The rockefeller university

We studied a family with three male individuals across two generations affected by common variable immune deficiency (CVID). We identified a novel missense heterozygous variant (c.2602T>A:p.Y868N) of NFKB2 in all patients and not in healthy relatives. Functional studies of the mutant allele in an overexpression system and of the patients' cells confirmed the deleteriousness of the NFKB2 variant and genotype, respectively, on the activation of the non-canonical NF-kappa B signaling pathway. Impaired processing of p100 into p52 underlies p100 accumulation, which results in gain-of-function (GOF) of I kappa B delta inhibitory activity and loss-of-function (LOF) of p52 transcriptional activity. The three patients' plasma contained autoantibodies that neutralized IFN-alpha 2 and/or IFN-omega, accounting for the severe or recurrent viral diseases of the patients, including influenza pneumonia in one sibling, and severe COVID-19 and recurrent herpes labialis in another. Our results confirm that NFKB2 alleles that are I kappa B delta GOF and p52 LOF can underlie CVID and drive the production of autoantibodies neutralizing type I IFNs, thereby predisposing to severe viral diseases.

The diversity of germ cell developmental strategies has been well documented across many vertebrate clades. However, much of our understanding of avian primordial germ cell (PGC) specification and differentiation has derived from only one species, the chicken ( Gallus gallus). ). Of the three major classes of birds, chickens belong to Galloanserae, representing less than 4% of species, while nearly 95% of extant bird species belong to Neoaves. This represents a significant gap in our knowledge of germ cell development across avian species, hampering efforts to adapt genome editing and reproductive technologies developed in chicken to other birds. We therefore applied single-cell RNA sequencing to investigate inter-species differences in germ cell development between chicken and zebra finch ( Tae- niopygia castanotis), ), a Neoaves songbird species and a common model of vocal learning. Analysis of early embryonic male and female gonads revealed the presence of two distinct early germ cell types in zebra finch and only one in chicken. Both germ cell types expressed zebra finch Germline Restricted Chromosome (GRC) genes, present only in songbirds among birds. One of the zebra finch germ cell types expressed the canonical PGC markers, as did chicken, but with expression differences in several signaling pathways and biological processes. The second zebra finch germ cell cluster was marked by proliferation and fate determination markers, indicating beginning of differentiation. Notably, these two zebra finch germ cell populations were present in both male and female zebra finch gonads as early as HH25. Using additional chicken developmental stages, similar germ cell heterogeneity was identified in the more developed gonads of females, but not males. Overall, our study demonstrates a substantial heterochrony in zebra finch germ cell development compared to chicken, indicating a richer diversity of avian germ cell developmental strategies than previously known.

Spinal cord injury (SCI) is a devastating condition with 250,000 to 500,000 new cases globally each year. Respiratory infections, e.g., pneumonia and influenza are the leading cause of death after SCI. Unfortunately, there is a poor understanding of how altered neuro-immune communication impacts an individual's outcome to infection. In humans and rodents, SCI leads to maladaptive changes in the spinal-sympathetic reflex (SSR) circuit which is crucial to sympathetic function. The cause of the impaired immune function may be related to harmful neuroinflammation which is detrimental to homeostatic neuronal function, aberrant plasticity, and hyperexcitable circuits. Soluble tumor necrosis factor (sTNF) is a pro-inflammatory cytokine that is elevated in the CNS after SCI and remains elevated for several months after injury. By pharmacologically attenuating sTNF in the CNS after SCI we were able to demonstrate improved immune function. Furthermore, when we investigated the specific cellular population which may be involved in altered neuro-immune communication we reported that excessive TNFR1 activity on excitatory INs promotes immune dysfunction. Furthermore, this observation is NFk beta dependent in VGluT2 + INs. Our data is the first report of a target within the CNS, TNFR1, that contributes to SCI-induced immune dysfunction after T9-SCI and is a potential avenue for future therapeutics.

Advanced genomic technologies such as whole exome or whole genome sequencing have improved diagnoses and disease outcomes for individuals with genetic diseases. Yet, variants of unknown significance (VUS) require rigorous validation to establish disease causality or modification, or to exclude them from further analysis. Here, we describe a young individual of Polynesian ancestry who in the first 13 mo of life presented with SARS-CoV-2 pneumonia, severe enterovirus meningitis and adenovirus gastroenteritis, and severe adverse reaction to MMR vaccination. Genomic analysis identified a previously reported pathogenic homozygous variant in IFNAR1 (c.1156G > T, p.Glu386* LOF), which is common in Western Polynesia. Moreover, a new and putatively deleterious canonical splice site variant in DOCK8 was also found in homozygosity (c.3234 + 2T > C). This DOCK8 variant is common in Polynesians and other under-represented ancestries in large genomic databases. Despite in silico bioinformatic predictions, extensive in vitro and ex vivo analysis revealed the DOCK8 variant likely be neutral. Thus, our study reports a novel case of IFNAR1 deficiency, but also highlights the importance of functional validation of VUS, including those predicted to be deleterious, and the pressing need to expand our knowledge of the genomic architecture and landscape of under-represented populations and ancestries.

The camelid single-domain antibody fragment, commonly referred to as a nanobody, achieves the targeting power of conventional monoclonal antibodies (mAbs) at only a fraction of their size. Isolated from camelid species (including llamas, alpacas, and camels), their small size at similar to 15 kDa, low structural complexity, and high stability compared with conventional antibodies have propelled nanobody technology into the limelight of biologic development. Nanobodies are proving themselves to be a potent complement to traditional mAb therapies, showing success in the treatment of, for example, autoimmune diseases and cancer, and more recently as therapeutic options to treat infectious diseases caused by rapidly evolving biological targets such as the SARS-CoV-2 virus. This review highlights the benefits of applying a proteomic approach to identify diverse nanobody sequences against a single antigen. This proteomic approach coupled with conventional yeast/phage display methods enables the production of highly diverse repertoires of nanobodies able to bind the vast epitope landscape of an antigen, with epitope sampling surpassing that of mAbs. Additionally, we aim to highlight recent findings illuminating the structural attributes of nanobodies that make them particularly amenable to comprehensive antigen sampling and to synergistic activity-underscoring the powerful advantage of acquiring a large, diverse nanobody repertoire against a single antigen. Lastly, we highlight the efforts being made in the clinical development of nanobodies, which have great potential as powerful diagnostic reagents and treatment options, especially when targeting infectious disease agents.

In insects, odorant receptors (ORs) are required for the detection of most olfactory cues. We investigated the function of a clade of four duplicated ORs in the hawkmoth Manduca sexta and found that these paralogs encode broadly tuned receptors with overlapping but distinct response spectra. Two paralogs, which arose after divergence from a related lineage, show high sensitivity to floral esters released by a nectar-rich plant frequently visited by M. sexta. Functional imaging in mutant moths lacking one of the paralogs suggests that olfactory sensory neurons expressing this OR target a previously identified feeding-associated glomerulus in the primary olfactory center of the brain. However, only the response of this glomerulus to the single ligand unique to the now mutated OR disappeared, suggesting neuronal coexpression of the paralogs. Our results suggest a link between the studied OR expansion and enhanced detection of odors emitted by valuable nectar sources in M. sexta.

It has been shown that excitotoxicity and tau-mediated toxicities are major contributing factors to neuronal death in Alzheimer's disease (AD). The excitatory amino acid transporter 2 (EAAT2 or GLT-1), the major glutamate transporter in the brain that regulates glutamate levels synaptically and extrasynaptically, has been shown to be deficient in AD brains, leading to excitotoxicity and subsequent cell death. Similarly, buildup of neurofibrillary tangles, which consist of hyperphosphorylated tau protein, correlates with cognitive decline and neuronal atrophy in AD. However, common genes and pathways that are critical in the aforementioned toxicities have not been well elucidated. To investigate the impact of glutamate dyshomeostasis and tau accumulation on translational profiles of affected hippocampal neurons, we used mouse models of excitotoxicity and tau-mediated toxicities (GLT-1(-/-) and P301S, respectively) in conjunction with BAC-TRAP technology. Our data show that GLT-1 deficiency in CA3 pyramidal neurons leads to translational signatures characterized by dysregulation of pathways associated with synaptic plasticity and neuronal survival, while the P301S mutation induces changes in endocytic pathways and mitochondrial dysfunction. Finally, the commonly dysregulated pathways include impaired ion homeostasis and metabolic pathways. These common pathways may shed light on potential therapeutic targets for ameliorating glutamate and tau-mediated toxicities in AD.

Folded RNAs contain tertiary contact motifs whose structures and energetics are conserved across different RNAs. The transferable properties of RNA motifs simplify the RNA folding problem, but measuring energetic and conformational properties of many motifs remains a challenge. Here, we use a high-throughput thermodynamic approach to investigate how sequence changes alter the binding properties of naturally occurring motifs, the GAAA tetraloop center dot tetraloop receptor (TLR) interactions. We measured the binding energies and conformational preferences of TLR sequences that span mutational pathways from the canonical 11ntR to two other natural TLRs, the IC3R and Vc2R. While the IC3R and Vc2R share highly similar energetic and conformational properties, the landscapes that map the sequence changes for their conversion from the 11ntR to changes in these properties differ dramatically. Differences in the energetic landscapes stem from the mutations needed to convert the 11ntR to the IC3R and Vc2R rather than a difference in the intrinsic energetic architectures of these TLRs. The conformational landscapes feature several nonnative TLR variants with conformational preferences that differ from both the initial and final TLRs; these species represent potential branching points along the multidimensional sequence space to sequences with greater fitness in other RNA contexts with alternative conformational preferences. Our high-throughput, quantitative approach reveals the complex nature of sequence-fitness landscapes and leads to models for their molecular origins. Systematic and quantitative molecular approaches provide critical insights into understanding the evolution of natural RNAs as they traverse complex landscapes in response to selective pressures.

Cell biology and genetic studies have demonstrated that DNA double-strand break (DSB) repair can be performed using an RNA transcript that spans the site of the DNA break as a template for repair. This type of DSB repair requires a reverse transcriptase to convert an RNA sequence into DNA to facilitate repair of the break, rather than copying from a DNA template as in canonical DSB repair. Translesion synthesis (TLS) DNA polymerases (Pol) are often more promiscuous than DNA Pols, raising the notion that reverse transcription could be performed by a TLS Pol. Indeed, several studies have demonstrated that human Pol h has reverse transcriptase activity, while others have suggested that the yeast TLS Polz is involved. Here, we purify all seven known nuclear DNA Pols of Saccharomyces cerevisiae and compare their reverse transcriptase activities. The comparison shows that Polz far surpasses Pol h and all other DNA Pols in reverse transcriptase activity. We fi nd that Polz reverse transcriptase activity is not affected by RPA or RFC/PCNA and acts distributively to make DNA complementary to an RNA template strand. Consistent with prior S. cerevisiae studies performed in vivo, we propose that Polz is the major DNA Pol that functions in the RNAtemplated DSB repair pathway.