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IntroductionProtein homeostasis is maintained by the opposing action of ubiquitin ligase and deubiquitinase, two important components of the ubiquitin-proteasome pathway, and contributes to both normal physiological and pathophysiological processes. The current study aims to delineate the roles of ubiquitin-specific protease 15 (USP15), a member of the largest deubiquitinase family, in HIV-1 gene expression and replication.MethodsWe took advantage of highly selective and specific ubiquitin variants (UbV), which were recently designed and developed for USP15, and ascertained the inhibitory effects of USP15 on HIV-1 gene expression and production by transfection and Western blotting. We also used real-time RT-PCR, transcription factor profiling, subcellular fractionation, immunoprecipitation followed by Western blotting to determine the transcription factors involved and the underlying molecular mechanisms.ResultsWe first confirmed the specificity of USP15-mediated HIV-1 gene expression and virus production. We then showed that the inhibition of HIV-1 production by USP15 occurred at the transcription level, associated with an increased protein level of YY1, a known HIV-1 transcription repressor. Moreover, we demonstrated that USP15 regulated YY1 deubiquitination and stability. Lastly, we demonstrated that YY1 siRNA knockdown significantly diminished the inhibition of USP15 on HIV-1 gene expression and virus production.ConclusionThese findings together demonstrate that stabilization of YY1 protein by USP15 deubiquitinating activity contributes to USP15-mediated inhibition of HIV-1 transcription and may help the development of USP15-specific UbV inhibitors as an anti-HIV strategy.

Samuel Maxwell Tom Williams,1 Rashid Ansumana,2 Jonathan Johnny,3 Ibrahim A Bakarr,3 Augustus Osborne1 1Department of Biological Sciences, School of Environmental Sciences, Njala University, Freetown, Sierra Leone; 2Department of Public Health, School of Community Health Sciences, Njala University, Freetown, Sierra Leone; 3Department of Wildlife Management and Conservation, School of Natural Resources Management, Njala University, Freetown, Sierra LeoneCorrespondence: Samuel Maxwell Tom Williams, Tel +23278462858, Email maxwell.williams@njala.edu.slBackground: Bats are considered wildlife species of public health concern, as they are known to host various pathogenic agents, and their interactions with humans are potential routes of pathogen spillover. A high level of knowledge on Bat-borne Zoonotic Diseases (BZD), their causative agents, signs, symptoms, mode and pattern of transmission, health attitudes, and practices towards the disorders are vital parameters in handling them. This study aimed to look into BZD knowledge, public attitudes, and behaviour.Methods: We surveyed the 14 chiefdoms of Moyamba district. A total of 421 participants were randomly sampled using closed-ended questionnaire. Simple linear regression analysis was used to determine the effects of gender, age, education, and livelihood opportunities on BZD knowledge (at 95% confidence interval and alpha value = 0.05). The findings were analysed and correlated with a scientific and public health perspective to assess the breadth of knowledge and awareness of BZD among the people of Moyamba district.Results: The findings from the study show a low level of knowledge on BZD among the people of the Moyamba district, with only 119 (28.3%) individuals that had some knowledge about BZD. Of those that knew about BZD, 94 (79.0%) had very little knowledge, 24 (20.2%) had a fair amount, and 1 (0.8%) had a great deal of knowledge about BZD. The primary mode of knowledge dissemination was through social media platforms.Conclusion: The level of knowledge about BZD is also very low. As a result of these findings, policymakers, health professionals, and environmental educators will be compelled to develop strategies to reduce the risk of BZD transmission in Sierra Leone’s population.Keywords: bat-borne zoonotic diseases, public health, transmission, Moyamba District, knowledge assessment, policymakers

Abstract Background Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating condition that can lead to severe impairment of physical, psychological, cognitive, social, and occupational functions. The cause of ME/CFS remains incompletely understood. There is no clinical diagnostic test for ME/CFS. Although many therapies have been used off-label to manage symptoms of ME/CFS, there are limited, if any, specific therapies or cure for ME/CFS. In this study, we investigated the expression of genes specific to key immune functions, and viral infection status in ME/CFS patients with an aim of identifying biomarkers for characterization and/or treatment of the disease. Methods In 2021, one-hundred and sixty-six (166) patients diagnosed with ME/CFS and 83 healthy controls in the US participated in this study via a social media-based application (app). The patients and heathy volunteers consented to the study and provided self-collected finger-stick blood and first morning void urine samples from home. RNA from the fingerstick blood was tested using DxTerity’s 51-gene autoimmune RNA expression panel (AIP). In addition, DNA from the same fingerstick blood sample was extracted to detect viral load of 4 known ME/CFS associated viruses (HHV6, HHV7, CMV and EBV) using a real-time PCR method. Results Among the 166 ME/CFS participants in the study, approximately half (49%) of the ME/CFS patients reported being house-bound or bedridden due to severe symptoms of the disease. From the AIP testing, ME/CFS patients with severe, bedridden conditions displayed significant increases in gene expression of IKZF2, IKZF3, HSPA8, BACH2, ABCE1 and CD3D, as compared to patients with mild to moderate disease conditions. These six aforementioned genes were further upregulated in the 22 bedridden participants who suffer not only from ME/CFS but also from other autoimmune diseases. These genes are involved in T cell, B cell and autoimmunity functions. Furthermore, IKZF3 (Aiolos) and IKZF2 (Helios), and BACH2 have been implicated in other autoimmune diseases such as systemic lupus erythematosus (SLE) and Rheumatoid Arthritis (RA). Among the 240 participants tested with the viral assays, 9 samples showed positive results (including 1 EBV positive and 8 HHV6 positives). Conclusions Our study indicates that gene expression biomarkers may be used in identifying or differentiating subsets of ME/CFS patients having different levels of disease severity. These gene targets may also represent opportunities for new therapeutic modalities for the treatment of ME/CFS. The use of social media engaged patient recruitment and at-home sample collection represents a novel approach for conducting clinical research which saves cost, time and eliminates travel for office visits.

The balance of microbial communities in the gut is extremely important for normal physiological function. Disruption of the balance is often associated with various disorders and diseases. Both HIV infection and cocaine use are known to change the gut microbiota and the epithelial barrier integrity, which contribute to inflammation and immune activation. Our recent study shows that Tat expression and cocaine exposure result in changes of genome-wide DNA methylation and gene expression and lead to worsen the learning and memory impairments. In the current study, we extended the study to determine effects of Tat and cocaine on the gut microbiota composition. We found that both Tat expression and cocaine exposure increased Alteromonadaceae in 6-month-old female/male mice. In addition, we found that Tat, cocaine, or both increased Alteromonadaceae, Bacteroidaceae, Cyanobiaceae, Erysipelotrichaceae, and Muribaculaceae but decreased Clostridiales_vadinBB60_group, Desulfovibrionaceae, Helicobacteraceae, Lachnospiraceae, and Ruminococcaceae in 12-month-old female mice. Lastly, we analyzed changes of metabolic pathways and found that Tat decreased energy metabolism and nucleotide metabolism, and increased lipid metabolism and metabolism of other amino acids while cocaine increased lipid metabolism in 12-month-old female mice. These results demonstrated that Tat expression and cocaine exposure resulted in significant changes of the gut microbiota in an age- and sex-dependent manner and provide additional evidence to support the bidirectional gut–brain axis hypothesis.

Summary: Cocaine use is a major comorbidity of HIV-associated neurocognitive disorder (HAND). In this study, we show that cocaine exposure worsens the learning and memory of doxycycline-inducible and brain-specific HIV Tat transgenic mice (iTat) and results in 14,838 hypermethylated CpG-related differentially methylated regions (DMRs) and 15,800 hypomethylated CpG-related DMRs, which are linked to 52 down- and 127 upregulated genes, respectively, in the hippocampus of iTat mice. These genes are mostly enriched at the neuronal function-, cell morphology-, and synapse formation-related extracellular matrix (ECM) receptor-ligand interaction pathway and mostly impacted in microglia. The accompanying neuropathological changes include swollen dendritic spines, increased synaptophysin expression, and diminished glial activation. We also find that sex (female) and age additively worsen the behavioral and pathological changes. These findings together indicate that chronic cocaine and long-term Tat expression interactively contribute to HAND, likely involving changes of DNA methylation and ECM receptor-ligand interactions.

To date, there are a small number of nuclear-restricted proteins that have been reported to play a role in NF-κB signaling. However, the exact molecular mechanisms are not fully understood. Tip110 is a nuclear protein that has been implicated in multiple biological processes. In a previous study, we have shown that Tip110 interacts with oncogenic ubiquitin specific peptidase 15 (USP15) and that ectopic expression of Tip110 leads to re-distribution of USP15 from the cytoplasm to the nucleus. USP15 is known to regulate NF-κB activity through several mechanisms including modulation of IκBα ubiquitination. These findings prompted us to investigate the role of Tip110 in the NF-κB signaling pathway. We showed that Tip110 regulates NF-κB activity. The expression of Tip110 potentiated TNF-α-induced NF-κB activity and deletion of the nuclear localization domain in Tip110 abrogated this potentiation activity. We then demonstrated that Tip110 altered IκBα phosphorylation and stability in the presence of TNF-α. Moreover, we found that Tip110 and USP15 opposingly regulated NF-κB activity by targeting IκBα protein stability. We further showed that Tip110 altered the expression of NF-κB-dependent proinflammatory cytokines. Lastly, by using whole-transcriptome analysis of Tip110 knockout mouse embryonic stem cells, we found several NF-κB and NF-κB-related pathways were dysregulated. Taken together, these findings add to the nuclear regulation of NF-κB activity by Tip110 through IκBα stabilization and provide new evidence to support the role of Tip110 in controlling cellular processes such as cancers that involve proinflammatory responses.

En este artículo, se desarrolló un algoritmo, basado en la técnica de enjambre de partículas (Siglas en inglés - PSO) para optimizar los procesos de fundición a presión y por compresión, utilizando modelos matemáticos para describir el comportamiento de ambos procesos. En la fundición por compresión el modelo matemático describe un problema con múltiples objetivos y restricciones, y en la fundición a presión el modelo describe un problema de un solo objetivo con restricciones. El desarrollo del algoritmo PSO se realizó con el software FORTRAN 90, y los resultados se compararon con los obtenidos usando un algoritmo de optimización basado en el proceso de enseñanza-aprendizaje (Siglas en inglés - TLBO), demostrando buenas capacidades en la optimización de parámetros en fundición a presión y por compresión. Se observó que con el algoritmo PSO se consigue una solución óptima en todos los procesos y los tiempos computacionales fueron mínimos.

En el presente estudio se resolvió, usando el método de los volúmenes finitos, un modelo matemático multifísico en 3D para predecir los fenómenos de flujo de fluidos y transferencia de calor en un molde de colada continua de palanquillas de acero 20CrMnTi. Los resultados mostraron la alta capacidad de enfriamiento del sistema para la formación de una capa sólida progresiva y uniforme que inicia muy cerca del menisco y alcanza a la salida del molde un espesor del 10 % de la sección de la pieza colada. A la salida de la boquilla de entrada sumergida (SEN) el acero experimentó un reflujo con una profundidad de 0,45 m medida desde el menisco. Parte del reflujo en ascenso llegó al menisco y descendió infiltrándose por las paredes del molde. Se observaron velocidades menores a 0,2 m/s en la zona superior del molde y gran penetración del chorro de acero en el centro del molde. Se concluyó, con base en lo descrito en la literatura, lo siguiente: a) El espesor de la costra solidificada, a la salida del molde, es suficiente para evitar roturas en la pieza debido a la presión ferrostática que ejerce el acero líquido, b) Las velocidades en el área del menisco generan baja turbulencia, lo que evita que se produzca atrapamiento de escoria en el acero líquido, c) Las condiciones fluidodinámicas que se presentan en la zona superior del molde, pueden ser contraproducentes para la transferencia de calor en el menisco y la disipación del sobrecalentamiento en el acero.

Background: The Philippines has one of the highest cigarette smoking rates in Southeast Asia. Tobacco prevention and control efforts should not be a one-size-fits-all approach. One of the most recent and highly marketed way of cutting down smoking is the use of E-cigarettes. But its use may also have potential harmful effects which would be similar to cigarette smoking. Purpose: This study aimed to determine the factors predicting electronic cigarette use among adults in a large metropolitan area in the Philippines. Methods:The study used a descriptive-correlational multivariate research design. Adults who are at least 18 years of age, who are electronic cigarettes users, dual users (electronic cigarette and cigarette users), and non-smokers were chosen for this study. The researchers utilized a researcher- made questionnaire based on the Theory of Planned Behavior (TPB) in gathering the data. Results: Multiple regression analysis suggests that positive attitude and high perceived behavioral control significantly predict intent to use electronic cigarettes. Moreover, intent to use is a significant predictor of actual e-cigarette use. Conclusion: People who have positive attitudes and high perceived behavioral control towards e-cigarette use are most likely to have higher intent to use e-cigarettes. Further, people who have high intent to use e-cigarettes will most likely use e-cigarettes. With reverence to the findings of this study, health managers and professionals should look into how e-cigarettes are being marketed to the public which may shape their attitude and behavior. Lastly, further studies should be conducted on other variables that may predict electronic cigarette use and measure health outcomes.

Literature tells how people experience and perceive their own aging may affect future health outcomes and health behaviors. There are also several sources by which these expectations are formed and reshaped as one ages. The study aimed to develop a theory on aging expectations and attribution through establishing axioms and constructing propositions using a deductive theory generation approach. The evolved theory suggests that a person’s expectation regarding aging would influence health behaviors and subsequently a person’s overall well-being. The theory further suggests that individual experiences during one’s lifetime influence how these personal aging expectations are constructed. This has major implications for health promotion programs and public health policies involving older adult populations.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, SARS2) remains a great global health threat and demands identification of more effective and SARS2-targeted antiviral drugs, even with successful development of anti-SARS2 vaccines. Viral replicons have proven to be a rapid, safe, and readily scalable platform for high-throughput screening, identification, and evaluation of antiviral drugs against positive-stranded RNA viruses. In the study, we report a unique robust HIV long terminal repeat (LTR)/T7 dual-promoter-driven and dual-reporter firefly luciferase (fLuc) and green fluorescent protein (GFP)-expressing SARS2 replicon. The genomic organization of the replicon was designed with quite a few features that were to ensure the replication fidelity of the replicon, to maximize the expression of the full-length replicon, and to offer the monitoring flexibility of the replicon replication. We showed the success of the construction of the replicon and expression of reporter genes fLuc and GFP and SARS structural N from the replicon DNA or the RNA that was in vitro transcribed from the replicon DNA. We also showed detection of the negative-stranded genomic RNA (gRNA) and subgenomic RNA (sgRNA) intermediates, a hallmark of replication of positive-stranded RNA viruses from the replicon. Lastly, we showed that expression of the reporter genes, N gene, gRNA, and sgRNA from the replicon was sensitive to inhibition by Remdesivir. Taken together, our results support use of the replicon for identification of anti-SARS2 drugs and development of new anti-SARS strategies targeted at the step of virus replication.

“Bugs as drugs” in medicine encompasses the use of microbes to enhance the efficacy of vaccination, such as the delivery of vaccines by Leishmania—the protozoan etiological agent of leishmaniasis. This novel approach is appraised in light of the successful development of vaccines for Covid-19. All relevant aspects of this pandemic are summarized to provide the necessary framework in contrast to leishmaniasis. The presentation is in a side-by-side matching format with particular emphasis on vaccines. The comparative approach makes it possible to highlight the timeframe of the vaccine workflows condensed by the caveats of pandemic urgency and, at the same time, provides the background of Leishmania behind its use as a vaccine carrier. Previous studies in support of the latter are summarized as follows. Leishmaniasis confers life-long immunity on patients after cure, suggesting the effective vaccination is achievable with whole-cell Leishmania. A new strategy was developed to inactivate these cells in vitro, rendering them non-viable, hence non-disease causing, albeit retaining their immunogenicity and adjuvanticity. This was achieved by installing a dual suicidal mechanism in Leishmania for singlet oxygen (1O2)-initiated inactivation. In vitro cultured Leishmania were genetically engineered for cytosolic accumulation of UV-sensitive uroporphyrin I and further loaded endosomally with a red light-sensitive cationic phthalocyanine. Exposing these doubly dye-loaded Leishmania to light triggers intracellular production of highly reactive but extremely short-lived 1O2, resulting in their rapid and complete inactivation. Immunization of susceptible animals with such inactivated Leishmania elicited immunity to protect them against experimental leishmaniasis. Significantly, the inactivated Leishmania was shown to effectively deliver transgenically add-on ovalbumin (OVA) to antigen-presenting cells (APC), wherein OVA epitopes were processed appropriately for presentation with MHC molecules to activate epitope-specific CD8+ T cells. Application of this approach to deliver cancer vaccine candidates, e.g., enolase-1, was shown to suppress tumor development in mouse models. A similar approach is predicted to elicit lasting immunity against infectious diseases, including complementation of the spike protein-based vaccines in use for COVID-19. This pandemic is devastating, but brings to light the necessity of considering many facets of the disease in developing vaccination programs. Closer collaboration is essential among those in diverse disciplinary areas to provide the roadmap toward greater success in the future. Highlighted herein are several specific issues of vaccinology and new approaches worthy of consideration due to the pandemic.

Abstract Background The current literature on single cell genomic analyses on the DNA level is conflicting regarding requirements for cell quality, amplification success rates, allelic dropouts and resolution, lacking a systematic comparison of multiple cell input down to the single cell. We hypothesized that such a correlation assay would provide an approach to address the latter issues, utilizing the leukemic cell line OCI-AML3 with a known set of genetic aberrations. Results By analyzing single and multiple cell replicates (2 to 50 cells) purified by micromanipulation and serial dilution we stringently assessed the signal-to-noise ratio (SNR) from single as well as a discrete number of cells based on a multiple displacement amplification method, with whole exome sequencing as signal readout. In this setting, known OCI-AML3 mutations as well as large copy number alterations could be identified, adding to the current knowledge of cytogenetic status. The presence of DNMT3A R882C, NPM1 W288 fs and NRAS Q61L was consistent, in spite of uneven allelic read depths. In contrast, at the level of single cells, we observed that one-third to half of all variants were not reproduced in the replicate sample, and this allelic mismatch displayed an exponential function of cell input. Large signature duplications were discernible from 5 cells, whereas deletions were visible down to the single cell. Thus, even under highly optimized conditions, single cell whole genome amplification and interpretation must be taken with considerable caution, given that allelic change is frequent and displays low SNR. Allelic noise is rapidly alleviated with increased cell input, and the SNR is doubled from 2 to 50 cells. Conclusions In conclusion, we demonstrate noisy allele distributions, when analyzing genetic aberrations within single cells relative to multiple cells. Based on the presented data we recommend that single cell analyses should include replicate cell dilution assays for a given setup for relative assessment of procedure-specific SNR to ensure that the resolution supports the specific hypotheses.

Mucopolysaccharidoses type IVA (Morquio disease) is a rare, autosomal recessive lysosomal storage disease that causes both obstructive and restrictive airway pathology, with respiratory failure being the primary cause of death. This article provides original data on the longitudinal characterization of pulmonary function changes in children with Mucopolysaccharidoses (MPS) IVA by presenting the data and nuanced trends of changes from sequential spirometry and oximetry. The sample size included 16 subjects, 13 had undergone enzyme replacement therapy (ERT), three had not undergone ERT treatment. A total of 180 individual plots are presented for spirometry variables (FEV1, FEV1 [%Pred] FVC, FVC [%Pred] and FEV1/FVC), 6MWT and oximetry variables (median %Spo2, ODI 3%, mean nadir 3%, ODI 4%, mean nadir 4% and min dip SpO2 [%]); over a nine-year period at a single quaternary paediatric metabolic centre. This data has been made public and has utility to clinicians and researchers due to the following: [1,2] by providing the first comprehensive report of detailed changes in pulmonary function in children with MPS IVA, with and without ERT; [1–3] as well as changes in pulmonary function following the institution of non-invasive ventilation (NIV) and adenotonsillectomy. The data presented is related to the research article by Kenth et al. “The Characterization of Pulmonary Function in Patients with Mucopolysaccharidoses IVA: A Longitudinal Analysis”. Keywords: Morquio syndrome, Mucopolysaccharidosis IVA, MPS, Respiratory changes, Enzyme replacement therapy, Sleep-disordered breathing

Abstract Tip110, an important regulator of several oncogenic proteins, was significantly downregulated in human metastatic melanoma cells exposed to a hypoxic condition. Therefore, in this study, we set to determine whether differential expression of Tip110 could be an important indicator for melanoma tumorigenesis and metastasis. We found that in melanoma, but not in other cancer types, Tip110 knockdown enhanced significant expression and secretion of IL-8 and melanoma cells invasions. This induction was further potentiated under hypoxia and by inflammatory cytokine and found independent of TNF-α autocrine signaling. We further showed that Tip110 knockdown-mediated IL-8 induction involved IL-8 mRNA stability. Furthermore, the transcriptomic profiling data and survival from 455 melanoma patients demonstrated that the correlation between Tip110 expression and the clinical outcomes in melanoma was stage-dependent. These findings uncover important roles of Tip110 in melanoma tumorigenesis and metastasis through regulation of IL-8 and hope to provide new clues for future therapeutic strategies.

Introduction: Mucopolysaccharidosis (MPS) type IVA is a rare, autosomal recessive lysosomal storage disease causing substrate accumulation in various organs and tissues. MPS IVA is associated with both obstructive and restrictive airway disease, with the former often resulting in sleep disordered breathing (SDB). Respiratory failure is a primary cause of death in this condition. The aim of this study was to characterise and catalogue the long-term respiratory changes in patients with MPS IVA treated with, or without, enzyme replacement therapy (ERT). Methods: In this retrospective, longitudinal, repeated-measures cohort study, descriptive statistics and non-parametric correlation were performed for demographic, respiratory function and oximetry variables over a study period from January 2009 to December 2018. Composite clinical endpoints used in this study for evaluating pulmonary function included spirometry variables (FEV1, FEV1 [%Pred] FVC, FVC [%Pred] and FEV1/FVC), oximetry variables (median %Spo2, ODI 3%, mean nadir 3%, ODI 4%, mean nadir 4% and min dip SpO2 [%]) and 6MWT to assess functional exercise capacity and thus integrated cardiopulmonary function. Results: Sequential spirometry and oximetry values were collected from 16 patients, of which 13/16 were ERT treated. In general, during the study period there was a global reduction in static spirometry values in all subjects, as well as cardiorespiratory function as assessed by the 6MWT, with the decline being delayed in the ERT group. Oximetry changed to a minor degree over time in the ERT group, whereas it declined in the non-ERT group. FEV1, FVC [%predicted] and ODI 3% exhibited a strong, combined positive correlation (r 0.74–95% CI 0.61 to 0.83; p

HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Several recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, how the intercellular Nef transfer occurs is in dispute. In the current study, we attempted to address this important issue using several complementary strategies, a panel of exosomal markers, and human CD4+ T lymphocyte cell line Jurkat and a commonly used cell line 293T. First, we showed that Nef was transferred from Nef-expressing or HIV-infected Jurkat to naïve Jurkat and other non-Jurkat cells and that the transfer required the membrane targeting function of Nef and was cell density-dependent. Then, we showed that Nef transfer was cell-cell contact-dependent, as exposure to culture supernatants or exosomes from HIV-infected Jurkat or Nef-expressing Jurkat and 293T led to little Nef detection in the target cells Jurkat. Thirdly, we demonstrated that Nef was only detected to be associated with HIV virions but not with acetylcholinesterase (AChE+) exosomes from HIV-infected Jurkat and not in the exosomes from Nef-expressing Jurkat. In comparison, when it was over-expressed in 293T, Nef was detected in detergent-insoluble AChE+/CD81 low/TSG101 low exosomes, but not in detergent-soluble AChE-/CD81 high/TSG101 high exosomes. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out 293T. Taken together, these results show that exosomes are unlikely involved in intercellular Nef transfer. In addition, this study reveals existence of two types of exosomes: AChE+/CD81 low/TSG101 low exosomes and AChE-/CD81 high/TSG101 high exosomes.

HIV-1 infection enhances HCV replication and as a consequence accelerates HCV-mediated hepatocellular carcinoma (HCC). However, the precise molecular mechanism by which this takes place is currently unknown. Our data showed that infectious HIV-1 failed to replicate in human hepatocytic cell lines. No discernible virus replication was observed, even when the cell lines transfected with HIV-1 proviral DNA were co-cultured with Jurkat T cells, indicating that the problem of liver deterioration in the co-infected patient is not due to the replication of HIV-1 in the hepatocytes of the HCV infected host. Instead, HIV-1 Nef protein was transferred from nef-expressing T cells to hepatocytic cells through conduits, wherein up to 16% (average 10%) of the cells harbored the transferred Nef, when the hepatocytic cells were co-cultured with nef-expressing Jurkat cells for 24 h. Further, Nef altered the size and numbers of lipid droplets (LD), and consistently up-regulated HCV replication by 1.5∼2.5 fold in the target subgenomic replicon cells, which is remarkable in relation to the initially indolent viral replication. Nef also dramatically augmented reactive oxygen species (ROS) production and enhanced ethanol-mediated up-regulation of HCV replication so as to accelerate HCC. Taken together, these data indicate that HIV-1 Nef is a critical element in accelerating progression of liver pathogenesis via enhancing HCV replication and coordinating modulation of key intra- and extra-cellular molecules for liver decay.

Rhinosporidiosis is a unique disease, which is seen to be endemic in certain places in India such as Tamil Nadu and Sri Lanka. The disease is caused by Rhinosporidium seebri and it is transmitted by bathing in ponds contaminated by cattle feces containing spores of the organism. The disease usually presents as multiple granulomatous bleeding polyps. The case described here is a unique presentation where it occurs only in a single site and that too in an uncommon location where the suspicion of rhinosporidiosis is a last possibility.

Hepatitis C virus (HCV) RNA replication involves complex interactions among the 3'x RNA element within the HCV 3' untranslated region, viral and host proteins. However, many of the host proteins remain unknown. In this study, we devised an RNA affinity chromatography /2D/MASS proteomics strategy and identified nine putative 3' X-associated host proteins; among them is oxysterol-binding protein-related protein 4 (ORP4), a cytoplasmic receptor for oxysterols. We determined the relationship between ORP4 expression and HCV replication. A very low level of constitutive ORP4 expression was detected in hepatocytes. Ectopically expressed ORP4 was detected in the endoplasmic reticulum and inhibited luciferase reporter gene expression in HCV subgenomic replicon cells and HCV core expression in JFH-1-infected cells. Expression of ORP4S, an ORP4 variant that lacked the N-terminal pleckstrin-homology domain but contained the C-terminal oxysterol-binding domain also inhibited HCV replication, pointing to an important role of the oxysterol-binding domain in ORP4-mediated inhibition of HCV replication. ORP4 was found to associate with HCV NS5B and its expression led to inhibition of the NS5B activity. ORP4 expression had little effect on intracellular lipid synthesis and secretion, but it induced lipid droplet formation in the context of HCV replication. Taken together, these results demonstrate that ORP4 is a negative regulator of HCV replication, likely via interaction with HCV NS5B in the replication complex and regulation of intracellular lipid homeostasis. This work supports the important role of lipids and their metabolism in HCV replication and pathogenesis.

The Clinic for Special Children (CSC) has integrated biochemical and molecular methods into a rural pediatric practice serving Old Order Amish and Mennonite (Plain) children. Among the Plain people, we have used single nucleotide polymorphism (SNP) microarrays to genetically map recessive disorders to large autozygous haplotype blocks (mean = 4.4 Mb) that contain many genes (mean = 79). For some, uninformative mapping or large gene lists preclude disease-gene identification by Sanger sequencing. Seven such conditions were selected for exome sequencing at the Broad Institute; all had been previously mapped at the CSC using low density SNP microarrays coupled with autozygosity and linkage analyses. Using between 1 and 5 patient samples per disorder, we identified sequence variants in the known disease-causing genes SLC6A3 and FLVCR1, and present evidence to strongly support the pathogenicity of variants identified in TUBGCP6, BRAT1, SNIP1, CRADD, and HARS. Our results reveal the power of coupling new genotyping technologies to population-specific genetic knowledge and robust clinical data.

Determining tends and issues is important for the health of any profession. The purpose of this research was to determine the current and future trends and issues facing technology and engineering education (TEE) in the United States (U.S.). The researchers used a three-round Modified-Delphi method to solicit information from technology and engineering education stakeholders across the U.S. In the first round, participants listed what they felt were current trends, future trends, current issues, and future issues facing TEE. The second rounds was designed to prioritize trends and issues. In the third rounds, participants were presented with one table for each current and future trend and issue and asked to identify if they felt each was essential or non-essential for technology and engineering leaders to ask. Two hundred sixty-eight participants responded in the third round, and the resulting trends and issues were categorized into eight themes: (1) teacher shortage; (2) secondary and university TEE programs; (3) funding programs and teachers; (4) curriculum; (5) technology and engineering education identity and relevance; (6) collaborative efforts; (7) teacher certification and development; and (8) student-centered foci.

BACKGROUND: Viruses interact with and exploit the host cellular machinery for their multiplication and propagation. The MEK/ERK signaling pathway positively regulates replication of many RNA viruses. However, whether and how this signaling pathway affects hepatitis C virus (HCV) replication and production is not well understood. METHODS AND RESULTS: In this study, we took advantage of two well-characterized MEK/ERK inhibitors and MEK/ERK dominant negative mutants and investigated the roles of the MEK/ERK signaling pathway in HCV gene expression and replication. We showed that inhibition of MEK/ERK signaling enhanced HCV gene expression, plus- and minus-strand RNA synthesis, and virus production. In addition, we showed that this enhancement was independent of interferon-alpha (IFN-alpha) antiviral activity and did not require prior activation of the MEK/ERK signaling pathway. Furthermore, we showed that only MEK and ERK-2 but not ERK-1 was involved in HCV replication, likely through regulation of HCV RNA translation. CONCLUSIONS: Taken together, these results demonstrate a negative regulatory role of the MEK/ERK signaling pathway in HCV replication and suggest a potential risk in targeting this signaling pathway to treat and prevent neoplastic transformation of HCV-infected liver cells.

BackgroundAstrocytes become activated with certain infections, and this might alter the brain to trigger or worsen depressed mood. Indeed, astrocytes are chronically activated in people with HIV infection (PWH), who are much more frequently depressed than people without HIV (PWoH). A particularly disabling component of depression in PWH is apathy, a loss of interest, motivation, emotion, and goal-directed behavior. We tested the hypothesis that depression and apathy in PWH would be associated with higher levels of a biomarker of astrocyte activation, glial fibrillary acidic protein (GFAP), in cerebrospinal fluid (CSF).MethodsWe evaluated PWH in a prospective observational study using the Beck Depression Inventory-II (BDI-II) and additional standardized assessments, including lumbar puncture. We measured GFAP in CSF with a customized direct sandwich ELISA method. Data were analyzed using ANOVA and multivariable regression.ResultsParticipants were 212 PWH, mean (SD) age 40.9±9.14 years, median (IQR) nadir and current CD4 199 (57, 326) and 411 (259, 579), 65.1% on ART, 67.3% virally suppressed. Higher CSF GFAP correlated with worse total BDI-II total scores (Pearson correlation r=0.158, p-value=0.0211), and with worse apathy scores (r=0.205, p=0.0027). The correlation between apathy/depression and GFAP was not in fluenced by other factors such as age or HIV suppression status.ConclusionsAstrocyte activation, reflected in higher levels of CSF GFAP, was associated with worse depression and apathy in PWH. Interventions to reduce astrocyte activation -- for example, using a peptide-1 receptor (GLP-1R) agonist -- might be studied to evaluate their impact on disabling depression in PWH.

This is the nineteenth in a series of articles entitled "The Legacy Project." The Legacy Project focuses on the lives and actions of leaders who have forged the technology and engineering teaching profession into what it is today. Members of the profession owe a debt of gratitude to these leaders. One simple way to demonstrate that gratitude is to recognize these leaders and some of their accomplishments. The focus in this issue is on Dr. Gerhard Salinger. Herein, Salinger responds to the following six questions: (1) Technology education funding through the National Science Foundation (NSF) was practically nonexistent until you started your career there. Why were you interested in developing the profession through various projects, and what did you do to start the long relationship that followed? (2) You helped provide experiences for a whole series of technology educators through the NSF rotators program. Who were those professionals, and what was the objective of having them work at NSF? What kinds of projects and experiences did they work on? (3) How did NSF determine the funding directions during your Program Officer years? Were they effective? Why? (4) You were the NSF Project Leader and a primary supporter for the Technology for All Americans Project that went on to create the "Technological Literacy" content standards, with supportive standards related to professional development, programs, and assessments. Did the project accomplish what you originally envisioned? How did the results position our profession both in and outside of our subject area? (5) What were some of the most important decisions that you made while working with NSF STEM educational programs, and what were some of the unrealized expectations? and (6) What leadership characteristics made you successful in promoting technology and engineering education, given the advent of STEM education and the inclusion of engineering education at the pre-university level?

An interview with Michael Hacker, co-director of the Center for Stem Research at Hofstra University in New York, is presented. Hacker describes his education while growing up in New York City and he talks about the industrial arts/technology education institutions in New York. He describes his career in education and the nature of the technology education curriculum in New York. He discusses his decision to move towards engineering education and several National Science Foundation projects.

In this quickly evolving digital world, the technology and education (T&E) education classroom can provide valuable instruction that creates a well-informed online citizenry through digital understanding and citizenship curriculum. For twenty years, "Standards for Technological Literacy" (STL) (ITEA/ ITEEA, 2000/2002/2007) provided technology education curriculum guidance. With STL's aging content, an increased focus on technology and engineering education, and new emphasis on integrative science, technology, engineering, and mathematics (STEM) education, technology and engineering leaders realized STL needed to be updated. The International Technology and Engineering Educators Association (ITEEA) and its Council on Technology and Engineering Teacher Education (CTETE) led the creation of "Standards for Technological and Engineering Literacy: The Role of Technology and Engineering in STEM Education" (STEL) (ITEEA, 2020). While maintaining the epistemological foundation contained in STL, STEL addresses technological, engineering, educational, and societal changes that have occurred since STL was last updated in 2007. The purpose of this article is to demonstrate how STEL addresses concerns found in the recent project "Current and Future Trends and Issues in Technology and Engineering Education" (Moye et al., 2020). The trends and issues project identified topics impacting the technology and engineering education profession and should not be confused with trends and issues that might be taught in a Technology and Engineering Education (TEE) laboratory classroom (i.e., current events such as emerging technologies, socio-technological issues, etc.). Specifically, this article looks at how STEL addresses curriculum trends and issues. The correlation between the two projects provides additional justification for technology educators to adopt "Standards for Technology and Engineering Literacy."

Many industrial arts, technology education, and now technology and engineering education leaders have made their mark on the technology and engineering teaching profession. Their legacy is something that members of the profession enjoy and have a responsibility to continue and build upon. This is the sixteenth in a series of articles entitled "The Legacy Project." The Legacy Project focuses on the lives and actions of leaders who have forged the the profession into what it is today. Members of the profession owe a debt of gratitude to these leaders. One simple way to demonstrate that gratitude is to recognize these leaders and some of their accomplishments. The focus in this issue is Dr. William David Greer, Jr. In this interview, Greer responds to the following two questions: (1) Like most people in the field of industrial arts, you started by being a teacher in the public schools. Please describe your teaching experience so that others will know what was being emphasized during that time in history; and (2) After teaching, you became an industrial arts administrator for a very large school system, the Fort Worth (TX) Independent School District. What were your responsibilities (such as curriculum development, etc.), the size of the system (number of teachers, number of students in I.A.), your nagging problems, and your successes?

Many industrial arts, technology education, and now technology and engineering education leaders have made their mark on our profession. Their legacy is something that members of the profession enjoy and have a responsibility to continue and build upon. This is the fifteenth in a series of articles entitled "The Legacy Project." The Legacy Project focuses on the lives and actions of leaders who have forged our profession into what it is today. Members of the profession owe a debt of gratitude to these leaders. One simple way to demonstrate that gratitude is to recognize these leaders and some of their accomplishments. The focus in this issue will be on Dr. Glenn Baker, Professor Emeritus, Educational Human Resource Development at Texas A&M University.

Despite the success of antiretroviral therapies to control systemic HIV-1 infection, the prevalence of HIV-associated neurocognitive disorders (HANDs) has not decreased among aging patients with HIV. Autophagy pathway alterations, triggered by HIV-1 proteins including gp120, Tat, and Nef, might contribute to the neurodegenerative process in aging patients with HAND. Although no treatments are currently available to manage HAND, we have previously shown that sunitinib, an anticancer drug that blocks receptor tyrosine-kinase and cyclin kinase pathways, might be of interest. Studies in cancer models suggest that sunitinib might also modulate autophagy, which is dysregulated in our models of Tat-induced neurotoxicity. We evaluated the efficacy of sunitinib to promote autophagy in the CNS and ameliorate neurodegeneration using LC3-GFP-expressing neuronal cells challenged with low concentrations of Tat and using inducible Tat transgenic mice. In neuronal cultures challenged with low levels of Tat, sunitinib increased markers of autophagy such as LC3-II and reduced p62 accumulation in a dose-dependent manner. In vivo, sunitinib treatment restored LC3-II, p62, and endophilin B1 (EndoB1) levels in doxycycline-induced Tat transgenic mice. Moreover, in these animals, sunitinib reduced the hyperactivation of CDK5, tau hyperphosphorylation, and p35 cleavage to p25. Restoration of CDK5 and autophagy were associated with reduced neurodegeneration and behavioral alterations. Alterations in autophagy in the Tat tg mice were associated with reduced levels of a CDK5 substrate, EndoB1, and levels of total EndoB1 were normalized by sunitinib treatment. We conclude that sunitinib might ameliorate Tat-mediated autophagy alterations and may decrease neurodegeneration in aging patients with HAND.

Today's world is shaped by technology and products engineered to serve society's needs and wants. The knowledge and skills required to develop and use these products are changing at an exponential rate (NASE&M, 2017; NRC, 2011). In order to understand and meet future challenges, it is vital that the United States produce technology- and engineering-literate citizens (NASE&M, 2017). Technology and engineering programs offer students the science, technology, engineering, and mathematics needed to be successful in STEM education and occupations (Moye, Dugger, & Starkweather, 2018). Technology and engineering students use hands-on problem-solving activities in class to learn about and address real-life problems (Ernst, & Clark, 2009). This article provides evidence illustrating how current technology and engineering programs provide students with the lessons and hands-on activities designed to produce technology- and engineering-literate students.

Many industrial arts, technology education, and now technology and engineering education leaders have made their mark on our profession. Their legacy is something that members of the profession enjoy and have a responsibility to continue and build upon. This is the thirteenth in a series of articles entitled "The Legacy Project." The Legacy Project focuses on the lives and actions of leaders who have forged our profession into what it is today. Members of the profession owe a debt of gratitude to these leaders. One simple way to demonstrate that gratitude is to recognize these leaders and some of their accomplishments. The focus in this issue will be on Dr. Rodney Custer, DTE.