Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices.

BACKGROUND: Nucleic acids, RNA among them, are widely used in biomedicine and Biotechnology. Because of their susceptibility to degradation by RNases, the handling and extraction process of RNA from cells and tissues require specialized personnel and standardized methods to guarantee high purity and integrity. Due to the diversity of techniques found in the market, a comparative study between different RNA extraction methods is useful to facilitate the best choice for the researcher or in research service platforms such as biobanks to see the traceability of the samples. METHODS AND RESULTS: In this study, we have compared seven different RNA extraction methods: manual (TRIzol™), semiautomated (QIAGEN™, Bio-Rad, Monarch®, and Canvax™), and fully automated (QIAcube™ and Maxwell®) processes, from two biological matrices: human Jurkat T cells and peripheral blood mononuclear cells (PBMC). Results showed marked differences in the RNA quality and functionality according to the method employed for RNA extraction and the matrix used. DISCUSSION: QIAcube™ and semi-automated extraction methods were perceived as the best options because of their lower variability, good functionality, and lower cost (P < 0.001). These data contribute to facilitating researchers or research service platforms (Biobanks) in decision-making practices and emphasize the relevance of the selection of the RNA extraction method in each experimental procedure or traceability study to guarantee both quality standards and its reproducibility.

Deficits in coordinated neuronal activity and network topology are striatal hallmarks in Huntington's disease.

BACKGROUND: Network alterations underlying neurodegenerative diseases often precede symptoms and functional deficits. Thus, their early identification is central for improved prognosis. In Huntington's disease (HD), the cortico-striatal networks, involved in motor function processing, are the most compromised neural substrate. However, whether the network alterations are intrinsic of the striatum or the cortex is not fully understood. RESULTS: In order to identify early HD neural deficits, we characterized neuronal ensemble calcium activity and network topology of HD striatal and cortical cultures. We used large-scale calcium imaging combined with activity-based network inference analysis. We extracted collective activity events and inferred the topology of the neuronal network in cortical and striatal primary cultures from wild-type and R6/1 mouse model of HD. Striatal, but not cortical, HD networks displayed lower activity and a lessened ability to integrate information. GABAA receptor blockade in healthy and HD striatal cultures generated similar coordinated ensemble activity and network topology, highlighting that the excitatory component of striatal system is spared in HD. Conversely, NMDA receptor activation increased individual neuronal activity while coordinated activity became highly variable and undefined. Interestingly, by boosting NMDA activity, we rectified striatal HD network alterations. CONCLUSIONS: Overall, our integrative approach highlights striatal defective network integration capacity as a major contributor of basal ganglia dysfunction in HD and suggests that increased excitatory drive may serve as a potential intervention. In addition, our work provides a valuable tool to evaluate in vitro network recovery after treatment intervention in basal ganglia disorders.

Batch mesophilic anaerobic co-digestion of spent goat batch mesophilic anaerobic co-digestion of spent goat straw bedding and goat cheese whey: Comparison with the mono-digestion of the two sole substrates.

Spent livestock bedding is a valuable resource for the production of green energy (methane) in rural areas. Comparison and evaluation of batch anaerobic digestion and co-digestion of different mixtures of goat straw bedding (SGSB) and goat cheese whey were carried out. Biochemical methane potential (BMP) tests of the 100% SGSB, 95% SGSB-5% whey, 90% SGSB-10% whey, 85% SGSB-15% whey and 100% whey were found to be 423 ± 7, 354 ± 9, 371 ± 2, 293 ± 1, 274 ± 2 mL CH4 g-1 VS. Two different kinetic models were evaluated. The logistic model revealed a decrease in the maximum methane production rate (Rm) from 34.7 ± 1.5 to 14.1 ± 0.9 mL CH4 g-1 VS·d-1 when the percentage of whey in the mixture increased from 0 to 15% as a consequence of the increased ammonia released during the co-digestion of increased concentrations of whey. The lowest value for the maximum methane production predicted by the model (P) was found for 100% whey (274 ± 10 mL CH4 g-1 VS). A two-substrate model was applied to describe the evident existence of rapid and slowly degradable material. Regarding the hydrolysis kinetic constants predicted by this model, considerable increases in the rapid biodegradation stage (krapid) were observed when comparing to the values found for the slow (kslow) biodegradation stage in all the cases tested. The increases between both constants rose from 5 to 42% when the percentage of whey increased.

Participative approach to elicit water quality monitoring needs from stakeholder groups - An application of integrated watershed management.

Water quality monitoring programs (WQMPs) must be based on monitoring objectives originating from the real knowledge needs of all stakeholders in a watershed and users of the resource. This paper proposes a participative approach to elicit knowledge needs and preferred modes of communication from citizens and representatives of organized stakeholders (ROS) on water quality and quantity issues. The participative approach includes six steps and is adaptable and transferable to different types of watersheds. These steps are: (1) perform a stakeholder analysis; (2) conduct an adaptable survey accompanied by a user-friendly public participation geographical information system (PPGIS); (3) hold workshops to meet with ROS to inform them of the results of the survey and PPGIS; discuss attainment of past monitoring objectives; exchange views on new knowledge needs and concerns on water quality and quantity; (4) meet with citizens to obtain the same type of input (as from ROS); (5) analyze the data and information collected to identify new knowledge needs and modes of communication and (6) identify, in collaboration with the individuals in charge of the WQMPs, the short-, medium- and long-term monitoring objectives and communication strategies to be pursued. The participative approach was tested on two distinct watersheds in the province of Quebec, Canada. It resulted in a series of optimization objectives of the existing WQMPs, new monitoring objectives and recommendations regarding communication strategies of the WQMPs' results. The results of this study show that the proposed methodology is appreciated by all parties and that the outcomes and monitoring objectives are acceptable. We also conclude that successful integrated watershed management is a question of scale, and that every aspect of integrated watershed management needs to be adapted to the surface watershed, the groundwater watershed (aquifers) and the human catchment area.

The clinical and immunological effects of Pru p 3 sublingual immunotherapy on peach and peanut allergy in patients with systemic reactions.

BACKGROUND: The peach non-specific lipid transfer protein, Pru p 3, is the primary sensitizer in fruits and responsible for severe reactions in the Mediterranean area. Peach allergy is frequently associated with other allergies such as peanut. Therefore, it is important to assess how specific immunotherapy to Pru p 3 could affect both peach and peanut tolerance. OBJECTIVES: To evaluate peach and peanut desensitization and immunological changes after 1 year of Pru p 3 sublingual immunotherapy (SLIT) in patients with systemic allergic reactions to peach and/or peanut. METHODS: Forty-eight peach allergic patients, 36 treated with SLIT and 12 non-treated, were monitored for 12 months. Treated patients were subclassified as peanut allergic (Group A), sensitized (Group B) or tolerant (Group C). SLIT effect was evaluated by skin prick test (SPT) reactivity and food challenge. Immunological changes were evaluated by monitoring sIgE and sIgG4 levels and basophil reactivity. RESULTS: After 1 year of SLIT, the weal area in SPT significantly decreased and a significant increase in peach threshold in treated patients was observed (P < 0.001). Patients in Group A showed a significant decrease in peanut SPT weal area and an increase in peanut threshold (P < 0.001). Immunological changes were observed in treated patients only, with a significant decrease in sIgE and a parallel increase in sIgG4, sIgG4/sIgE and basophil reactivity for both Pru p 3 and Ara h 9. CONCLUSIONS AND CLINICAL RELEVANCE: After 1 year, Pru p 3 SLIT induces both desensitization and immunological changes not only for peach but also for other food allergens relevant in the induction of severe reactions such as peanut.

Prevalence and distribution of hepatitis C virus genotypes in Spain during the 2000-2015 period (the GEHEP 005 study).

We report the largest study on the prevalence and distribution of HCV genotypes in Spain (2000-2015), and we relate them with clinical, epidemiological and virological factors. Patients from 29 hospitals in 10 autonomous communities (Andalusia, Aragon, Castilla-Leon, Catalonia, Galicia, Canary Islands, Madrid Community, Valencian Community, Murcia Region and Basque Country) have been studied. Annual distribution of HCV genotypes and subtypes, as well as gender, age, transmission route, HIV and/or HBV coinfection, and treatment details were recorded. We included 48595 chronically HCV-infected patients with the following characteristics: median age 51 years (IQR, 44-58), 67.9% male, 19.1% HIV-coinfected, 23.5% HBV-coinfected. Parenteral transmission route was the most frequent (58.7%). Genotype distribution was 66.9% GT1 (24.9% subtype 1a and 37.9% subtype 1b), 2.8% GT2, 17.3% GT3, 11.4% GT4 and 0.1% GT5 and 0.02% GT6. LiPA was the most widely HCV genotyping test used (52.4%). HCV subtype 1a and genotypes 3 and 4 were closely associated with male gender, parenteral route of infection and HIV and HBV coinfection; in contrast, subtype 1b and genotype 2 were associated with female gender, nonparenteral route and mono-infection. Age was related to genotype distribution, and different patterns of distribution and biodiversity index were observed between different geographical areas. Finally, we describe how treatment and changes in transmission routes may have affected HCV genotype prevalence and distribution patterns. We present the most recent data on molecular epidemiology of hepatitis C virus in Spain. This study confirms that genotype distributions vary with age, sex, HIV and HBV coinfection and within geographical areas and epidemiological groups.

The L-type voltage-gated calcium channel modulates microglial pro-inflammatory activity.

Under pathological conditions, microglia, the resident CNS immune cells, become reactive and release pro-inflammatory cytokines and neurotoxic factors. We investigated whether this phenotypic switch includes changes in the expression of the L-type voltage-gated calcium channel (VGCC) in a rat model of N-methyl-D-aspartate-induced hippocampal neurodegeneration. Double immunohistochemistry and confocal microscopy evidenced that activated microglia express the L-type VGCC. We then analyzed whether BV2 microglia express functional L-type VGCC, and investigated the latter's role in microglial cytokine release and phagocytic capacity. Activated BV2 microglia express the CaV1.2 and CaV1.3 subunits of the L-type VGCC determined by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry. Depolarization with KCl induced a Ca2+ entry facilitated by Bay k8644 and partially blocked with nifedipine, which also reduced TNF-α and NO release by 40%. However, no nifedipine effect on BV2 microglia viability or phagocytic capacity was observed. Our results suggest that in CNS inflammatory processes, the L-type VGCC plays a specific role in the control of microglial secretory activity.

Immunologic responses to the major allergen of Olea europaea in local and systemic allergic rhinitis subjects.

OBJECTIVE: To evaluate the in vivo and in vitro responses to nOle e 1 in allergic rhinitis (AR) and local allergic rhinitis (LAR) patients sensitized to olive tree pollen (OL) confirmed by nasal allergen provocation test (NAPT). METHODS: Twelve subjects with AR, 12 with LAR and 12 subjects as control group (CG) were selected. Skin testing and NAPT with nOle e 1 were performed. Eosinophilic cationic protein (ECP) and tryptase were measured in nasal lavages before and after NAPT. Serum IgE to OL allergens was measured by ELISA. Basophil activation tests (BAT) with OL and nOle e 1 and dendritic cell maturation/proliferation studies were carried out. RESULTS: All AR (12/12) and 10/12 (83%) of LAR had a +NAPT to nOle e 1. ECP levels in nasal lavages were significantly increased after NAPT in both AR and LAR compared with CG at 15 min (P < 0.05). Serum IgE was positive only in AR. All AR had +BAT responses to OL and 10/12 to nOle e 1 (83%); 8/12 LAR (66.6%) had a +BAT to OL and 4/12 (33%) to nOle e 1, with only one subject of the CG with a +BAT to both OL and nOle e 1 (8%). Dendritic cell proliferation to nOle e 1 was increased in AR compared to LAR and CG (P = 0.019 and P = 0.001, respectively). CONCLUSION: Both AR and LAR had a similar in vivo response to nOle e 1 with release of inflammatory mediators. Specific basophil activation with OL and nOle e 1 was observed in LAR confirming previous data obtained with dust mites.

Periodic nanostructures on unpolished substrates and their integration in solar cells.

We present a novel fabrication process based on laser interference lithography, lift-off and reactive ion etching, which allows us to fabricate periodic nanostructures on photovoltaic substrates with an average root mean square (RMS) roughness of 750 nm. We fabricate nanostructures on unpolished crystalline silicon substrates, which reduces their reflectance 30% as fabricated. When an additional passivation layer is deposited, the light trapping grows, achieving a reflectance reduction of 60%. In addition, we have successfully integrated the nanostructured substrates in silicon wafer-based solar cells following standard processes, achieving a final efficiency of 15.56%.

TMPRSS4 regulates levels of integrin α5 in NSCLC through miR-205 activity to promote metastasis.

BACKGROUND: TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown. METHODS: miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC. RESULTS: miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G0/G1 cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin α5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin α5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin α5 levels. CONCLUSION: We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin α5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC.

Glucose pathways adaptation supports acquisition of activated microglia phenotype.

With its capacity to survey the environment and phagocyte debris, microglia assume a diversity of phenotypes to respond specifically through neurotrophic and toxic effects. Although these roles are well accepted, the underlying energetic mechanisms associated with microglial activation remain largely unclear. This study investigates microglia metabolic adaptation to ATP, NADPH, H(+) , and reactive oxygen species production. To this end, in vitro studies were performed with BV-2 cells before and after activation with lipopolysaccharide + interferon-γ. Nitric oxide (NO) was measured as a marker of cell activation. Our results show that microglial activation triggers a metabolic reprogramming based on an increased glucose uptake and a strengthening of anaerobic glycolysis, as well as of the pentose pathway oxidative branch, while retaining the mitochondrial activity. Based on this energy commitment, microglial defense capacity increases rapidly as well as ribose-5-phosphate and nucleic acid formation for gene transcription, essential to ensure the newly acquired functions demanded by central nervous system signaling. We also review the role of NO in this microglial energy commitment that positions cytotoxic microglia within the energetics of the astrocyte-neuron lactate shuttle.

Systematic review and meta-analysis of performance of second-trimester nasal bone assessment in detection of fetuses with Down syndrome.

OBJECTIVE: To review systematically the literature on diagnostic tests and performance of second-trimester sonographic assessment of nasal bone (NB) in identifying fetuses affected by Down syndrome. METHODS: A search of studies involving screening tests for NB evaluation and measurements was carried out in the main international bibliographic databases (MEDLINE, EMBASE and CINAHL). Those considered to be relevant were then subjected to critical reading, following Critical Appraisal Skills Programme (CASP) criteria, by at least three independent observers. All data were extracted and tabulated by two independent investigators. A statistical synthesis of sensitivity, specificity and likelihood ratios was performed using specific software (Meta-DiSc). RESULTS: From an initial list of 852 articles referring to ultrasound markers for Down syndrome, 207 relevant papers were selected. Following exclusions, 21 studies were included in the quantitative synthesis. The pooled estimates of positive and negative likelihood ratios were 40.08 (95% CI, 18.10-88.76) and 0.71 (95% CI, 0.64-0.79), respectively, for absent NB and 15.15 (95% CI, 8.15-28.16) and 0.47 (95% CI, 0.34-0.64), respectively, for hypoplastic NB. No relevant differences were found between the various means of defining nasal hypoplasia (multiples of the median (MoM) or percentiles). The biparietal diameter/nasal bone length (BPD/NBL) ratio showed somewhat higher sensitivity but lower specificity with a threshold effect. CONCLUSIONS: NB absence or hypoplasia show high specificity and low but acceptable sensitivity in identifying fetuses with Down syndrome. Screening performance is better with NB measurements as a function of MoM or percentiles rather than as the BPD/NBL ratio. Classification of women into various risk groups for Down syndrome does not affect diagnostic performance.

Characterization of Escherichia fergusonii and Escherichia albertii isolated from water.

AIMS: The aim of this study was to characterize Escherichia fergusonii and Escherichia albertii isolated from water. METHODS AND RESULTS: The characterization of E. fergusonii and E. albertii isolated from water was determined using an Escherichia coli-specific uidA PCR, a tuf PCR, and with phylogenetic analysis using three housekeeping genes (adk, gyrB, and recA) from the E. coli MLST scheme, selected for their ability to discriminate among all Escherichia species. Among the 527 isolates tested, 25 (4·7%) were uidA PCR negative and tuf PCR positive. Phylogenetic analysis using adk, gyrB and recA genes showed that 6, 18 and 1 of these 25 non-E. coli Escherichia spp. isolates grouped with reference strains of E. fergusonii, E. albertii, and E. coli, respectively. Finally, the 25 non-E. coli Escherichia spp. strains isolated were investigated for the presence of pathogenic factors, comprising intimin (eae gene), cytolethal distending toxin (cdtB gene) and shiga toxin (stx gene). With the PCR primers used, the presence of eae and stx genes was not detected. However, cdtB genes types I/IV were detected for 3 (16·7%) E. albertii strains, whereas 15 of 18 (83·3%) possessed the cdtB gene types II/III/V. CONCLUSIONS: These results showed that MLST scheme allows a more accurate identification of non-E. coli species than phenotypic tests. We also showed that E. fergusonii and E. albertii represent, respectively, 0·8 and 2·5% of all Escherichia species isolated and the pathogenic cdtB genes were present in 83·3% of these strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented in this study provided an efficient way to correctly identify non-E. coli species contributing to our understanding of the risks associated with Escherichia species in water consumed by humans and animals. Furthermore, the results give an insight about the natural habitats of these species.

Physiological strategies contributing to the coexistence of two predatory species of stoneflies: Dinocras cephalotes and Perla bipunctata.

Our study focuses on the oxidative state of two aquatic insects of the order Plecoptera belonging to the family Perlidae, namely Dinocras cephalotes (Curtis, 1827) and Perla bipunctata Pictet, 1833. These species are widely distributed throughout the Western Palearctic region and coexist in the stream where individuals for this study come from. We highlight the physiological strategies of these two different predator species of stoneflies, showing a higher accumulation of lipid reserves in P. bipunctata, higher glucose levels in the body tissues of D. cephalotes and a higher capacity of the antioxidant enzymes in P. bipunctata, what provides it a protection against oxidation of lipids, which are greater in this species. This leads to a similar oxidative state in both species. Based on these results is discussed how two close related species developing a very similar ecological role in the same habitat can achieve a similar fitness with differences in their physiological strategies.

Relationship between brain and liver oxidative state and maximum lifespan potential of different fish species.

The aim of the present study is to determine whether a relationship exists between the maximum lifespan potential (MLSP) of different fish species (Squalius pyrenaicus, Cyprinus carpio, Carassius auratus, Luciobarbus sclateri and Oncorhynchus mykiss) and the oxidative state of brain and liver. For this purpose, the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), glutathione reductase (GR), glutathione S-transferase (GST) and DT-diaphorase (NAD(P)H:quinone oxidoreductase1) were measured together with the lipid peroxidation degree (malondialdehyde, MDA) in liver and brain tissues of the collected species. Only the brain tissue manifested a higher activity of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and glutathione S-transferase in addition to a negative correlation between the values of MDA and MLSP among the different fish species. Hypotheses are proposed from the analysis of the obtained results that open new areas of research and converge on the importance of the theory of free radicals in the processes that condition the maximum life expectancy of an animal species.

Radiological predictors of final infarct volume in patients with proximal vascular occlusion.

INTRODUCTION: Various clinical and radiologic variables impact the neurologic prognosis of patients with ischemic cerebrovascular accidents. About 30% of ischemic cerebrovascular accidents are caused by proximal obstruction of the anterior circulation; in these cases, systemic thrombolysis is of limited usefulness. CT angiography is indicated in candidates for endovascular treatment. Various radiologic factors, including the grade of leptomeningeal collateral circulation, as well as the length, density, and extension of the thrombus, have been identified as predictors of neurologic prognosis after anterior ischemic cerebrovascular accidents due to proximal vascular obstruction. Final infarct volume correlations with mortality and long-term functional outcome in these patients. This study aimed to determine the best predictors of final infarct volume on CT angiography in patients with ischemic cerebral accidents due to proximal occlusion. MATERIALS AND METHODS: This retrospective observational study included adults with ischemic cerebrovascular accidents due to obstruction of the anterior circulation diagnosed by CT angiography in the period comprising June 2009 through December 2019. We measured the length and density of the thrombus in unenhanced CT images, and we used the clot burden score to record the grade of leptomeningeal collateral circulation and the extension of the thrombus. Then we measured the final infarct volume on follow-up CT and analyzed the correlations among these radiologic factors in the infarct volume. RESULTS: We included 54 patients [mean age, 82 y; 41 (75%) women] with ischemic cerebrovascular accidents due to proximal occlusion. About 60% of the cerebrovascular accidents affected the right cerebral hemisphere, and the most commonly affected vessel was the M1 segment of the medial cerebral artery (40.7%). Final infarct volume correlated with the grade of leptomeningeal collateral circulation (p=0.03) and with the clot burden score (p=0.01). Neither the length nor the density of the thrombus correlated with final infarct volume. CONCLUSION: The final infarct volume can be estimated on the initial CT angiogram. Nevertheless, we found no useful predictive factors in unenhanced CT images. The best independent radiologic predictors of the final infarct volume are the grade of collateral circulation and the clot burden score, especially in patients who did not undergo mechanical thrombectomy, because mechanical thrombectomy improves outcomes. These factors are important for decision making in the management of patients with ischemic cerebrovascular accidents due to proximal occlusion.

[COVID-19 epidemiology in health and social health workers in the health area of A Coruña and Cee]. / Epidemiología de la COVID-19 entre el personal de centros sanitarios y sociosanitarios del área sanitaria de A Coruña y Cee.

OBJECTIVE: Since the beginning of the pandemic, it has become necessary to know the epidemiological evolution of SARS-CoV-2. Therefore, this study aims to describe the characteristics of the casuistry of COVID-19 in health and social-health workers in the health area of A Coruña and Cee during the first epidemic wave, as well as to determine the association between the clinical profile and/or its duration and the condition of RT-PCR repositivization. MATERIALS AND METHODS: During the study period, 210 cases belonging to healthcare and social-healthcare workers from the healthcare area of A Coruña and Cee were diagnosed. A descriptive analysis of sociodemographic factors was carried out, as well as the search for association between the clinical picture and the duration of detection of a positive RT-PCR. RESULTS: The most affected categories were nursing (33.3%) and nursing assistants (16.2%). The mean time taken for cases to become RT-PCR negative was 18.3±9.1 days, with a median of 17. It was observed that 26 cases (13.8%) had a positive result in a subsequent RT-PCR, without meeting criteria for reinfection. The existence of skin manifestations and arthralgias was associated with repositivization after adjusting for age and sex (OR=4.6 and OR=6.5, respectively). CONCLUSIONS: In healthcare professionals diagnosed with COVID-19 during the first wave, symptoms such as dyspnea, skin manifestations and arthralgias led to RT-PCR repositivization after a previous negative result and without meeting criteria for reinfection.

RENEB Inter-Laboratory Comparison 2021: The FISH-Based Translocation Assay.

Translocation analysis using fluorescence in situ hybridization (FISH) is the method of choice for dose assessment in case of chronic or past exposures to ionizing radiation. Although it is a widespread technique, unlike dicentrics, the number of FISH-based inter-laboratory comparisons is small. For this reason, although the current Running the European Network of Biological and Physical retrospective Dosimetry (RENEB) inter-laboratory comparison 2021 was designed as a fast response to a real emergency scenario, it was considered a good opportunity to perform an inter-laboratory comparison using the FISH technique to gain further experience. The Bundeswehr Institute of Radiobiology provided peripheral blood samples from one healthy human volunteer. Three test samples were irradiated with blinded doses of 0, 1.2, and 3.5 Gy, respectively. Samples were then sent to the seven participating laboratories. The FISH technique was applied according to the standard procedure of each laboratory. Both, the frequency of translocations and the estimated dose for each sample were sent to the coordinator using a special scoring sheet for FISH. All participants sent their results in due time. However, although it was initially requested to send the results based on the full analysis, evaluating 500 equivalent cells, most laboratories only sent the results based on triage, with a smaller number of analyzed cells. In the triage analysis, there was great heterogeneity in the number of equivalent cells scored. On the contrary, for the full analysis, this number was more homogeneous. For all three samples, one laboratory showed outlier yields compared to the other laboratories. Excluding these results, in the triage analysis, the frequency of translocations in sample no. 1 ranged from 0 to 0.013 translocations per cell, and for samples no. 2 and no. 3 the genomic mean frequency were 0.27 ± 0.03 and 1.47 ± 0.14, with a coefficient of variation of 0.29 and 0.23 respectively. Considering only results obtained in the triage analysis for sample no. 1, all laboratories, except one, classified this sample as the non-irradiated one. For sample no. 2, excluding the outlier value, the mean reported dose was 1.74 ± 0.16 Gy indicating a mean deviation of about 0.5 Gy to the delivered dose of 1.2 Gy. For sample no. 3 the mean dose estimated was 4.21 ± 0.21 Gy indicating a mean deviation of about 0.7 Gy to the delivered dose of 3.5 Gy. In the frame of RENEB, this is the second FISH-based inter-laboratory comparison. The whole exercise was planned as a response to an emergency, therefore, a triage analysis was requested for all the biomarkers except for FISH. Although a full analysis was initially requested for FISH, most of the laboratories reported only a triage-based result. The main reason is that it was not clearly stated what was required before starting the exercise. Results show that most of the laboratories successfully discriminated unexposed and irradiated samples from each other without any overlap. A good agreement in the observed frequencies of translocations was observed but there was a tendency to overestimate the delivered doses. Efforts to improve the harmonization of this technique and subsequent exercises to elucidate the reason for this trend should be promoted.

Isolated cardiomyopathy caused by a DMD nonsense mutation in somatic mosaicism: genetic normalization in skeletal muscle.

X-linked dilated cardiomyopathy is a pure cardiac dystrophinopathy phenotype mainly caused by DMD mutations that present a specific transcription effect in cardiac tissue. We report a 26-year-old male who presented with severe dilated cardiomyopathy and high creatine kinase. The patient did not complain of skeletal muscle weakness. A muscle biopsy showed mild dystrophic changes and a low proportion of dystrophin-negative fibres. A molecular study identified a nonsense DMD mutation (p.Arg2098X) in somatic mosaicism. The ratio of mutant versus normal allele in blood and skeletal muscle suggests selective pressure against mutant muscle cells, a process known as genetic normalization. We hypothesize that this process may have mitigated skeletal muscle symptoms in this patient. This is the second report of a DMD somatic mosaic with evidence of genetic normalization in muscle. Somatic DMD mutations should be considered in patients presenting with idiopathic dilated cardiomyopathy.

Digestive enzyme activity and trophic behavior in two predator aquatic insects (Plecoptera, Perlidae): a comparative study.

Plecoptera (Perlidae) are among the major macroinvertebrate predators in stream ecosystems and one of the insect families with lower tolerance to environmental alterations, being usually employed as bioindicators of high water ecological quality. The differences in the trophic roles of the coexisting species have been exclusively studied from their gut contents, while no data are available on the comparative digestive capacity. In the present paper, we make a comparative study of the activity of several digestive enzymes, namely proteases (at different pH), amylase, lipase, trypsin and chymotrypsin, in two species of stoneflies, Perla bipunctata and Dinocras cephalotes, which cohabit in the same stream. The study of digestive enzyme activity together with the analysis of gut contents can contribute to a better understanding of the ecology of these aquatic insects and their role in freshwater food webs. Thus, our results show that the two studied predator species inhabiting in the same stream present specializations on their feeding behaviors, facilitating their coexistence, and also differences in their capacity of use the resources. One of the main findings of this study is that D. cephalotes is able to assimilate a wider trophic resource spectrum and this could be one of the reasons why this species has a wider global distribution in all its geographical range.