Differentiation Drives Widespread Rewiring of the Neural Stem Cell Chaperone Network.

Neural stem and progenitor cells (NSPCs) are critical for continued cellular replacement in the adult brain. Lifelong maintenance of a functional NSPC pool necessitates stringent mechanisms to preserve a pristine proteome. We find that the NSPC chaperone network robustly maintains misfolded protein solubility and stress resilience through high levels of the ATP-dependent chaperonin TRiC/CCT. Strikingly, NSPC differentiation rewires the cellular chaperone network, reducing TRiC/CCT levels and inducing those of the ATP-independent small heat shock proteins (sHSPs). This switches the proteostasis strategy in neural progeny cells to promote sequestration of misfolded proteins into protective inclusions. The chaperone network of NSPCs is more effective than that of differentiated cells, leading to improved management of proteotoxic stress and amyloidogenic proteins. However, NSPC proteostasis is impaired by brain aging. The less efficient chaperone network of differentiated neural progeny may contribute to their enhanced susceptibility to neurodegenerative diseases characterized by aberrant protein misfolding and aggregation.

High-resolution mapping reveals the mechanism and contribution of genome insertions and deletions to RNA virus evolution.

RNA viruses rapidly adapt to selective conditions due to the high intrinsic mutation rates of their RNA-dependent RNA polymerases (RdRps). Insertions and deletions (indels) in viral genomes are major contributors to both deleterious mutational load and evolutionary novelty, but remain understudied. To characterize the mechanistic details of their formation and evolutionary dynamics during infection, we developed a hybrid experimental-bioinformatic approach. This approach, called MultiMatch, extracts insertions and deletions from ultradeep sequencing experiments, including those occurring at extremely low frequencies, allowing us to map their genomic distribution and quantify the rates at which they occur. Mapping indel mutations in adapting poliovirus and dengue virus populations, we determine the rates of indel generation and identify mechanistic and functional constraints shaping indel diversity. Using poliovirus RdRp variants of distinct fidelity and genome recombination rates, we demonstrate tradeoffs between fidelity and Indel generation. Additionally, we show that maintaining translation frame and viral RNA structures constrain the Indel landscape and that, due to these significant fitness effects, Indels exert a significant deleterious load on adapting viral populations. Conversely, we uncover positively selected Indels that modulate RNA structure, generate protein variants, and produce defective interfering genomes in viral populations. Together, our analyses establish the kinetic and mechanistic tradeoffs between misincorporation, recombination, and Indel rates and reveal functional principles defining the central role of Indels in virus evolution, emergence, and the regulation of viral infection.

Lessons Learned From Developing a Mobile App to Assist in Patient Recovery After Weight Loss Surgery.

BACKGROUND: Weight loss surgery is safe and effective, but fluid and electrolyte balance remains problematic postoperatively. We developed a mobile app to help patients recover after weight loss surgery. MATERIAL AND METHODS: Single-center prospective, mixed-methods, participatory research design study of eligible English-speaking adults undergoing weight loss surgery was used. Patients used the app for 30 d after surgery. We developed and refined the app from July 2017 to October 2018. The principal component of the app is a daily survey designed to assess patients' recovery. The app was revised based on patient feedback, and we compared app utilization between initial and updated versions of the app. Primary outcome was successful patient engagement, which we defined as 80% of patients completing at least 70% of the surveys. RESULTS: Ten patients completed the trial period, four with the initial version of the app, and six with the updated version. All patients expressed satisfaction with the app and most frequently reported that push notifications were helpful. We found that one (25%) patient completed at least 70% of the surveys in the initial version of the app. In the updated version, five (83.3%) of patients completed at least 70% of the surveys, passing our criteria for successful engagement. CONCLUSIONS: Participatory research design in app development requires continuous evaluation and refinement to patient and clinician needs. This effort is essential as we observed significant improvement in app utilization. Our next step is to pilot the app in a larger set of patients to assess utility and feasibility.

Structure and dynamics of enterovirus genotype networks.

Like all biological populations, viral populations exist as networks of genotypes connected through mutation. Mapping the topology of these networks and quantifying population dynamics across them is crucial to understanding how populations adapt to changes in their selective environment. The influence of mutational networks is especially profound in viral populations that rapidly explore their mutational neighborhoods via high mutation rates. Using a single-cell sequencing method, scRNA-seq-enabled acquisition of mRNA and consensus haplotypes linking individual genotypes and host transcriptomes (SEARCHLIGHT), we captured and assembled viral haplotypes from hundreds of individual infected cells, revealing the complexity of viral population structures. We obtained these genotypes in parallel with host cell transcriptome information, enabling us to link host cell transcriptional phenotypes to the genetic structures underlying virus adaptation. Our examination of these structures reveals the common evolutionary dynamics of enterovirus populations and illustrates how viral populations reach through mutational "tunnels" to span evolutionary landscapes and maintain connection with multiple adaptive genotypes simultaneously.

Uncovering Structural Plasticity of Enterovirus A through Deep Insertional and Deletional Scanning.

Insertions and deletions (InDels) are essential sources of novelty in protein evolution. In RNA viruses, InDels cause dramatic phenotypic changes contributing to the emergence of viruses with altered immune profiles and host engagement. This work aimed to expand our current understanding of viral evolution and explore the mutational tolerance of RNA viruses to InDels, focusing on Enterovirus A71 (EV-A71) as a prototype for Enterovirus A species (EV-A). Using newly described deep InDel scanning approaches, we engineered approximately 45,000 insertions and 6,000 deletions at every site across the viral proteome, quantifying their effects on viral fitness. As a general trend, most InDels were lethal to the virus. However, our screen reproducibly identified a set of InDel-tolerant regions, demonstrating our ability to comprehensively map tolerance to these mutations. Tolerant sites highlighted structurally flexible and mutationally plastic regions of viral proteins that avoid core structural and functional elements. Phylogenetic analysis on EV-A species infecting diverse mammalian hosts revealed that the experimentally-identified hotspots overlapped with sites of InDels across the EV-A species, suggesting structural plasticity at these sites is an important function for InDels in EV speciation. Our work reveals the fitness effects of InDels across EV-A71, identifying regions of evolutionary capacity that require further monitoring, which could guide the development of Enterovirus vaccines.

C1q enables influenza hemagglutinin stem binding antibodies to block viral attachment and broadens the antibody escape repertoire.

Antigenic drift, the gradual accumulation of amino acid substitutions in the influenza virus hemagglutinin (HA) receptor protein, enables viral immune evasion. Antibodies (Abs) specific for the drift-resistant HA stem region are a promising universal influenza vaccine target. Although anti-stem Abs are not believed to block viral attachment, here we show that complement component 1q (C1q), a 460-kilodalton protein with six Ab Fc-binding domains, confers attachment inhibition to anti-stem Abs and enhances their fusion and neuraminidase inhibition. As a result, virus neutralization activity in vitro is boosted up to 30-fold, and in vivo protection from influenza PR8 infection in mice is enhanced. These effects reflect increased steric hindrance and not increased Ab avidity. C1q greatly expands the anti-stem Ab viral escape repertoire to include residues throughout the HA, some of which cause antigenic alterations in the globular region or modulate HA receptor avidity. We also show that C1q enhances the neutralization activity of non-receptor binding domain anti-SARS-CoV-2 spike Abs, an effect dependent on spike density on the virion surface. These findings demonstrate that C1q can greatly expand Ab function and thereby contribute to viral evolution and immune escape.

Widespread and dynamic expression of granzyme C by skin-resident antiviral T cells.

After recognition of cognate antigen (Ag), effector CD8+ T cells secrete serine proteases called granzymes in conjunction with perforin, allowing granzymes to enter and kill target cells. While the roles for some granzymes during antiviral immune responses are well characterized, the function of others, such as granzyme C and its human ortholog granzyme H, is still unclear. Granzyme C is constitutively expressed by mature, cytolytic innate lymphoid 1 cells (ILC1s). Whether other antiviral effector cells also produce granzyme C and whether it is continually expressed or responsive to the environment is unknown. To explore this, we analyzed granzyme C expression in different murine skin-resident antiviral lymphocytes. At steady-state, dendritic epidermal T cells (DETCs) expressed granzyme C while dermal γδ T cells did not. CD8+ tissue-resident memory T cells (TRM) generated in response to cutaneous viral infection with the poxvirus vaccinia virus (VACV) also expressed granzyme C. Both DETCs and virus-specific CD8+ TRM upregulated granzyme C upon local VACV infection. Continual Ag exposure was not required for maintained TRM expression of granzyme C, although re-encounter with cognate Ag boosted expression. Additionally, IL-15 treatment increased granzyme C expression in both DETCs and TRM. Together, our data demonstrate that granzyme C is widely expressed by antiviral T cells in the skin and that expression is responsive to both environmental stimuli and TCR engagement. These data suggest that granzyme C may have functions other than killing in tissue-resident lymphocytes.

Nuclear and cytoplasmic spatial protein quality control is coordinated by nuclear-vacuolar junctions and perinuclear ESCRT.

Effective protein quality control (PQC), essential for cellular health, relies on spatial sequestration of misfolded proteins into defined inclusions. Here we reveal the coordination of nuclear and cytoplasmic spatial PQC. Cytoplasmic misfolded proteins concentrate in a cytoplasmic juxtanuclear quality control compartment, while nuclear misfolded proteins sequester into an intranuclear quality control compartment (INQ). Particle tracking reveals that INQ and the juxtanuclear quality control compartment converge to face each other across the nuclear envelope at a site proximal to the nuclear-vacuolar junction marked by perinuclear ESCRT-II/III protein Chm7. Strikingly, convergence at nuclear-vacuolar junction contacts facilitates VPS4-dependent vacuolar clearance of misfolded cytoplasmic and nuclear proteins, the latter entailing extrusion of nuclear INQ into the vacuole. Finding that nuclear-vacuolar contact sites are cellular hubs of spatial PQC to facilitate vacuolar clearance of nuclear and cytoplasmic inclusions highlights the role of cellular architecture in proteostasis maintenance.

Colonic Stents as a Bridge to Surgery Compared with Immediate Resection in Patients with Malignant Large Bowel Obstruction in a NY State Database.

BACKGROUND: There is controversy surrounding the efficacy and safety of colonic stents as a bridge to surgery compared with immediate resection in patients presenting with an acute malignant large bowel obstruction. METHODS: Retrospective longitudinal cohort study using the NYS SPARCS Database. Patients with acute malignant large bowel obstruction who either had stent followed by elective surgery within 3 weeks (bridge to surgery) or underwent immediate resection between October 2009 and June 2016 in the state of New York were included. The primary outcome was rate of stoma creation at index resection. Secondary outcomes were 90-day readmission, reoperation, procedural complications, and discharge disposition. RESULTS: A total of 3059 patients were included, n = 2917 (95.4%) underwent an immediate resection and n = 142 (4.6%) underwent bridge to surgery. We analyzed 139 patients in propensity score-matched groups. Patients in the bridge to surgery group were less likely than those in the immediate resection group to get a stoma at the time of surgery (OR 0.33, 95% CI 0.18-0.60). They were also less likely to be discharged to a rehabilitation facility or require a home health aide upon discharge (OR 0.36, 95% CI 0.22-0.61). There were no differences in rates of 90-day readmission, reoperation, or procedural complications between groups. DISCUSSION: Colonic stenting as a bridge to surgery leads to less stoma creation, a significant quality of life advantage, compared with immediate resection. Patients should be counseled regarding these potential benefits when the technology is available.

Principles of dengue virus evolvability derived from genotype-fitness maps in human and mosquito cells.

Dengue virus (DENV) cycles between mosquito and mammalian hosts. To examine how DENV populations adapt to these different host environments, we used serial passage in human and mosquito cell lines and estimated fitness effects for all single-nucleotide variants in these populations using ultra-deep sequencing. This allowed us to determine the contributions of beneficial and deleterious mutations to the collective fitness of the population. Our analysis revealed that the continuous influx of a large burden of deleterious mutations counterbalances the effect of rare, host-specific beneficial mutations to shape the path of adaptation. Beneficial mutations preferentially map to intrinsically disordered domains in the viral proteome and cluster to defined regions in the genome. These phenotypically redundant adaptive alleles may facilitate host-specific DENV adaptation. Importantly, the evolutionary constraints described in our simple system mirror trends observed across DENV and Zika strains, indicating it recapitulates key biophysical and biological constraints shaping long-term viral evolution.

Viruses are constantly evolving as a result of mutations in their genetic material and environmental pressures. Viruses switching between insects and mammals face unique evolutionary pressures because they must retain their ability to infect both types of organisms. Yet, the mutations in a virus that may be beneficial in an insect may be different from the ones that may be beneficial in a mammal. Mutations in one host may be even harmful in the other. To learn more about how such viruses thrive as they switch between hosts, Dolan, Taguwa et al. studied the dengue virus, which causes over 390 million infections and over 10,000 deaths each year around the globe. They compared the mutations that occurred as the virus multiplied in human and mosquito cells grown in a laboratory. In the experiments, they used a method called ultra-deep RNA sequencing to identify every change that occurred in the genetic material of the virus each time it multiplied. They determined whether the mutations were beneficial or harmful based on whether they became more common ­ suggesting they helped the virus survive ­ or whether they did not persist because they were likely harmful or even fatal to the virus. The experiments showed that many harmful mutations constantly occur in the virus, in both human and mosquito cells. Beneficial changes happen rarely, and those that do are usually only helpful in one type of cell. Fatal mutations tended to occur in parts of the genetic material that encodes regions in the viral proteins that must remain the same. These structural elements appear to be essential to the virus's survival and unable to undergo change, which makes them good targets for antiviral drugs or vaccines. The techniques used in the study may be useful for investigating other viruses and for understanding the evolutionary constraints on viruses more generally. This may help scientists develop antiviral drugs or vaccines that will remain effective even as viruses continue to evolve and mutate.

Association of Demographic and Program Factors With American Board of Surgery Qualifying and Certifying Examinations Pass Rates.

Importance: American Board of Surgery board certification requires passing both a written qualifying examination and an oral certifying examination. No studies have been conducted assessing the effect of sociodemographic variables on board passage rates. Objective: To evaluate if trainee sociodemographic factors are associated with board passage rates. Design, Setting, and Participants: This national and multi-institutional prospective observational cohort study of 1048 categorical general surgery trainees starting in 2007-2008 were surveyed. Data collection began in June 2007, follow-up was completed on December 31, 2016, and analysis began September 2018. Main Outcomes and Measures: Survey responses were linked to American Board of Surgery board passage data. Results: Of 662 examinees who had complete survey and follow-up data, 443 (65%) were men and 459 (69%) were white, with an overall board passage rate of 87% (n = 578). In a multinomial regression model, trainees of Hispanic ethnicity were more likely to not attempt the examinations (vs passed both) than non-Hispanic trainees (odds ratio [OR], 4.7; 95% CI, 1.5-14). Compared with examinees who were married with children during internship, examinees who were married without children (OR, 0.3; 95% CI, 0.1-0.8) or were single (OR, 0.4; 95% CI, 0.2-0.9) were less likely to fail the examinations. Logistic regression showed white examinees compared with nonwhite examinees (black individuals, Asian individuals, and individuals of other races) (OR, 1.8; 95% CI, 1.03-3.0) and examinees who performed better on their first American Board of Surgery In-Training Examination (OR, 1.03; 95% CI, 1.02-1.05) were more likely to pass the qualifying examination on the first try. White examinees compared with nonwhite examinees (OR, 1.8; 95% CI, 1.1-2.8), non-Hispanic compared with Hispanic examinees (OR, 2.4; 95% CI, 1.2-4.7), and single women compared with women who were married with children during internship (OR, 10.3; 95% CI, 2.1-51) were more likely to pass the certifying examination on the first try. Conclusions and Relevance: Resident race, ethnicity, sex, and family status at internship were observed to be associated with board passage rates. There are multiple possible explanations for these worrisome observations that need to be explored. Tracking demographics of trainees to help understand passage rates based on demographics will be important. The American Board of Surgery already has begun addressing the potential for unconscious bias among board examiners by increasing diversity and adding implicit bias training.

National prospective cohort study describing how financial stresses are associated with attrition from surgical residency.

BACKGROUND: Attrition from general surgery residency is high with a national rate of 20%. We evaluated potential associations between financial considerations and attrition. METHODS: National prospective cohort study of categorical general surgery trainees. RESULTS: Of the 1048 interns who started training in 2007, 681 (65%) had complete survey and follow-up data. In logistic regression, those with higher starting attending salary expectations (>$300K) were more likely to leave training (OR 2.9, 95% CI 1.2-6.9). Women with a partner who earned more (>$50K/year) were more likely to leave training (OR 4.1, 95% CI 1.6-10.5). In a subgroup of interns undecided about their future practice setting (academic, community, private practice, industry), those with less debt (≤$100K) were more likely to leave training (OR 2.4, 95% CI 1.1-5.2). CONCLUSIONS: Several financial matters were associated with attrition. Addressing these financial concerns may help decrease attrition in surgical training and improve surgical training.

Engineering the Live-Attenuated Polio Vaccine to Prevent Reversion to Virulence.

The live-attenuated oral poliovirus vaccine (OPV or Sabin vaccine) replicates in gut-associated tissues, eliciting mucosa and systemic immunity. OPV protects from disease and limits poliovirus spread. Accordingly, vaccination with OPV is the primary strategy used to end the circulation of all polioviruses. However, the ability of OPV to regain replication fitness and establish new epidemics represents a significant risk of polio re-emergence should immunization cease. Here, we report the development of a poliovirus type 2 vaccine strain (nOPV2) that is genetically more stable and less likely to regain virulence than the original Sabin2 strain. We introduced modifications within at the 5' untranslated region of the Sabin2 genome to stabilize attenuation determinants, 2C coding region to prevent recombination, and 3D polymerase to limit viral adaptability. Prior work established that nOPV2 is immunogenic in preclinical and clinical studies, and thus may enable complete poliovirus eradication.

Control of RNA viruses in mosquito cells through the acquisition of vDNA and endogenous viral elements.

Aedes aegypti transmit pathogenic arboviruses while the mosquito itself tolerates the infection. We examine a piRNA-based immunity that relies on the acquisition of viral derived cDNA (vDNA) and how this pathway discriminates between self and non-self. The piRNAs derived from these vDNAs are essential for virus control and Piwi4 has a central role in the pathway. Piwi4 binds preferentially to virus-derived piRNAs but not to transposon-targeting piRNAs. Analysis of episomal vDNA from infected cells reveals that vDNA molecules are acquired through a discriminatory process of reverse-transcription and recombination directed by endogenous retrotransposons. Using a high-resolution Ae. aegypti genomic sequence, we found that vDNAs integrated in the host genome as endogenous viral elements (EVEs), produce antisense piRNAs that are preferentially loaded onto Piwi4. Importantly, EVE-derived piRNAs are specifically loaded onto Piwi4 to inhibit virus replication. Thus, Ae. aegypti employs a sophisticated antiviral mechanism that promotes viral persistence and generates long-lasting adaptive immunity.

The Plasmodium falciparum MESA erythrocyte cytoskeleton-binding (MEC) motif binds to erythrocyte ankyrin.

The MESA erythrocyte cytoskeleton binding (MEC) motif is a 13-amino acid sequence found in 14 exported Plasmodium falciparum proteins. First identified in the P. falciparum Mature-parasite-infected Erythrocyte Surface Antigen (MESA), the MEC motif is sufficient to target proteins to the infected red blood cell cytoskeleton. To identify host cell targets, purified MESA MEC motif was incubated with a soluble extract from uninfected erythrocytes, precipitated and subjected to mass spectrometry. The most abundant co-purifying protein was erythrocyte ankyrin (ANK1). A direct interaction between the MEC motif and ANK1 was independently verified using co-purification experiments, the split-luciferase assay, and the yeast two-hybrid assay. A systematic mutational analysis of the core MEC motif demonstrated a critical role for the conserved aspartic acid residue at the C-terminus of the MEC motif for binding to both erythrocyte inside-out vesicles and to ANK1. Using a panel of ANK1 constructs, the MEC motif binding site was localized to the ZU5C domain, which has no known function. The MEC motif had no impact on erythrocyte deformability when introduced into uninfected erythrocyte ghosts, suggesting the MEC motif's primary function is to target exported proteins to the cytoskeleton. Finally, we show that PF3D7_0402100 (PFD0095c) binds to ANK1 and band 4.1, likely through its MEC and PHIST motifs, respectively. In conclusion, we have provided multiple lines of evidence that the MEC motif binds to erythrocyte ANK1.

Mechanisms and Concepts in RNA Virus Population Dynamics and Evolution.

RNA viruses are unique in their evolutionary capacity, exhibiting high mutation rates and frequent recombination. They rapidly adapt to environmental changes, such as shifts in immune pressure or pharmacological challenge. The evolution of RNA viruses has been brought into new focus with the recent developments of genetic and experimental tools to explore and manipulate the evolutionary dynamics of viral populations. These studies have uncovered new mechanisms that enable viruses to overcome evolutionary challenges in the environment and have emphasized the intimate relationship of viral populations with evolution. Here, we review some of the emerging viral and host mechanisms that underlie the evolution of RNA viruses. We also discuss new studies that demonstrate that the relationship between evolutionary dynamics and virus biology spans many spatial and temporal scales, affecting transmission dynamics within and between hosts as well as pathogenesis.

Mapping the Evolutionary Potential of RNA Viruses.

The deterministic force of natural selection and stochastic influence of drift shape RNA virus evolution. New deep-sequencing and microfluidics technologies allow us to quantify the effect of mutations and trace the evolution of viral populations with single-genome and single-nucleotide resolution. Such experiments can reveal the topography of the genotype-fitness landscapes that shape the path of viral evolution. By combining historical analyses, like phylogenetic approaches, with high-throughput and high-resolution evolutionary experiments, we can observe parallel patterns of evolution that drive important phenotypic transitions. These developments provide a framework for quantifying and anticipating potential evolutionary events. Here, we examine emerging technologies that can map the selective landscapes of viruses, focusing on their application to pathogenic viruses. We identify areas where these technologies can bolster our ability to study the evolution of viruses and to anticipate and possibly intervene in evolutionary events and prevent viral disease.

Surgical time out: Our counts are still short on racial diversity in academic surgery.

BACKGROUND: This study provides an updated description of diversity along the academic surgical pipeline to determine what progress has been made. METHODS: Data was extracted from a variety of publically available data sources to determine proportions of minorities in medical school, general surgery training, and academic surgery leadership. RESULTS: In 2014-2015, Blacks represented 12.4% of the U.S. population, but only 5.7% graduating medical students, 6.2% general surgery trainees, 3.8% assistant professors, 2.5% associate professors and 2.0% full professors. From 2005-2015, representation among Black associate professors has gotten worse (-0.07%/year, p < 0.01). Similarly, in 2014-2015, Hispanics represented 17.4% of the U.S. population but only 4.5% graduating medical students, 8.5% general surgery trainees, 5.0% assistant professors, 5.0% associate professors and 4.0% full professors. There has been modest improvement in Hispanic representation among general surgery trainees (0.2%/year, p < 0.01), associate (0.12%/year, p < 0.01) and full professors (0.13%/year, p < 0.01). CONCLUSION: Despite efforts to promote diversity in surgery, Blacks and Hispanics remain underrepresented. A multi-level national focus is imperative to elucidate effective mechanisms to make academic surgery more reflective of the US population.

The Diversity, Structure, and Function of Heritable Adaptive Immunity Sequences in the Aedes aegypti Genome.

The Aedes aegypti mosquito transmits arboviruses, including dengue, chikungunya, and Zika virus. Understanding the mechanisms underlying mosquito immunity could provide new tools to control arbovirus spread. Insects exploit two different RNAi pathways to combat viral and transposon infection: short interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) [1, 2]. Endogenous viral elements (EVEs) are sequences from non-retroviral viruses that are inserted into the mosquito genome and can act as templates for the production of piRNAs [3, 4]. EVEs therefore represent a record of past infections and a reservoir of potential immune memory [5]. The large-scale organization of EVEs has been difficult to resolve with short-read sequencing because they tend to integrate into repetitive regions of the genome. To define the diversity, organization, and function of EVEs, we took advantage of the contiguity associated with long-read sequencing to generate a high-quality assembly of the Ae. aegypti-derived Aag2 cell line genome, an important and widely used model system. We show EVEs are acquired through recombination with specific classes of long terminal repeat (LTR) retrotransposons and organize into large loci (>50 kbp) characterized by high LTR density. These EVE-containing loci have increased density of piRNAs compared to similar regions without EVEs. Furthermore, we detected EVE-derived piRNAs consistent with a targeted processing of persistently infecting virus genomes. We propose that comparisons of EVEs across mosquito populations may explain differences in vector competence, and further study of the structure and function of these elements in the genome of mosquitoes may lead to epidemiological interventions.