Paternal microbiome perturbations impact offspring fitness.

The gut microbiota operates at the interface of host-environment interactions to influence human homoeostasis and metabolic networks1-4. Environmental factors that unbalance gut microbial ecosystems can therefore shape physiological and disease-associated responses across somatic tissues5-9. However, the systemic impact of the gut microbiome on the germline-and consequently on the F1 offspring it gives rise to-is unexplored10. Here we show that the gut microbiota act as a key interface between paternal preconception environment and intergenerational health in mice. Perturbations to the gut microbiota of prospective fathers increase the probability of their offspring presenting with low birth weight, severe growth restriction and premature mortality. Transmission of disease risk occurs via the germline and is provoked by pervasive gut microbiome perturbations, including non-absorbable antibiotics or osmotic laxatives, but is rescued by restoring the paternal microbiota before conception. This effect is linked with a dynamic response to induced dysbiosis in the male reproductive system, including impaired leptin signalling, altered testicular metabolite profiles and remapped small RNA payloads in sperm. As a result, dysbiotic fathers trigger an elevated risk of in utero placental insufficiency, revealing a placental origin of mammalian intergenerational effects. Our study defines a regulatory 'gut-germline axis' in males, which is sensitive to environmental exposures and programmes offspring fitness through impacting placenta function.

Microscopy-Based Chromosome Conformation Capture Enables Simultaneous Visualization of Genome Organization and Transcription in Intact Organisms.

Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. Here we present a multiplexed, sequential imaging approach (Hi-M) that permits simultaneous detection of chromosome organization and transcription in single nuclei. This allowed us to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome unpairing during awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understanding the mechanisms and consequences of the 4D organization of the genome.

The emotional impact of COVID-19 on Spanish nurses and potential strategies to reduce it.

Background: Although the emotional and psychological impact of nurses' work had been identified before the COVID-19 pandemic, the pandemic aggravated risk indicators for their mental health. Aim: The objective of this study was to analyse the levels of anxiety, depression, post-traumatic stress and burnout of nurses in the Balearic Islands (Spain) during the pandemic to identify possible sociodemographic and related occupational factors. Design: A cross-sectional study of 892 nurses was conducted during four weeks from February to March 2021. Methods: Sociodemographic data related to the pandemic were collected and anxiety, depression, burnout and post-traumatic stress were measured with validated scales. A multivariate and predictive analysis was carried out with risk estimates. Findings: About 75.6% of the nurses had experience in COVID-19 units, and 49.1% had worked for more than 10 months in a COVID-19 unit. Nurses in COVID-19 units (hospital ward or ICU) were more likely to report emotional fatigue (OR 1.9, p < 0.001) and anxiety (OR 1.5, p = 0.021). In general, moderate post-traumatic stress was evident in general nurses (p = 0.027), and severe post-traumatic stress was evident in ICU nurses (p = 0.027). A 1.24-month reduction in COVID-19 patient care predicted reduced levels of emotional fatigue (5.45 points), depersonalisation (1.87 points) and post-traumatic stress (4.65 points) in nurses. Conclusion: Given the occurrence of new waves of COVID-19, the need to establish preventive strategies that focus on the personal and occupational characteristics related to these indicators and to implement urgent psychological support strategies is demonstrated. Impact: Given these findings, it is imperative solutions are urgently applied in order to prevent compounding risk to the health system.

Evolutionary fates of universal stress protein paralogs in Platyhelminthes.

BACKGROUND: Universal stress proteins (USPs) are present in all domains of life. Their expression is upregulated in response to a large variety of stress conditions. The functional diversity found in this protein family, paired with the sequence degeneration of the characteristic ATP-binding motif, suggests a complex evolutionary pattern for the paralogous USP-encoding genes. In this work, we investigated the origin, genomic organization, expression patterns and evolutionary history of the USP gene family in species of the phylum Platyhelminthes. RESULTS: Our data showed a cluster organization, a lineage-specific distribution, and the presence of several pseudogenes among the USP gene copies identified. The absence of a well conserved -CCAATCA- motif in the promoter region was positively correlated with low or null levels of gene expression, and with amino acid changes within the ligand binding motifs. Despite evidence of the pseudogenization of various USP genes, we detected an important functional divergence at several residues, mostly located near sites that are critical for ligand interaction. CONCLUSIONS: Our results provide a broad framework for the evolution of the USP gene family, based on the emergence of new paralogs that face very contrasting fates, including pseudogenization, subfunctionalization or neofunctionalization. This framework aims to explain the sequence and functional diversity of this gene family, providing a foundation for future studies in other taxa in which USPs occur.

Structured Illumination Microscopy Improves Spot Detection Performance in Spatial Transcriptomics.

Spatial biology is a rapidly growing research field that focuses on the transcriptomic or proteomic profiling of single cells within tissues with preserved spatial information. Imaging-based spatial transcriptomics uses epifluorescence microscopy, which has shown remarkable results for the identification of multiple targets in situ. Nonetheless, the number of genes that can be reliably visualized is limited by the diffraction of light. Here, we investigate the effect of structured illumination (SIM), a super-resolution microscopy approach, on the performance of single-gene transcript detection in spatial transcriptomics experiments. We performed direct mRNA-targeted hybridization in situ sequencing for multiple genes in mouse coronal brain tissue sections. We evaluated spot detection performance in widefield and confocal images versus those with SIM in combination with 20×, 25× and 60× objectives. In general, SIM increases the detection efficiency of gene transcript spots compared to widefield and confocal modes. For each case, the specific fold increase in localizations is dependent on gene transcript density and the numerical aperture of the objective used, which has been shown to play an important role, especially for densely clustered spots. Taken together, our results suggest that SIM has the capacity to improve spot detection and overall data quality in spatial transcriptomics.

Multiple parameters shape the 3D chromatin structure of single nuclei at the doc locus in Drosophila.

The spatial organization of chromatin at the scale of topologically associating domains (TADs) and below displays large cell-to-cell variations. Up until now, how this heterogeneity in chromatin conformation is shaped by chromatin condensation, TAD insulation, and transcription has remained mostly elusive. Here, we used Hi-M, a multiplexed DNA-FISH imaging technique providing developmental timing and transcriptional status, to show that the emergence of TADs at the ensemble level partially segregates the conformational space explored by single nuclei during the early development of Drosophila embryos. Surprisingly, a substantial fraction of nuclei display strong insulation even before TADs emerge. Moreover, active transcription within a TAD leads to minor changes to the local inter- and intra-TAD chromatin conformation in single nuclei and only weakly affects insulation to the neighboring TAD. Overall, our results indicate that multiple parameters contribute to shaping the chromatin architecture of single nuclei at the TAD scale.

Clinical Impact of the Capacity-Motivation-Opportunity Pharmacist-Led Intervention in People Living with HIV in Spain, 2019-2020.

Background: People living with HIV (PLWH) have significantly enhanced their life expectancy. Consequently, age-associated comorbidities and related health conditions are increasingly found in PLWH complicating their clinical management. Objective: To determine the effect of the capacity-motivation-opportunity (CMO) structured pharmaceutical care intervention for improving clinical health-care results frequently associated to PLWH. Methods: Multicenter, prospective, pre-post intervention study evaluating the CMO pharmacist-led program in adult PLWH was conducted between September 2019 and September 2020 with six months of follow-up. The primary objective of this study was to determine differences in clinical outcomes (total cholesterol, triglycerides, HDL, blood pressure and glycosylated hemoglobin) and variation in the patient's activation measure before and after the intervention. Results: A total of 61 patients were included, 72% were men with a median age of 53 years. After the implementation of the pharmacist-driven program, the percentage of patients with high levels of total cholesterol decreased significantly (18% to 4.9%; p < 0.001). Similarly, the prevalence of patients with high levels of triglycerides, HDL or with hypertension was significantly lower post intervention (13.1% to 6.6%, p < 0.001; 47.5% to 6.6%, p = 0.019 and 24% to 4%, p = 0.009, respectively). The number of patients who achieved the highest activation level increased from 69% to 77.6% (p < 0.001). Conclusion: The CMO program resulted in significantly better health outcomes during the six months following the pharmacist-led intervention as well as improved activation in PLWH.

Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development.

Acquisition of cell fate is thought to rely on the specific interaction of remote cis-regulatory modules (CRMs), for example, enhancers and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly within multicellular developing organisms. In the present study, we employ Hi-M, a single-cell spatial genomics approach, to detect CRM-promoter looping interactions within topologically associating domains (TADs) during early Drosophila development. By comparing cis-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses reveal that multiple CRMs spatially coalesce to form hubs. Loops and CRM hubs are established early during development, before the emergence of TADs. Moreover, CRM hubs are formed, in part, via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach provides insight into the role of CRM-promoter interactions in defining transcriptional states, as well as distinct cell types.

Application of the CMO methodology to the improvement of primary adherence to concomitant medication in people living with-HIV. The PRICMO Project.

OBJECTIVE: To determine the effectiveness of a pharmaceutical care intervention based on the CMO methodology (Capacity, Motivation  and Opportunity) in improving primary adherence to concomitant  treatment in HIV+ patients on antiretroviral treatment. METHOD: This was a longitudinal prospective multicenter study carried out  between September 2019 and September 2020, which included HIV+  patients older than 18 years who were on antiretroviral treatment and  were taking concomitant medications. Demographic, clinical, and pharmacotherapeutic variables were collected. As required by the CMO methodology, all patients were followed for 6 months and stratified  into three levels of care. Individualized pharmaceutical care was provided according to the interventions established for each level. At every consultation, a motivational interview was conducted based on each  patient's alignment with and achievement of their pharmacotherapeutic  objectives. A website was developed to deal with the opportunity pillar.  The main variable was the percentage of patients considered primary  adherents to the prescribed concomitant medication. Adherence over the  six months prior to the study was compared to adherence at the end of the study. Additionally, the percentage of patients considered secondary  adherents to concomitant treatment and antiretroviral treatment during  the 6 months prior to the start of the study was compared to the  percentage of such patients at the end of the study. Adherence was  measured based on dispensation records and specific validated  questionnaires. Patients were only considered adherent if they were  deemed adherent by both methods. RESULTS: A total of 61 patients were included in the study, 72% male. Median age was 53 years and the median number of concomitant drugs prescribed was 7. A total of 60.6% of patients were polymedicated. The percentage of patients considered primary non-adherent was 52.5% at baseline (n = 32) and 4.9% (n = 3, p < 0.001) at the end of the study. Secondary adherence to both concomitant medication (41.6% vs 88.3%) and antiretroviral treatment (85.2% vs 95.1%) improved at the end of the study (p < 0.0001). CONCLUSIONS: Pharmaceutical care based on the CMO methodology significantly improved both primary and secondary  dherence to concomitant drugs and to antiretroviral treatment.

Objetivo: Determinar la efectividad de una intervención farmacéutica, basada en la metodología CMO (Capacidad, Motivación,  portunidad), para mejorar la adherencia primaria al tratamiento  concomitante en pacientes VIH+ en tratamiento antirretroviral.Método: Estudio longitudinal, prospectivo, multicéntrico, realizado entre septiembre de 2019 y septiembre de 2020. Se incluyeron pacientes VIH+ mayores de 18 años, en tratamiento antirretroviral y  prescripción de fármacos concomitantes. Se recogieron variables  demográficas, clínicas y farmacoterapéuticas. Se realizó atención  farmacéutica durante 6 meses según el modelo CMO en cada paciente,  basado en su nivel de estratificación y las intervenciones establecidas para cada umbral. En cada consulta se realizó una entrevista motivacional  basada en el alcance de los objetivos farmacoterapéuticos para cada  paciente. Para desarrollar el pilar de oportunidad se creó y desarrolló la  web: www.proyecto-pricmo.com. La variable principal fue el porcentaje de  pacientes considerados adherentes primarios a la medicación concomitante  prescrita, comparando los 6 meses previos al estudio, frente al mismo valor al finalizar el estudio. Adicionalmente, se comparó el  porcentaje de pacientes adherentes secundarios al tratamiento  concomitante y al tratamiento antirretroviral durante los 6 meses previos  al inicio del estudio frente al mismo valor en los pacientes al finalizar el  estudio. Para medir la adherencia se consideraron dos métodos: registros  y cuestionarios validados específicos. Solo se consideraron adherentes si lo fueron a ambos métodos.Resultados: Se incluyeron 61 pacientes. El 72,0% fueron hombres, con una mediana de edad de 53 años. La mediana de fármacos  oncomitantes fue de 7. El 60,6% de los pacientes tenían presencia de  polifarmacia. El porcentaje de pacientes considerados no adherentes  primarios basalmente fue del 52,5% (n = 32), mientras que a la  finalización fue del 4,9% (n = 3, p < 0,001). Tanto la adherencia  secundaria a la medicación concomitante (41,6% versus 88,3%) como al  tratamiento antirretroviral (85,2% versus 95,1%) mejoraron al finalizar el  estudio (p < 0,001).Conclusiones: La intervención farmacéutica basada en la metodología CMO mejoró significativamente tanto la adherencia primaria  como secundaria a la medicación concomitante y la secundaria al  tratamiento antirretroviral.

Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M.

Simultaneous observation of 3D chromatin organization and transcription at the single-cell level and with high spatial resolution may hold the key to unveiling the mechanisms regulating embryonic development, cell differentiation and even disease. We recently developed Hi-M, a technology that enables the sequential labeling, 3D imaging and localization of multiple genomic DNA loci, together with RNA expression, in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe rehybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos in microfluidic chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4-5 d, including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1-2 d to complete all rounds of labeling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells or organization of chromatin within complex tissues.

Identification and profiling of microRNAs in two developmental stages of the model cestode parasite Mesocestoides corti.

MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in fundamental biological processes such as metabolism and development. The particular developmental characteristics of cestode parasites highlight the importance of studying miRNA gene regulation in these organisms. Here, we performed a comprehensive analysis of miRNAs in two developmental stages of the model cestode Mesocestoides corti. Using a high-throughput sequencing approach, we found transcriptional evidence of 42 miRNA loci in tetrathyridia larvae and strobilated worms. Tetrathyridium and strobilated worm-specific miRNAs were found, as well as differentialy expressed miRNAs between these developmental stages, suggesting miRNA regulation of stage-specific features. Moreover, it was shown that uridylation is a differential mechanism of post-transcriptional modification of M. corti miRNAs. The whole set of M. corti miRNAs represent 33 unique miRNA families, and confirm the remarkable loss of conserved miRNA families within platyhelminth parasites, reflecting their relatively low morphological complexity and high adaptation to parasitism. Overall, the presented results provide a valuable platform to studies aiming to identify and characterize novel miRNA-based molecular mechanisms of post-transcriptional gene regulation in cestodes, necessary for the elucidation of developmental aspects of the complex biology of these parasites.

Perceptions about HIV pre-exposure prophylaxis among healthcare professionals in Spain (PERPPRES Study).

OBJECTIVE: To determine the level of support, knowledge and perceptions of pre-exposure prophylaxis (PrEP) by Infectious Disease Specialists and Hospital Pharmacists in Spain. METHODS: Cross-sectional study through an on-line 31-item survey (sociodemographical data, employment status/experience, knowledge of PrEP, use, identified barriers and economic issues). A univariate analysis was performed to evaluate the variables associated with support for PrEP, and compare the assessments by Specialists and Pharmacists. The questions about support for PrEP and agreement with the indication approval were repeated after showing data from published studies. The significance of the change in the answers was analyzed using the McNemar Test. RESULTS: 211 questionnaires were received (80.1% from Pharmacists). 40.3% had low/no familiarity with PrEP (46.2% Pharmacists vs. 16.7% Physicians; p < 0.01). A 53.6% of them would support the use of PrEP (49.7% Pharmacists vs. 69% Physicians; p = 0.038). The minimum acceptable efficacy in order to support PrEP was 85.0 ± 15.5% (82.6 ± 12.1% by Physicians vs. 85.6 ± 15.0% by Pharmacists; p = 0.02). The variables associated with support were: medical profession (OR = 2.26; 95%CI 1.1-4.6; p = 0.038) and lower demand for efficacy (difference = 10.5%; 95%CI 6.9 to 14.1; p < 0.001). After receiving the information, there was an increase in their support for use and indication approval. Most participants (81.5%) did not support its reimbursement. The main barriers identified were: an increase in risk behaviour (24.1%), increase in sexually transmitted diseases (19.0%), resistance (16.6%) and cost (16.0%). CONCLUSIONS: More than half of participants were familiar with PrEP. The majority of them would support its use and the approval of the indication, but would not reimburse it. The use of PrEP in real practice is currently low.

Objetivo: Determinar el grado de apoyo, conocimientos y percepciones respecto a la profilaxis preexposición (PrEP) de los médicos infectólogos y farmacéuticos hospitalarios en España. Métodos: Estudio transversal mediante encuesta de 31 ítems (datos sociodemográficos, situación laboral/experiencia, conocimiento sobre PrEP, uso, opiniones, barreras detectadas y aspectos financieros). Se realizó un análisis univariante para evaluar las variables relacionadas con el apoyo a PrEP y comparar las valoraciones de médicos y farmacéuticos. Las preguntas sobre apoyo a la PrEP y el acuerdo sobre aprobar la indicación se repitieron tras mostrar datos de estudios publicados. Se analizó la significación del cambio en la respuesta mediante la prueba de McNemar. Resultados: Se recibieron 211 cuestionarios (80,1% farmacéuticos). El 40,3% estuvieron nada/poco familiarizados con la PrEP (46,2% farmacéuticos vs. 16,7% médicos; p < 0,01). El 53,6% apoyaría su uso (49,7% farmacéuticos vs. 69% médicos; p = 0,038). La eficacia mínima considerada aceptable fue 85,0 ± 15,5% (82,6 ± 12,1% médicos vs. 85,6 ± 15,0% farmacéuticos; p = 0,02). Las variables relacionadas con el apoyo fueron: profesión médica (OR = 2,26 IC95% 1,1-4,6; p = 0,038) y menor exigencia de eficacia (diferencia 10,5% IC95% 6,9-14,1; p < 0,001). Tras recibir la información, aumentaron el apoyo al uso y la aprobación. El 81,5% no apoyaron la financiación. Las principales barreras señaladas fueron: aumento de conductas de riesgo (24,1%), aumento de enfermedades de transmisión sexual (19,0%), resistencias (16,6%) y coste (16,0%). Conclusiones: Más de la mitad de los encuestados estaban familiarizados con la PrEP. La mayoría apoyaría su uso y la aprobación de la indicación, pero no la financiaría. El uso en la práctica real de la PrEP es escaso en la actualidad.

Protection but maintained dysfunction of nigral dopaminergic nerve cell bodies and striatal dopaminergic terminals in MPTP-lesioned mice after acute treatment with the mGluR5 antagonist MPEP.

The mGluR5 antagonist MPEP was used to study the role of mGluR5 in MPTP-induced injury of the nigrostriatal DA neurons. The findings indicate that acute blockade of mGluR5 may result in neuroprotective actions against MPTP neurotoxicity on nigral DA cell bodies and striatal DA terminals using stereological analysis of TH immunoreactivity and microdensitometry. Biochemical analysis showed no restoration of DA levels and metabolism indicating a maintained reduction of DA transmission.

Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages.

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (ßTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, ßACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, ßACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus.

Rapid detection of Echinococcus species by a high-resolution melting (HRM) approach.

BACKGROUND: High-resolution melting (HRM) provides a low-cost, fast and sensitive scanning method that allows the detection of DNA sequence variations in a single step, which makes it appropriate for application in parasite identification and genotyping. The aim of this work was to implement an HRM-PCR assay targeting part of the mitochondrial cox1 gene to achieve an accurate and fast method for Echinococcus spp. differentiation. FINDINGS: For melting analysis, a total of 107 samples from seven species were used in this study. The species analyzed included Echinococcus granulosus (n = 41) and Echinococcus ortleppi (n = 50) from bovine, Echinococcus vogeli (n = 2) from paca, Echinococcus oligarthra (n = 3) from agouti, Echinococcus multilocularis (n = 6) from monkey and Echinococcus canadensis (n = 2) and Taenia hydatigena (n = 3) from pig. DNA extraction was performed, and a 444-bp fragment of the cox1 gene was amplified. Two approaches were used, one based on HRM analysis, and a second using SYBR Green Tm-based. In the HRM analysis, a specific profile for each species was observed. Although some species exhibited almost the same melting temperature (Tm) value, the HRM profiles could be clearly discriminated. The SYBR Green Tm-based analysis showed differences between E. granulosus and E. ortleppi and between E. vogeli and E. oligarthra. CONCLUSIONS: In this work, we report the implementation of HRM analysis to differentiate species of the genus Echinococcus using part of the mitochondrial gene cox1. This method may be also potentially applied to identify other species belonging to the Taeniidae family.