A superior heterologous prime-boost vaccination strategy against COVID-19: A bivalent vaccine based on yeast-derived RBD proteins followed by a heterologous vaccine.

Various vaccines have been challenged by SARS-CoV-2 variants. Here, we reported a yeast-derived recombinant bivalent vaccine (Bivalent wild-type [Wt]+De) based on the wt and Delta receptor-binding domain (RBD). Yeast derived RBD proteins based on the wt and Delta mutant were used as the prime vaccine. It was found that, in the presence of aluminium hydroxide (Alum) and unmethylated CpG-oligodeoxynucleotides (CpG) adjuvants, more cross-protective immunity against SARS-CoV-2 prototype and variants were elicited by bivalent vaccine than monovalent wtRBD or Delta RBD. Furthermore, a heterologous boosting strategy consisting of two doses of bivalent vaccines followed by one dose adenovirus vectored vaccine exhibited cross-neutralization capacity and specific T cell responses against Delta and Omicron (BA.1 and BA.4/5) variants in mice, superior to a homologous vaccination strategy. This study suggested that heterologous prime-boost vaccination with yeast-derived bivalent protein vaccine could be a potential approach to address the challenge of emerging variants.

Sentinel Surveillance System Implementation and Evaluation for SARS-CoV-2 Genomic Data, Washington, USA, 2020-2021.

Genomic data provides useful information for public health practice, particularly when combined with epidemiologic data. However, sampling bias is a concern because inferences from nonrandom data can be misleading. In March 2021, the Washington State Department of Health, USA, partnered with submitting and sequencing laboratories to establish sentinel surveillance for SARS-CoV-2 genomic data. We analyzed available genomic and epidemiologic data during presentinel and sentinel periods to assess representativeness and timeliness of availability. Genomic data during the presentinel period was largely unrepresentative of all COVID-19 cases. Data available during the sentinel period improved representativeness for age, death from COVID-19, outbreak association, long-term care facility-affiliated status, and geographic coverage; timeliness of data availability and captured viral diversity also improved. Hospitalized cases were underrepresented, indicating a need to increase inpatient sampling. Our analysis emphasizes the need to understand and quantify sampling bias in phylogenetic studies and continue evaluation and improvement of public health surveillance systems.

Deleterious Variants in ABCC12 are Detected in Idiopathic Chronic Cholestasis and Cause Intrahepatic Bile Duct Loss in Model Organisms.

BACKGROUND & AIMS: The etiology of cholestasis remains unknown in many children. We surveyed the genome of children with chronic cholestasis for variants in genes not previously associated with liver disease and validated their biological relevance in zebrafish and murine models. METHOD: Whole-exome (n = 4) and candidate gene sequencing (n = 89) was completed on 93 children with cholestasis and normal serum γ-glutamyl transferase (GGT) levels without pathogenic variants in genes known to cause low GGT cholestasis such as ABCB11 or ATP8B1. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing was used to induce frameshift pathogenic variants in the candidate gene in zebrafish and mice. RESULTS: In a 1-year-old female patient with normal GGT cholestasis and bile duct paucity, we identified a homozygous truncating pathogenic variant (c.198delA, p.Gly67Alafs∗6) in the ABCC12 gene (NM_033226). Five additional rare ABCC12 variants, including a pathogenic one, were detected in our cohort. ABCC12 encodes multidrug resistance-associated protein 9 (MRP9) that belongs to the adenosine 5'-triphosphate-binding cassette transporter C family with unknown function and no previous implication in liver disease. Immunohistochemistry and Western blotting revealed conserved MRP9 protein expression in the bile ducts in human, mouse, and zebrafish. Zebrafish abcc12-null mutants were prone to cholangiocyte apoptosis, which caused progressive bile duct loss during the juvenile stage. MRP9-deficient mice had fewer well-formed interlobular bile ducts and higher serum alkaline phosphatase levels compared with wild-type mice. They exhibited aggravated cholangiocyte apoptosis, hyperbilirubinemia, and liver fibrosis upon cholic acid challenge. CONCLUSIONS: Our work connects MRP9 with bile duct homeostasis and cholestatic liver disease for the first time. It identifies a potential therapeutic target to attenuate bile acid-induced cholangiocyte injury.

Multi-project and Multi-profile joint Non-negative Matrix Factorization for cancer omic datasets.

MOTIVATION: The integration of multi-omic data using machine learning methods has been focused on solving relevant tasks such as predicting sensitivity to a drug or subtyping patients. Recent integration methods, such as joint Non-negative Matrix Factorization, have allowed researchers to exploit the information in the data to unravel the biological processes of multi-omic datasets. RESULTS: We present a novel method called Multi-project and Multi-profile joint Non-negative Matrix Factorization capable of integrating data from different sources, such as experimental and observational multi-omic data. The method can generate co-clusters between observations, predict profiles and relate latent variables. We applied the method to integrate low-grade glioma omic profiles from The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia projects. The method allowed us to find gene clusters mainly enriched in cancer-associated terms. We identified groups of patients and cell lines similar to each other by comparing biological processes. We predicted the drug profile for patients, and we identified genetic signatures for resistant and sensitive tumors to a specific drug. AVAILABILITY AND IMPLEMENTATION: Source code repository is publicly available at https:/bitbucket.org/dsalazarb/mmjnmf/-Zenodo DOI: 10.5281/zenodo.5150920. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Tuning the magnetic anisotropy energy by external electric fields of CoPt dimers deposited on graphene.

In the framework of first-principles calculations, we comprehensively investigate the external electric-field (EF) manipulation of the magnetic anisotropy energy (MAE) of alloyed CoPt dimers deposited on graphene. In particular, we focus on the possibility of tuning the MAE barriers under the action of external EFs and on the effects of Co-substitution. Among the various considered structures, the lowest-energy configurations were the hollow-upright and top-upright, having the Co-atom closest to the graphene layer. The optimal and higher energy configurations were related to the electronic structure through the local density of states and hybridizations between the transition-metal (TM) atoms of the dimer and graphene. In contrast to Co2/graphene [M. Tanveer, J. Dorantes-Dávila and G. M. Pastor, Phys. Rev. B, 2017, 96(22), 224413.], the CoPt dimer having the hollow-upright ground-state configuration, exhibits a much lower value of the MAE (about |ΔE| ≃ 4.5 meV per atom) and the direction of the magnetization lies in the graphene layer. Moreover, we observe a spin-reorientation transition occurring at εz ≃ 0.5 V Å-1, which opens the possibility of inducing magnetization switching by external electric fields. The microscopic origin of the changes of the MAE associated with changes in the EF has been qualitatively related to the details of the electronic structure by analyzing the local density of states and to the spin-dependent electronic densities close to the Fermi energy. Finally, the role of local environment was quantified by performing electronic structure and magnetic calculations on several higher-energy structure configurations.

A single NGS-based assay covering the entire genomic sequence of the DMD gene facilitates diagnostic and newborn screening confirmatory testing.

Molecular diagnosis for Duchenne and Becker muscular dystrophies (DMD/BMD) involves a two-tiered approach for detection of deletions/duplications using MLPA or array CGH, followed by sequencing of coding and flanking intronic regions to detect sequence variants, which is time-consuming and expensive. We have developed a comprehensive next-generation sequencing (NGS)-based single-step assay to sequence the entire 2.2 Mb of the DMD gene to detect all copy number and sequence variants in both index males and carrier females. Assay validation was 100% concordant with other methodologies. A total of 772 samples have been tested, of which 62% (N = 480) were index cases with a clinical suspicion of DMD. Carrier testing females account for 38% (N = 292). Molecular diagnosis was confirmed in 86% (N = 413) of the index cases. Intragenic deletions and duplications (single-exon or multi-exon) were detected in 60% (N = 247) and 14% (N = 58) of the index cases, respectively. Full-sequence analysis of the entire gene allows for detection of deep intronic pathogenic variants and accurate breakpoint detection of CNVs involving similar exons, which could have an impact on the outcome of clinical trials. This comprehensive assay is highly sensitive for diagnostic testing for DMD and is also suitable for confirmatory testing for newborn screening for DMD.

Biparental Inheritance of Mitochondrial DNA in Humans.

Although there has been considerable debate about whether paternal mitochondrial DNA (mtDNA) transmission may coexist with maternal transmission of mtDNA, it is generally believed that mitochondria and mtDNA are exclusively maternally inherited in humans. Here, we identified three unrelated multigeneration families with a high level of mtDNA heteroplasmy (ranging from 24 to 76%) in a total of 17 individuals. Heteroplasmy of mtDNA was independently examined by high-depth whole mtDNA sequencing analysis in our research laboratory and in two Clinical Laboratory Improvement Amendments and College of American Pathologists-accredited laboratories using multiple approaches. A comprehensive exploration of mtDNA segregation in these families shows biparental mtDNA transmission with an autosomal dominantlike inheritance mode. Our results suggest that, although the central dogma of maternal inheritance of mtDNA remains valid, there are some exceptional cases where paternal mtDNA could be passed to the offspring. Elucidating the molecular mechanism for this unusual mode of inheritance will provide new insights into how mtDNA is passed on from parent to offspring and may even lead to the development of new avenues for the therapeutic treatment for pathogenic mtDNA transmission.

Gene and protein expression of myosin heavy chain in Nellore cattle comparing growth or meat tenderness traits.

The objective was to investigate gene and protein expression of myosin heavy chain (MyHC) in Nellore cattle slaughtered at different weights (BW) or degrees of meat tenderness. Ninety animals with initial BW 370 ± 37 kg, 24 months of age, were slaughtered after 95 days on feed. We evaluated shear force (SF), myofibrillar fragmentation index, ribeye area, backfat thickness, marbling, color, and cooking losses. Subsequently, 24 animals were selected and divided into four contrasting groups, in which light (BW = 504.58 ± 32.36 kg) versus heavy animals (BW = 604.83 ± 42.97 kg) and animals with tender (SF = 3.88 ± 0.57 kg) versus tough meat (SF = 7.95 ± 1.04 kg) were compared. The MYH7, MYH2 and MYH1 genes were analyzed by real-time PCR. The MyHC isoforms (MyHC-I, MyHC-IIa, and MyHC-IIx) were quantified by SDS-PAGE electrophoresis. We found lower expression of MYH2 and MYH1 genes in heavy compared to light animals and a higher amount of MyHC-I isoform in the tough meat group compared to the tender meat group. Protein expression of MyHC-IIa was higher in the tender meat group. A negative correlation was found of this protein and SF (tenderness), suggesting MyHC-IIa as a biomarker of meat quality.

An international telemedicine program for diagnosis of genetic disorders: Partnership of pediatrician and geneticist.

There is a shortage of genetics providers worldwide and access is limited to large academic centers. Telemedicine programs can facilitate access to genetic services to patients living in remote locations. The goal of this study was to improve access to genetic services in the Dominican Republic by creating a partnership model between a pediatrician and geneticist. This approach has been used within the United States but not in the setting of two different countries, healthcare system, and cultures. Patients were referred to the Centro de Obstetricia y Ginecologia program if a syndromic or genetic etiology was suspected by their local provider. Pediatrician first evaluated all patients prior to telemedicine appointment to review family and medical history. All genetic visits were scheduled within 2 weeks of referral in collaboration with telehealth program at Cincinnati Children's Hospital Medical Center. A total of 66 individuals were evaluated during a period of 5 years. Fifty-seven individuals underwent genetic studies, and a molecular diagnosis was made in 39 individuals. Exome sequencing was the most common first line test when differential diagnosis was broad (n = 40). The most common inheritance was autosomal recessive in 15 individuals, followed by 13 individuals with autosomal dominant disorders, 7 individuals X-linked disorders, and 4 individuals with chromosomal abnormalities. This study provides data to support utility of geneticist and pediatrician partnership to provide outreach telemedicine diagnostics and management services for rare diseases in an international setting.

Identification of glucocorticoid receptor in Drosophila melanogaster.

BACKGROUND: Vertebrate glucocorticoid receptor (GR) is an evolutionary-conserved cortisol-regulated nuclear receptor that controls key metabolic and developmental pathways. Upon binding to cortisol, GR acts as an immunosuppressive transcription factor. Drosophila melanogaster, a model organism to study innate immunity, can also be immunosuppressed by glucocorticoids. However, while the genome of fruit fly harbors 18 nuclear receptor genes, the functional homolog of vertebrate GR has not been identified. RESULTS: In this study, we demonstrated that while D. melanogaster is susceptible to Saccharomyces cerevisiae oral infection, the oral exposure to cortisol analogs, cortisone acetate or estrogen, increases fly sensitivity to yeast challenge. To understand the mechanism of this steroid-induced immunosuppression, we identified the closest genetic GR homolog as D. melanogaster Estrogen Related Receptor (ERR) gene. We discovered that Drosophila ERR is necessary for cortisone acetate- and estrogen-mediated increase in sensitivity to fungal infection: while ERR mutant flies are as sensitive to the fungal challenge as the wildtype flies, the yeast-sensitivity of ERR mutants is not increased by these steroids. Interestingly, the fungal cortisone analog, ergosterol, did not increase the susceptibility of Drosophila to yeast infection. The immunosuppressive effect of steroids on the sensitivity of flies to fungi is evolutionary conserved in insects, as we show that estrogen significantly increases the yeast-sensitivity of Culex quinquefasciatus mosquitoes, whose genome contains a close ortholog of the fly ERR gene. CONCLUSIONS: This study identifies a D. melanogaster gene that structurally resembles vertebrate GR and is functionally necessary for the steroid-mediated immunosuppression to fungal infections.

Biallelic mutations in the ferredoxin reductase gene cause novel mitochondriopathy with optic atrophy.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, including mitochondrial respiration, DNA repair, and iron homeostasis. A steadily increasing number of disorders are being associated with disrupted biogenesis of Fe-S clusters. Here, we conducted whole-exome sequencing of patients with optic atrophy and other neurological signs of mitochondriopathy and identified 17 individuals from 13 unrelated families with recessive mutations in FDXR, encoding the mitochondrial membrane-associated flavoprotein ferrodoxin reductase required for electron transport from NADPH to cytochrome P450. In vitro enzymatic assays in patient fibroblast cells showed deficient ferredoxin NADP reductase activity and mitochondrial dysfunction evidenced by low oxygen consumption rates (OCRs), complex activities, ATP production and increased reactive oxygen species (ROS). Such defects were rescued by overexpression of wild-type FDXR. Moreover, we found that mice carrying a spontaneous mutation allelic to the most common mutation found in patients displayed progressive gait abnormalities and vision loss, in addition to biochemical defects consistent with the major clinical features of the disease. Taken together, these data provide the first demonstration that germline, hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans.

Zebrafish abcb11b mutant reveals strategies to restore bile excretion impaired by bile salt export pump deficiency.

Bile salt export pump (BSEP) adenosine triphosphate-binding cassette B11 (ABCB11) is a liver-specific ABC transporter that mediates canalicular bile salt excretion from hepatocytes. Human mutations in ABCB11 cause progressive familial intrahepatic cholestasis type 2. Although over 150 ABCB11 variants have been reported, our understanding of their biological consequences is limited by the lack of an experimental model that recapitulates the patient phenotypes. We applied CRISPR/Cas9-based genome editing technology to knock out abcb11b, the ortholog of human ABCB11, in zebrafish and found that these mutants died prematurely. Histological and ultrastructural analyses showed that abcb11b mutant zebrafish exhibited hepatocyte injury similar to that seen in patients with progressive familial intrahepatic cholestasis type 2. Hepatocytes of mutant zebrafish failed to excrete the fluorescently tagged bile acid that is a substrate of human BSEP. Multidrug resistance protein 1, which is thought to play a compensatory role in Abcb11 knockout mice, was mislocalized to the hepatocyte cytoplasm in abcb11b mutant zebrafish and in a patient lacking BSEP protein due to nonsense mutations in ABCB11. We discovered that BSEP deficiency induced autophagy in both human and zebrafish hepatocytes. Treatment with rapamycin restored bile acid excretion, attenuated hepatocyte damage, and extended the life span of abcb11b mutant zebrafish, correlating with the recovery of canalicular multidrug resistance protein 1 localization. CONCLUSIONS: Collectively, these data suggest a model that rapamycin rescues BSEP-deficient phenotypes by prompting alternative transporters to excrete bile salts; multidrug resistance protein 1 is a candidate for such an alternative transporter. (Hepatology 2018;67:1531-1545).

Genetic variants in acute, acute recurrent and chronic pancreatitis affect the progression of disease in children.

BACKGROUND/OBJECTIVES: Acute pancreatitis (AP) is emerging in pediatrics. A subset of children with AP progresses to acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). The role of extensive gene testing in the progression has not been investigated previously. We have followed children enrolled in the registry and at our center for progression to ARP and CP after the first attack. METHODS: This study utilizes an extensive gene sequencing panel as a platform to evaluate the role of genetics in first attack AP, and the progression over time, from first attack to ARP and CP in children. RESULTS: Genes, with corresponding variants were involved in the 3 groups studied: AP, ARP and CP. We have shown that the presence of gene variants from the eight tested genes is enriched in the CP group compared to the AP and ARP groups. The presence of more than one gene was associated with CP (p = 0.01). SPINK1 mutation(s) was significantly associated with faster progression to ARP, (p = 0.04). Having a variant from CFTR, SPINK1 or PRSS1, was associated with the faster progression from AP to CP over time (p < 0.05). CONCLUSIONS: This study shows that genetics have a significant role in progression to ARP and CP from the first attack of pancreatitis.

Comparison of medical management and genetic counseling options pre- and post-whole exome sequencing for patients with positive and negative results.

Whole exome sequencing (WES) is expected to impact patient management, but data surrounding the types of downstream effects and how frequently these effects are observed depending on the type of WES results received is limited. This study investigated changes to medical management and genetic counseling (GC) options following WES for individuals with positive and negative results. Electronic medical records of patients who had positive (n = 37) or negative (n = 41) WES results from Cincinnati Children's Hospital were retrospectively reviewed. Pre- and post-WES management and GC options were analyzed as were differences between positive and negative results. Almost all participants (97%) were observed to have at least one difference in medical management and/or GC options following WES. Comparing pre- and post-WES detected significant differences (p ≤ 0.05) in genetic testing, imaging, and metabolic testing regardless of WES results. Participants with positive results also had significant differences in recurrence risk, reproductive options, testing for family members, and support groups. Pre- to post-WES differences were significantly different between participants with positive and negative results in specialist referrals, lifestyle recommendations, recurrence risk, and all GC options (p ≤ 0.05); specifically, participants with positive results were more likely to have differences in these categories. Overall, differences in medical management and/or GC options were observed for participants with both types of WES results (positive and negative). Results from this study may contribute to the understanding of how WES impacts patients and their care and thus improve its utilization.

Pediatric Whole Exome Sequencing: an Assessment of Parents' Perceived and Actual Understanding.

Whole exome sequencing (WES) is an integral tool in the diagnosis of genetic conditions in pediatric patients, but concerns have been expressed about the complexity of the information and the possibility for secondary findings that need to be conveyed to those deciding about WES. Currently, there is no validated tool to assess parental understanding of WES. We developed and implemented a survey to assess perceived and actual understanding of WES in parents who consented to clinical WES for their child between July 2013 and May 2015. Fifty-three eligible surveys were returned (57% response rate). Areas with both low perceived and actual understanding about WES included how genes are analyzed and lack of protection against life insurance discrimination. Parents also had low actual understanding for two questions related to secondary findings - reporting of secondary findings in a parent (if tested) and whether secondary findings can be related to traits such as height and hair color. Further work to develop a validated tool to assess understanding of WES would be beneficial as WES is integrated more frequently into clinical care.

Gallstone ileus: An overview of the literature. / El íleo biliar: una revisión de la literatura médica.

INTRODUCTION: Gallstone ileus represents 4% of the causes of bowel obstruction in the general population, but increases to 25% in patients above the age of 65 years. Gallstone ileus does not present with unique symptoms, making diagnosis difficult. Its management is surgical, but there is no consensus as to which of the different surgical techniques is the procedure of choice. At present, there is no recent review of this pathology. AIM: To conduct an up-to-date review of this disease. MATERIALS AND METHODS: Articles published within the time frame of 2000 to 2014 were found utilizing the PUBMED, EMBASE, and Cochrane Library search engines with the terms "gallstone ileus" plus "review" and the following filters: "review", "full text", and "humans". RESULTS: The results of this review showed that gallstone ileus etiology was due to intestinal obstruction from a gallstone that migrated into the intestinal lumen through a bilioenteric fistula. The presence of 2 of the 3 Rigler's triad signs was considered diagnostic. Abdominal tomography was the imaging study of choice for gallstone ileus diagnosis and the surgical procedures for management were enterolithotomy, one-stage surgery, and two-stage surgery. Enterolithotomy had lower morbidity and mortality than the other 2 procedures. CONCLUSIONS: The aim of gallstone ileus treatment is to release the obstruction, which is done through enterolithotomy. It is the recommended technique for gallstone ileus management because of its lower morbidity and mortality, compared with the other techniques.

Synergistic defects of different molecules in the cytotoxic pathway lead to clinical familial hemophagocytic lymphohistiocytosis.

Several molecules (LYST, AP3, RAB27A, STX11, STXBP2, MUNC13-4, and PRF1) have been associated with the function of cytotoxic lymphocytes. Biallelic defects in all of these molecules have been associated with familial hemophagocytic lymphohistiocytosis (FHL). We retrospectively reviewed the genetic and immunology test results from 2701 patients with a clinically suspected diagnosis of hemophagocytic lymphohistiocytosis and found 28 patients with single heterozygous mutations in 2 FHL-associated genes. Of these patients, 21 had mutations within PRF1 and a degranulation gene, and 7 were found to have mutations within 2 genes involved in the degranulation pathway. In patients with combination defects involving 2 genes in the degranulation pathway, CD107a degranulation was decreased, comparable to patients with biallelic mutations in one of the genes in the degranulation pathway. This suggests a potential digenic mode of inheritance of FHL as a result of a synergistic function effect within genes involved in cytotoxic lymphocyte degranulation.