shown in monocytic cell lines. Finally, we found that about 1 in 10,000 individuals in the UK Biobank were heterozygous for CBL LOF variants and that these carriers were at high risk of hematological and inflammatory conditions.

Infants with biallelic IL7R loss-of-function variants have severe combined immune deficiency (SCID) characterized by the absence of autologous T lymphocytes, but normal counts of circulating B and NK cells (T-B+NK+ SCID). We report 6 adults (aged 22 to 59 years) from 4 kindreds and 3 ancestries (Colombian, Israeli Arab, Japanese) carrying homozygous IL7 loss-of-function variants resulting in combined immunodeficiency (CID). Deep immunophenotyping revealed relatively normal counts and/or proportions of myeloid, B, NK, and innate lymphoid cells. By contrast, the patients had profound T cell lymphopenia, with low proportions of innate-like adaptive mucosal-associated invariant T and invariant NK T cells. They also had low blood counts of T cell receptor (TCR) excision circles, recent thymic emigrant T cells and naive CD4+ T cells, and low overall TCR repertoire diversity, collectively indicating impaired thymic output. The proportions of effector memory CD4+ and CD8+ T cells were high, indicating IL-7-independent homeostatic T cell proliferation in the periphery. Intriguingly, the proportions of other T cell subsets, including TCRγδ+ T cells and some TCRαβ+ T cell subsets (including Th1, Tfh, and Treg) were little affected. Peripheral CD4+ T cells displayed poor proliferation, but normal cytokine production upon stimulation with mitogens in vitro. Thus, inherited IL-7 deficiency impairs T cell development less severely and in a more subset-specific manner than IL-7R deficiency. These findings suggest that another IL-7R-binding cytokine, possibly thymic stromal lymphopoietin, governs an IL-7-independent pathway of human T cell development.

Human inborn errors of the type I IFN response pathway and auto-Abs neutralizing IFN-α, -β, and/or -ω can underlie severe viral illnesses. We report a simple assay for the detection of both types of condition. We stimulate whole blood from healthy individuals and patients with either inborn errors of type I IFN immunity or auto-Abs against type I IFNs with glycosylated human IFN-α2, -β, or -ω. As controls, we add a monoclonal antibody (mAb) blocking the type I IFN receptors and stimulated blood with IFN-γ (type II IFN). Of the molecules we test, IP-10 (encoded by the interferon-stimulated gene (ISG) CXCL10 ) is the molecule most strongly induced by type I and type II IFNs in the whole blood of healthy donors in an ELISA-like assay. In patients with inherited IFNAR1, IFNAR2, TYK2, or IRF9 deficiency, IP-10 is induced only by IFN-γ, whereas, in those with auto-Abs neutralizing specific type I IFNs, IP-10 is also induced by the type I IFNs not neutralized by the auto-Abs. The measurement of type I and type II IFN-dependent IP-10 induction therefore constitutes a simple procedure for detecting rare inborn errors of the type I IFN response pathway and more common auto-Abs neutralizing type I IFNs., Competing Interests: Competing interests statement:J.L.-C. is an inventor on patent application PCT/US2021/042741, filed July 22, 2021, submitted by The Rockefeller University and covering the diagnosis of susceptibility to, and the treatment of, viral disease, and viral vaccines, including COVID-19 and vaccine-associated diseases. Reviewer S.R.O. is a co-author of two recent papers with J.L.C. Reviewer P.H. has a collaboration with the Casanova group in the exchange of reagents, but it does not cover the topic of this collaboration.

Severe defects in human IFNγ immunity predispose individuals to both Bacillus Calmette-Guérin disease and tuberculosis, whereas milder defects predispose only to tuberculosis 1 . Here we report two adults with recurrent pulmonary tuberculosis who are homozygous for a private loss-of-function TNF variant. Neither has any other clinical phenotype and both mount normal clinical and biological inflammatory responses. Their leukocytes, including monocytes and monocyte-derived macrophages (MDMs) do not produce TNF, even after stimulation with IFNγ. Blood leukocyte subset development is normal in these patients. However, an impairment in the respiratory burst was observed in granulocyte-macrophage colony-stimulating factor (GM-CSF)-matured MDMs and alveolar macrophage-like (AML) cells 2 from both patients with TNF deficiency, TNF- or TNFR1-deficient induced pluripotent stem (iPS)-cell-derived GM-CSF-matured macrophages, and healthy control MDMs and AML cells differentiated with TNF blockers in vitro, and in lung macrophages treated with TNF blockers ex vivo. The stimulation of TNF-deficient iPS-cell-derived macrophages with TNF rescued the respiratory burst. These findings contrast with those for patients with inherited complete deficiency of the respiratory burst across all phagocytes, who are prone to multiple infections, including both Bacillus Calmette-Guérin disease and tuberculosis 3 . Human TNF is required for respiratory-burst-dependent immunity to Mycobacterium tuberculosis in macrophages but is surprisingly redundant otherwise, including for inflammation and immunity to weakly virulent mycobacteria and many other infectious agents., (© 2024. The Author(s).)

UNC93B1 is a transmembrane domain protein mediating the signaling of endosomal Toll-like receptors (TLRs). We report five families harboring rare missense substitutions (I317M, G325C, L330R, R466S, and R525P) in UNC93B1 causing systemic lupus erythematosus (SLE) or chilblain lupus (CBL) as either autosomal dominant or autosomal recessive traits. As for a D34A mutation causing murine lupus, we recorded a gain of TLR7 and, to a lesser extent, TLR8 activity with the I317M (in vitro) and G325C (in vitro and ex vivo) variants in the context of SLE. Contrastingly, in three families segregating CBL, the L330R, R466S, and R525P variants were isomorphic with respect to TLR7 activity in vitro and, for R525P, ex vivo. Rather, these variants demonstrated a gain of TLR8 activity. We observed enhanced interaction of the G325C, L330R, and R466S variants with TLR8, but not the R525P substitution, indicating different disease mechanisms. Overall, these observations suggest that UNC93B1 mutations cause monogenic SLE or CBL due to differentially enhanced TLR7 and TLR8 signaling., (© 2024 David et al.)

BACKGROUNDWeakly virulent environmental mycobacteria (EM) can cause severe disease in HLA-DRB1*15:02 or 16:02 adults harboring neutralizing anti-IFN-γ autoantibodies (nAIGAs). The overall prevalence of nAIGAs in the general population is unknown, as are the penetrance of nAIGAs in HLA-DRB1*15:02 or 16:02 individuals and the proportion of patients with unexplained, adult-onset EM infections carrying nAIGAs.METHODSThis study analyzed the detection and neutralization of anti-IFN-γ autoantibodies (auto-Abs) from 8,430 healthy individuals of the general population, 257 HLA-DRB1*15:02 or 16:02 carriers, 1,063 patients with autoimmune disease, and 497 patients with unexplained severe disease due to EM.RESULTSWe found that anti-IFN-γ auto-Abs detected in 4,148 of 8,430 healthy individuals (49.2%) from the general population of an unknown HLA-DRB1 genotype were not neutralizing. Moreover, we did not find nAIGAs in 257 individuals carrying HLA-DRB1* 15:02 or 16:02. Additionally, nAIGAs were absent in 1,063 patients with an autoimmune disease. Finally, 7 of 497 patients (1.4%) with unexplained severe disease due to EM harbored nAIGAs.CONCLUSIONThese findings suggest that nAIGAs are isolated and that their penetrance in HLA-DRB1*15:02 or 16:02 individuals is low, implying that they may be triggered by rare germline or somatic variants. In contrast, the risk of mycobacterial disease in patients with nAIGAs is high, confirming that these nAIGAs are the cause of EM disease.FUNDINGThe Laboratory of Human Genetics of Infectious Diseases is supported by the Howard Hughes Medical Institute, the Rockefeller University, the St. Giles Foundation, the National Institutes of Health (NIH) (R01AI095983 and U19AIN1625568), the National Center for Advancing Translational Sciences (NCATS), the NIH Clinical and Translational Science Award (CTSA) program (UL1 TR001866), the French National Research Agency (ANR) under the "Investments for the Future" program (ANR-10-IAHU-01), the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID), ANR-GENMSMD (ANR-16-CE17-0005-01), ANR-MAFMACRO (ANR-22-CE92-0008), ANRSECTZ170784, the French Foundation for Medical Research (FRM) (EQU201903007798), the ANRS-COV05, ANR GENVIR (ANR-20-CE93-003), and ANR AI2D (ANR-22-CE15-0046) projects, the ANR-RHU program (ANR-21-RHUS-08-COVIFERON), the European Union's Horizon 2020 research and innovation program under grant agreement no. 824110 (EASI-genomics), the Square Foundation, Grandir - Fonds de solidarité pour l'enfance, the Fondation du Souffle, the SCOR Corporate Foundation for Science, the Battersea & Bowery Advisory Group, William E. Ford, General Atlantic's Chairman and Chief Executive Officer, Gabriel Caillaux, General Atlantic's Co-President, Managing Director, and Head of business in EMEA, and the General Atlantic Foundation, Institut National de la Santé et de la Recherche Médicale (INSERM) and of Paris Cité University. JR was supported by the INSERM PhD program for doctors of pharmacy (poste d'accueil INSERM). JR and TLV were supported by the Bettencourt-Schueller Foundation and the MD-PhD program of the Imagine Institute. MO was supported by the David Rockefeller Graduate Program, the Funai Foundation for Information Technology (FFIT), the Honjo International Scholarship Foundation (HISF), and the New York Hideyo Noguchi Memorial Society (HNMS).

Magnesium alloys play an essential role in metallic lightweight construction for modern mobility applications due to their low density, excellent specific strength, and very good castability. For some years now, degradable implants have also been made from magnesium alloys, which, thanks to this special functionality, save patients a second surgery for explantation. New additive manufacturing processes, which are divided into powder-based and wire-based processes depending on the feedstock used, can be utilized for these applications. Therefore, magnesium alloys should also be used here, but this is hardly ever implemented, and few literature reports exist on this subject. This is attributable to the high affinity of magnesium to oxygen, which makes the use of powders difficult. Therefore, magnesium wires are likely to be used. In this paper, a magnesium-based nanocomposite wire is made from an AM60 (Mg-6Al-0.4Mn) (reinforced with 1 wt% AlN nanoparticles and containing calcium to reduce flammability), using a high-shear process and then extruded into wires. These wires are then used as feedstock to build up samples by wire-arc directed energy deposition, and their mechanical properties and microstructure are examined. Our results show that although the ductility is reduced by adding calcium and nanoparticles, the yield strength in the welding direction and perpendicular to it is increased to 131 MPa.

Ocular graft-versus-host disease (oGVHD) affects ~50% of post-stem cell transplant patients and is the only form of GVHD diagnosed without a biopsy. As it must be distinguished from other dry eye diseases, there is a need to identify oGVHD biomarkers to improve diagnosis and treatment. We conducted a systematic review of 19 scholarly articles published from 2018 to 2023 including articles focused on adult patients diagnosed with oGVHD following allogeneic hematopoietic stem cell transplant and used biomarkers as the outcome measure. Articles that were not original investigations or were not published in English were excluded. These clinical investigations explored different molecular oGVHD biomarkers and were identified on 3 October 2023 from the Scopus, PubMed, and Embase databases by using search terms including ocular graft-versus-host disease, biomarkers, cytokines, proteomics, genomics, immune response, imaging techniques, and dry-eye-related key terms. The Newcastle-Ottawa scale for case-control studies was used to assess bias. From the 19 articles included, cytokine, proteomic, lipid, and leukocyte profiles were studied in tear film, as well as ocular surface microbiota and fluorescein staining. Our findings suggest that cytokine profiling is the most studied oGVHD biomarker. Additionally, variations correlating these biomarkers with disease state may lead to a more targeted diagnosis and therapeutic approach. Limitations include language bias, publication bias, and sampling bias, as well as a lack of appropriate controls for included studies.

Human inherited disorders of interferon-gamma (IFN-γ) immunity underlie severe mycobacterial diseases. We report X-linked recessive MCTS1 deficiency in men with mycobacterial disease from kindreds of different ancestries (from China, Finland, Iran, and Saudi Arabia). Complete deficiency of this translation re-initiation factor impairs the translation of a subset of proteins, including the kinase JAK2 in all cell types tested, including T lymphocytes and phagocytes. JAK2 expression is sufficiently low to impair cellular responses to interleukin-23 (IL-23) and partially IL-12, but not other JAK2-dependent cytokines. Defective responses to IL-23 preferentially impair the production of IFN-γ by innate-like adaptive mucosal-associated invariant T cells (MAIT) and γδ T lymphocytes upon mycobacterial challenge. Surprisingly, the lack of MCTS1-dependent translation re-initiation and ribosome recycling seems to be otherwise physiologically redundant in these patients. These findings suggest that X-linked recessive human MCTS1 deficiency underlies isolated mycobacterial disease by impairing JAK2 translation in innate-like adaptive T lymphocytes, thereby impairing the IL-23-dependent induction of IFN-γ., Competing Interests: Declaration of interests J.-L.C. serves on the scientific advisory boards of ADMA Biologics Inc., Kymera Therapeutics, and Elixiron Immunotherapeutics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Asexual reproduction can be triggered by interspecific hybridization, but its emergence is supposedly rare, relying on exceptional combinations of suitable genomes. To examine how genomic and karyotype divergence between parental lineages affect the incidence of asexual gametogenesis, we experimentally hybridized fishes (Cobitidae) across a broad phylogenetic spectrum, assessed by whole exome data. Gametogenic pathways generally followed a continuum from sexual reproduction in hybrids between closely related evolutionary lineages to sterile or inviable crosses between distant lineages. However, most crosses resulted in a combination of sterile males and asexually reproducing females. Their gametes usually experienced problems in chromosome pairing, but females also produced a certain proportion of oocytes with premeiotically duplicated genomes, enabling their development into clonal eggs. Interspecific hybridization may thus commonly affect cell cycles in a specific way, allowing the formation of unreduced oocytes. The emergence of asexual gametogenesis appears tightly linked to hybrid sterility and constitutes an inherent part of the extended speciation continuum., Competing Interests: AM, TT, OB, JK, MS, VB, JB, KH, LC, MS, DD, KJ No competing interests declared, (© 2023, Marta et al.)

Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency produce autoantibodies that neutralize type I interferons (IFNs) 1,2 , conferring a predisposition to life-threatening COVID-19 pneumonia 3 . Here we report that patients with autosomal recessive NIK or RELB deficiency, or a specific type of autosomal-dominant NF-κB2 deficiency, also have neutralizing autoantibodies against type I IFNs and are at higher risk of getting life-threatening COVID-19 pneumonia. In patients with autosomal-dominant NF-κB2 deficiency, these autoantibodies are found only in individuals who are heterozygous for variants associated with both transcription (p52 activity) loss of function (LOF) due to impaired p100 processing to generate p52, and regulatory (IκBδ activity) gain of function (GOF) due to the accumulation of unprocessed p100, therefore increasing the inhibitory activity of IκBδ (hereafter, p52 LOF /IκBδ GOF ). By contrast, neutralizing autoantibodies against type I IFNs are not found in individuals who are heterozygous for NFKB2 variants causing haploinsufficiency of p100 and p52 (hereafter, p52 LOF /IκBδ LOF ) or gain-of-function of p52 (hereafter, p52 GOF /IκBδ LOF ). In contrast to patients with APS-1, patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies. However, their thymuses have an abnormal structure, with few AIRE-expressing medullary thymic epithelial cells. Human inborn errors of the alternative NF-κB pathway impair the development of AIRE-expressing medullary thymic epithelial cells, thereby underlying the production of autoantibodies against type I IFNs and predisposition to viral diseases., (© 2023. The Author(s).)

Single-molecule experiments have changed the way we explore the physical world, yet data analysis remains time-consuming and prone to human bias. Here, we introduce Deep-LASI (Deep-Learning Assisted Single-molecule Imaging analysis), a software suite powered by deep neural networks to rapidly analyze single-, two- and three-color single-molecule data, especially from single-molecule Förster Resonance Energy Transfer (smFRET) experiments. Deep-LASI automatically sorts recorded traces, determines FRET correction factors and classifies the state transitions of dynamic traces all in ~20-100 ms per trajectory. We benchmarked Deep-LASI using ground truth simulations as well as experimental data analyzed manually by an expert user and compared the results with a conventional Hidden Markov Model analysis. We illustrate the capabilities of the technique using a highly tunable L-shaped DNA origami structure and use Deep-LASI to perform titrations, analyze protein conformational dynamics and demonstrate its versatility for analyzing both total internal reflection fluorescence microscopy and confocal smFRET data., (© 2023. Springer Nature Limited.)

To approach the taxonomy of large and complex animal groups it is of advantage to focus on species groups with shared derived character state. We investigate the composition, morphological characteristics and relationships of and within the Schistura cincticauda species group, whose members are small freshwater fishes that inhabit streams and rivers in eastern Myanmar and western and southern Thailand. A phylogenetic analysis using molecular genetic markers demonstrated the monophyly of this group; a combined genetic and morphological analysis revealed the inclusion of at least twelve species. They share the presence of a pair of black marks on the lower lip, one on each side of the median interruption (these marks may be reduced to few melanophores or even missing in some individuals). Additionally, all species share a small body size (max. 60 mm SL), an incomplete lateral line reaching at most to vertical through anal-fin base, and the absence of sexual dimorphism. Each of the 12 species is diagnosed by a unique combination of character states in fin ray numbers, anus position, presence/absence of an axillary pelvic lobe, and colour pattern. The distribution areas of several species overlap and five cases of syntopic occurrence are known. Five unnamed species are described herein., (© 2023. Springer Nature Limited.)

As whole-genome sequencing has become pervasive, some have suggested that reduced genomic representation approaches, for example, sequence capture, are becoming obsolete. In the present study, we argue that these techniques still provide excellent tools in terms of price and quality of data as well as in their ability to provide markers with specific features, as required, for example, in phylogenomics. A potential drawback of the wide-scale application of reduced representation approaches could be their drop in efficiency with increasing phylogenetic distance from the reference species. While some studies have focused on the degree and performance of reduced representation techniques in such situations, to our knowledge, none of them evaluated their applicability to inter-specific hybrids and polyploids. This highlights a significant gap in current knowledge since there is increasing evidence for the frequent occurrence of natural hybrids and polyploids, as well as for the major importance of both phenomena in evolution. The main aim of the present study was to carry out a thorough validation of SEQcap applicability to (1) a set of non-model taxa with a wide range of phylogenetic relatedness and (2) inter-specific hybrids of various ploidies and genomic compositions. Considering the latter point, we especially focused on mechanisms causing allelic bias and consequent allelic dropout, as these could have confounding effects with respect to the evolutionary genomic dynamics of hybrids, especially in asexuals, which virtually reproduce as a frozen F1 generation., (© 2023 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

The karyotype of the freshwater fish Sabanejewiabulgarica (Drensky, 1928), from the Danube Delta, was studied by conventional Giemsa staining and the C-banding technique. The diploid chromosome number was 2n = 50. The karyotype contained 2 pairs of metacentric (the first one was much larger than the second one), 6 pairs of submetacentric and 17 pairs of subtelocentric to acrocentric chromosomes. Pericentromeric blocks of heterochromatin were revealed in most of the chromosome pairs. The karyotype phenotype of S.bulgarica was the same as found for S.balcanica from Northern Carpathian Mountains., (Eva Hnátková, Zuzana Majtánová, Vendula Bohlen Šlechtová, Joerg Bohlen, Petr Ráb.)

The processability during massive deformation of magnesium-wrought products is hampered by the low formability of magnesium alloys. The research results of recent years demonstrate that rare earth elements as alloying elements improve the properties of magnesium sheets, such as formability, strength and corrosion resistance. The substitution of rare earth elements by Ca in Mg-Zn-based alloys results in a similar texture evolution and mechanical behaviour as RE-containing alloys. This work is an approach to understanding the influence of Mn as an alloying element to increase the strength of a Mg-Zn-Ca alloy. For this aim, a Mg-Zn-Mn-Ca alloy is used to investigate how Mn affects the process parameters during rolling and the subsequent heat treatment. The microstructure, texture and mechanical properties of rolled sheets and heat treatment at different temperatures are compared. The outcome of casting and the thermo-mechanical treatment are used to discuss how to adapt the mechanical properties of magnesium alloy ZMX210. The alloy ZMX210 behaves very similarly to the ternary Mg-Zn-Ca alloys. The influence of the process parameter rolling temperature on the properties of the ZMX210 sheets was investigated. The rolling experiments show that the ZMX210 alloy has a relatively narrow process window.

Roughly half of animal mRNAs contain upstream open reading frames (uORFs). These uORFs can represent an impediment to translation of the main ORF since ribosomes usually bind the mRNA cap at the 5' end and then scan for ORFs in a 5'-to-3' fashion. One way for ribosomes to bypass uORFs is via leaky scanning, whereby the ribosome disregards the uORF start codon. Hence leaky scanning is an important instance of post-transcriptional regulation that affects gene expression. Few molecular factors regulating or facilitating this process are known. Here we show that the PRRC2 proteins PRRC2A, PRRC2B and PRRC2C impact translation initiation. We find that they bind eukaryotic translation initiation factors and preinitiation complexes, and are enriched on ribosomes translating mRNAs with uORFs. We find that PRRC2 proteins promote leaky scanning past translation start codons, thereby promoting translation of mRNAs containing uORFs. Since PRRC2 proteins have been associated with cancer, this provides a mechanistic starting point for understanding their physiological and pathophysiological roles., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

3D super-resolution microscopy with nanometric resolution is a key to fully complement ultrastructural techniques with fluorescence imaging. Here, we achieve 3D super-resolution by combining the 2D localization of pMINFLUX with the axial information of graphene energy transfer (GET) and the single-molecule switching by DNA-PAINT. We demonstrate <2 nm localization precision in all 3 dimension with axial precision reaching below 0.3 nm. In 3D DNA-PAINT measurements, structural features, i.e., individual docking strands at distances of 3 nm, are directly resolved on DNA origami structures. pMINFLUX and GET represent a particular synergetic combination for super-resolution imaging near the surface such as for cell adhesion and membrane complexes as the information of each photon is used for both 2D and axial localization information. Furthermore, we introduce local PAINT (L-PAINT), in which DNA-PAINT imager strands are equipped with an additional binding sequence for local upconcentration improving signal-to-background ratio and imaging speed of local clusters. L-PAINT is demonstrated by imaging a triangular structure with 6 nm side lengths within seconds., (© 2023. The Author(s).)

Patients with autosomal recessive (AR) IL-12p40 or IL-12Rβ1 deficiency display Mendelian susceptibility to mycobacterial disease (MSMD) due to impaired IFN-γ production and, less commonly, chronic mucocutaneous candidiasis (CMC) due to impaired IL-17A/F production. We report six patients from four kindreds with AR IL-23R deficiency. These patients are homozygous for one of four different loss-of-function IL23R variants. All six patients have a history of MSMD, but only two suffered from CMC. We show that IL-23 induces IL-17A only in MAIT cells, possibly contributing to the incomplete penetrance of CMC in patients unresponsive to IL-23. By contrast, IL-23 is required for both baseline and Mycobacterium -inducible IFN-γ immunity in both Vδ2 + γδ T and MAIT cells, probably contributing to the higher penetrance of MSMD in these patients. Human IL-23 appears to contribute to IL-17A/F-dependent immunity to Candida in a single lymphocyte subset but is required for IFN-γ-dependent immunity to Mycobacterium in at least two lymphocyte subsets.

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/β-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/β immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/β. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/β-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/β-dependent antiviral immunity., Competing Interests: Declaration of interests J.-L.C. serves on the scientific advisory boards of ADMA Biologics Inc., Kymera Therapeutics, and Elixiron Immunotherapeutics., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)

Fluorescence correlation spectroscopy is a versatile tool for studying fast conformational changes of biomolecules especially when combined with Förster resonance energy transfer (FRET). Despite the many methods available for identifying structural dynamics in FRET experiments, the determination of the forward and backward transition rate constants and thereby also the equilibrium constant is difficult when two intensity levels are involved. Here, we combine intensity correlation analysis with fluorescence lifetime information by including only a subset of photons in the autocorrelation analysis based on their arrival time with respect to the excitation pulse (microtime). By fitting the correlation amplitude as a function of microtime gate, the transition rate constants from two fluorescence-intensity level systems and the corresponding equilibrium constants are obtained. This shrinking-gate fluorescence correlation spectroscopy (sg-FCS) approach is demonstrated using simulations and with a DNA origami-based model system in experiments on immobilized and freely diffusing molecules. We further show that sg-FCS can distinguish photophysics from dynamic intensity changes even if a dark quencher, in this case graphene, is involved. Finally, we unravel the mechanism of a FRET-based membrane charge sensor indicating the broad potential of the method. With sg-FCS, we present an algorithm that does not require prior knowledge and is therefore easily implemented when an autocorrelation analysis is carried out on time-correlated single-photon data.

Ancient genomics can directly detect human genetic adaptation to environmental cues. However, it remains unclear how pathogens have exerted selective pressures on human genome diversity across different epochs and affected present-day inflammatory disease risk. Here, we use an ancestry-aware approximate Bayesian computation framework to estimate the nature, strength, and time of onset of selection acting on 2,879 ancient and modern European genomes from the last 10,000 years. We found that the bulk of genetic adaptation occurred after the start of the Bronze Age, <4,500 years ago, and was enriched in genes relating to host-pathogen interactions. Furthermore, we detected directional selection acting on specific leukocytic lineages and experimentally demonstrated that the strongest negatively selected candidate variant in immunity genes, lipopolysaccharide-binding protein ( LBP ) D283G, is hypomorphic. Finally, our analyses suggest that the risk of inflammatory disorders has increased in post-Neolithic Europeans, possibly because of antagonistic pleiotropy following genetic adaptation to pathogens., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Inborn errors of IFN-γ immunity can underlie tuberculosis (TB). We report three patients from two kindreds without EBV viremia or disease but with severe TB and inherited complete ITK deficiency, a condition associated with severe EBV disease that renders immunological studies challenging. They have CD4+ αβ T lymphocytopenia with a concomitant expansion of CD4-CD8- double-negative (DN) αβ and Vδ2- γδ T lymphocytes, both displaying a unique CD38+CD45RA+T-bet+EOMES- phenotype. Itk-deficient mice recapitulated an expansion of the γδ T and DN αβ T lymphocyte populations in the thymus and spleen, respectively. Moreover, the patients' T lymphocytes secrete small amounts of IFN-γ in response to TCR crosslinking, mitogens, or forced synapse formation with autologous B lymphocytes. Finally, the patients' total lymphocytes secrete small amounts of IFN-γ, and CD4+, CD8+, DN αβ T, Vδ2+ γδ T, and MAIT cells display impaired IFN-γ production in response to BCG. Inherited ITK deficiency undermines the development and function of various IFN-γ-producing T cell subsets, thereby underlying TB., (© 2022 Ogishi et al.)

Homomorphic sex chromosomes and their turnover are common in teleosts. We investigated the evolution of nascent sex chromosomes in several populations of two sister species of African annual killifishes, Nothobranchius furzeri and N. kadleci, focusing on their under-studied repetitive landscape. We combined bioinformatic analyses of the repeatome with molecular cytogenetic techniques, including comparative genomic hybridization, fluorescence in situ hybridization with satellite sequences, ribosomal RNA genes (rDNA) and bacterial artificial chromosomes (BACs), and immunostaining of SYCP3 and MLH1 proteins to mark lateral elements of synaptonemal complexes and recombination sites, respectively. Both species share the same heteromorphic XY sex chromosome system, which thus evolved prior to their divergence. This was corroborated by sequence analysis of a putative master sex determining (MSD) gene gdf6Y in both species. Based on their divergence, differentiation of the XY sex chromosome pair started approximately 2 million years ago. In all populations, the gdf6Y gene mapped within a region rich in satellite DNA on the Y chromosome long arms. Despite their heteromorphism, X and Y chromosomes mostly pair regularly in meiosis, implying synaptic adjustment. In N. kadleci, Y-linked paracentric inversions like those previously reported in N. furzeri were detected. An inversion involving the MSD gene may suppress occasional recombination in the region, which we otherwise evidenced in the N. furzeri population MZCS-121 of the Limpopo clade lacking this inversion. Y chromosome centromeric repeats were reduced compared with the X chromosome and autosomes, which points to a role of relaxed meiotic drive in shaping the Y chromosome repeat landscape. We speculate that the recombination rate between sex chromosomes was reduced due to heterochiasmy. The observed differences between the repeat accumulations on the X and Y chromosomes probably result from high repeat turnover and may not relate closely to the divergence inferred from earlier SNP analyses., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5' UTR structures and upstream near-cognate start codons., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In the field of magnesium-based degradable implantable devices, the Mg-Y-RE-Zr alloying system (WE-type) has gained popularity due to its satisfying degradation rate together with mechanical strength. However, utilization of RE and Zr in the WE-type alloys was originally driven to improve Mg-based alloys for high-temperature applications in the industry, while for medical purposes, there is a question of whether the amount of alloying elements may be further optimized. For this reason, our paper presents the Mg-3Y (W3) magnesium alloy as an alternative to the WE43 alloy. This study shows that the omission of RE and Zr elements did not compromise the corrosion resistance and the degradation rate of the W3 alloy when compared with the WE43 alloy; appropriate biocompatibility was preserved as well. It was shown that the decrease in the mechanical strength caused by the omission of RE and Zr from the WE43 alloy could be compensated for by severe plastic deformation, as achieved in this study, by equal channel angular pressing. Ultrafine-grained W3 alloy exhibited compression yield strength of 362 ± 6 MPa and plastic deformation at maximum stress of 18 ± 1%. Overall, the early results of this study put forward the motion of avoiding RE elements and Zr in magnesium alloy as a suitable material for biodegradable applications and showed that solo alloying of yttrium is sufficient for maintaining desirable properties of the material at once.

Human cells homozygous for rare loss-of-expression (LOE) TYK2 alleles have impaired, but not abolished, cellular responses to IFN-α/β (underlying viral diseases in the patients) and to IL-12 and IL-23 (underlying mycobacterial diseases). Cells homozygous for the common P1104A TYK2 allele have selectively impaired responses to IL-23 (underlying isolated mycobacterial disease). We report three new forms of TYK2 deficiency in six patients from five families homozygous for rare TYK2 alleles (R864C, G996R, G634E, or G1010D) or compound heterozygous for P1104A and a rare allele (A928V). All these missense alleles encode detectable proteins. The R864C and G1010D alleles are hypomorphic and loss-of-function (LOF), respectively, across signaling pathways. By contrast, hypomorphic G996R, G634E, and A928V mutations selectively impair responses to IL-23, like P1104A. Impairment of the IL-23-dependent induction of IFN-γ is the only mechanism of mycobacterial disease common to patients with complete TYK2 deficiency with or without TYK2 expression, partial TYK2 deficiency across signaling pathways, or rare or common partial TYK2 deficiency specific for IL-23 signaling., (© 2022 Ogishi et al.)

Multiple aspects of mRNA translation are subject to regulation. Here we present a ribosome footprinting protocol to determine the location and composition of 40S and 80S ribosome complexes on endogenous mRNAs transcriptome-wide in vivo in yeast and mammalian cells. We present an extension of the translation complex profiling (TCP-seq) protocol, originally developed in yeast, by including an immunoprecipitation step to assay the location of both 40S and 80S ribosome complexes containing proteins of interest. This yields information on where along mRNAs the ribosome-bound protein of interest joins the ribosome to act, and where it leaves again, thereby monitoring the sequential steps of translation and the roles of various translation factors therein. Rapid fixation of live cells ensures the integrity of all translation complexes bound to mRNA at native positions. Two procedures are described, differing mainly in the fixation conditions and the library preparation. Depending on the research question, either procedure offers advantages. Execution of a Sel-TCP-seq experiment takes 5-10 working days, and initial data analysis can be completed within 2 days., (© 2022. Springer Nature Limited.)

Metal matrix nanocomposites are attracting attention because of their great potential for improved mechanical properties and possible functionalization. These hybrid materials are often produced by casting processes, but they can also develop their property profile after hot working, e.g., by forging or extrusion. In this study, a commercial cast magnesium alloy AM60 was enriched with 1 wt.% AlN nanoparticles and extruded into round bars with varied extrusion rates. The same process was carried out with unreinforced AM60 in order to determine the influences of the AlN nanoparticles in direct comparison. The influence of extrusion speed on the recrystallization behavior as well the effect of nanoparticles on the microstructure evolution and the particle-related strengthening are discussed and assessed with respect to the resulting mechanical performance.

Recessive or dominant inborn errors of type I interferon (IFN) immunity can underlie critical COVID-19 pneumonia in unvaccinated adults. The risk of COVID-19 pneumonia in unvaccinated children, which is much lower than in unvaccinated adults, remains unexplained. In an international cohort of 112 children (<16 yr old) hospitalized for COVID-19 pneumonia, we report 12 children (10.7%) aged 1.5-13 yr with critical (7 children), severe (3), and moderate (2) pneumonia and 4 of the 15 known clinically recessive and biochemically complete inborn errors of type I IFN immunity: X-linked recessive TLR7 deficiency (7 children) and autosomal recessive IFNAR1 (1), STAT2 (1), or TYK2 (3) deficiencies. Fibroblasts deficient for IFNAR1, STAT2, or TYK2 are highly vulnerable to SARS-CoV-2. These 15 deficiencies were not found in 1,224 children and adults with benign SARS-CoV-2 infection without pneumonia (P = 1.2 × 10-11) and with overlapping age, sex, consanguinity, and ethnicity characteristics. Recessive complete deficiencies of type I IFN immunity may underlie ∼10% of hospitalizations for COVID-19 pneumonia in children., (© 2022 ZHANG et al.)

This paper reports on the dilemma of the strength and forming behavior of magnesium alloy sheets due to hot rolling and precipitation aging as an obstacle for property adjustment. The effect of the Zn content on the age-hardenability and formability of Mg-Zn-Al-Ca-Mn sheets was investigated. Sheets of two alloys with 2 or 4 wt.% Zn, respectively, were produced by casting and subsequent hot rolling and their microstructure development, precipitation behavior and formability were examined. With higher Zn content the age-hardenability was increased, but at the same time the formability of the sheet decreased, concurrent to the basal-type texture development during rolling. On the other hand, the sheet containing a lower amount of Zn exhibited a weak rolling texture and rather high formability but low age-hardenability. The addition of a larger amount of Zn improved the age-hardenability through the formation of β1' and β2' phases. The basal texture was exhibited due to the consumption of solute Ca due to the formation of the Ca 2 Mg 6 Zn 3 phase. This study suggests that this contradictory exhibition of the age-hardenability and formability of Ca-containing and Zn-based alloy sheets requires a strategical approach in alloy and process design, which allows tailoring the alloying elements and processing for the respective purpose.

Background: Autosomal recessive (AR) complete IRF8 deficiency is a rare severe inborn error of immunity underlying an absence of blood myeloid mononuclear cells, intracerebral calcifications, and multiple infections. Only three unrelated patients have been reported., Materials and Methods: We studied an Argentinian child with multiple infectious diseases and severe pulmonary alveolar proteinosis (PAP). We performed whole-exome sequencing (WES) and characterized his condition by genetic, immunological, and clinical means., Results: The patient was born and lived in Argentina. He had a history of viral pulmonary diseases, disseminated disease due to bacillus Calmette-Guérin (BCG), PAP, and cerebral calcifications. He died at the age of 10 months from refractory PAP. WES identified two compound heterozygous variants in IRF8: c.55del and p.R111*. In an overexpression system, the p.R111* cDNA was loss-of-expression, whereas the c.55del cDNA yielded a protein with a slightly lower molecular weight than the wild-type protein. The mutagenesis of methionine residues downstream from c.55del revealed a re-initiation of translation. However, both variants were loss-of-function in a luciferase assay, suggesting that the patient had AR complete IRF8 deficiency. The patient had no blood monocytes or dendritic cells, associated with neutrophilia, and normal counts of NK and other lymphoid cell subsets., Conclusion: We describe the fourth patient with AR complete IRF8 deficiency. This diagnosis should be considered in children with PAP, which is probably due to the defective development or function of alveolar macrophages., (© 2022. The Author(s).)

A rectangular DNA origami structure is one of the most studied and often used motif for applications in DNA nanotechnology. Here, we present two assays to study structural changes in DNA nanostructures and reveal a reversible rolling-up of the rectangular DNA origami structure induced by bivalent cations such as magnesium or calcium. First, we applied one-color and two-color superresolution DNA-PAINT with protruding strands along the long edges of the DNA origami rectangle. At increasing salt concentration, a single line instead of two lines is observed as a first indicator of rolling-up. Two-color measurements also revealed different conformations with parallel and angled edges. Second, we placed a gold nanoparticle and a dye molecule at different positions on the DNA origami structure. Distance dependent fluorescence quenching by the nanoparticle reports on dynamic transitions as well as it provides evidence that the rolling-up occurs preferentially along the diagonal of the DNA origami rectangle. The results will be helpful to test DNA structural models and the assays presented will be useful to study further structural transitions in DNA nanotechnology.

DENR and MCTS1 have been identified as oncogenes in several different tumor entities. The heterodimeric DENR·MCTS1 protein complex promotes translation of mRNAs containing upstream Open Reading Frames (uORFs). We show here that DENR is phosphorylated on Serine 73 by Cyclin B/CDK1 and Cyclin A/CDK2 at the onset of mitosis, and then dephosphorylated as cells exit mitosis. Phosphorylation of Ser73 promotes mitotic stability of DENR protein and prevents its cleavage at Asp26. This leads to enhanced translation of mRNAs involved in mitosis. Indeed, we find that roughly 40% of all mRNAs with elevated translation in mitosis are DENR targets. In the absence of DENR or of Ser73 phosphorylation, cells display elevated levels of aberrant mitoses and cell death. This provides a mechanism how the cell cycle regulates translation of a subset of mitotically relevant mRNAs during mitosis., (© 2022. The Author(s).)

E2F transcription factors are master regulators of the eukaryotic cell cycle. In Drosophila , the sole activating E2F, E2F1, is both required for and sufficient to promote G1→S progression. E2F1 activity is regulated both by binding to RB Family repressors and by posttranscriptional control of E2F1 protein levels by the EGFR and TOR signaling pathways. Here, we investigate cis-regulatory elements in the E2f1 messenger RNA (mRNA) that enable E2f1 translation to respond to these signals and promote mitotic proliferation of wing imaginal disc and intestinal stem cells. We show that small upstream open reading frames (uORFs) in the 5' untranslated region (UTR) of the E2f1 mRNA limit its translation, impacting rates of cell proliferation. E2f1 transgenes lacking these 5'UTR uORFs caused TOR-independent expression and excess cell proliferation, suggesting that TOR activity can bypass uORF-mediated translational repression. EGFR signaling also enhanced translation but through a mechanism less dependent on 5'UTR uORFs. Further, we mapped a region in the E2f1 mRNA that contains a translational enhancer, which may also be targeted by TOR signaling. This study reveals translational control mechanisms through which growth signaling regulates cell cycle progression., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Large animal families with unresolved taxonomy are notoriously difficult to handle with respect to their biodiversity, systematics, and evolutionary history. We approach a large and taxonomically unresolved family of freshwater fishes (Nemacheilidae, >600 species) by proposing, on the basis of morphologic data, a species group within the family and study its phylogeny with conclusions regarding its diversity, taxonomy, and biogeographic history. Phylogenetic analyses of two mitochondrial and three nuclear genes of 139 specimens, representing about 46 species (17 candidate species from the proposed species-group, plus 29 comparative species), revealed that the proposed species group does not form a distinct monophyletic lineage, but that the candidate and comparative species mixed in three different lineages. However, the results revealed more than 20% of undescribed species within the ingroup and showed that species do not cluster according to the presently recognised genera. At least one of the genetic clades shows signs of an eastward range expansion during the second half of Miocene from north India via Myanmar into Laos, western China, and western Thailand. We conclude that the approach of picking monophyletic lineages to study biodiversity, systematics, and evolutionary history helps to open the door to large animal families.

Phosphorylation of Ribosomal Protein S6 (RPS6) was the first post-translational modification of the ribosome to be identified and is a commonly-used readout for mTORC1 activity. Although the cellular and organismal functions of RPS6 phosphorylation are known, the molecular consequences of RPS6 phosphorylation on translation are less well understood. Here we use selective ribosome footprinting to analyze the location of ribosomes containing phosphorylated RPS6 on endogenous mRNAs in cells. We find that RPS6 becomes progressively dephosphorylated on ribosomes as they translate an mRNA. As a consequence, average RPS6 phosphorylation is higher on mRNAs with short coding sequences (CDSs) compared to mRNAs with long CDSs. We test whether RPS6 phosphorylation differentially affects mRNA translation based on CDS length by genetic removal of RPS6 phosphorylation. We find that RPS6 phosphorylation promotes translation of mRNAs with short CDSs more strongly than mRNAs with long CDSs. Interestingly, RPS6 phosphorylation does not promote translation of mRNAs with 5' TOP motifs despite their short CDS lengths, suggesting they are translated via a different mode. In sum this provides a dynamic view of RPS6 phosphorylation on ribosomes as they translate mRNAs and the functional consequence on translation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sex chromosomes are generally derived from a pair of classical type-A chromosomes, and relatively few alternative models have been proposed up to now. 1 , 2 B chromosomes (Bs) are supernumerary and dispensable chromosomes with non-Mendelian inheritance found in many plant and animal species 3 , 4 that have often been considered as selfish genetic elements that behave as genome parasites. 5 , 6 The observation that in some species Bs can be either restricted or predominant in one sex 7-14 raised the interesting hypothesis that Bs could play a role in sex determination. 15 The characterization of putative B master sex-determining (MSD) genes, however, has not yet been provided to support this hypothesis. Here, in Astyanax mexicanus cavefish originating from Pachón cave, we show that Bs are strongly male predominant. Based on a high-quality genome assembly of a B-carrying male, we characterized the Pachón cavefish B sequence and found that it contains two duplicated loci of the putative MSD gene growth differentiation factor 6b (gdf6b). Supporting its role as an MSD gene, we found that the Pachón cavefish gdf6b gene is expressed specifically in differentiating male gonads, and that its knockout induces male-to-female sex reversal in B-carrying males. This demonstrates that gdf6b is necessary for triggering male sex determination in Pachón cavefish. Altogether these results bring multiple and independent lines of evidence supporting the conclusion that the Pachón cavefish B is a "B-sex" chromosome that contains duplicated copies of the gdf6b gene, which can promote male sex determination in this species., Competing Interests: Declaration of interests The authors declare no competing interests, (Copyright © 2021 Elsevier Inc. All rights reserved.)