The rockefeller university

Raw vegetables are a key food for a healthy diet, but their increased consumption brings a higher risk for foodborne disease. Contamination of salad greens with Shiga toxin-producing Escherichia coli (STEC) O157:H7 has caused severe disease and important economic losses almost yearly in the United States over the last 10 years. To curb the risk of infections from contaminated produce, approaches based on bacterial virus - commonly known as bacteriophage or phage - have recently started to draw interest among other antimicrobial strategies. Phages enter bacterial cells to reproduce and cause cellular lysis to release their phage progeny at the end of their infection cycle. This lytic effect is caused by lysins, phage-encoded enzymes that have evolved to degrade the bacterial cell wall resulting in hypotonic lysis. When applied externally in their purified form, such enzymes are able to kill sensitive bacteria on contact in a similar way. Their unique bactericidal properties have made lysins effective antimicrobial agents in a variety of applications, from treating multidrug-resistant infections in humans to controlling bacterial contamination in several areas, including microbiological food safety. Here we describe a novel lysin, namely PlyEc2, with potent bactericidal activity against key gram-negative pathogens including E. coli, Salmonella, Shigella, Acinetobacter and Pseudomonas. PlyEc2 displayed high bactericidal activity against STEC to a concentration of 12.5 mu g/ml under different pH conditions. This lysin was also able to reduce the bacterial titer of several pathogenic strains in vitro by more than 5 logarithmic units, resulting in complete sterilization. Importantly, PlyEc2 proved to be a powerful produce decontamination agent in its ability to clear 99.7% of contaminating STEC O157:H7 in our Romaine lettuce leaf model. PlyEc2 was also able to eradicate 99.8% of the bacteria contaminating the washing solution, drastically reducing the risk of cross-contamination during the washing process. A sensory evaluation panel found that treatment with PlyEc2 did not alter the visual and tactile quality of lettuce leaves compared to the untreated leaves. Our study is the first to describe a highly effective lysin treatment to control gram-negative pathogenic contamination on fresh lettuce without the addition of membrane destabilizing agents.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models(1,2). Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory B cells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory B cells remains unchanged at 6.2 months after infection. Memory B cells display clonal turnover after 6.2 months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of coronavirus disease 2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 individuals. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

The anaerobic bacterium Cutibacterium acnes has been increasingly linked to the development of degenerative disc disease (DDD), although causality is yet to be conclusively proven. To better study how this organism could contribute to the aetiology of DDD, improved animal models that are more reflective of human disc anatomy, biology and mechanical properties are required. Against this background, our proof-of concept study aimed to be the first demonstration that C. acnes could be safely administered percutaneously into sheep intervertebral discs (IVDs) for in vivo study. Following our protocol, two sheep were successfully injected with a strain of C. acnes (8.3 x 10(6) CFU/disc) previously recovered from a human degenerative disc. No adverse reactions were noted, and at one-month post inoculation all triplicate infected discs in our first animal grew C. acnes, albeit at a reduced load (5.12 x 10(4) to 6.67 x 10(4) CFU/disc). At six months, no growth was detected in discs from our second animal indicating bacterial clearance. This pilot study has demonstrated the feasibility of safe percutaneous injection of C. acnes into sheep IVDs under fluoroscopic guidance. The design of follow-up sheep studies to investigate the potential of C. acnes to drive pathological changes within infected discs should now be pursued.

The formation of cellular microtubule networks is regulated by the gamma-tubuhn ring complex (gamma-TuRC). This similar to 2.3 MD assembly of >31 proteins includes y-tubuhn and GCP2-6, as well as MZT1 and an actin-like protein in a "lumenal bridge" (LB). The challenge of reconstituting the gamma-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human gamma-TuRC (gamma-TuRC-GFP) as a similar to 35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, gamma-TuRC(Delta LB)-GFP, which lacks MZT1 and actin. We show that gamma-TuRC(Delta LB)-GFP nucleates microtubules in a guanine nucleotide-dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that gamma-TuRC-GFP resembles the native gamma-TuRC architecture, while gamma-TuRC(Delta LB)-GFP adopts a partial cone shape presenting only 8-10 gamma-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the gamma-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating "core" in the holocomplex.

Staphylococcus saprophyticus is a primary cause of community-acquired urinary tract infections (UTIs) in young women. S. saprophyticus colonizes humans and animals but basic features of its molecular epidemiology are undetermined. We conducted a phylogenomic analysis of 321 S. saprophyticus isolates collected from human UTIs worldwide during 1997-2017 and 232 isolates from human UTIs and the pig-processing chain in a confined region during 2016-2017. We found epidemiologic and genomic evidence that the meat-production chain is a major source of S. saprophyticus causing human UTIs; human microbiota is another possible origin. Pathogenic S. saprophyticus belonged to 2 lineages with distinctive generic features that are globally and locally disseminated. Pangenome-wide approaches identified a strong association between pathogenicity and antimicrobial resistance, phages, platelet binding proteins, and an increased recombination rate. Our study provides insight into the origin, transmission, and population structure of pathogenic S. saprophyticus and identifies putative new virulence factors.

Chromosome segregation relies on centromeres, yet their repetitive DNA is often prone to aberrant rearrangements under pathological conditions. Factors that maintain centromere integrity to prevent centromere-associated chromosome translocations are unknown. Here, we demonstrate the importance of the centromere-specific histone H3 variant CENP-A in safeguarding DNA replication of alpha-satellite repeats to prevent structural aneuploidy. Rapid removal of CENP-A in S phase, but not other cell-cycle stages, caused accumulation of R loops with increased centromeric transcripts, and interfered with replication fork progression. Replication without CENP-A causes recombination at alpha-satellites in an R loop-dependent manner, unfinished replication, and anaphase bridges. In turn, chromosome breakage and translocations arise specifically at centromeric regions. Our findings provide insights into how specialized centromeric chromatin maintains the integrity of transcribed noncoding repetitive DNA during S phase.

CD8(+) T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8(+) T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-gamma production and cytotoxic activity of CD8(+) T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8(+) T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8(+) T cell surveillance through point mutations in MHC-I-restricted viral epitopes.

The motor thalamus relays signals from subcortical structures to the motor cortical areas. Previous studies in songbirds and rodents suggest that cortical feedback inputs crucially contribute to the generation of movement-related activity in the motor thalamus. In primates, however, it remains uncertain whether the corticothalamic projections may play a role in shaping neuronal activity in the motor thalamus. Here, using an optogenetic inactivation technique with the viral vector system expressing halorhodopsin, we investigated the role of cortical input in modulating thalamic neuronal activity during goal-directed behavior. In particular, we assessed whether the suppression of signals originating from the supplementary eye field at the corticothalamic terminals could change the task-related neuronal modulation in the oculomotor thalamus in monkeys performing a self-initiated saccade task. We found that many thalamic neurons exhibited changes in their firing rates depending on saccade direction or task event, indicating that optical stimulation exerted task-specific effects on neuronal activity beyond the global changes in baseline activity. These results suggest that the corticothalamic projections might be actively involved in the signal processing necessary for goal-directed behavior. However, we also found that some thalamic neurons exhibited overall, non-task -specific changes in the firing rate during optical stimulation, even in control animals without vector injections. The stimulation effects in these animals started with longer latency, implying a possible thermal effect on neuronal activity. Thus, our results not only reveal the importance of direct cortical input in neuronal activity in the primate motor thalamus, but also provide useful information for future optogenetic studies.

The superfamily 1 helicase nonstructural protein 13 (nsp13) is required for SARS-CoV-2 replication. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the RNA substrate. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be activated >50-fold by piconewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, such as hepatitis C virus NS3, and instead draws stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

SARS-CoV-2, the causative agent of COVID-19, has been responsible for over 42 million infections and 1 million deaths since its emergence in December 2019. There are few therapeutic options and no approved vaccines. Here, we examine the properties of highly potent human monoclonal antibodies (hu-mAbs) in a Syrian hamster model of SARS-CoV-2 and in a mouse-adapted model of SARS-CoV-2 infection (SARS-CoV-2 MA). Antibody combinations were effective for prevention and in therapy when administered early. However, in vitro antibody neutralization potency did not uniformly correlate with in vivo protection, and some hu-mAbs were more protective in combination in vivo. Analysis of antibody Fc regions revealed that binding to activating Fc receptors contributes to optimal protection against SARS-CoV-2 MA. The data indicate that intact effector function can affect hu-mAb protective activity and that in vivo testing is required to establish optimal hu-mAb combinations for COVID-19 prevention.