Enhancement of erythropoietic output by Cas9-mediated insertion of a natural variant in haematopoietic stem and progenitor cells.

Some gene polymorphisms can lead to monogenic diseases, whereas other polymorphisms may confer beneficial traits. A well-characterized example is congenital erythrocytosis-the non-pathogenic hyper-production of red blood cells-that is caused by a truncated erythropoietin receptor. Here we show that Cas9-mediated genome editing in CD34+ human haematopoietic stem and progenitor cells (HSPCs) can recreate the truncated form of the erythropoietin receptor, leading to substantial increases in erythropoietic output. We also show that combining the expression of the cDNA of a truncated erythropoietin receptor with a previously reported genome-editing strategy to fully replace the HBA1 gene with an HBB transgene in HSPCs (to restore normal haemoglobin production in cells with a ß-thalassaemia phenotype) gives the edited HSPCs and the healthy red blood cell phenotype a proliferative advantage. Combining knowledge of human genetics with precise genome editing to insert natural human variants into therapeutic cells may facilitate safer and more effective genome-editing therapies for patients with genetic diseases.

A simultaneous knockout knockin genome editing strategy in HSPCs potently inhibits CCR5- and CXCR4-tropic HIV-1 infection.

Allogeneic hematopoietic stem and progenitor cell transplant (HSCT) of CCR5 null (CCR5Δ32) cells can be curative for HIV-1-infected patients. However, because allogeneic HSCT poses significant risk, CCR5Δ32 matched bone marrow donors are rare, and CCR5Δ32 transplant does not confer resistance to the CXCR4-tropic virus, it is not a viable option for most patients. We describe a targeted Cas9/AAV6-based genome editing strategy for autologous HSCT resulting in both CCR5- and CXCR4-tropic HIV-1 resistance. Edited human hematopoietic stem and progenitor cells (HSPCs) maintain multi-lineage repopulation capacity in vivo, and edited primary human T cells potently inhibit infection by both CCR5-tropic and CXCR4-tropic HIV-1. Modification rates facilitated complete loss of CCR5-tropic replication and up to a 2,000-fold decrease in CXCR4-tropic replication without CXCR4 locus disruption. This multi-factor editing strategy in HSPCs could provide a broad approach for autologous HSCT as a functional cure for both CCR5-tropic and CXCR4-tropic HIV-1 infections.

Combining Cell-Intrinsic and -Extrinsic Resistance to HIV-1 By Engineering Hematopoietic Stem Cells for CCR5 Knockout and B Cell Secretion of Therapeutic Antibodies.

Autologous transplantation of CCR5 null hematopoietic stem and progenitor cells (HSPCs) is the only known cure for HIV-1 infection. However, this treatment is limited because of the rarity of CCR5 -null matched donors, the morbidities associated with allogeneic transplantation, and the prevalence of HIV-1 strains resistant to CCR5 knockout (KO) alone. Here, we propose a one-time therapy through autologous transplantation of HSPCs genetically engineered ex vivo to produce both CCR5 KO cells and long-term secretion of potent HIV-1 inhibiting antibodies from B cell progeny. CRISPR-Cas9-engineered HSPCs maintain engraftment capacity and multi-lineage potential in vivo and can be engineered to express multiple antibodies simultaneously. Human B cells engineered to express each antibody secrete neutralizing concentrations capable of inhibiting HIV-1 pseudovirus infection in vitro . This work lays the groundwork for a potential one-time functional cure for HIV-1 through combining the long-term delivery of therapeutic antibodies against HIV-1 and the known efficacy of CCR5 KO HSPC transplantation.

Factors Influencing Parental Willingness to Consent to a Survey Study for Patients in the Pediatric Emergency Department.

OBJECTIVE: To identify factors that impact parental willingness to consent to research studies conducted for their children during visits to pediatric emergency departments (EDs). METHODS: Parents and guardians of children receiving care in our pediatric ED were approached and asked if they would be willing to let their child participate in a research study requiring the child to complete an electronic questionnaire. No such questionnaire existed, however, because the primary purpose was to ascertain the parent's willingness to let their child participate. All parents were debriefed and informed of the true purpose of the study and asked to complete a survey themselves to help understand factors that influenced their initial decision of whether to consent. Bivariate tests and logistic regression were used to evaluate unadjusted and adjusted associations between parent and patient characteristics and parental consent decision. RESULTS: We approached 431 eligible parents about the hypothetical research study involving their children, and 386 (89.6%) consented for their children to participate. After the debriefing, 392 (91.0%) parents consented to complete the parental survey. We observed statistically significant associations between shorter length of ED stay to approach for consent for the study ( P = 0.048) as well as longer travel time ( P = 0.03) and willingness to consent in bivariate analysis, though this did not hold in regression analysis. Regression analysis revealed parents of children who have previously participated in research had 79 times lower odds of consenting to participate in our study adjusted for parent race, ethnicity, actual and perceived length of stay, travel time to the ED, and altruism. CONCLUSIONS: A high proportion of parents consented to their child participating in research in our ED with previous child participation in research being associated with lower odds of parental consent even when adjusted for other factors. Our findings may inform future research practices and studies investigating parental perceptions and motivations surrounding research studies.

High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition.

Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool.

Occurrence, transport, and detection techniques of emerging pollutants in groundwater.

Emerging pollutants (EPs) are a group of different contaminants, such as hormones, pesticides, heavy metals, and drugs, usually found in concentrations between the order of ng and µg per liter. The global population's daily city and agro-industrial activities release EPs into the environment.  Due to the chemical nature of EPs and deficient wastewater treatment and management, they are transported to superficial and groundwater through the natural water cycle, where they can potentially cause harmful effects on living organisms. Recent efforts have focused on developing technology that allows EPs quantification and monitoring in real-time and in situ. The newly developed technology aims to provide accessible groundwater management that detects and treats EPs while avoiding their contact with living beings and their toxic effects. This review presents some of the recently reported techniques that have been applied to advance the detection of EPs in groundwater and potential technologies that can be used for EP removal.

Aberrant activation of TCL1A promotes stem cell expansion in clonal haematopoiesis.

Mutations in a diverse set of driver genes increase the fitness of haematopoietic stem cells (HSCs), leading to clonal haematopoiesis1. These lesions are precursors for blood cancers2-6, but the basis of their fitness advantage remains largely unknown, partly owing to a paucity of large cohorts in which the clonal expansion rate has been assessed by longitudinal sampling. Here, to circumvent this limitation, we developed a method to infer the expansion rate from data from a single time point. We applied this method to 5,071 people with clonal haematopoiesis. A genome-wide association study revealed that a common inherited polymorphism in the TCL1A promoter was associated with a slower expansion rate in clonal haematopoiesis overall, but the effect varied by driver gene. Those carrying this protective allele exhibited markedly reduced growth rates or prevalence of clones with driver mutations in TET2, ASXL1, SF3B1 and SRSF2, but this effect was not seen in clones with driver mutations in DNMT3A. TCL1A was not expressed in normal or DNMT3A-mutated HSCs, but the introduction of mutations in TET2 or ASXL1 led to the expression of TCL1A protein and the expansion of HSCs in vitro. The protective allele restricted TCL1A expression and expansion of mutant HSCs, as did experimental knockdown of TCL1A expression. Forced expression of TCL1A promoted the expansion of human HSCs in vitro and mouse HSCs in vivo. Our results indicate that the fitness advantage of several commonly mutated driver genes in clonal haematopoiesis may be mediated by TCL1A activation.

Prevalence of Anxiety Symptoms and Associated Clinical and Sociodemographic Factors in Mexican Adults Seeking Psychological Support for Grief During the COVID-19 Pandemic: A Cross-Sectional Study.

The COVID-19 pandemic is one of the greatest challenges in modern history, with more than four million confirmed deaths worldwide. To date, evidence regarding the psychological impact of the COVID-19 pandemic on grievers is scarce for developing countries such as Mexico. This study aimed to assess the levels of anxiety and associated concerns in a sample of Mexican adults bereaved during the COVID-19 outbreak. A cross-sectional study was conducted through the Duelo COVID (COVID Grief) platform, which is a self-guided online treatment. A total of 5,224 participants reported their anxiety, depression, sleep quality, avoidance, and arousal, prolonged grief symptoms, and medication consumption. Independent sample Mann-Whitney U-tests, chi-square tests, and Kruskal-Wallis tests, as well as multinomial logistic regression, were conducted. Results indicated that 90.4% of the participants reported clinical levels of anxiety, depression, and sleep affectations. The people who lost someone during the last 5 months scored higher in normal grief symptoms compared to the people whose loss was 6 months ago or more, and 9.8% of individuals reported the use of prescription medication, with anxiolytics and antidepressants being the most common. Females, younger respondents, unemployed people with a lower educational level, and participants who disclosed a recent suicide attempt were among those who reported medication consumption. Sleep problems were more frequent in older participants.

A phase 1/2 open label nonrandomized clinical trial of intravenous 2-hydroxypropyl-ß-cyclodextrin for acute liver disease in infants with Niemann-Pick C1.

INTRODUCTION: Niemann-Pick C (NPC) is an autosomal recessive disease due to defective NPC1 or NPC2 proteins resulting in endo-lysosomal storage of unesterified cholesterol in the central nervous system and liver. Acute liver disease in the newborn period may be self-limited or fatal. 2-hydroxypropyl-ß-cyclodextrin (2HPBCD) is a cholesterol-binding agent that reduces lysosomal cholesterol storage. We have enrolled 3 infants 0-6 months old with direct hyperbilirubinemia due to NPC1 or NPC2 liver disease in a Phase I/II open label clinical trial of intravenous 2HPBCD. METHODS: Infants received intravenous 2HPBCD twice a week for 6 weeks, followed by monthly infusion for 6-months. Primary outcome measure was reduction of plasma (3ß,5α,6ß-trihydroxy-cholan-24-oyl) glycine (TCG), a bile acid generated from cholesterol sequestered in lysosome. RESULTS: Three participants completed this protocol. A fourth patient received intravenous 2HPBCD under an emergency investigational new drug study but later expired from her underlying condition. The three protocol patients are living and have improved liver enzymes and TCG. No patient has experienced a drug-related adverse event. CONCLUSION: Intravenous 2HPBCD was tolerated in three infants with liver disease due to NPC.

Whole exome sequencing and functional characterization increase diagnostic yield in siblings with a 46, XY difference of sexual development (DSD).

Pathogenic biallelic variants in HSD17B3 result in 17ß-hydroxysteroid dehydrogenase 3 (17ß-HSD3) deficiency, variable disruption of testosterone production, and phenotypic diversity among 46, XY individuals with differences of sexual development (DSDs). We performed quad whole exome sequencing (WES) on two male siblings with microphallus, perineal hypospadias, and bifid scrotum and their unaffected parents. Both male siblings were compound heterozygous for a rare pathogenic HSD17B3 variant (c.239 G > A, p.R80Q) previously identified among individuals with 17ß-HSD3 deficiency and a HSD17B3 variant (c.641A > G, p.E214 G) of uncertain significance. Following WES, the siblings underwent hCG stimulation testing with measurement of testosterone, androstenedione, and dihydrotestosterone which was non-diagnostic. To confirm pathogenicity of the HSD17B3 variants, we performed transient transfection of HEK-293 cells and measured conversion of radiolabeled androstenedione to testosterone. Both HSD17B3 variants decreased conversion of radiolabeled androstenedione to testosterone. As pathogenic HSD17B3 variants are rare causes of 46, XY DSD and hCG stimulation testing may not be diagnostic for 17ß-HSD3 deficiency, WES in 46, XY individuals with DSDs can increase diagnostic yield and identify genomic variants for functional characterization of disruption of testosterone production.

A Self-Applied Multi-Component Psychological Online Intervention Based on UX, for the Prevention of Complicated Grief Disorder in the Mexican Population During the COVID-19 Outbreak: Protocol of a Randomized Clinical Trial.

Background: COVID-19 has taken many lives worldwide and due to this, millions of persons are in grief. When the grief process lasts longer than 6 months, the person is in risk of developing Complicated Grief Disorder (CGD). The CGD is related to serious health consequences. To reduce the probability of developing CGD a preventive intervention could be applied. In developing countries like Mexico, the psychological services are scarce, self-applied interventions could provide support to solve this problem and reduce the health impact even after the pandemic has already finished. Aims: To design and implement a self-applied intervention composed of 12 modules focused on the decrease of the risk of developing CGD, and increasing the life quality, and as a secondary objective to reduce the symptomatology of anxiety, depression, and increase of sleep quality. The Intervention Duelo COVID (Grief COVID) follows the principles of User Experience (UX) and is designed according to the needs and desires of a sample of the objective participants, to increase the adherence to the self-applied intervention, considered one of the main weaknesses of online interventions. Methods: A Randomized Controlled Trial will be conducted from the 22nd of December of 2020 to the first of June 2021. The participants will be assigned to an intervention with elements of Cognitive Behavioral Therapy, Acceptance and Commitment Therapy, Mindfulness and Positive Psychology. The control group will be a wait-list condition, that will receive the intervention 1.5-2 months after the pre-measurement were taken. The Power Size Calculation conducted through G*Power indicated the need for a total of 42 participants, which will be divided by 21 participants in each group. The platform will be delivered through responsive design assuring with this that the intervention will adapt to the screen size of cellphones, tablets, and computers. Ethics and Dissemination: The study counts with the approval of the Research Ethics Committee of the Autonomous University of Ciudad Juárez, México, and it is registered in Clinical Trials (NCT04638842). The article is sent and registered in clinical trials before the recruitment started. The results will be reported in future conferences, scientific publications, and media.

A Self-Administered Multicomponent Web-Based Mental Health Intervention for the Mexican Population During the COVID-19 Pandemic: Protocol for a Randomized Controlled Trial.

BACKGROUND: The COVID-19 pandemic has become a public health emergency of international concern; it has not only threatened people's physical health but has also affected their mental health and psychological well-being. It is necessary to develop and offer strategies to reduce the psychological impact of the outbreak and promote adaptive coping. OBJECTIVE: This study protocol aims to describe a self-administered web-based intervention (Mental Health COVID-19) based on the principles of positive psychology supported by elements of cognitive behavioral therapy and behavioral activation therapy to reduce the symptoms of anxiety and depression and increase positive emotions and sleep quality during and after the COVID-19 outbreak through a telepsychology system. METHODS: A randomized controlled clinical superiority trial with two independent groups will be performed, with intrasubject measures at four evaluation periods: pretest, posttest, 3-month follow-up, and 6-month follow-up. Participants will be randomly assigned to one of two groups: self-administered intervention with assistance via chat or self-administered intervention without assistance via chat. The total required sample size will be 166 participants (83 per group). RESULTS: The clinical trial is ongoing. This protocol was approved by the Research Ethics Board of the Free School of Psychology-University of Behavioral Sciences (Escuela libre de Psicología-Universidad de Ciencias del Comportamiento). The aim is to publish the preliminary results in December 2020. A conservative approach will be adopted, and the size effect will be estimated using the Cohen d index with a significance level (α) of .05 (95% reliability) and a conventional 80% power statistic. CONCLUSIONS: The central mechanism of action will be to investigate the effectiveness of an intervention based on positive psychology through a web platform that can be delivered through computers and tablets, with content that has been rigorously contextualized to the Mexican culture to provide functional strategies to help the target users cope with the COVID-19 pandemic. TRIAL REGISTRATION: ClinicalTrials.gov NCT04468893; https://clinicaltrials.gov/ct2/show/NCT04468893. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/23117.

Effectiveness of behavioral activation for depression treatment in medical students: Study protocol for a quasi-experimental design.

INTRODUCTION: Depression is a frequent mood disorder among medical students that can lead to multiple negative consequences at individual and social level (such as academic achievement and interpersonal conflicts) as well as patient care performance. Therefore, the need of depression decreasing treatments in medical students is important. This study is designed to evaluate the effectiveness of the Behavioral Activation Treatment for Depression in a sample of Mexican medical students. METHODS: This study will be performed under a quasi-experimental design to verify the effectiveness of the Behavioral Activation Treatment for Depression to reduce depressive symptoms in medical students from two public universities in northwestern Mexico. The participants will be assessed with the Center for Epidemiologic Studies Depression Scale, the Depression Anxiety Stress Scales, the Pittsburgh Sleep Quality Index, and the Plutchik Suicide Risk Scale. In addition to the psychometric assessment, there will be an electroencephalogram evaluation using the EMOTIV (v 1.1) device. RESULTS: A pre-post intervention of 10 Behavioral Activation Treatment for Depression sessions will be implemented. The results of the effectiveness of the Behavioral Activation Treatment for Depression will be analyzed in five measures at pre-post intervention and two follow-ups of 3 and 6 months. CONCLUSIONS: This study looks for evidence regarding the efficacy and feasibility of the Behavioral Activation Treatment for Depression in a sample of medical students from two public universities in Mexico with high levels of depression along with stress and anxiety.

Respiratory health assessment and exposure to polycyclic aromatic hydrocarbons in Mexican indigenous population.

Indoor air pollution is an important risk factor for the generation of lung diseases in developing countries. The indigenous population is particularly susceptible to be exposed to the mixture of pollutants from the biomass burning, among them, polycyclic aromatic hydrocarbons (PAHs). The objective of this study was to assess respiratory health and exposure to PAHs in indigenous populations of the Huasteca Potosina in Mexico. The urinary metabolite 1-hydroxypyrene (1-OHP) was evaluated by HPLC with fluorescence detector, the forced expiratory volume in one second (FEV1) and the FEV1/FVC ratio (forced vital capacity) by spirometry in the Teenek indigenous adult population of the communities from Tocoy (TOC), Xolol (XOL), and Tanjajnec (TAN). A total of 134 subjects participated in the study: 64 from TOC, 30 from XOL, and 40 from TAN; in all the communities, high percentages of overweight and obesity were presented (from 50 to 73%). The average hours of firewood usage per year were 281.06, 284.6, and 206.6 in TOC, XOL, and TAN, respectively. The average of the three communities of the % FEV1 post-bronchodilator was 86.1%. There were identified from 4.5 to 6.6% and from 12.5 to 15.5% of spirometric obstructive and restrictive patterns respectively, in all communities. The highest exposure levels reported as median were found in TOC (1.15 µmol/mol of creatinine) followed by TAN (0.94 µmol/mol of creatinine) and XOL (0.65 µmol/mol of creatinine). Considering the magnitude of the indigenous population exposed to pollutants from the biomass burning and the possible effects on respiratory health, it is important to design strategies that mitigate exposure and evaluate the effectiveness through biological monitoring and effects.