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Somatic mutations in cancer genes have been detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, because mutated and wild-type cells are admixed, we have limited ability to link genotypes with phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing genotype, transcriptomes and methylomes in progenitors from individuals with DNMT3A R882 mutated clonal hematopoiesis. DNMT3A mutations result in myeloid over lymphoid bias, and an expansion of immature myeloid progenitors primed toward megakaryocytic-erythroid fate, with dysregulated expression of lineage and leukemia stem cell markers. Mutated DNMT3A leads to preferential hypomethylation of polycomb repressive complex 2 targets and a specific CpG flanking motif. Notably, the hypomethylation motif is enriched in binding motifs of key hematopoietic transcription factors, serving as a potential mechanistic link between DNMT3A mutations and aberrant transcriptional phenotypes. Thus, single-cell multi-omics paves the road to defining the downstream consequences of mutations that drive clonal mosaicism. Multi-modality single-cell sequencing determines genotype, transcriptome and methylome information in cells from individuals with DNMT3A R882 mutated clonal hematopoiesis, allowing for the comparison of mutant and wild-type cells from the same individuals.

Studies of red swamp crayfish (Procambarus clarkii) outside of the United States confirm the presence of a variety of zoonotic pathogens, but it is unknown whether these same pathogens occur in P. clarkii in the United States. The U.S. commercial crayfish industry generates $200 million yearly, underscoring the need to evaluate this consumer commodity. The study objectives were to evaluate specific zoonotic pathogens present on P. clarkii from Alabama and Louisiana, states in the southeastern United States, and to determine the effectiveness of traditional food preparation methods to reduce pathogens. Experiment A evaluated the presence of Escherichia coli, Salmonella, Staphylococcus aureus, and Vibrio spp. in crayfish and environmental samples over a 2-month collection period (May to June 2021). Crayfish sampling consisted of swabbing the cephalothorax region; 15 samples were tested for E. coli, Salmonella, and S. aureus, and an additional 15 samples for Vibrio spp. Additionally, crayfish shipping materials were sampled. In experiment B, 92 crayfish were evaluated for Paragonimus kellicotti. Experiment C compared live and boiled crayfish for the presence of Vibrio spp. In experiments A and B, all 60 (100%) crayfish samples and 13 (81.25%) of 16 environmental samples showed growth characteristic of Vibrio spp. Three (5%) of 60 samples showed E. coli growth, with no statistical difference (P = 0.5536) between farms. P. kellicotti, Salmonella, and S. aureus were not recovered from any samples. In experiment C, all 10 (100%) of the live preboiled crayfish samples showed characteristic growth, whereas 1 (10%) of 10 samples of crayfish boiled in unseasoned water showed Vibrio growth (P , 0.0001). These results confirm that Vibrio spp. and E. coli may be present on U.S. commercial crayfish and that care should be taken when handling any materials that come into contact with live crayfish because they can potentially be contaminated.

Convening a national bioethics commission has historically been one of the most powerful bioethical legacies a US presidential administration can undertake. The Biden Administration has not yet created such a commission; here we argue that centering health equity and healthcare access would provide a vital framework for a new commission's legacy. Moreover, we demonstrate two crucial historical episodes when American presidents appointed commissions to examine the practical and ethical implications of these very issues. We turn first to the 1952 President's Commission report on "Building America's Health," a lofty vision of universal healthcare access stymied by both political conflict and unaddressed problems of racism in the era's legislation. Its rich yet incomplete account of American health inequities serves as a valuable forerunner to questions of justice in bioethics. We then explore the President's Commission's 1983 report "Securing Access to Healthcare: A Report on the Ethical Implications of Differences in the Availability of Health Services." This report took up the mantle of equity in healthcare access, again with mixed results. Only by understanding the checkered history of these overlooked, practically "lost" reports can a new era in American bioethics successfully re-center the goal of equitable health for all.

Attenuation of a virulent virus is a proven approach for generating vaccines but can be unpredictable. For example, synonymous recoding of viral genomes can attenuate replication but sometimes results in pleiotropic effects that confound rational vaccine design. To enable specific, conditional attenuation of viruses, we examined target RNA features that enable zinc finger antiviral protein (ZAP) function. ZAP recognized CpG dinucleotides and targeted CpG-rich RNAs for depletion, but RNA features such as CpG numbers, spacing and surrounding nucleotide composition that enable specific modulation by ZAP were undefined. Using synonymously mutated HIV-1 genomes, we defined several sequence features that govern ZAP sensitivity and enable stable attenuation. We applied rules derived from experiments with HIV-1 to engineer a mutant enterovirus A71 genome whose attenuation was stable and strictly ZAP-dependent, both in cell culture and in mice. The conditionally attenuated enterovirus A71 mutant elicited neutralizing antibodies that were protective against wild-type enterovirus A71 infection and disease in mice. ZAP sensitivity can thus be readily applied for the rational design of conditionally attenuated viral vaccines. Rational design of live-attenuated RNA viruses with potential as vaccines is enabled by identification of sequence rules for zinc finger antiviral protein.

Protein or peptide-based subunit vaccines have generated excitement and renewed interest in combating human cancer or COVID-19 outbreak. One major concern for subunit vaccine application is the weak immune responses induced by protein or peptides. Developing novel and effective vaccine adjuvants are critical for the success of subunit vaccines. Here we explored the potential of heat-inactivated MVA (heat-iMVA) as a vaccine adjuvant. Heat-iMVA dramatically enhances T cell responses and antibodies responses, mainly toward Th1 immune responses when combined with protein or peptide-based immunogen. The adjuvant effect of Heat-iMVA is stronger than live MVA and is dependent on the cGAS/STING-mediated cytosolic DNA-sensing pathway. In a therapeutic vaccination model based on tumor neoantigen peptide vaccine, Heat-iMVA significantly extended the survival and delayed tumor growth. When combined with SARS-CoV-2 spike protein, Heat-iMVA induced more robust spike-specific antibody production and more potent neutralization antibodies. Our results support that Heat-iMVA can be developed as a safe and potent vaccine adjuvant for subunit vaccines against cancer or SARS-CoV-2.

Background The contact system is initiated by factor (F) XII activation and the assembly of high molecular weight kininogen (HK) with either FXI or prekallikrein (PK) on a negatively charged surface. Overactivation of this system contributes to thrombosis and inflammation in numerous diseases. To develop effective therapeutics for contact system disorders, a detailed understanding of this pathway is needed. Methods We performed coagulation assays in normal human plasma and various factor-deficient plasmas. To evaluate how HK-mediated PK and FXI activation contributes to coagulation, we used an anti-HK antibody to block access to domain 6 of HK, the region required for efficient activation of PK and FXI. Results FXI's binding to HK and its subsequent activation by activated FXII contributes to coagulation. We found that the 3E8 anti-HK antibody can inhibit the binding of FXI or PK to HK, delaying clot formation in human plasma. Our data show that in the absence of FXI, however, PK can substitute for FXI in this process. Addition of activated FXI (FXIa) or activated PK (PKa) abolished the inhibitory effect of 3E8. Moreover, the requirement of HK in intrinsic coagulation can be largely bypassed by adding FXIa. Like FXIa, exogenous PKa shortened the clotting time in HK-deficient plasma, which was not due to feedback activation of FXII. Conclusions This study improves our understanding of HK-mediated coagulation and provides an explanation for the absence of bleeding in HK-deficient individuals. 3E8 specifically prevented HK-mediated FXI activation; therefore, it could be used to prevent contact activation-mediated thrombosis without altering hemostasis.

Autosomal-recessive cerebellar ataxias (ARCAs) are heterogeneous rare disorders mainly affecting the cerebellum and manifest as movement disorders in children and young adults. To date, ARCA causing mutations have been identified in nearly 100 genes; however, they account for less than 50% of all cases. We studied a multiplex, consanguineous Pakistani family presenting with a slowly progressive gait ataxia, body imbalance, and dysarthria. Cerebellar atrophy was identified by magnetic resonance imaging of brain. Using whole exome sequencing, a novel homozygous missense mutation ERCC8:c.176T>C (p.M59T) was identified that co-segregated with the disease. Previous studies have identified homozygous mutations in ERCC8 as causal for Cockayne Syndrome type A (CSA), a UV light-sensitive syndrome, and several ARCAs. ERCC8 plays critical roles in the nucleotide excision repair complex. The p.M59T, a substitution mutation, is located in a highly conserved WD1 beta-transducin repeat motif. In silico modeling showed that the structure of this protein is significantly affected by the p.M59T mutation, likely impairing complex formation and protein-protein interactions. In cultured cells, the p.M59T mutation significantly lowered protein stability compared to wildtype ERCC8 protein. These findings expand the role of ERCC8 mutations in ARCAs and indicate that ERCC8-related mutations should be considered in the differential diagnosis of ARCAs.

Tuberculosis (TB) is the leading cause of death from any bacterial infection, causing 1.5 million deaths worldwide each year. Due to the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) there have been significant efforts aimed at developing novel drugs to treat TB. One promising drug target in Mtb is the arabinogalactan biosynthetic enzyme DprE1, and there have been over a dozen unique chemical scaffolds identified which inhibit the activity of this protein. Among the most promising lead compounds are the benzothiazinones BTZ043 and PBTZ169, both of which are currently in or have completed phase IIa clinical trials. Due to the potential clinical utility of these drugs, we sought to identify potential synergistic interactions and new mechanisms of resistance using a genome-scale CRISPRi chemical-genetic screen with PBTZ169. We found that knockdown of rv0678, the negative regulator of the mmpS5/L5 drug efflux pump, confers resistance to PBTZ169. Mutations in rv0678 are the most common form of resistance to bedaquiline and there is already abundant evidence of these mutations emerging in bedaquiline-treated patients. We confirmed that rv0678 mutations from clinical isolates confer low level cross-resistance to BTZ043 and PBTZ169. While it is yet unclear whether rv0678 mutations would render benzothiazinones ineffective in treating TB, these results highlight the importance of monitoring for clinically prevalent rv0678 mutations during ongoing BTZ043 and PBTZ169 clinical trials.

Type 1 secretion systems (T1SSs) are widespread in pathogenic Gram-negative bacteria, extruding protein substrates following synthesis of the entire polypeptide. The Escherichia coli hemolysin A secretion system has long been considered a prototype in structural and mechanistic studies of T1SSs. Three membrane pro-teins-an inner membrane ABC transporter HlyB, an adaptor protein HlyD, and an outer membrane porin TolC-are required for secretion. However, the stoichiometry and structure of the complex are unknown. Here, cryo-electron microscopy (cryo-EM) structures determined in two conformations reveal that the inner membrane complex is a hetero-dodecameric assembly comprising three HlyB homodimers and six HlyD subunits. Functional studies indicate that oligomerization of HlyB and HlyD is essential for protein secretion and that polypeptides translocate through a canonical ABC transporter pathway in HlyB. Our data suggest that T1SSs entail three ABC transporters, one that functions as a protein channel and two that allosterically power the translocation process.

Anterior face patches in the macaque have been assumed to represent face identity in a holistic manner. Here the authors show that the neural encoding of face identity in the anterior medial and anterior fundus face patches are instead driven principally by local features. Humans and other primates recognize one another in part based on unique structural details of the face, including both local features and their spatial configuration within the head and body. Visual analysis of the face is supported by specialized regions of the primate cerebral cortex, which in macaques are commonly known as face patches. Here we ask whether the responses of neurons in anterior face patches, thought to encode face identity, are more strongly driven by local or holistic facial structure. We created stimuli consisting of recombinant photorealistic images of macaques, where we interchanged the eyes, mouth, head, and body between individuals. Unexpectedly, neurons in the anterior medial (AM) and anterior fundus (AF) face patches were predominantly tuned to local facial features, with minimal neural selectivity for feature combinations. These findings indicate that the high-level structural encoding of face identity rests upon populations of neurons specialized for local features.