Mine water as a source of energy: an application in a coalfield in Laciana Valley (León, NW Spain).

Mine water can be a renewable and economical source of geothermal and hydraulic energy. Nine discharges from closed and flooded coal mines in the Laciana Valley (León, NW Spain) have been studied. Various technologies for the energy use of mine water, as well as the influence of factors such as temperature, the need for water treatment, investment, potential customers and expansion capacity, have been evaluated by means of a decision-making tool. It is concluded that the most advantageous option is an open-loop geothermal system using the waters of a mountain mine, the temperature of which exceeds 14 °C and whose distance to customers is less than 2 km. A technical-economic viability study for a district heating network designed to supply heating and hot water to six public buildings in the nearby town of Villablino is presented. The proposed use of mine water might help areas that have been greatly affected socioeconomically by the closure of the mines and has other advantages compared to conventional energy systems, such as the reduction of CO2 emissions. Graphical Abstract: It showing the advantages of using mine water as an energy source for district heating and a simplified layout. Supplementary Information: The online version contains supplementary material available at 10.1007/s10098-023-02526-y.

Severe COVID-19 patients have severe vitamin D deficiency in Northeast Mexico / Los pacientes graves con COVID-19 tienen deficiencia grave de vitamina D en el noreste de México

Objective: the association between vitamin D and COVID-19 severity is not consistent. We compared prevalences and analyzed the association between vitamin D deficiency and COVID-19 severity in Northeast Mexico. Methods: this was a cross-sectional study with individuals consecutively included at a referral diagnostic center during March-September 2020 (n = 181). Concurrently, every patient admitted to intensive care was also consecutively included (n = 116). Serum 25(OH)D < 20 ng/mL was considered vitamin D deficiency. Descriptive, ANOVA, and multivariate ordinal regression analyses were performed. Results: vitamin D deficiency prevalence was 63.8 % (95 % CI, 54.7, 72.0) in severe COVID-19; 25.6 % (95 % CI, 17.4, 36.0) in mild COVID-19; and 42.4 % (95 % CI, 33.2, 52.3) in non-diseased individuals. Vitamin D deficiency increased 5 times the odds of severe COVID-19 (95 % CI, 1.1, 24.3), independently of sex, age, body mass index, and inflammatory markers. Conclusions: this study is the first report of vitamin D deficiency in Northeast Mexico. Vitamin D deficiency was associated with COVID-19 severity (AU)

Objetivo: la asociación entre la vitamina D y la gravedad de la COVID-19 no es consistente. Se comparó la prevalencia y se analizó la asociación de la deficiencia de vitamina D con la gravedad de los pacientes con COVID-19 en el noreste de México. Métodos: este fue un estudio transversal. Se incluyó consecutivamente a individuos de un centro de diagnóstico de referencia durante marzo-septiembre de 2020 (n = 181). Paralelamente, se reclutó a todos los pacientes que ingresaron a cuidados intensivos en ese mismo periodo (n = 116). Se consideró que había deficiencia de vitamina D ante cifras de 25(OH)D sérica < 20 ng/ml. Se realizaron un análisis descriptivo, un ANOVA y una regresión ordinal multivariante. Resultados: la prevalencia de la deficiencia de vitamina D fue del 63,8 % (IC del 95 %: 54,7; 72,0) en la COVID-19 grave, del 25,6 % (IC del 95 %: 17,4; 36,0) en la COVID-19 leve y del 42,4 % (IC del 95 %: 33,2; 52,3) sin COVID-19. La deficiencia aumentó 5 veces las probabilidades de una COVID-19 grave (IC del 95 %: 1,1; 23,9) independientemente del sexo, la edad, el índice de masa corporal y los marcadores inflamatorios. Conclusiones: este estudio es el primer informe de la deficiencia de vitamina D en el noreste de México. La deficiencia de vitamina D se asoció con la gravedad de la COVID-19 (AU)

A de novo paradigm for male infertility.

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10-5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10-4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

Current Choroidal Imaging Findings in Central Serous Chorioretinopathy.

BACKGROUND: Central serous chorioretinopathy (CSCR) is a chorioretinal disease affecting mostly middle age males. It is marked by the serous detachment of the neurosensory layer at the macula. This review of the literature provides a framework of the current characteristic/relevant imaging findings of CSCR. Although the pathogenesis of CSCR is unclear, the choroid plays a major role and its changes are fundamental to the diagnosis and treatment of CSCR. METHODS: A systematic literature search focusing on current multimodal imaging for CSCR was performed. Only articles reporting on original clinical data were selected, studies in a language other than English were included only if an English abstract was provided. Additional sources included articles cited in the references list of the first selected articles. We deduced imaging findings based on current and relevant literature on the topic. RESULTS: We found that sub foveal choroidal thickness (SFCT) and choroidal vascularity index (CVI) were greater in eyes with acute CSCR than in eyes with chronic CSCR or normal eyes. There was increased choroidal thickness (CT) in the macula compared to peripapillary region. In healthy eyes, the highest CVI was found in the nasal region followed by the inferior, temporal, and superior quadrant. The area with the least CVI was the macula. In eyes with CSCR, 100% had asymmetric dominant vortex veins compared to 38% in normal eyes. CONCLUSION: Choroidal imaging has advanced the diagnosis of CSCR. This has led to numerous imaging biomarkers like CVI, CT, and hyper-reflective dots for early detection and possible prognostication of CSCR. More techniques like wide field scans and en face imaging are being employed to characterize the choroid in CSCR.

Fractional order controllers increase the robustness of closed-loop deep brain stimulation systems.

We studied the effects of using fractional order proportional, integral, and derivative (PID) controllers in a closed-loop mathematical model of deep brain stimulation. The objective of the controller was to dampen oscillations from a neural network model of Parkinson's disease. We varied intrinsic parameters, such as the gain of the controller, and extrinsic variables, such as the excitability of the network. We found that in most cases, fractional order components increased the robustness of the model multi-fold to changes in the gains of the controller. Similarly, the controller could be set to a fixed set of gains and remain stable to a much larger range, than for the classical PID case, of changes in synaptic weights that otherwise would cause oscillatory activity. The increase in robustness is a consequence of the properties of fractional order derivatives that provide an intrinsic memory trace of past activity, which works as a negative feedback system. Fractional order PID controllers could provide a platform to develop stand-alone closed-loop deep brain stimulation systems.

Absceso pulmonar con fístula neumocutánea / Pulmonary abscess with pneumocutaneous fistula

Lung abscess is a rare entity in pediatric age, but it generates significant morbidity. Even less frequent is the presence of this with spontaneous drainage to the skin, generating an abscess in the chest wall, reason for consultation, of the present clinical case. Subsequently, the presence of lung abscess with extension to the chest wall without pleural involvement was documented by imaging studies, an extremely rare and unusual entity, with only one case described in the world literature within our reach and in an adult patient.

El absceso pulmonar es una entidad infrecuente en la edad pediátrica, pero que genera una morbilidad importante. Aún menos frecuente es la presencia de este con drenaje espontáneo a piel, generando un absceso en pared torácica, motivo de consulta, del presente caso clínico. Posteriormente y por estudios imagenológicos se documentó la presencia de absceso pulmonar con extensión a pared torácica sin afectación pleural, una entidad extremadamente rara e inusual, con un solo caso descrito en la literatura mundial a nuestro alcance y en un paciente adulto.

Microscopical Techniques to Analyze the Hepatic and Peritoneal Changes Caused by Fasciola hepatica Infection.

The helminth parasite Fasciola hepatica modulates the host immune response at early stages of infection (Rodríguez et al., PLoS Negl Trop Dis 9:e0004234, 2015; Vukman et al., J Immunol 190:2873-2879, 2013). Nevertheless, little is known about the cell composition of the peritoneal fluid at these early stages of infection.In this chapter, we describe a method to perform peritoneal lavages and to recover peritoneal fluid from sheep experimentally infected and noninfected with F. hepatica at early stages of infection. In addition, with the aim to characterize the peritoneal fluid immune cell phenotype, we describe a procedure to obtain the total leukocyte count, the differential leukocyte count and the preparation and storage of peritoneal fluid smears, together with the application of an immunocytochemical technique and an automatic method to count the immunoreactive cells. Finally, the present protocol describes the evaluation of the gross and the histopathological lesions together with the immunohistochemical analysis of the hepatic tissue.

Sequencing of a 'mouse azoospermia' gene panel in azoospermic men: identification of RNF212 and STAG3 mutations as novel genetic causes of meiotic arrest.

STUDY QUESTION: What is the diagnostic potential of next generation sequencing (NGS) based on a 'mouse azoospermia' gene panel in human non-obstructive azoospermia (NOA)? SUMMARY ANSWER: The diagnostic performance of sequencing a gene panel based on genes associated with mouse azoospermia was relatively successful in idiopathic NOA patients and allowed the discovery of two novel genes involved in NOA due to meiotic arrest. WHAT IS KNOWN ALREADY: NOA is a largely heterogeneous clinical entity, which includes different histological pictures. In a large proportion of NOA, the aetiology remains unknown (idiopathic NOA) and yet, unknown genetic factors are likely to play be involved. The mouse is the most broadly used mammalian model for studying human disease because of its usefulness for genetic manipulation and its genetic and physiological similarities to man. Mouse azoospermia models are available in the Mouse Genome Informatics database (MGI: http://www.informatics.jax.org/). STUDY DESIGN, SIZE, DURATION: The first step was to design of a 'mouse azoospermia' gene panel through the consultation of MGI. The second step was NGS analysis of 175 genes in a group of highly selected NOA patients (n = 33). The third step was characterization of the discovered gene defects in human testis tissue, through meiotic studies using surplus testicular biopsy material from the carriers of the RNF212 and STAG3 pathogenic variants. The final step was RNF212 and STAG3 expression analysis in a collection of testis biopsies. PARTICIPANTS/MATERIALS, SETTING, METHODS: From a total of 1300 infertile patients, 33 idiopathic NOA patients were analysed in this study, including 31 unrelated men and 2 brothers from a consanguineous family. The testis histology of the 31 unrelated NOA patients was as follows: 20 Sertoli cell-only syndrome (SCOS), 11 spermatogenic arrest (6 spermatogonial arrest and 5 spermatocytic arrest). The two brothers were affected by spermatocytic arrest. DNA extracted from blood was used for NGS on Illumina NextSeq500 platform. Generated sequence data was filtered for rare and potentially pathogenic variants. Functional studies in surplus testicular tissue from the carriers included the investigation of meiotic entry, XY body formation and metaphases by performing fluorescent immunohistochemical staining and immunocytochemistry. mRNA expression analysis through RT-qPCR of RNF212 and STAG3 was carried out in a collection of testis biopsies with different histology. MAIN RESULTS AND THE ROLE OF CHANCE: Our approach was relatively successful, leading to the genetic diagnosis of one sporadic NOA patient and two NOA brothers. This relatively high diagnostic performance is likely to be related to the stringent patient selection criteria i.e. all known causes of azoospermia were excluded and to the relatively high number of patients with rare testis histology (spermatocytic arrest). All three mutation carriers presented meiotic arrest, leading to the genetic diagnosis of three out of seven cases with this specific testicular phenotype. For the first time, we report biallelic variants in STAG3, in one sporadic patient, and a homozygous RNF212 variant, in the two brothers, as the genetic cause of NOA. Meiotic studies allowed the detection of the functional consequences of the mutations and provided information on the role of STAG3 and RNF212 in human male meiosis. LIMITATIONS, REASONS FOR CAUTION: All genes, with the exception of 5 out of 175, included in the panel cause azoospermia in mice only in the homozygous or hemizygous state. Consequently, apart from the five known dominant genes, heterozygous variants (except compound heterozygosity) in the remaining genes were not taken into consideration as causes of NOA. We identified the genetic cause in approximately half of the patients with spermatocytic arrest. The low number of analysed patients can be considered as a limitation, but it is a very rare testis phenotype. Due to the low frequency of this specific phenotype among infertile men, our finding may be considered of low clinical impact. However, at an individual level, it does have relevance for prognostic purposes prior testicular sperm extraction. WIDER IMPLICATIONS OF THE FINDINGS: Our study represents an additional step towards elucidating the genetic bases of early spermatogenic failure, since we discovered two new genes involved in human male meiotic arrest. We propose the inclusion of RNF212 and STAG3 in a future male infertility diagnostic gene panel. Based on the associated testis phenotype, the identification of pathogenic mutations in these genes also confers a negative predictive value for testicular sperm retrieval. Our meiotic studies provide novel insights into the role of these proteins in human male meiosis. Mutations in STAG3 were first described as a cause of female infertility and ovarian cancer, and Rnf212 knock out in mice leads to male and female infertility. Hence, our results stimulate further research on shared genetic factors causing infertility in both sexes and indicate that genetic counselling should involve not only male but also female relatives of NOA patients. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Spanish Ministry of Health Instituto Carlos III-FIS (grant number: FIS/FEDER-PI14/01250; PI17/01822) awarded to CK and AR-E, and by the European Commission, Reproductive Biology Early Research Training (REPROTRAIN, EU-FP7-PEOPLE-2011-ITN289880), awarded to CK, WB, and AE-M. The authors have no conflict of interest.

Genetics of ncHH: from a peculiar inheritance of a novel GNRHR mutation to a comprehensive review of the literature.

BACKGROUND: Normosmic congenital hypogonadotropic hypogonadism (ncHH) is caused by the deficient production, secretion, or action of gonadotropin-releasing hormone (GnRH). Its typical clinical manifestation is delayed puberty and azoospermia. Homozygous and compound heterozygous mutations in the GNRHR gene (4q13.2) are the most frequent genetic causes of ncHH. OBJECTIVES: (i) Characterization at the molecular level (genetic origin and functional effect) of a unique homozygous mutation (p.Gly99Glu) in a ncHH man; (ii) to provide a comprehensive catalog of GNRHR mutations with genotype-phenotype correlation and comparison of in vitro studies vs. in silico prediction tools. MATERIAL AND METHODS: A ncHH man and his parents, in whom we performed the following: (i) Sanger sequencing, qPCR of the GNRHR gene; (ii) chromosome 4 SNP array; and (iii) competition binding assay and inositol phosphate signaling assay. PubMed and Human Genome Mutation Database (HGMD) search for GNRHR mutations. Bioinformatic analysis of 55 reported variants. RESULTS: qPCR showed two GNRHR copies in the index case. SNP array revealed the inheritance of two homologous chromosomes 4 from the mother (maternal heterodisomy; hUPD) with two loss of heterozygosity regions, one of them containing the mutated gene (maternal isodisomy; iUPD). Functional studies for the p.Gly99Glu mutation demonstrated a right-shifted GnRH-stimulated signaling response. Bioinformatic tools show that commonly used in silico tools are poor predictors of the function of ncHH-associated GNRHR variants. DISCUSSION: Functional analysis of the p.Gly99Glu mutation is consistent with severely decreased GnRH binding affinity (a severe partial loss-of-function mutation). Complete LOF variants are associated with severe and severe/moderate phenotype, whereas partial LOF variants show wide range of clinical manifestations. CONCLUSION: This is the first ncHH patient carrying a novel causative missense mutation of GNRHR with proven 'severe pLOF' due to maternal hUPD/iUPD of chromosome 4. Our literature review shows that functional studies remain essential both for diagnostic and potential therapeutic purposes.

Pathological, immunological and parasitological study of sheep vaccinated with the recombinant protein 14-3-3z and experimentally infected with Fasciola hepatica.

In this study, the immunogenicity and protective capacity of a new recombinant vaccine candidate, the rFh14-3-3z protein was analysed in sheep experimentally challenged with Fasciola hepatica, in terms of fluke burden, faecal egg counts, hepatic damage and humoral immune response. Three groups of 8 animals each were used for study, group 1 was immunised with the rFh14-3-3z in Montanide adjuvant, whereas group 2 and 3 remained as adjuvant control and infection control groups, respectively. The parasitological analysis showed that no significant reduction in fluke burden, fluke size and faecal egg counts was detected. The extent of hepatic damage was very similar between groups. Nonetheless, animals immunised with the rFh14-3-3z protein induced the development of specific IgG1 and IgG2, being the IgG1 the predominant antibody; which confirms the immunogenicity of this protein in sheep. This is the first report of the 14-3-3z proteins as vaccine against the infection with F. hepatica.

Study of peritoneal macrophage immunophenotype in sheep experimentally infected with Fasciola hepatica.

During Fasciola hepatica infection, the parasite has the capability to modulate the host immune response towards a non-protector Th2 type instead of Th1. This type of immune response is closely related to the alternative activation of macrophages (M2 profile) as has been shown in vivo in murine models. In this study, an experiment was carried out in order to evaluate the expression of CD68, CD14, CD206 and iNOS in cells present in the peritoneal fluid of sheep during early stages of infection with F. hepatica (1, 3, 9 and 18 days post-infection, dpi) by immunocytochemistry. To the authors' knowledge, this is the first report that studies the in vivo immunophenotype of macrophages from the peritoneal fluid of sheep infected with F. hepatica. Throughout the experiments the absolute number of leucocytes progressively increased, reaching its highest value at 18 dpi, mainly due to the increase of eosinophils. This immunocytochemical study had two purposes: 1) CD68 expression was assessed with Hansel counterstaining, to optimally identify peritoneal macrophages, eosinophils and lymphocytes; 2) expression of CD14, CD206 and iNOS was evaluated to identify alternative or classical pathways of macrophage activation. The results showed a significant increase in CD14 from day 3 dpi compared with the non-infected group. CD206 expression at all time-points showed a significant and dramatic increase in comparison with the uninfected group. On the other hand, iNOS expression showed little variation, and was significantly decreased at 18 dpi in comparison with the uninfected group. These results suggest that F. hepatica induces an alternative activation of peritoneal macrophages of sheep from the first day post-infection, which may facilitate parasite survival. This is the first report describing M2 activation of peritoneal macrophages in ruminants infected with F. hepatica.

Evaluation of sperm DNA quality in men presenting with testicular cancer and lymphoma using alkaline and neutral Comet assays.

Despite more cancers in young men over the past two decades, improvements in therapies give a greater chance to live full lives following treatment. Sperm genomic quality is variable following cancer diagnosis, so its assessment is important if sperm cryopreservation is being considered. Here, we evaluated DNA damage using two DNA damage assays: an alkaline and for the first time, a neutral Comet assays in men presenting with testicular cancer (n = 19 for alkaline and 13 for neutral group) and lymphoma (n = 13 for alkaline and 09 for neutral group) compared with fertile donors (n = 20 for alkaline and 14 for neutral group). No significant differences were observed in any semen analysis parameters. In contrast, sperm DNA damage was higher in men with testicular cancer than in donors as assessed by both the alkaline (12.4% vs. 37.4%, p < 0.001) and neutral (7.5% vs. 13.4%; p < 0.05) Comet assays. Similar trends were observed in men with lymphoma. Here, sperm DNA damage was higher using both the alkaline (35.0% vs. 12.4%) and neutral (10.7% against 7.5% (p < 0.05) Comet assays. Moreover, the DNA strand breaks (particularly double-strand breaks) were significantly more prominent in men with cancer having abnormal seminal parameters than normozoospermic ones. This study showed that sperm DNA testing using alkaline and neutral Comet assays is more sensitive than semen analysis in detecting impaired sperm quality in men presenting with cancer. It may provide a useful adjunct when considering storage prior to cancer investigations and assisted reproductive techniques (ART)-based treatment.

Fractional observer to estimate periodical forces.

In the present work we propose a fractional state observer with constant gain to estimate the periodical force exerted on a mechanical system by measuring only its displacement. The state observer is designed from both the Fourier series that approximates the periodical force and the equations of the damped harmonic oscillator that represents the behavior of the system. Specifically, the reconstruction of the force is carried out from the estimates of the series coefficients, which in fact are part of the dynamical system that composes the observer. Adams-Bashforth-Moulton method is used to compute the fractional derivatives of the observer in the Liouville-Caputo sense. Experiments based on real data are presented to show the advantages of using a fractional observer in the reconstruction of forces.

Th1/Th2 balance in the liver and hepatic lymph nodes of vaccinated and unvaccinated sheep during acute stages of infection with Fasciola hepatica.

The expression of IFNγ and IL4 was quantified using q-PCR in the liver and hepatic lymph nodes (HLN) of sheep during early stages of infection with Fasciola hepatica (1, 3, 9 and 18days post-infection, dpi). A group of animals (Group 1) were vaccinated with Fasciola hepatica recombinant cathepsin L1 (FhCL1) in montanide 70 VG prior to infection, a second group (group 2) was used as infected control and a third (group 3) was used as uninfected control. To study vaccine efficacy three additional groups were sacrificed 19 weeks post-infection (group 4 immunized with CL1, group 5 with the adjuvant and group 6 was used as infected control). The vaccinated group did not show significant fluke reduction compared to the adjuvant group and infected control group. IL4 expression was observed to increase at 9 dpi and was further elevated at 18 dpi in the liver and HLN of vaccinated and infected control groups compared to the uninfected group. IFNγ expression exhibited different dynamics in the liver and HLN compared to IL4; thus, in the liver this cytokine increased at 9 dpi in the vaccinated and at 18 dpi in vaccinated and infected control groups, while in the HLN it decreased gradually and significantly from 1 dpi onwards. These results suggest that a marked Th2 polarization is present from 9 dpi in HLN and from 18 dpi in the liver. The increase of IFNγ in the liver may correspond with tissue damage response with granuloma formation. The FhCL1 vaccine did not alter the Th1/Th2 balance when compared to unvaccinated and infected sheep. The study of IFNγ and IL4 in the various tissue compartments in sheep could facilitate selection of new adjuvants inducing a strong Th1 response for a more rationale vaccine formulation.

Apoptosis of peritoneal leucocytes during early stages of Fasciola hepatica infections in sheep.

Several immunomodulatory properties have been described in Fasciola hepatica infections. Apoptosis has been shown to be an effective mechanism to avoid the immune response in helminth infections. The aim of the present work was to study apoptosis in peritoneal leucocytes of sheep experimentally infected with F. hepatica during the early stages of infection. Five groups (n=5) of sheep were used. Groups 2-5 were orally infected with 200 metacercariae (mc) and sacrificed at 1, 3, 9 and 18days post-infection (dpi), respectively. Group 1 was used as the uninfected control (UC). Apoptosis was detected using three different methods 1) immunocytochemistry (ICC) with a polyclonal antibody anti-active caspase-3; 2) an annexin V flow cytometry assay using the Annexin V-FITC/propidium iodide (PI); and 3) transmission electron microscopy (TEM). The differential leucocyte count revealed that the majority of peritoneal granulocytes were eosinophils, which increased significantly at 9 and 18 dpi with respect to the uninfected controls. The ICC study revealed that the percentage of caspase-3+ apoptotic peritoneal leucocytes increased significantly from 3 dpi onwards with respect to the uninfected controls. The flow cytometry annexin V assay detected a very significant (P<0.001) increase of apoptotic peritoneal macrophages, lymphocytes and granulocytes, which remained higher than in the UC until 18 dpi. Transmission electron microscopy studies also confirmed the presence of apoptosis in peritoneal eosinophils at 18 dpi. This is the first report of apoptosis induced by F. hepatica in the peritoneal leucocytes of sheep in vivo. The results of this work suggest the importance of apoptosis induction for the survival of the juvenile parasites in the peritoneal migratory stages of infection.

Distribution of Foxp3+ T cells in the liver and hepatic lymph nodes of goats and sheep experimentally infected with Fasciola hepatica.

Foxp3 regulatory T cells (Tregs) are now considered to play a key role in modulation of immune responses during parasitic helminth infections. Immunomodulation is a key factor in Fasciola hepatica infection; however, the distribution and role of Foxp3+ Tregs cells have not been investigated in F. hepatica infected ruminants. The aim of this study was to evaluate the presence of Foxp3+ Tregs in the liver and hepatic lymph nodes from experimentally infected sheep and goats during acute and chronic stages of infection. Three groups of goats (n=6) and three groups of sheep (n=6) were used in this study. Goats in groups 1-2 and sheep in groups 4-5 were orally infected with metacercarie of ovine origin. Groups 1 and 4 were killed during the acute stage of the infection, at nine days post infection (dpi); groups 2 and 5 were killed during the chronic stage, at 15 and19 weeks post infection respectively (wpi). Groups 3 (goats) and 6 (sheep) were left as uninfected controls. Fluke burdens and liver damage were assessed and the avidin-biotin-complex method was used for the immunohistochemical study. At nine dpi in acute hepatic lesions, the number of both Foxp3+ and CD3+ T lymphocytes increased significantly in goats and sheep. In the chronic stages of infection (15-19wpi), the number of Foxp3+ and CD3+ T lymphocytes were also significantly increased with respect to control livers, particularly in portal spaces with severely enlarged bile ducts (response to adult flukes) while the increase was lower in granulomas, chronic tracts and smaller portal spaces (response to tissue damage). Foxp3+ Tregs were increased in the cortex of hepatic lymph nodes of sheep (chronic infection) and goats (acute and chronic infection). The estimated proportion of T cells which were Foxp3+ was significantly increased in the large bile ducts and hepatic lymph node cortex of chronically infected goats but not sheep. This first report of the expansion of Foxp3+ Tregs in acute and chronic hepatic lesions in ruminants suggests that these cells may be involved in both parasite survival and modulation of hepatic damage. Future studies should be focused on the investigation of parasite molecules and cytokines involved in this process.

Fasciola hepatica induces eosinophil apoptosis in the migratory and biliary stages of infection in sheep.

The aim of the present work was to evaluate the number of apoptotic eosinophils in the livers of sheep experimentally infected with Fasciola hepatica during the migratory and biliary stages of infection. Four groups (n=5) of sheep were used; groups 1-3 were orally infected with 200 metacercariae (mc) and sacrificed at 8 and 28 days post-infection (dpi), and 17 weeks post-infection (wpi), respectively. Group 4 was used as an uninfected control. Apoptosis was detected using immunohistochemistry with a polyclonal antibody against anti-active caspase-3, and transmission electron microscopy (TEM). Eosinophils were identified using the Hansel stain in serial sections for caspase-3, and by ultrastructural features using TEM. At 8 and 28 dpi, numerous caspase-3(+) eosinophils were mainly found at the periphery of acute hepatic necrotic foci. The percentage of caspase -3(+) apoptotic eosinophils in the periphery of necrotic foci was high (46.1-53.9) at 8 and 28 dpi, respectively, and decreased in granulomas found at 28 dpi (6%). Transmission electron microscopy confirmed the presence of apoptotic eosinophils in hepatic lesions at 8 and 28 dpi. At 17 wpi, apoptotic eosinophils were detected in the infiltrate surrounding some enlarged bile ducts containing adult flukes. This is the first report of apoptosis induced by F. hepatica in sheep and the first study reporting apoptosis in eosinophils in hepatic inflammatory infiltrates in vivo. The high number of apoptotic eosinophils in acute necrotic tracts during the migratory and biliary stages of infection suggests that eosinophil apoptosis may play a role in F. hepatica survival during different stages of infection.

Comprehensive investigation in patients affected by sperm macrocephaly and globozoospermia.

The aim of this study was to provide a comprehensive genetic/phenotypic characterization of subjects suffering infertility owing to sperm macrocephaly (n = 3) or globozoospermia (n = 9) and to investigate whether the patients' genetic status was correlated with the alteration of various sperm parameters. AURKC was sequenced in case of sperm macrocephaly while the DPY19L2 status has been analyzed by multiple approaches including a novel qPCR-based copy number assay in case of globozoospermia. Globozoospermic patients were also analyzed for SPACA1, a novel candidate gene herein tested for the first time in humans. The effect of the patients' genetic status was interrogated by implementing the molecular screening with the characterization of several sperm parameters: (i) routine sperm analysis, integrated with transmission electron microscopy; (ii) sperm fluorescent in situ hybridization (FISH) analysis; (iii) sperm DNA fragmentation (DF) analysis. Moreover, for the first time, we performed microsatellite instability analysis as a marker of genome instability in men with sperm macrocephaly and globozoospermia. Finally, artificial reproductive technology (ART) history has been reported for those patients who underwent the treatment. Macrocephalic patients had an AURKC mutation and >89% tetraploid, highly fragmented spermatozoa. DPY19L2 was mutated in all patients with >80% globozoospermia: the two homozygous deleted men and the compound heterozygous showed the severest phenotype (90-100%). The newly developed qPCR method was fully validated and has the potential of detecting also yet undiscovered deletions. DPY19L2 status is unlikely related to FISH anomalies and DF, although globozoospermic men showed a higher disomy rate and DF compared with internal reference values. No patient was mutated for SPACA1. Our data support the general agreement on the negative correlation between macro/globozoospermia and conventional intracytoplasmic sperm injection outcomes. Microsatellites were stable in all patients analyzed. The comprehensive picture provided on these severe phenotypes causing infertility is of relevance in the management of patients undergoing ART.

Biocatalytic synthesis of polypyrrole powder, colloids, and films using horseradish peroxidase.

Polypyrrole was synthesized in high yield by a biocatalytic method in mild aqueous media using hydrogen peroxide as oxidizer. A redox mediator, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) diammonium salt, was used to oxidize the pyrrole. ABTS is a very effective peroxidase substrate, which was enzymatically oxidized to generate a radical cation that in turn was able to chemically oxidize pyrrole. This indirect biocatalytic method was implemented because pyrrole is not a substrate of horseradish peroxidase, however, the polymerization process was successfully optimized and later adapted to prepare also polypyrrole thin films and water dispersible polypyrrole colloids. The polypyrrole powder and colloids were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, electrical conductivity, and thermogravimetric analysis. In addition, the deposition of the polypyrrole thin film was monitored using a quartz-crystal microbalance and its morphology studied by optical and scanning electron microscopy. The biocatalytic polymerization of pyrrole results in a polymer spectroscopically very similar to chemically synthesized polypyrrole.