Apolipoprotein L1 (APOL1) risk variants greatly elevate the risk of kidney disease in African Americans. Here we report a cohort of patients who developed collapsing focal segmental ...glomerulosclerosis while receiving therapeutic interferon, all of whom carried the APOL1 high-risk genotype. This finding raised the possibility that interferons and the molecular pattern recognition receptors that stimulate interferon production may contribute to APOL1-associated kidney disease. In cell culture, interferons and Toll-like receptor (TLR) agonists increased APOL1 expression by up to 200-fold, in some cases with the appearance of transcripts not detected under basal conditions. PolyI:C, a double-stranded RNA TLR3 agonist, increased APOL1 expression by upregulating interferons directly or through an interferon-independent, IFN-regulatory factor 3 (IRF3)-dependent pathway. Using pharmacological inhibitors, small hairpin RNA knockdown, and chromatin immunoprecipitation, we found that the interferon-independent TLR3 pathway relied on signaling through TBK1, NF-κB, and Jak kinases, and on binding of IRF1, IRF2, and STAT2 at the APOL1 transcription start site. We also demonstrate that overexpression of the APOL1 risk variants is more injurious to cells than overexpression of the wild-type APOL1 protein. Our study illustrates that antiviral pathways may be important inducers of kidney disease in individuals with the APOL1 high-risk genotype and identifies potential targets for prevention or treatment.
The spatial presentation of mechanical information is a key parameter for cell behavior. We have developed a method of polymerization control in which the differential diffusion distance of unreacted ...cross-linker and monomer into a prepolymerized hydrogel sink results in a tunable stiffness gradient at the cell–matrix interface. This simple, low-cost, robust method was used to produce polyacrylamide hydrogels with stiffness gradients of 0.5, 1.7, 2.9, 4.5, 6.8, and 8.2 kPa/mm, spanning the in vivo physiological and pathological mechanical landscape. Importantly, three of these gradients were found to be nondurotactic for human adipose-derived stem cells (hASCs), allowing the presentation of a continuous range of stiffnesses in a single well without the confounding effect of differential cell migration. Using these nondurotactic gradient gels, stiffness-dependent hASC morphology, migration, and differentiation were studied. Finally, the mechanosensitive proteins YAP, Lamin A/C, Lamin B, MRTF-A, and MRTF-B were analyzed on these gradients, providing higher-resolution data on stiffness-dependent expression and localization.
Background
Alterations in the intestinal microbiome are prospectively associated with the development of asthma; less is known regarding the role of microbiome alterations in food allergy ...development.
Methods
Intestinal microbiome samples were collected at age 3‐6 months in children participating in the follow‐up phase of an interventional trial of high‐dose vitamin D given during pregnancy. At age 3, sensitization to foods (milk, egg, peanut, soy, wheat, walnut) was assessed. Food allergy was defined as caretaker report of healthcare provider‐diagnosed allergy to the above foods prior to age 3 with evidence of IgE sensitization. Analysis was performed using Phyloseq and DESeq2; P‐values were adjusted for multiple comparisons.
Results
Complete data were available for 225 children; there were 87 cases of food sensitization and 14 cases of food allergy. Microbial diversity measures did not differ between food sensitization and food allergy cases and controls. The genera Haemophilus (log2 fold change −2.15, P=.003), Dialister (log2 fold change −2.22, P=.009), Dorea (log2 fold change −1.65, P=.02), and Clostridium (log2 fold change −1.47, P=.002) were underrepresented among subjects with food sensitization. The genera Citrobacter (log2 fold change −3.41, P=.03), Oscillospira (log2 fold change −2.80, P=.03), Lactococcus (log2 fold change −3.19, P=.05), and Dorea (log2 fold change −3.00, P=.05) were underrepresented among subjects with food allergy.
Conclusions
The temporal association between bacterial colonization and food sensitization and allergy suggests that the microbiome may have a causal role in the development of food allergy. Our findings have therapeutic implications for the prevention and treatment of food allergy.
We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule, real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation ...of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenine, 5-methylcytosine and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We used these kinetic signatures to identify adenine methylation in genomic samples and found that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns in even highly repetitive genomic regions.
Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory ...sequelae. Challenges with gene delivery have limited attempts to treat CF using in vivo gene therapy, and low correction levels have hindered ex vivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the ΔF508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%–50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients and observed 20%–50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF.
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•Cas9 RNP and AAV can be used to efficiently gene edit human airway basal stem cells•This method yields >30% allelic correction without selection markers or antibiotics•Correction of >30% ΔF508 alleles restores CFTR function to near non-CF levels•Corrected stem cells can differentiate after embedding in the scaffold for engraftment
Vaidyanathan et al. use Cas9 and AAV to correct the CF causing ΔF508 mutation in >30% of alleles in airway basal stem cells from CF patients and embed corrected cells on a scaffold for engraftment. This method restores physiologic CFTR function and provides an ex vivo strategy to treat cystic fibrosis.
•Parents projected the benefits of AVs to be advancing mobility and safety.•Mothers and parents with younger children had more concerns than their counterparts.•Parents’ perceived concerns should ...guide the design of AV mobility features.•Using AVs to transport children is a likely ridership scenario.
Prior research has estimated the impact of an autonomous vehicle (AV) environment on the mobility of underserved populations such as adult non-drivers. What is currently unknown is the impact of AVs on enhancing the mobility of children who are also mobility disadvantaged, as child passengers are likely part of AV ridership scenarios in the perceivable future. To address this question, our study collected perceived benefits and concerns of AVs from a US convenience sample of parents whose children relied on them for mobility. We found that parents’ intentions to travel in AV and their technology readiness as well as parent (sex, residence area) and child (age, restraint system) demographic profiles were important determinants of potential AV acceptance and impact. In addition, two groups of potential AV users emerged from the data: the curious and the practical. This study addresses a gap in the literature by assessing parents’ perspectives on using AVs to transport children. The results have great potentials to guide the design of mobility features, safety evaluations, and implementation policies, as a decline in public interest in AVs has been recently documented.
Inefficiency and inequity are two challenges that plague humanitarian operations and health delivery in resource‐limited regions. Increasing capacity in humanitarian and health delivery supply chains ...is one option that has the potential to improve equity while maintaining efficiency. For example, the nonprofit organization Riders for Health has worked to increase capacity by providing reliable transportation to health workers in rural parts of sub‐Saharan Africa; with more motorcycle hours at their disposal, health workers can perform more outreach to outlying communities. We develop a model using a family of fairness function to quantify the efficiency and equity of health delivery as capacity is increased via development programs. We present optimal resource allocations under utilitarian, proportionally fair, and egalitarian objectives and extend the model to include dual modes of transport and diminishing returns of subsequent outreach visits. Finally, we demonstrate how to apply our model at a regional level to provide support for humanitarian decision makers such as Riders for Health. We use data from the baseline phase of our evaluation trial of Riders for Health in Zambia to quantify efficiency and equity for one real‐world scenario.
Antibody recognition of antigens is a critical element of adaptive immunity. One key class of antibody-antigen complexes is comprised of antibodies targeting linear epitopes of proteins, which in ...some cases are conserved elements of viruses and pathogens of relevance for vaccine design and immunotherapy. Here we report a detailed analysis of the structural and interface features of this class of complexes, based on a set of nearly 200 nonredundant high resolution antibody-peptide complex structures that were assembled from the Protein Data Bank. We found that antibody-bound peptides adopt a broad range of conformations, often displaying limited secondary structure, and that the same peptide sequence bound by different antibodies can in many cases exhibit varying conformations. Propensities of contacts with antibody loops and extent of antibody binding conformational changes were found to be broadly similar to those for antibodies in complex with larger protein antigens. However, antibody-peptide interfaces showed lower buried surface areas and fewer hydrogen bonds than antibody-protein antigen complexes, while calculated binding energy per buried interface area was found to be higher on average for antibody-peptide interfaces, likely due in part to a greater proportion of buried hydrophobic residues and higher shape complementarity. This dataset and these observations can be of use for future studies focused on this class of interactions, including predictive computational modeling efforts and the design of antibodies or epitope-based vaccine immunogens.
BACKGROUNDInitial reports from the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic described children as being less susceptible to coronavirus disease 2019 (COVID-19) than adults. ...Subsequently, a severe and novel pediatric disorder termed multisystem inflammatory syndrome in children (MIS-C) emerged. We report on unique hematologic and immunologic parameters that distinguish between COVID-19 and MIS-C and provide insight into pathophysiology.METHODSWe prospectively enrolled hospitalized patients with evidence of SARS-CoV-2 infection and classified them as having MIS-C or COVID-19. Patients with COVID-19 were classified as having either minimal or severe disease. Cytokine profiles, viral cycle thresholds (Cts), blood smears, and soluble C5b-9 values were analyzed with clinical data.RESULTSTwenty patients were enrolled (9 severe COVID-19, 5 minimal COVID-19, and 6 MIS-C). Five cytokines (IFN-γ, IL-10, IL-6, IL-8, and TNF-α) contributed to the analysis. TNF-α and IL-10 discriminated between patients with MIS-C and severe COVID-19. The presence of burr cells on blood smears, as well as Cts, differentiated between patients with severe COVID-19 and those with MIS-C.CONCLUSIONPediatric patients with SARS-CoV-2 are at risk for critical illness with severe COVID-19 and MIS-C. Cytokine profiling and examination of peripheral blood smears may distinguish between patients with MIS-C and those with severe COVID-19.FUNDINGFinancial support for this project was provided by CHOP Frontiers Program Immune Dysregulation Team; National Institute of Allergy and Infectious Diseases; National Cancer Institute; the Leukemia and Lymphoma Society; Cookies for Kids Cancer; Alex's Lemonade Stand Foundation for Childhood Cancer; Children's Oncology Group; Stand UP 2 Cancer; Team Connor; the Kate Amato Foundations; Burroughs Wellcome Fund CAMS; the Clinical Immunology Society; the American Academy of Allergy, Asthma, and Immunology; and the Institute for Translational Medicine and Therapeutics.
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