Nonsense CD247 mutations show dominant-negative features in TCR expression and function.

BACKGROUND: The invariant TCRζ/CD247 homodimer is crucial for TCR/CD3 expression and signaling through its three immunoreceptor tyrosine-based activation motifs (ITAMs). Homozygous null mutations in CD247 lead to immunodeficiency, while carriers exhibit 50% reduced surface CD3. It is unclear whether carriers of other CD247 variants show dominant-negative effects. OBJECTIVE: To analyze and model the potential impact on TCR expression and function of heterozygous nonsense CD247 mutations found in patients with signs of immunodeficiency or autoimmunity. METHODS: Jurkat T cells, either wild-type (WT) or CRISPR/Cas9-edited CD247-deficient (ZKO), were lentivirally transduced with wild-type CD247 or mutations ablating one (Q142X), two (Q101X), or three (Q70X) ITAMs. RESULTS: Three patients from unrelated families were studied. Two heterozygous nonsense CD247 mutations were identified (p.Y152X and p.Q101X), which affected ITAM-3 and ITAM-2+3, respectively. Both mutations were associated with low surface CD3 expression, normal intracellular CD247 levels using a transmembrane-specific antibody but very low intracellular CD247 levels using an ITAM-3-specific one, suggesting the presence of truncated variants in T cells. Transduction of the mutations lacking 1, 2, or 3 ITAMs into ZKO could not restore normal surface CD3 expression (only 60%, 22% and 10%, respectively), whereas in WT they reduced it (to 39%, 19% and 9% of normal levels), and both effects were ITAM number dependent. All six transfectants showed reduced CD69 induction (25-50%), indicating that they were unable to signal downstream properly neither isolated nor associated with wild-type CD247. CONCLUSION: Our results suggest that CD247 variants lacking ITAMs due to nonsense, but not null, mutations are defective for normal TCR assembly and exert a dominant-negative effect on TCR expression and signaling in vitro. This, in turn, may correlate with clinical features in vivo.

Lymphocyte integration of complement cues.

We address current data, views and puzzles on the emerging topic of regulation of lymphocytes by complement proteins or fragments. Such regulation is believed to take place through complement receptors (CR) and membrane complement regulators (CReg) involved in cell function or protection, respectively, including intracellular signalling. Original observations in B cells clearly support that complement cues through CR improve their performance. Other lymphocytes likely integrate complement-derived signals, as most lymphoid cells constitutively express or regulate CR and CReg upon activation. CR-induced signals, particularly by anaphylatoxins, clearly regulate lymphoid cell function. In contrast, data obtained by CReg crosslinking using antibodies are not always confirmed in human congenital deficiencies or knock-out mice, casting doubts on their physiological relevance. Unsurprisingly, human and mouse complement systems are not completely homologous, adding further complexity to our still fragmentary understanding of complement-lymphocyte interactions.

Perspectives, Experiences, and Practices in the Use of Digital Information Technologies in the Management of Depression and Alcohol Use Disorder in Health Care Systems in Colombia.

Digital information technologies are increasingly used in the treatment of mental health disorders. Through this qualitative study, researchers illuminated perspectives, experiences, and practices among diverse stakeholders in the use of digital information technologies in the management of depression and alcohol use disorders in Colombia. In-depth interviews and focus groups were conducted in five primary care institutions across Colombia. Thematic analysis was used to analyze the data. The use of technology in the treatment of mental health disorders can facilitate the evaluation and diagnosis, treatment, and promotion and prevention of mental health disorders, as well as multiple nonmental health applications in the primary care setting. Potential barriers to the use of technology in this setting include challenges of digital literacy, access to technology, confidentiality, and financing. This study can inform the implementation of digital information technologies in the care of depression and problematic alcohol use within health care systems in Colombia.

Formative and Summative Assessment in Veterinary Pathology and Other Courses at a Mexican Veterinary College.

The Faculty of Veterinary Medicine and Animal Science of the National Autonomous University of Mexico (UNAM) uses the Moodle learning management system for formative and summative computer assessment. The authors of this article-the teacher primarily responsible for Moodle implementation and a researcher who is a recent Moodle adopter-describe and discuss the students' and teachers' attitudes to summative and formative computer assessment in Moodle. Item analysis of quiz results helped us to identify and fix poorly performing questions, which greatly reduced student complaints and improved objective assessment. The use of certainty-based marking (CBM) in formative assessment in veterinary pathology was well received by the students and should be extended to more courses. The importance of having proficient computer support personnel should not be underestimated. A properly translated language pack is essential for the use of Moodle in a language other than English.

RecX facilitates homologous recombination by modulating RecA activities.

The Bacillus subtilis recH342 strain, which decreases interspecies recombination without significantly affecting the frequency of transformation with homogamic DNA, carried a point mutation in the putative recX (yfhG) gene, and the mutation was renamed as recX342. We show that RecX (264 residues long), which shares partial identity with the Proteobacterial RecX (<180 residues), is a genuine recombination protein, and its primary function is to modulate the SOS response and to facilitate RecA-mediated recombinational repair and genetic recombination. RecX-YFP formed discrete foci on the nucleoid, which were coincident in time with RecF, in response to DNA damage, and on the poles and/or the nucleoid upon stochastic induction of programmed natural competence. When DNA was damaged, the RecX foci co-localized with RecA threads that persisted for a longer time in the recX context. The absence of RecX severely impaired natural transformation both with plasmid and chromosomal DNA. We show that RecX suppresses the negative effect exerted by RecA during plasmid transformation, prevents RecA mis-sensing of single-stranded DNA tracts, and modulates DNA strand exchange. RecX, by modulating the "length or packing" of a RecA filament, facilitates the initiation of recombination and increases recombination across species.

The immunopathological landscape of human pre-TCRα deficiency: From rare to common variants.

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-α T cell receptor (pre-TCRα) expression. Low circulating naive αß T cell counts at birth persisted over time, with normal memory αß and high γδ T cell counts. Their TCRα repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue αß T cell development. Only a minority of these individuals were sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naive αß T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αß T cells, autoimmune conditions were more frequent in these patients compared with the general population.

Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins.

Bacillus subtilis pnpA gene product, polynucleotide phosphorylase (PNPase), is involved in double-strand break (DSB) repair via homologous recombination (HR) or non-homologous end-joining (NHEJ). RecN is among the first responders to localize at the DNA DSBs, with PNPase facilitating the formation of a discrete RecN focus per nucleoid. PNPase, which co-purifies with RecA and RecN, was able to degrade single-stranded (ss) DNA with a 3' → 5' polarity in the presence of Mn(2+) and low inorganic phosphate (Pi) concentration, or to extend a 3'-OH end in the presence dNDP · Mn(2+). Both PNPase activities were observed in evolutionarily distant bacteria (B. subtilis and Escherichia coli), suggesting conserved functions. The activity of PNPase was directed toward ssDNA degradation or polymerization by manipulating the Pi/dNDPs concentrations or the availability of RecA or RecN. In its dATP-bound form, RecN stimulates PNPase-mediated polymerization. ssDNA phosphorolysis catalyzed by PNPase is stimulated by RecA, but inhibited by SsbA. Our findings suggest that (i) the PNPase degradative and polymerizing activities might play a critical role in the transition from DSB sensing to end resection via HR and (ii) by blunting a 3'-tailed duplex DNA, in the absence of HR, B. subtilis PNPase might also contribute to repair via NHEJ.

Detection of the early stage of recombinational DNA repair by silicon nanowire transistors.

A silicon nanowire-based biosensor has been designed and applied for label-free and ultrasensitive detection of the early stage of recombinational DNA repair by RecA protein. Silicon nanowires transistors were fabricated by atomic force microscopy nanolithography and integrated into a microfluidic environment. The sensor operates by measuring the changes in the resistance of the nanowire as the biomolecular reactions proceed. We show that the nanoelectronic sensor can detect and differentiate several steps in the binding of RecA to a single-stranded DNA filament taking place on the nanowire-aqueous interface. We report relative changes in the resistance of 3.5% which are related to the interaction of 250 RecA·single-stranded DNA complexes. Spectroscopy data confirm the presence of the protein-DNA complexes on the functionalized silicon surfaces.

Working and non-working conditions related to the presence of depressive symptoms in women workers with subsistence jobs in the centre of Medellín, 2015-2019.

OBJECTIVE: To determine the working and non-working conditions related to depressive symptoms in informal workers in the centre of Medellín in 2015-2019. METHODS: Cross-sectional study with analytical intention and primary sources of information. Data collection with assisted survey in one of its union headquarters in 2016, after a pilot test and standardisation of pollsters. A total of 291 women workers were taken as a census, and their working and non-working conditions were explored. RESULTS: The women had an average age of 45 years, ≤5 years of schooling, low and middle-income housing, and income below the 2016 minimum wage. About 60% suffered moderate-severe food insecurity, and received no state benefits. They were mainly the head of the family, with 1 or 2 dependents, and were responsible for the work at home. They worked at least 8 h a day, 6 or 7 days a week, with parents or relatives selling in the street, and at least 20 years in their work. About 60% had a partner, 21.6% with family dysfunction, and 15.4% moderate-severe depressive symptoms. Living in one room or a slum, with a low socioeconomic status and moderate-severe family dysfunction were associated with, and contributed to the explanation of, moderate-severe depressive symptoms. CONCLUSIONS: The non-working conditions that are associated with and explain the moderate-severe depressive symptoms of female workers can be modified with actions that impact on the social determinants of health.

Bacillus subtilis polynucleotide phosphorylase 3'-to-5' DNase activity is involved in DNA repair.

In the presence of Mn(2+), an activity in a preparation of purified Bacillus subtilis RecN degrades single-stranded (ss) DNA with a 3' --> 5' polarity. This activity is not associated with RecN itself, because RecN purified from cells lacking polynucleotide phosphorylase (PNPase) does not show the exonuclease activity. We show here that, in the presence of Mn(2+) and low-level inorganic phosphate (P(i)), PNPase degrades ssDNA. The limited end-processing of DNA is regulated by ATP and is inactive in the presence of Mg(2+) or high-level P(i). In contrast, the RNase activity of PNPase requires Mg(2+) and P(i), suggesting that PNPase degradation of RNA and ssDNA occur by mutually exclusive mechanisms. A null pnpA mutation (DeltapnpA) is not epistatic with Delta recA, but is epistatic with DeltarecN and Delta ku, which by themselves are non-epistatic. The addA5, Delta recO, Delta recQ (Delta recJ), Delta recU and Delta recG mutations (representative of different epistatic groups), in the context of DeltapnpA, demonstrate gain- or loss-of-function by inactivation of repair-by-recombination, depending on acute or chronic exposure to the damaging agent and the nature of the DNA lesion. Our data suggest that PNPase is involved in various nucleic acid metabolic pathways, and its limited ssDNA exonuclease activity plays an important role in RecA-dependent and RecA-independent repair pathways.

Barriers and facilitators to the diagnosis and treatment of depression in primary care in Colombia: Perspectives of providers, healthcare administrators, patients and community representatives.

INTRODUCTION: Depression represents a major disease burden in Colombia. To better understand opportunities to improve access to mental healthcare in Colombia, a research team at Javeriana University conducted formative qualitative research to explore stakeholders' experiences with the integration of mental healthcare into the primary care system. METHODS: The research team conducted 16 focus groups and 4 in-depth interviews with patients, providers, health administrators and representatives of community organisations at five primary care clinics in Colombia, and used thematic analysis to study the data. RESULTS: Themes were organised into barriers and facilitators at the level of patients, providers, organisations and facilities. Barriers to the treatment of depression included stigma, lack of mental health literacy at the patient and provider level, weak links between care levels, and continued need for mental health prioritization at the national level. Facilitators to the management of depression in primary care included patient support systems, strong patient-provider relationships, the targeting of depression interventions and national depression guidelines. DISCUSSION: This study elucidates the barriers to depression care in Colombia, and highlights action items for further integrating depression care into the primary care setting.

Working and Non-Working Conditions Related to the Presence of Depressive Symptoms in Women Workers with Subsistence Jobs in the Centre of Medellín, 2015-2019. / Condiciones laborales y extralaborales relacionadas con la presencia de síntomas depresivos en mujeres trabajadoras con empleos de subsistencia en el centro de Medellín, 2015-2019.

OBJECTIVE: To determine the working and non-working conditions related to depressive symptoms in informal workers in the centre of Medellín in 2015-2019. METHODS: Cross-sectional study with analytical intention and primary sources of information. Data collection with assisted survey in one of its union headquarters in 2016, after a pilot test and standardisation of pollsters. A total of 291 women workers were taken as a census, and their working and non-working conditions were explored. RESULTS: The women had an average age of 45 years, ≤5 years of schooling, low and middle-income housing, and income below the 2016 minimum wage. About 60% suffered moderate-severe food insecurity, and received no state benefits. They were mainly the head of the family, with 1 or 2 dependents, and were responsible for the work at home. They worked at least 8hours a day, 6 or 7 days a week, with parents or relatives selling in the street, and at least 20 years in their work. About 60% had a partner, 21.6% with family dysfunction, and 15.4% moderate-severe depressive symptoms. Living in one room or a slum, with a low socioeconomic status and moderate-severe family dysfunction were associated with, and contributed to the explanation of, moderate-severe depressive symptoms. CONCLUSIONS: The non-working conditions that are associated with and explain the moderate-severe depressive symptoms of female workers can be modified with actions that impact on the social determinants of health.

Addressing harmful alcohol use in primary care in Colombia: Understanding the sociocultural context.

Harmful alcohol use is a public health problem worldwide, contributing to an estimated 5.1% of the global burden of illness. Screening and addressing at-risk drinking in primary care settings is an empirically supported health care intervention strategy to help reduce the burden of alcohol-use problems. In preparation for introducing screening and treatment for at-risk drinking in primary care clinics in Colombia, we conducted interviews with clinicians, clinic administrators, patients, and participants in Alcoholics Anonymous. Interviews were conducted within the framework of the Detección y Atención Integral de Depresión y Abuso de Alcohol en Atención Primaria (DIADA, [Detection and Integrated Care for Depression and Alcohol Use in Primary Care] www.project-diada.org) research project, and its qualitative phase that consisted of the collection of data from 15 focus groups, 6 interviews and field observations in 5 regional settings. All participants provided informed consent to participate in this research. Findings revealed the association of harmful alcohol use with a culture of consumption, within which it is learned and socially accepted practice. Recognition of harmful alcohol consumption includes a social context that influences its screening, diagnosis and prevention. The discussion highlights how, despite the existence of institutional strategies in healthcare settings and the awareness of the importance of at-risk drinking among health personnel, the recognition of the harmful use of alcohol as a pathology should be embedded in an understanding of historical, social and cultural dimensions that may affect different identification and care scenarios.

CD3G or CD3D Knockdown in Mature, but Not Immature, T Lymphocytes Similarly Cripples the Human TCRαß Complex.

The human αß T-cell receptor (TCR) is composed of a variable heterodimer (TCRαß) and three invariant dimers (CD3γε, CD3δε, and ζζ/CD2472). The role of each invariant chain in the stepwise interactions among TCR chains along the assembly is still not fully understood. Despite the high sequence homology between CD3γ and CD3δ, the clinical consequences of the corresponding immunodeficiencies (ID) in humans are very different (mild and severe, respectively), and mouse models do not recapitulate findings in human ID. To try to understand such disparities, we stably knocked down (KD) CD3D or CD3G expression in the human Jurkat T-cell line and analyzed comparatively their impact on TCRαß assembly, transport, and surface expression. The results indicated that TCR ensembles were less stable and CD3ε levels were lower when CD3γ, rather than CD3δ, was scarce. However, both defective TCR ensembles were strongly retained in the ER, lacked ζζ/CD2472, and barely reached the T-cell surface (<11% of normal controls) in any of the CD3 KD cells. This is in sharp contrast to human CD3γ ID, whose mature T cells express higher levels of surface TCR (>30% vs. normal controls). CD3 KD of human T-cell progenitors followed by mouse fetal thymus organ cultures showed high plasticity in emerging immature polyclonal T lymphocytes that allowed for the expression of significant TCR levels which may then signal for survival in CD3γ, but not in CD3δ deficiency, and explain the immunological and clinical disparities of such ID cases.

Access barriers, self-recognition, and recognition of depression and unhealthy alcohol use: A qualitative study.

INTRODUCTION: Access to healthcare services involves a complex dynamic, where mental health conditions are especially disadvantaged, due to multiple factors related to the context and the involved stakeholders. However, a characterisation of this phenomenon has not been carried out in Colombia, and this motivates the present study. OBJECTIVES: The objective of this study was to explore the causes that affect access to health services for depression and unhealthy alcohol use in Colombia, according to various stakeholders involved in the care process. METHODS: In-depth interviews and focus groups were conducted with health professionals, administrative professionals, users, and representatives of community health organisations in five primary and secondary-level institutions in three regions of Colombia. Subsequently, to describe access to healthcare for depression and unhealthy alcohol use, excerpts from the interviews and focus groups were coded through content analysis, expert consensus, and grounded theory. Five categories of analysis were created: education and knowledge of the health condition, stigma, lack of training of health professionals, culture, and structure or organisational factors. RESULTS: We characterised the barriers to a lack of illness recognition that affected access to care for depression or unhealthy alcohol use according to users, healthcare professionals and administrative staff from five primary and secondary care centres in Colombia. The groups identified that lack of recognition of depression was related to low education and knowledge about this condition within the population, stigma, and lack of training of health professionals, as well as to culture. For unhealthy alcohol use, the participants identified that low education and knowledge about this condition, lack of training of healthcare professionals, and culture affected its recognition, and therefore, healthcare access. Neither structural nor organisational factors seemed to play a role in the recognition or self-recognition of these conditions. CONCLUSIONS: This study provides essential information for the search for factors that undermine access to mental health in the Colombian context. Likewise, it promotes the generation of hypotheses that can lead to the development and implementation of tools to improve care in the field of mental illness.

Overexpression of the recA gene decreases oral but not intraperitoneal fitness of Salmonella enterica.

Transcription of the Salmonella enterica recA gene is negatively controlled by the LexA protein, the repressor of the SOS response. The introduction of a mutation (recAo6869) in the LexA binding site, in the promoter region of the S. enterica ATCC 14028 recA gene, allowed the analysis of the effect that RecA protein overproduction has on the fitness of this virulent strain. The fitness of orally but not intraperitoneally inoculated recAo6869 cells decreased dramatically. However, the SOS response of this mutant was induced normally, and there was no increase in the sensitivity of the strain toward DNA-damaging agents, bile salts, or alterations in pH. Nevertheless, S. enterica recAo6869 cells were unable to swarm and their capacity to cross the intestinal epithelium was significantly reduced. The swarming deficiency in recAo6869 cells is independent of the flagellar phase. Moreover, swimming activity of the recAo6869 strain was not diminished with respect to the wild type, indicating that the flagellar synthesis is not affected by RecA protein overproduction. In contrast, swarming was recovered in a recAo6869 derivative that overproduced CheW, a protein known to be essential for this function. These data demonstrate that an equilibrium between the intracellular concentrations of RecA and CheW is necessary for swarming in S. enterica. Our results are the first to point out that the SOS response plays a critical role in the prevention of DNA damage by abolishing bacterial swarming in the presence of a genotoxic compound.

Dynamic structures of Bacillus subtilis RecN-DNA complexes.

Genetic and cytological evidences suggest that Bacillus subtilis RecN acts prior to and after end-processing of DNA double-strand ends via homologous recombination, appears to participate in the assembly of a DNA repair centre and interacts with incoming single-stranded (ss) DNA during natural transformation. We have determined the architecture of RecN-ssDNA complexes by atomic force microscopy (AFM). ATP induces changes in the architecture of the RecN-ssDNA complexes and stimulates inter-complex assembly, thereby increasing the local concentration of DNA ends. The large CII and CIII complexes formed are insensitive to SsbA (counterpart of Escherichia coli SSB or eukaryotic RPA protein) addition, but RecA induces dislodging of RecN from the overhangs of duplex DNA molecules. Reciprocally, in the presence of RecN, RecA does not form large RecA-DNA networks. Based on these results, we hypothesize that in the presence of ATP, RecN tethers the 3'-ssDNA ends, and facilitates the access of RecA to the high local concentration of DNA ends. Then, the resulting RecA nucleoprotein filaments, on different ssDNA segments, might promote the simultaneous genome-wide homology search.

Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein.

Ubiquitous RarA AAA+ ATPases play crucial roles in the cellular response to blocked replication forks in pro- and eukaryotes. Here, we provide evidence that absence of RarA reduced the viability of ΔrecA, ΔrecO, and recF15 cells during unperturbed growth. The rarA gene was epistatic to recO and recF genes in response to H2O2- or MMS-induced DNA damage. Conversely, the inactivation of rarA partially suppressed the HR defect of mutants lacking end-resection (ΔaddAB, ΔrecJ, ΔrecQ, ΔrecS) or branch migration (ΔruvAB, ΔrecG, ΔradA) activity. RarA contributes to RecA thread formation, that are thought to be the active forms of RecA during homology search. The absence of RarA reduced RecA accumulation, and the formation of visible RecA threads in vivo upon DNA damage. When ΔrarA was combined with mutations in genuine RecA accessory genes, RecA accumulation was further reduced in ΔrarA ΔrecU and ΔrarA ΔrecX double mutant cells, and was blocked in ΔrarA recF15 cells. These results suggest that RarA contributes to the assembly of RecA nucleoprotein filaments onto single-stranded DNA, and possibly antagonizes RecA filament disassembly.

Uso de aromaterapia en gestantes: una revisión de la literatura / Uso da aromaterapia em mulheres grávidas: uma revisão da literatura / Use of aromatherapy in pregnant women: a review of the literature

Introducción: Algunas mujeres, durante su gestación pueden padecer de sintomatología molesta que interfiere con su cotidianidad, molestias como náuseas, vómitos, dolores lumbares, y fluctuaciones emocionales. Tales molestias podrían manejarse con técnicas como la aromaterapia, propendiendo por ofrecer bienestar sin posibles efectos adversos para el binomio madre e hijo. El objetivo de este artículo es identificar los beneficios que produce la aromaterapia en la gestación mediante una revisión documental. Metodología: Se realizó una búsqueda de artículos científicos en bases de datos como: Science direct, google academic, EBSCO, Pubmed, Gale Power Search, BASE (Bielefeld academic search engine), Proquest; utilizando los DECS Aromatherapy, Pregnancy, Lavandula, Citrus, Jengibre y Rosa en inglés y español, usando el conector booleano AND, de esta búsqueda se obtuvo 50 artículos de investigación que cumplían con el objetivo propuesto. Resultados: se identificó la utilización de la aromaterapia como recurso para las náuseas, vómitos y el dolor, además de lograr mejorar el sueño y disminuir la fatiga. Conclusiones: Se determina entonces que los aceites esenciales usados en los estudios fueron eficaces disminuyendo síntomas molestos en la mayoría de las gestantes participantes de los estudios analizados

Introdução: algumas mulheres, durante a sua gestação podem apresentar sintomas incômodos que interferem com a sua vida diária tais como: náusea, vômitos, dor na lombar e mudanças emocionais. Ditos sintomas podem tratar-se com terapias como a aromaterapia, fornecendo bem-estar, sem efeitos adversos para a mãe e o feto. Objetivo: identificar os benefícios da aromaterapia em mulheres grávidas através de uma revisão documental. Materiais e métodos: procuraram-se artigos científicos em Science direct, Google academic, EBSCO, Pubmed, Gale Power Search, BASE (Bielefeld academic search engine) e Proquest; utilizando os DeCs Aromatherapy, Pregnancy, Lavandula, Citrus, Gengibre e Rosa em inglês e espanhol. Dessa pesquisa surgiram 50 artigos que cumpriam com o objetivo do estudo. Resultados: identificou-se o uso da aromaterapia como recurso para evitar a náusea, o vomito e a dor; melhorando também o sono e a fadiga. Conclusão: determinou-se que os aceites essenciais usados nos estudos foram eficazes porque reduziram os sintomas incômodos na maioria das grávidas dos estudos realizados.