Enhanced oxidative stress resistance in Ustilago maydis and its implications on the virulence.

The phytopathogenic fungus Ustilago maydis causes corn smut by suppressing host plant defenses, including the oxidative burst response. While many studies have investigated how U. maydis responds to oxidative stress during infection, the consequences of heightened resistance to oxidative stress on virulence remain understudied. This study aimed to identify the effects on virulence in U. maydis strains exhibiting enhanced resistance to hydrogen peroxide (H2O2).To achieve this, we exposed U. maydis SG200 to 20 escalating H2O2 shocks, resulting in an adapted strain resistant to concentrations as high as 60 mM of H2O2, a lethal dose for the initial strain. Genetic analysis of the adapted strain revealed five nucleotide substitutions, two minor copy number variants, and a large amplification event on chromosome nine (1-149 kb) encompassing the sole catalase gene. Overexpressing catalase increased resistance to H2O2; however, this resistance was lower than that observed in the adapted strain. Additionally, virulence was reduced in both strains with enhanced H2O2 resistance.In summary, enhanced H2O2 resistance, achieved through either continuous exposure to the oxidative agent or through catalase overexpression, decreased virulence. This suggests that the response to the oxidative stress burst in U. maydis is optimal and that increasing the resistance to H2O2 does not translate into increased virulence. These findings illuminate the intricate relationship between oxidative stress resistance and virulence in U. maydis, offering insights into its infection mechanisms.

Modulation of tumor microenvironment by targeting histone acetylation in bladder cancer.

Alterations in the epigenetic machinery in both tumor and immune cells contribute to bladder cancer (BC) development, constituting a promising target as an alternative therapeutic option. Here, we have explored the effects of a novel histone deacetylase (HDAC) inhibitor CM-1758, alone or in combination with immune checkpoint inhibitors (ICI) in BC. We determined the antitumor effects of CM-1758 in various BC cell lines together with the induction of broad transcriptional changes, with focus on the epigenetic regulation of PD-L1. Using an immunocompetent syngeneic mouse model of metastatic BC, we studied the effects of CM-1758 alone or in combination with anti-PD-L1 not only on tumor cells, but also in the tumor microenvironment. In vitro, we found that CM-1758 has cytotoxic and cytostatic effects either by inducing apoptosis or cell cycle arrest in BC cells at low micromolar levels. PD-L1 is epigenetically regulated by histone acetylation marks and is induced after treatment with CM-1758. We also observed that treatment with CM-1758 led to an important delay in tumor growth and a higher CD8 + T cell tumor infiltration. Moreover, anti-PD-L1 alone or in combination with CM-1758 reprogramed macrophage differentiation towards a M1-like polarization state and increased of pro-inflammatory cytokines systemically, yielding potential further antitumor effects. Our results suggest the possibility of combining HDAC inhibitors with immunotherapies for the management of advanced metastatic BC.

Yeast diversity in open agave fermentations across Mexico.

Yeasts are a diverse group of fungal microorganisms that are widely used to produce fermented foods and beverages. In Mexico, open fermentations are used to obtain spirits from agave plants. Despite the prevalence of this traditional practice throughout the country, yeasts have only been isolated and studied from a limited number of distilleries. To systematically describe the diversity of yeast species from open agave fermentations, here we generate the YMX-1.0 culture collection by isolating 4524 strains from 68 sites with diverse climatic, geographical, and biological contexts. We used MALDI-TOF mass spectrometry for taxonomic classification and validated a subset of the strains by ITS and D1/D2 sequencing, which also revealed two potential novel species of Saccharomycetales. Overall, the composition of yeast communities was weakly associated with local variables and types of climate, yet a core set of six species was consistently isolated from most producing regions. To explore the intraspecific variation of the yeasts from agave fermentations, we sequenced the genomes of four isolates of the nonconventional yeast Kazachstania humilis. The genomes of these four strains were substantially distinct from a European isolate of the same species, suggesting that they may belong to different populations. Our work contributes to the understanding and conservation of an open fermentation system of great cultural and economic importance, providing a valuable resource to study the biology and genetic diversity of microorganisms living at the interface of natural and human-associated environments.

Management of Penis Fracture: Clinical Cases Treated in Our Center and Comparison with Literature.

OBJECTIVE: To expose our clinical experience in the management of the penis fracture and make a literature review about this topic. METHODS: We present a case of a 49 years old man diagnosticated penis fracture. We expose the results of our clinical cases diagnosticated and treated from October 2018 to October 2020 and make a literature review. RESULTS: A 49 years old man that presented swallow and sensation of snap during a sexual intercourse. He was diagnosticated of penis fracture with the help of ultrasound and was performed an urgent reparation. The results of our serial of 4 cases were: The 75% (3) presented pain and sensation of snap during the sexual intercourse, 50% (2) detumescence, the 100% (4) ecchymosis and the 25% (1) present an actual lateral deviation. Anyone presents erectile dysfunction nowadays. CONCLUSIONS: The penis fracture has a clinical diagnosis but the ultrasound could be useful. The early surgical repair has a good result with low tase of complications.

Management of Penis Fracture: Clinical Cases Treated in Our Center and Comparison with Literature

Abstract Objective: To expose our clinical experience in the management of the penis fracture and make a literature review about this topic. Methods: We present a case of a 49 years old man diagnosticated penis fracture. We expose the results of our clinical cases diagnosticated and treated from October 2018 to October 2020 and make a literature review. Results: A 49 years old man that presented swallow and sensation of snap during a sexual intercourse. He was diagnosticated of penis fracture with the help of ultrasound and was performed an urgent reparation. The results of our serial of 4 cases were: The 75% (3) presented pain and sensation of snap during the sexual intercourse, 50% (2) detumescence, the 100% (4) ecchymosis and the 25% (1) present an actual lateral deviation. Anyone presents erectile dysfunction nowadays. Conclusions: The penis fracture has a clinical diagnosis but the ultrasound could be useful. The early surgical repair has a good result with low tase of complications (AU)

Responsabilidad profesional derivada del ejercicio de la odontología en Costa Rica: I Parte / Professional responsibility derived from the practice of dentistry in Costa Rica: Part I

El aumento a nivel mundial de las denuncias por responsabilidad profesional en Ciencias de la Salud hace imprescindible que quienes ejerzan esta profesión deban conocer las implicaciones de su labor, así como cumplir con los principios bioéticos y jurídicos para garantizar una sana relación odontólogo-paciente. Es importante comprender que el ejercicio de la odontología en Costa Rica está regulado por normas, códigos y leyes como cualquier otra actividad humana en un Estado de Derecho.

The number of complaints related to professional liability in health sciences has increased worldwide, it is essential for dental professionals to be aware of the implications of their labor, as well as to comply with bioethical and legal principles to ensure a healthy dentist-patient relationship. It is important to understand that the practice of dentistry in Costa Rica is regulated by norms, codes, and laws like any other human activity in a State governed by the rule of law.

Draft Genome Sequence of a Kazachstania humilis Strain Isolated from Agave Fermentation.

The ascomycetous yeast Kazachstania humilis is an active species in backslopped sourdough and in the spontaneous fermentation of several traditional foods and beverages. Here, we report the draft genome sequence of a K. humilis strain isolated from agave must from a traditional distillery in Mexico.

Contribution of Host miRNA-223-3p to SARS-CoV-Induced Lung Inflammatory Pathology.

Severe acute respiratory syndrome coronavirus (SARS-CoV) and the closely related SARS-CoV-2 are emergent highly pathogenic human respiratory viruses causing acute lethal disease associated with lung damage and dysregulated inflammatory responses. SARS-CoV envelope protein (E) is a virulence factor involved in the activation of various inflammatory pathways. Here, we study the contribution of host miRNAs to the virulence mediated by E protein. Small RNAseq analysis of infected mouse lungs identified miRNA-223 as a potential regulator of pulmonary inflammation, since it was significantly increased in SARS-CoV-WT virulent infection compared to the attenuated SARS-CoV-ΔE infection. In vivo inhibition of miRNA-223-3p increased mRNA levels of pro-inflammatory cytokines and NLRP3 inflammasome, suggesting that during lung infection, miRNA-223 might contribute to restrict an excessive inflammatory response. Interestingly, miRNA-223-3p inhibition also increased the levels of the CFTR transporter, which is involved in edema resolution and was significantly downregulated in the lungs of mice infected with the virulent SARS-CoV-WT virus. At the histopathological level, a decrease in the pulmonary edema was observed when miR-223-3p was inhibited, suggesting that miRNA-223-3p was involved in the regulation of the SARS-CoV-induced inflammatory pathology. These results indicate that miRNA-223 participates in the regulation of E protein-mediated inflammatory response during SARS-CoV infection by targeting different host mRNAs involved in the pulmonary inflammation, and identify miRNA-223 as a potential therapeutic target in SARS-CoV infection. IMPORTANCE The SARS-CoV-2 pandemic has emphasized the need to understand the mechanisms of severe lung inflammatory pathology caused by human deadly coronaviruses in order to design new antiviral therapies. Here, we identify miRNA-223-3p as a host miRNA involved in the regulation of lung inflammatory response mediated by envelope (E) protein during SARS-CoV infection. miRNAs downregulate the expression of cellular mRNAs and participate in complex networks of mRNA-miRNA interactions that regulate cellular processes. The inhibition of miRNA-223 in infected mice by intranasal administration of antisense RNAs led to changes in the expression of host factors involved in inflammation (cytokines, chemokines, and NLRP3 inflammasome) and in the resolution of lung edema ion transporter CFTR. These results confirmed the contribution of miRNA-223 to the regulation of SARS-CoV-induced pathogenic processes and support the therapeutic potential of inhibiting miRNAs during coronavirus infection using RNA interference approaches.

Toward Tumor Fight and Tumor Microenvironment Remodeling: PBA Induces Cell Cycle Arrest and Reduces Tumor Hybrid Cells' Pluripotency in Bladder Cancer.

Bladder cancer (BC) is the second most frequent cancer of the genitourinary system. The most successful therapy since the 1970s has consisted of intravesical instillations of Bacillus Calmette-Guérin (BCG) in which the tumor microenvironment (TME), including macrophages, plays an important role. However, some patients cannot be treated with this therapy due to comorbidities and severe inflammatory side effects. The overexpression of histone deacetylases (HDACs) in BC has been correlated with macrophage polarization together with higher tumor grades and poor prognosis. Herein we demonstrated that phenylbutyrate acid (PBA), a HDAC inhibitor, acts as an antitumoral compound and immunomodulator. In BC cell lines, PBA induced significant cell cycle arrest in G1, reduced stemness markers and increased PD-L1 expression with a corresponding reduction in histone 3 and 4 acetylation patterns. Concerning its role as an immunomodulator, we found that PBA reduced macrophage IL-6 and IL-10 production as well as CD14 downregulation and the upregulation of both PD-L1 and IL-1ß. Along this line, PBA showed a reduction in IL-4-induced M2 polarization in human macrophages. In co-cultures of BC cell lines with human macrophages, a double-positive myeloid-tumoral hybrid population (CD11b+EPCAM+) was detected after 48 h, which indicates BC cell-macrophage fusions known as tumor hybrid cells (THC). These THC were characterized by high PD-L1 and stemness markers (SOX2, NANOG, miR-302) as compared with non-fused (CD11b-EPCAM+) cancer cells. Eventually, PBA reduced stemness markers along with BMP4 and IL-10. Our data indicate that PBA could have beneficial properties for BC management, affecting not only tumor cells but also the TME.

Genomic Landscape of Vinflunine Response in Metastatic Urothelial Cancer.

BACKGROUND AND AIMS: Metastatic urothelial carcinoma (mUC) remains an incurable disease with limited treatment options after platinum-based chemotherapy and immune checkpoint blockade (ICB). Vinflunine has shown a modest increase in overall survival and remains a therapeutic option for chemo- and immunotherapy refractory tumours. However, biomarkers that could identify responding patients to vinflunine and possible alternative therapies after failure to treatment are still missing. In this study, we aimed to identify potential genomic biomarkers of vinflunine response in mUC patient samples and potential management alternatives. METHODS: Formalin-fixed paraffin-embedded samples of mUC patients (n = 23) from three university hospitals in Spain were used for genomic targeted-sequencing and transcriptome (using the Immune Profile panel by NanoString) analyses. Patients who received vinflunine after platinum-based chemotherapy failure were classified in non-responders (NR: progressive disease ≤ 3 months; n= 11) or responders (R: response ≥ 6 months; n = 12). RESULTS: Genomic characterization revealed that the most common alteration, TP53 mutations, had comparable frequency in R (6/12; 50%) and NR (4/11; 36%). Non-synonymous mutations in KTM2C (4/12; 33.3%), PIK3CA (3/12; 25%) and ARID2 (3/12; 25%) were predominantly associated with response. No significant difference was observed in tumour mutational burden (TMB) between R and NR patients. The NR tumours showed increased expression of diverse immune-related genes and pathways, including various interferon gamma-related genes. We also identified increased MAGEA4 expression as a potential biomarker of non-responding tumours to vinflunine treatment. CONCLUSIONS: Our data may help to identify potential genomic biomarkers of response to vinflunine. Moreover, tumours refractory to vinflunine showed immune signatures potentially associated with response to ICB. Extensive validation studies, including longitudinal series, are needed to corroborate these findings.

Cell Therapies in Bladder Cancer Management.

Currently, bladder cancer (BC) represents a challenging problem in the field of Oncology. The high incidence, prevalence, and progression of BC have led to the exploration of new avenues in its management, in particular in advanced metastatic stages. The recent inclusion of immune checkpoint blockade inhibitors as a therapeutic option for BC represents an unprecedented advance in BC management. However, although some patients show durable responses, the fraction of patients showing benefit is still limited. Notwithstanding, cell-based therapies, initially developed for the management of hematological cancers by infusing immune or trained immune cells or after the engineering of chimeric antigen receptor (CAR) expressing cells, are promising tools to control, or even cure, solid tumors. In this review, we summarize recent cell-based immunotherapy studies, with a special focus on BC.

Perfect Match Genomic Landscape strategy: Refinement and customization of reference genomes.

When addressing a genomic question, having a reliable and adequate reference genome is of utmost importance. This drives the necessity to refine and customize reference genomes (RGs). Our laboratory has recently developed a strategy, the Perfect Match Genomic Landscape (PMGL), to detect variation between genomes [K. Palacios-Flores et al.Genetics 208, 1631-1641 (2018)]. The PMGL is precise and sensitive and, in contrast to most currently used algorithms, is nonstatistical in nature. Here we demonstrate the power of PMGL to refine and customize RGs. As a proof-of-concept, we refined different versions of the Saccharomyces cerevisiae RG. We applied the automatic PMGL pipeline to refine the genomes of microorganisms belonging to the three domains of life: the archaea Methanococcus maripaludis and Pyrococcus furiosus; the bacteria Escherichia coli, Staphylococcus aureus, and Bacillus subtilis; and the eukarya Schizosaccharomyces pombe, Aspergillus oryzae, and several strains of Saccharomyces paradoxus. We analyzed the reference genome of the virus SARS-CoV-2 and previously published viral genomes from patients' samples with COVID-19. We performed a mutation-accumulation experiment in E. coli and show that the PMGL strategy can detect specific mutations generated at any desired step of the whole procedure. We propose that PMGL can be used as a final step for the refinement and customization of any haploid genome, independently of the strategies and algorithms used in its assembly.

High-Throughput Task to Study Memory Recall During Spatial Navigation in Rodents.

Spatial navigation is one of the most frequently used behavioral paradigms to study memory formation in rodents. Commonly used tasks to study memory are labor-intensive, preventing the simultaneous testing of multiple animals with the tendency to yield a low number of trials, curtailing the statistical power. Moreover, they are not tailored to be combined with neurophysiology recordings because they are not based on overt stereotyped behavioral responses that can be precisely timed. Here we present a novel task to study long-term memory formation and recall during spatial navigation. The task consists of learning sessions during which mice need to find the rewarding port that changes from day to day. Hours after learning, there is a recall session during which mice search for the location of the memorized rewarding port. During the recall sessions, the animals repeatedly poke the remembered port over many trials (up to ∼20) without receiving a reward (i.e., no positive feedback) as a readout of memory. In this task, mice show memory of port locations learned on up to three previous days. This eight-port maze task requires minimal human intervention, allowing for simultaneous and unsupervised testing of several mice in parallel, yielding a high number of recall trials per session over many days, and compatible with recordings of neural activity.

Prediction and identification of recurrent genomic rearrangements that generate chimeric chromosomes in Saccharomyces cerevisiae.

Genomes are dynamic structures. Different mechanisms participate in the generation of genomic rearrangements. One of them is nonallelic homologous recombination (NAHR). This rearrangement is generated by recombination between pairs of repeated sequences with high identity. We analyzed rearrangements mediated by repeated sequences located in different chromosomes. Such rearrangements generate chimeric chromosomes. Potential rearrangements were predicted by localizing interchromosomal identical repeated sequences along the nuclear genome of the Saccharomyces cerevisiae S288C strain. Rearrangements were identified by a PCR-based experimental strategy. PCR primers are located in the unique regions bordering each repeated region of interest. When the PCR is performed using forward primers from one chromosome and reverse primers from another chromosome, the break point of the chimeric chromosome structure is revealed. In all cases analyzed, the corresponding chimeric structures were found. Furthermore, the nucleotide sequence of chimeric structures was obtained, and the origin of the unique regions bordering the repeated sequence was located in the expected chromosomes, using the perfect-match genomic landscape strategy (PMGL). Several chimeric structures were searched in colonies derived from single cells. All of the structures were found in DNA isolated from each of the colonies. Our findings indicate that interchromosomal rearrangements that generate chimeric chromosomes are recurrent and occur, at a relatively high frequency, in cell populations of S. cerevisiae.

Análisis de huellas labiales en una muestra de estudiantes de odontología de la Universidad de Costa Rica mediante la clasificación de Renaud: estudio piloto / Lip prints analysis in a sample of dentistry students of the University of Costa Rica based in Renaud's classification: pilot study

Resumen El presente estudio, tuvo como objetivo, analizar los distintos patrones morfológicos presentes en las huellas labiales de una determinada población costarricense y su correlación con las variables, sexo, edad y afinidad biológica. Se analizaron 105 huellas labiales pertenecientes a estudiantes de quinto y sexto año de la carrera de odontología de la Universidad de Costa Rica durante el II semestre del 2016. De las 105 huellas labiales analizadas mediante el método de Renaud, el patrón más prevalente fue el tipo C de manera general, mientras que, de manera individual, en labio superior predomina el patrón tipo A y en labio inferior predomina el patrón tipo C. De la totalidad de muestras analizadas, se destaca que todas revelaron patrones distintos y la correlación estadística entre las variables, sexo, edad y afinidad biológica, permitió conocer la existencia de elementos que podrían ser útiles para identificación de individuos en el ámbito forense. Los resultados demuestran que, con la aplicación de las técnicas adecuadas en la toma de muestras y un análisis estandarizado, las huellas labiales pueden ser utilizadas en el ámbito forense como método de identificación de individuos. Este estudio, además, pone en evidencia la necesidad de realizar más investigación sobre huellas labiales en Costa Rica, no sólo por la necesidad de un aumento en el número de muestras y así obtener mayores resultados estadísticos, si no también, para desarrollar las herramientas necesarias en la toma, revelado y análisis de huellas labiales, así como su aplicabilidad al servicio de la justicia costarricense.

Abstract The objective of this study was to analyze the different morphological patterns present in the lip prints of a certain Costa Rican population and its correlation with the variables, sex, age and biological affinity. 105 lip prints were analyzed belonging to fifth and sixth year students of the dental career of the University of Costa Rica during the second semester of 2016. Of the 105 labials analyzed by the Renaud method, the most prevalent pattern was type C in general, while, individually, in the upper lip, predominantly, the type of pattern, A and the lower lip, predominantly the type C pattern. Of the total of the analyzed samples, the different patterns and the statistical correlation between the variables, sex, age and biological affinity, remarks information that can be useful to identify individuals in the forensic field. The results show that, with the application of the appropriate techniques in the taking of samples and a standardized analysis, the lip prints can be used in the field of forensic science as a method of identification of individuals. This study also highlights the need to conduct more research on lip prints in Costa Rica, not only to increase the number of samples, but also to obtain more extended statistical results. Besides, this research can be used to develop the necessary tools in the taking, development and analysis of lip print. Moreover, the results obtained may be taken into consideration in order to improve forensic research in the Costa Rican justice system.

A Perfect Match Genomic Landscape Provides a Unified Framework for the Precise Detection of Variation in Natural and Synthetic Haploid Genomes.

We present a conceptually simple, sensitive, precise, and essentially nonstatistical solution for the analysis of genome variation in haploid organisms. The generation of a Perfect Match Genomic Landscape (PMGL), which computes intergenome identity with single nucleotide resolution, reveals signatures of variation wherever a query genome differs from a reference genome. Such signatures encode the precise location of different types of variants, including single nucleotide variants, deletions, insertions, and amplifications, effectively introducing the concept of a general signature of variation. The precise nature of variants is then resolved through the generation of targeted alignments between specific sets of sequence reads and known regions of the reference genome. Thus, the perfect match logic decouples the identification of the location of variants from the characterization of their nature, providing a unified framework for the detection of genome variation. We assessed the performance of the PMGL strategy via simulation experiments. We determined the variation profiles of natural genomes and of a synthetic chromosome, both in the context of haploid yeast strains. Our approach uncovered variants that have previously escaped detection. Moreover, our strategy is ideally suited for further refining high-quality reference genomes. The source codes for the automated PMGL pipeline have been deposited in a public repository.

Molecular diversity and frequency of the diarrheagenic enteric protozoan Giardia duodenalis and Cryptosporidium spp. in a hospital setting in Northern Spain.

BACKGROUND: Human giardiosis and cryptosporidiosis are caused by the enteric protozoan parasites Giardia duodenalis and Cryptosporidium spp. Both pathogens are major contributors to the global burden of diarrhoeal disease, affecting primarily children and immunodebilitated individuals in resource-poor settings. Giardiosis and cryptosporidiosis also represent an important, often underestimate, public health threat in developed countries. In Spain only limited information is currently available on the epidemiology of these infections. Molecular data on the diversity, frequency, geographical distribution, and seasonality of G. duodenalis assemblages/sub-assemblages and Cryptosporidium species/sub-genotypes are particularly scarce. METHODS: A longitudinal molecular epidemiological survey was conducted between July 2015 to September 2016 in patients referred to or attended at the Hospital San Pedro (La Rioja, Northern Spain) that tested positive for G. duodenalis (N = 106) or Cryptosporidium spp. (N = 103) by direct microscopy and/or a rapid lateral flow immunochromatographic assay. G. duodenalis infections were subsequently confirmed by real-time PCR and positive isolates assessed by multi-locus sequence genotyping of the glutamate dehydrogenase and ß-giardin genes of the parasite. Cryptosporidium species and sub-genotypes were investigated at the 60 kDa glycoprotein or the small subunit ribosomal RNA genes of the parasite. Sociodemographic and clinical parameters of infected patients were also gathered and analysed. PRINCIPAL FINDINGS: Out of 90 G. duodenalis-positive isolates by real-time PCR a total of 16 isolates were successfully typed. AII (44%, 7/16) was the most prevalent sub-assemblage found, followed by BIV (31%, 5/16) and BIII (19%, 3/16). A discordant genotype result AII/AIII was identified in an additional (6%, 1/16) isolate. No mixed infections A+B were detected. Similarly, a total of 81 Cryptosporidium spp. isolates were successfully typed, revealing the presence of C. hominis (81%, 66/81) and C. parvum (19%, 15/81). Obtained GP60 sequences were assigned to sub-type families Ib (73%, 59/81) within C. hominis, and IIa (7%, 6/81) and IId (2%, 2/81) within C. parvum. A marked inter-annual variation in Cryptosporidium cases was observed. CONCLUSIONS: Human giardiasis and cryptosporidiosis are commonly identified in patients seeking medical care in Northern Spain and represent a more important health concern than initially thought. Assemblage A within G. duodenalis and sub-genotype IbA10G2 within C. hominis were the genetic variants of these parasite species more frequently found circulating in the population under study. Molecular data presented here seem to suggest that G. duodenalis and Cryptosporidium infections arise through anthroponotic rather than zoonotic transmission in this Spanish region.

Association between enteric protozoan parasites and gastrointestinal illness among HIV- and tuberculosis-infected individuals in the Chowke district, southern Mozambique.

Human immune deficiency virus (HIV) and tuberculosis (TB) infections remain major public health issues globally, particularly in sub-Saharan Africa. Impairment of both cell-mediated and humoral immunity by HIV and/or TB infections may limit the host's defences against other pathogens, including the diarrheagenic protozoan Cryptosporidium spp., Giardia intestinalis, and Entamoeba histolytica. During September-December 2015 a cross-sectional study was conducted to assess the prevalence and molecular diversity of these enteric parasites among HIV- and/or TB-infected patients at a medical reference centre in Chowke district, southern Mozambique. A total of 99 stool specimens were initially screened by direct microscopy and further confirmed and characterised by molecular methods. DNA sequence analyses of the genes encoding the small subunit ribosomal RNA and the 60-kDa glycoprotein were used for the typing and sub-typing of Cryptosporidium isolates, respectively. G. intestinalis-positive isolates by real-time PCR were subsequently typed at the glutamate dehydrogenase locus. Differential diagnosis of E. histolytica/dispar was achieved by real-time PCR. G. intestinalis (8.1%) was the enteric protozoan more frequently detected, followed by Cryptosporidium spp. (7.1%), and Entamoeba histolytica/dispar (6.1%). Two HIV-infected (but not TB-infected) patients harbour G. intestinalis and Cryptosporidium spp. co-infections. Two (29%) G. intestinalis isolates were successfully characterised, revealing the presence of known AII and novel BIV genotypes. Four (57%) Cryptosporidium isolates were unmistakeable assigned to C. hominis, identifying two (IbA10G2 and IdA22) sub-types. Cryptosporidium infections were not associated to diarrhoea in HIV-positive patients, probably because improved immune function in the affected individuals due to antiretroviral therapy. G. intestinalis was considered a non-opportunistic pathogen, whereas the presence of E. histolytica could not be confirmed by molecular methods. Based on their common presence in the studied clinical population, we recommend the effective diagnosis and treatment of these enteropathogens for improving the management of HIV and TB patients.

SARS-CoV-Encoded Small RNAs Contribute to Infection-Associated Lung Pathology.

Severe acute respiratory syndrome coronavirus (SARS-CoV) causes lethal disease in humans, which is characterized by exacerbated inflammatory response and extensive lung pathology. To address the relevance of small non-coding RNAs in SARS-CoV pathology, we deep sequenced RNAs from the lungs of infected mice and discovered three 18-22 nt small viral RNAs (svRNAs). The three svRNAs were derived from the nsp3 (svRNA-nsp3.1 and -nsp3.2) and N (svRNA-N) genomic regions of SARS-CoV. Biogenesis of CoV svRNAs was RNase III, cell type, and host species independent, but it was dependent on the extent of viral replication. Antagomir-mediated inhibition of svRNA-N significantly reduced in vivo lung pathology and pro-inflammatory cytokine expression. Taken together, these data indicate that svRNAs contribute to SARS-CoV pathogenesis and highlight the potential of svRNA-N antagomirs as antivirals.

Occurrence and molecular genotyping of Giardia duodenalis and Cryptosporidium spp. in wild mesocarnivores in Spain.

There is a surprisingly scarce amount of epidemiological and molecular data on the prevalence, frequency, and diversity of the intestinal protozoan parasites Giardia duodenalis and Cryptosporidium spp. in wildlife in general and mesocarnivore species in particular. Consequently, the extent of the cyst/oocyst environmental contamination attributable to these wild host species and their potential implications for public veterinary health remain largely unknown. In this molecular epidemiological survey a total of 193 individual faecal samples from badgers (Meles meles, n=70), ferrets (Mustela putorius furo, n=2), genets (Genetta genetta, n=6), Iberian lynxes (Lynx pardinus, n=6), beech martens (Martes foina, n=8), mongooses (Herpestes ichneumon, n=2), otters (Lutra lutra, n=2), polecats (Mustela putorius, n=2), red foxes (Vulpes vulpes, n=87), wildcats (Felis silvestris, n=2), and wolves (Canis lupus, n=6) were obtained from road-killed, hunted, and accidentally found carcasses, and from camera-trap surveys or animals entering rescue shelters, during the period December 2003-April 2016. Investigated specimens were collected in five Spanish autonomous regions including Andalusia (n=1), Asturias (n=69), Basque Country (n=49), Castile-La Mancha (n=38), and Extremadura (n=36). The presence of cysts/oocysts was confirmed by PCR-based methods targeting the small subunit (ssu) ribosomal RNA gene of these parasite species. Genotyping of the obtained isolates were attempted at appropriate markers including the glutamate dehydrogenase (G. duodenalis) and the 60-kDa glycoprotein (C. parvum and C. ubiquitum) loci. Overall, G. duodenalis was detected in 8% (7/87) of red foxes, a single beech marten, and a single wolf, respectively. Cryptosporidium was identified in 3% (2/70) of badgers, 8% (7/87) of red foxes, a single genet, and a single mongoose, respectively. None of the nine G. duodenalis isolates generated could be genotyped at the assemblage/sub-assemblage level. Out of the nine Cryptosporidium isolates successfully characterized, three were identified as C. canis (one in a mongoose and two in red foxes), and three as C. parvum (one in a badger and three in red foxes). The remaining three isolates were assigned to C. felis (in a red fox), C. hominis (in a badger), and C. ubiquitum (in a red fox), respectively. Two additional Cryptosporidium isolates infecting a badger and a genet, respectively, were untypable. The red fox was confirmed as a suitable host of potentially zoonotic Cryptosporidium species, mainly C. parvum and C. ubiquitum. The high mobility and wide home range of red foxes, together with their increasing presence in urban and peri-urban settings, may led to the overlapping of sylvatic and domestic cycles of the parasite, and consequently, to an increased risk of cryptosporidiosis in production animals and humans. The detection of C. hominis oocysts in a badger raises the question of whether this finding represents a true infection or a sporadic event of mechanical passage of C. hominis oocyst of anthroponotic origin.