Host genetic variation guides hepacivirus clearance, chronicity, and liver fibrosis in mice.

BACKGROUND AIMS: Human genetic variation is thought to guide the outcome of HCV infection, but model systems within which to dissect these host genetic mechanisms are limited. Norway rat hepacivirus, closely related to HCV, causes chronic liver infection in rats but causes acute self-limiting hepatitis in typical strains of laboratory mice, which resolves in 2 weeks. The Collaborative Cross (CC) is a robust mouse genetics resource comprised of a panel of recombinant inbred strains, which model the complexity of the human genome and provide a system within which to understand diseases driven by complex allelic variation. APPROACH RESULTS: We infected a panel of CC strains with Norway rat hepacivirus and identified several that failed to clear the virus after 4 weeks. Strains displayed an array of virologic phenotypes ranging from delayed clearance (CC046) to chronicity (CC071, CC080) with viremia for at least 10 months. Body weight loss, hepatocyte infection frequency, viral evolution, T-cell recruitment to the liver, liver inflammation, and the capacity to develop liver fibrosis varied among infected CC strains. CONCLUSIONS: These models recapitulate many aspects of HCV infection in humans and demonstrate that host genetic variation affects a multitude of viruses and host phenotypes. These models can be used to better understand the molecular mechanisms that drive hepacivirus clearance and chronicity, the virus and host interactions that promote chronic disease manifestations like liver fibrosis, therapeutic and vaccine performance, and how these factors are affected by host genetic variation.

Proximal and Distal Bronchioles Contribute to the Pathogenesis of Non-Cystic Fibrosis Bronchiectasis.

Rationale: Non-cystic fibrosis bronchiectasis (NCFB) may originate in bronchiolar regions of the lung. Accordingly, there is a need to characterize the morphology and molecular characteristics of NCFB bronchioles. Objectives: Test the hypothesis that NCFB exhibits a major component of bronchiolar disease manifest by mucus plugging and ectasia. Methods: Morphologic criteria and region-specific epithelial gene expression, measured histologically and by RNA in situ hybridization and immunohistochemistry, identified proximal and distal bronchioles in excised NCFB lungs. RNA in situ hybridization and immunohistochemistry assessed bronchiolar mucus accumulation and mucin gene expression. CRISPR-Cas9-mediated IL-1R1 knockout in human bronchial epithelial cultures tested IL-1α and IL-1ß contributions to mucin production. Spatial transcriptional profiling characterized NCFB distal bronchiolar gene expression. Measurements and Main Results: Bronchiolar perimeters and lumen areas per section area were increased in proximal, but not distal, bronchioles in NCFB versus control lungs, suggesting proximal bronchiolectasis. In NCFB, mucus plugging was observed in ectatic proximal bronchioles and associated nonectatic distal bronchioles in sections with disease. MUC5AC and MUC5B mucins were upregulated in NCFB proximal bronchioles, whereas MUC5B was selectively upregulated in distal bronchioles. Bronchiolar mucus plugs were populated by IL-1ß-expressing macrophages. NCFB sterile sputum supernatants induced human bronchial epithelial MUC5B and MUC5AC expression that was >80% blocked by IL-1R1 ablation. Spatial transcriptional profiling identified upregulation of genes associated with secretory cells, hypoxia, interleukin pathways, and IL-1ß-producing macrophages in mucus plugs and downregulation of epithelial ciliogenesis genes. Conclusions: NCFB exhibits distinctive proximal and distal bronchiolar disease. Both bronchiolar regions exhibit bronchiolar secretory cell features and mucus plugging but differ in mucin gene regulation and ectasia.

The environmental impact of beef and ultra-processed food consumption in Brazil.

OBJECTIVE: This study evaluated the independent and combined environmental impacts of the consumption of beef and ultra-processed foods in Brazil. DESIGN: Cross-sectional study. SETTING: Brazil. PARTICIPANTS: We used food purchases data from a national household budget survey conducted between July 2017 and July 2018, representing all Brazilian households. Food purchases were converted into energy, carbon footprints and water footprints. Multiple linear regression models were used to assess the association between quintiles of beef and ultra-processed foods in total energy purchases and the environmental footprints, controlling for sociodemographic variables. RESULTS: Both beef and ultra-processed foods had a significant linear association with carbon and water footprints (P < 0·01) in crude and adjusted models. In the crude upper quintile of beef purchases, carbon and water footprints were 47·7 % and 30·8 % higher, respectively, compared to the lower quintile. The upper quintile of ultra-processed food purchases showed carbon and water footprints 14·4 % and 22·8 % higher, respectively, than the lower quintile. The greatest reduction in environmental footprints would occur when both beef and ultra-processed food purchases are decreased, resulting in a 21·1 % reduction in carbon footprint and a 20·0 % reduction in water footprint. CONCLUSIONS: Although the environmental footprints associated with beef consumption are higher, dietary patterns with lower consumption of beef and ultra-processed foods combined showed the greatest reduction in carbon and water footprints in Brazil. The high consumption of beef and ultra-processed foods is harmful to human health, as well as to the environment; thus, their reduction is beneficial to both.

Cereal bar enriched with ora-pro-nóbis (Pereskia aculeata Miller): physicochemical and sensory characterization.

Ora-pro-nobis (OPN) is an unconventional food plant with high nutritional value, and its nutritional composition can be altered according to cultivation. Cereal bars are a popular nutrient-poor foods, and OPN could be incorporated to improve the nutritional quality. This study aimed to evaluate the physicochemical characteristics and sensory acceptability of cereal bars enriched with OPN flour (OpnF) from different forms of cultivation. OpnF was obtained by dehydrating and grinding OPN leaves collected in rural (ROpnF) and urban (UOpnF) municipalities. Two formulations of cereal bars, peanut flavor (Bpn) and mango flavor (Bmg), each with 10% OpnF, were prepared. The macronutrients and mineral composition, oxalate content, water activity, texture, color profile, and acceptability were evaluated. ROpnF had the highest protein, iron, and manganese content, whereas UOpnF had the highest ash and magnesium content. The oxalic acid/calcium ratio was 1.43 and did not imply calcium bioavailability. In addition to nutritional and protein values, Bpn and Bmg presented a good sensory acceptability index of > 77.5% with market potential. Bmg has the highest mineral content and is a source of iron, manganese, and magnesium. OpnF can be used in cereal bars and potentially improve nutritional attributes and used in other foods in a similar way.

Prevalence and Mechanisms of Mucus Accumulation in COVID-19 Lung Disease.

Rationale: The incidence and sites of mucus accumulation and molecular regulation of mucin gene expression in coronavirus (COVID-19) lung disease have not been reported. Objectives: To characterize the incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease. Methods: Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by Alcian blue and periodic acid-Schiff staining, immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected human bronchial epithelial (HBE) cultures were used to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures. Measurements and Main Results: MUC5B and variably MUC5AC RNA concentrations were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the subacute/chronic disease phase after SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of subjects with COVID-19 in both morphologically identified bronchioles and microcysts, and MUC5B accumulated in damaged alveolar spaces. SARS-CoV-2-infected HBE cultures exhibited peak titers 3 days after inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days after inoculation. SARS-CoV-2 infection of HBE cultures induced expression of epidermal growth factor receptor (EGFR) ligands and inflammatory cytokines (e.g., IL-1α/ß) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways or administration of dexamethasone reduced SARS-CoV-2-induced mucin expression. Conclusions: SARS-CoV-2 infection is associated with a high prevalence of distal airspace mucus accumulation and increased MUC5B expression in COVID-19 autopsy lungs. HBE culture studies identified roles for EGFR and IL-1R signaling in mucin gene regulation after SARS-CoV-2 infection. These data suggest that time-sensitive mucolytic agents, specific pathway inhibitors, or corticosteroid administration may be therapeutic for COVID-19 lung disease.

Barriers and facilitators to anal cancer screening among people living with HIV in Puerto Rico.

BACKGROUND: Anal cancer (AC) disproportionally affects people living with HIV (PLWH). Although there are no consensus-based AC screening guidelines, experts recommend anal pap as a primary screening tool in settings where high-resolution anoscopy (HRA) is available. We aimed to assess barriers and facilitators to anal cancer screening in a sample of Hispanic PLWH in Puerto Rico. METHODS: To assess their knowledge and attitudes, we conducted a cross-sectional survey from 2020-2021 among PLWH in Puerto Rico (n = 212). Data was collected through a telephone interview that assessed information on sociodemographics, knowledge, and attitudes about AC, and the history of AC screening. The chi-square test, Fisher exact test, and logistic regression models were used to assess factors associated with screening uptake. RESULTS: Anal Pap and HRA awareness were 60.4% and 30.7%, respectively. Anal Pap and HRA uptake was 51.5% and 19.3%, respectively. The most common barriers for anal Pap and HRA were lack of knowledge about the test and lack of physician recommendation. MSM were more likely to have heard of anal Pap (OR: 2.15, 95% CI:1.30-3.54) than MSW. MSM (OR: 3.04, 95% CI: 1.79-5.19) and women (OR: 3.00, 95% CI: 1.72-5.20) were also more likely to have undergone anal Pap. Similarly, individuals with a history of genital warts were more likely to have heard of anal Pap and HRA and have undergone anal Pap and HRA. Awareness of where to go for concerns about anal health was positively associated with having received anal Pap and HRA. CONCLUSIONS: With emerging evidence on the effectiveness of screening and treatment for anal cancer, several organizations are steering toward generating consensus-based anal cancer screening recommendations. Our study provides foundational data on barriers and facilitators to anal cancer screening in Puerto Rico that will be critical to informing screening implementation in this US territory.

Quantitative Imaging Analysis of the Spatial Relationship between Antiretrovirals, Reverse Transcriptase Simian-Human Immunodeficiency Virus RNA, and Fibrosis in the Spleens of Nonhuman Primates.

Although current antiretroviral therapy (ART) has increased life expectancy, a cure for human immunodeficiency virus (HIV) remains elusive due to the persistence of the virus in tissue reservoirs. In the present study, we sought to elucidate the relationship between antiretrovirals (ARVs) and viral expression in the spleen. We performed mass spectrometry imaging (MSI) of 6 different ARVs, RNAscope in situ hybridization of viral RNA, and immunohistochemistry of three different fibrosis markers in the spleens of 8 uninfected and 10 reverse transcriptase simian-human immunodeficiency virus (RT-SHIV)-infected rhesus macaques (infected for 6 weeks) that had been dosed for 10 days with combination ART. Using MATLAB, computational quantitative imaging analysis was performed to evaluate the spatial and pharmacological relationships between the 6 ARVs, viral RNA, and fibrotic deposition. In these spleens, >50% of the spleen tissue area was not covered by any detectable ARV response (any concentration above the limits of detection for individual ARVs). The median spatial ARV coverage across all tissues was driven by maraviroc followed by efavirenz. Yet >50% of RNA-positive cells were not exposed to any detectable ARV. Quantifiable maraviroc and efavirenz colocalization with RNA-positive cells was usually greater than the in vitro concentration inhibiting 50% replication (IC50). Fibrosis markers covered more than 50% of the spleen tissue area and had negative relationships with cumulative ARV coverages. Our findings suggest that a heterogeneous ARV spatial distribution must be considered when evaluating viral persistence in lymphoid tissue reservoirs.

Pulmonary exposure of mice to ammonium perfluoro(2-methyl-3-oxahexanoate) (GenX) suppresses the innate immune response to carbon black nanoparticles and stimulates lung cell proliferation.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) have been associated with respiratory diseases in humans, yet the mechanisms through which PFAS cause susceptibility to inhaled agents is unknown. Herein, we investigated the effects of ammonium perfluoro(2-methyl-3-oxahexanoate) (GenX), an emerging PFAS, on the pulmonary immune response of mice to carbon black nanoparticles (CBNP). We hypothesized that pulmonary exposure to GenX would increase susceptibility to CBNP through suppression of innate immunity. METHODS: Male C57BL/6 mice were exposed to vehicle, 4 mg/kg CBNP, 10 mg/kg GenX, or CBNP and GenX by oropharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) was collected at 1 and 14 days postexposure for cytokines and total protein. Lung tissue was harvested for histopathology, immunohistochemistry (Ki67 and phosphorylated (p)-STAT3), western blotting (p-STAT3 and p-NF-κB), and qRT-PCR for cytokine mRNAs. RESULTS: CBNP increased CXCL-1 and neutrophils in BALF at both time points evaluated. However, GenX/CBNP co-exposure reduced CBNP-induced CXCL-1 and neutrophils in BALF. Moreover, CXCL-1, CXCL-2 and IL-1ß mRNAs were increased by CBNP in lung tissue but reduced by GenX. Western blotting showed that CBNP induced p-NF-κB in lung tissue, while the GenX/CBNP co-exposed group displayed decreased p-NF-κB. Furthermore, mice exposed to GenX or GenX/CBNP displayed increased numbers of BALF macrophages undergoing mitosis and increased Ki67 immunostaining. This was correlated with increased p-STAT3 by western blotting and immunohistochemistry in lung tissue from mice co-exposed to GenX/CBNP. CONCLUSIONS: Pulmonary exposure to GenX suppressed CBNP-induced innate immune response in the lungs of mice yet promoted the proliferation of macrophages and lung epithelial cells.

Quantitative Imaging Analysis of the Spatial Relationship between Antiretrovirals, Reverse Transcriptase Simian-Human Immunodeficiency Virus RNA, and Collagen in the Mesenteric Lymph Nodes of Nonhuman Primates.

Human immunodeficiency virus (HIV) persistence in tissue reservoirs is a major barrier to HIV cure. While antiretrovirals (ARVs) suppress viral replication, antiretroviral therapy (ART) interruption results in rapid rebound viremia that may originate from lymphoid tissues. To understand the relationship between anatomic distribution of ARV exposure and viral expression in lymph nodes, we performed mass spectrometry imaging (MSI) of 6 ARVs, RNAscope in situ hybridization for viral RNA (vRNA), and immunohistochemistry of collagen in mesenteric lymph nodes from 8 uninfected and 10 reverse transcriptase simian/human immunodeficiency virus (RT-SHIV)-infected rhesus macaques dosed to steady state with combination ART. MATLAB-based quantitative imaging analysis was used to evaluate spatial and pharmacological relationships between these ARVs, viral RNA (both vRNA+ cells and follicular dendritic cell [FDC]-bound virions), and collagen deposition. Using MSI, 31% of mesenteric lymph node tissue area was found to be not covered by any ARV. Additionally, 28% of FDC-trapped virions and 21% of infected cells were not exposed to any detected ARV. Of the 69% of tissue area that was covered by cumulative ART exposure, nearly 100% of concentrations were greater than in vitro 50% inhibitory concentration (IC50) values; however, 52% of total tissue coverage was from only one ARV, primarily maraviroc. Collagen covered ∼35% of tissue area but did not influence ARV distribution heterogeneity. Our findings are consistent with our hypothesis that ARV distribution, in addition to total-tissue drug concentration, must be considered when evaluating viral persistence in lymph nodes and other reservoir tissues.

The current state of headache medicine education in the United States and Canada: An observational, survey-based study of neurology clerkship directors and curriculum deans.

OBJECTIVE: To investigate the current headache medicine education paradigm in allopathic and osteopathic medical schools in the United States and Canada. BACKGROUND: There is a disparity in the number of clinicians specially trained to treat patients with headache disorders and the number of people who have them. Early education and exposure to headache medicine is crucial to address this disparity. However, the current state of headache education within medical schools across the United States and Canada is unknown. METHODS: The authors created a medical student headache education survey, which is a 20-question REDCap survey that was distributed via email to the neurology clerkship director, curriculum dean, or similar role at each US and Canadian MD or DO conferring medical school. The email listserv was created using the American Academy of Neurology Clerkship Directory, the Association of American Medical Colleges Organization Directory, the American Association of College of Osteopathic Medicine Organization Directory, manual searches of the institutions' websites, and phone calls and emails to administrators as needed. RESULTS: Of the 249 individuals contacted, 78 completed the survey, yielding a response rate of 31.3%. Of those responses, 84.6% of respondents (66/78) reported that their institution has at least one mandatory session on headache disorders. Many of these sessions (72.7% (48/78)) occurred during preclinical training, and 74.2% (49/78) occurred as part of the clinical curricula. Of respondents, 44.9% (39/78) reported that their institutions coordinate headache education across training levels (i.e., from preclinical to clinical), and only 17.9% (14/78) coordinate across clinical rotations. The most common topics covered were headache red flags, migraine, pharmacologic management, and differentiating primary versus secondary headache. 65.4% of respondents (51/78) felt that the preclinical headache curriculum prepares their students for the clinical experience, and 55.1% (43/78) felt that medical students were learning enough about headache medicine at their institution. Barriers to educating medical students about headache included insufficient time during courses, lack of administrative support in curricula development, lack of available resources, and lack of student interest. Case-based learning modules and online lectures were the most desired educational materials to improve medical student headache education at their institution. CONCLUSIONS: The majority of medical schools report incorporating headache medicine education into preclinical or clinical curricula and cover a range of topics in headache medicine. Yet there remains a lack of consistency, with some reporting limited headache education, citing barriers such as lack of administrative support and available educational resources. There is also variation in what is being taught at the medical student level. Future projects should aim to address said barriers, with the goal of providing a standardized headache medicine curriculum for use across medical schools.

Isolated postaxial polydactyly: Epidemiologic characteristics from a multicenter birth defects study.

Isolated postaxial polydactyly (I-PAP), as a single defect, is a frequent malformation, characterized by an extra digit placed on the ulnar or fibular side of the limbs. Worldwide prevalence varies from as high as 225/10,000 in Nigerians to so low as 6.08/10,000 in Argentinians. Genetic-ethnic background significantly affects worldwide prevalence and type of I-PAP. Herein we describe the epidemiological characteristics of I-PAP in 697 newborns, 383 males and 314 females identified in 1,178,993 examined live births from a multicenter case-control hospital-based population study, the Mexican program of Registry and Epidemiological Surveillance of Congenital Malformations (RYVEMCE). The main characteristics analyzed included total I-PAP, stratified in Types A and B, defined as complete or incomplete extra-digit formation, respectively, sex prevalence, affected limb, laterality, parity, prematurity, delivery-type, twinning, consanguinity, and parental age. Males (6.35/10,000) are significantly more frequently affected than females (5.45/10,000), hands more than feet, left more than right limbs, and Type B (74.50%) more than A (25.50%). Prematurity and forceps use were significantly more frequent in cases than controls. An evident decreasing time-trend prevalence was present. Similar findings with other studies were males, upper and left limbs more frequently affected. Findings that were not previously reported include prematurity, forceps use, a significant decreasing time trend and an inverse ethnic prevalence for Types A (75%) and B (25%) in the Mayan population in contrast to other worldwide ethnic groups.

Antiretroviral concentrations and surrogate measures of efficacy in the brain tissue and CSF of preclinical species.

1. Antiretroviral concentrations in cerebrospinal fluid (CSF) are used as surrogate for brain tissue, although sparse data support this. We quantified antiretrovirals in brain tissue across preclinical models, compared them to CSF, and calculated 90% inhibitory quotients (IQ90) for nonhuman primate (NHP) brain tissue. Spatial distribution of efavirenz was performed by mass-spectrometry imaging (MSI). 2. HIV or RT-SHIV-infected and uninfected animals from two humanized mouse models (hemopoietic-stem cell/RAG2-, n = 36; bone marrow-liver-thymus/BLT, n =13) and an NHP model (rhesus macaque, n =18) were dosed with six antiretrovirals. Brain tissue, CSF (NHPs), and plasma were collected at necropsy. Drug concentrations were measured by LC-MS/MS. Rapid equilibrium dialysis determined protein binding in NHP brain. 3. Brain tissue penetration of most antiretrovirals were >10-fold lower (p < 0.02) in humanized mice than NHPs. NHP CSF concentrations were >13-fold lower (p <0.02) than brain tissue with poor agreement except for efavirenz (r = 0.91, p = 0.001). Despite 97% brain tissue protein binding, efavirenz achieved IQ90>1 in all animals and 2-fold greater white versus gray matter concentration. 4. Brain tissue penetration varied across animal models for all antiretrovirals except raltegravir, and extrapolating brain tissue concentrations between models should be avoided. With the exception of efavirenz, CSF is not a surrogate for brain tissue concentrations.

Human Schwann cells exhibit long-term cell survival, are not tumorigenic and promote repair when transplanted into the contused spinal cord.

The transplantation of rodent Schwann cells (SCs) provides anatomical and functional restitution in a variety of spinal cord injury (SCI) models, supporting the recent translation of SCs to phase 1 clinical trials for human SCI. Whereas human (Hu)SCs have been examined experimentally in a complete SCI transection paradigm, to date the reported behavior of SCs when transplanted after a clinically relevant contusive SCI has been restricted to the use of rodent SCs. Here, in a xenotransplant, contusive SCI paradigm, the survival, biodistribution, proliferation and tumorgenicity as well as host responses to HuSCs, cultured according to a protocol analogous to that developed for clinical application, were investigated. HuSCs persisted within the contused nude rat spinal cord through 6 months after transplantation (longest time examined), exhibited low cell proliferation, displayed no evidence of tumorigenicity and showed a restricted biodistribution to the lesion. Neuropathological examination of the CNS revealed no adverse effects of HuSCs. Animals exhibiting higher numbers of surviving HuSCs within the lesion showed greater volumes of preserved white matter and host rat SC and astrocyte ingress as well as axon ingrowth and myelination. These results demonstrate the safety of HuSCs when employed in a clinically relevant experimental SCI paradigm. Further, signs of a potentially positive influence of HuSC transplants on host tissue pathology were observed. These findings show that HuSCs exhibit a favorable toxicity profile for up to 6 months after transplantation into the contused rat spinal cord, an important outcome for FDA consideration of their use in human clinical trials.

Challenges facing European agriculture and possible biotechnological solutions.

Agriculture faces many challenges to maximize yields while it is required to operate in an environmentally sustainable manner. In the present study, we analyze the major agricultural challenges identified by European farmers (primarily related to biotic stresses) in 13 countries, namely Belgium, Bulgaria, the Czech Republic, France, Germany, Hungary, Italy, Portugal, Romania, Spain, Sweden, UK and Turkey, for nine major crops (barley, beet, grapevine, maize, oilseed rape, olive, potato, sunflower and wheat). Most biotic stresses (BSs) are related to fungi or insects, but viral diseases, bacterial diseases and even parasitic plants have an important impact on yield and harvest quality. We examine how these challenges have been addressed by public and private research sectors, using either conventional breeding, marker-assisted selection, transgenesis, cisgenesis, RNAi technology or mutagenesis. Both national surveys and scientific literature analysis followed by text mining were employed to evaluate genetic engineering (GE) and non-GE approaches. This is the first report of text mining of the scientific literature on plant breeding and agricultural biotechnology research. For the nine major crops in Europe, 128 BS challenges were identified with 40% of these addressed neither in the scientific literature nor in recent European public research programs. We found evidence that the private sector was addressing only a few of these "neglected" challenges. Consequently, there are considerable gaps between farmer's needs and current breeding and biotechnology research. We also provide evidence that the current political situation in certain European countries is an impediment to GE research in order to address these agricultural challenges in the future. This study should also contribute to the decision-making process on future pertinent international consortia to fill the identified research gaps.

Immobilization of Aspergillus niger lipase on chitosan-coated magnetic nanoparticles using two covalent-binding methods.

Aspergillus niger lipase immobilization by covalent binding on chitosan-coated magnetic nanoparticles (CMNP), obtained by one-step co-precipitation, was studied. Hydroxyl and amino groups of support were activated using glycidol and glutaraldehyde, respectively. Fourier transform infrared spectrometry, high-resolution transmission electron microscopy and thermogravimetric analysis confirmed reaction of these coupling agents with the enzyme and achievement of a successful immobilization. The derivatives showed activities of 309.5 ± 2.0 and 266.2 ± 2.8 U (g support)(-1) for the CMNP treated with glutaraldehyde and with glycidol, respectively. Immobilization enhanced the enzyme stability against changes of pH and temperature, compared to free lipase. Furthermore, the kinetic parameters K m and V max were determined for the free and immobilized enzyme. K m value quantified for enzyme immobilized by means of glutaraldehyde was 1.7 times lowers than for free lipase. High storage stability during 50 days was observed in the immobilized derivatives. Finally, immobilized derivatives retained above 80% of their initial activity after 15 hydrolytic cycles. The immobilized enzyme can be applied in various biotechnological processes involving magnetic separation.

Analysis of KIAA1549-BRAF fusion gene expression and IDH1/IDH2 mutations in low grade pediatric astrocytomas.

Low-grade astrocytomas comprise about 30 % of the central nervous system tumors in children. Several investigations have searched a correlation between the BRAF gene fusions alterations and mutations at IDH1 and IDH2 genes in low grade pediatric astrocytomas. This study identified the expression of KIAA1549-BRAF fusion gene and BRAF V600E mutation, mutations at exon 4 of the IDH1 and IDH2 genes in samples of pilocytic astrocytomas (PA) and grade-II astrocytomas (A-II) pediatric patients. The correlation between these alterations and the clinical profile of the patients was also evaluated. Eighty-two samples of low-grade astrocytomas (65 PA and 17 A-II) were analyzed by PCR and sequencing for each of the targets identified. We identified the KIAA1549-BRAF fusion transcript in 45 % of the samples. BRAF V600E and BRAFins598T mutations were detected in 7 and 1 % of the samples, respectively. Mutations in the R132/R172 residues of the IDH1/IDH2 genes were detected in only two samples, and the G105G polymorphism (rs11554137:C>T) was identified in ten patients. Additionally, we observed two mutations out of the usual hotspots at IDH1 and IDH2 genes. We observed a smaller frequency of mutations in IDHs genes than previously described, but since the prior studies were composed of adult or mixed (adults and children) samples, we believe that our results represent a relevant contribution to the growing knowledge in low grade childhood astrocytomas.

[Evaluation of vaginal dysfunction in symptomatic and asymptomatic pregnant women by using the analysis of basic vaginal states (BVS) and its comparison with the conventional microbiological study]. / Evaluación de la disfunción vaginal en mujeres embarazadas sintomáticas y asintomáticas mediante la utilización de los estados vaginales básicos (EVB) y su comparación con el estudio microbiológico convencional.

Infections of the lower genital tract associated to maternal and perinatal complications frequently occur during pregnancy. The aim of this study was to evaluate vaginal dysfunction through the analysis of basic vaginal states (BVS) using the methodology of balance of the vaginal content (BAVACO) and to compare it with the microbiological study of candidiasis, trichomoniasis and bacterial vaginosis (BV). Pregnant patients (1238) were examined from 2010 to 2012. In asymptomatic (A) (n: 1046) and symptomatic pregnant women (S) (n: 192) BVS I was 59.5% and 26% of the patients, respectively. BVS II was observed in 19.7% of A and in 17.2% of S. BVS III was only detected in A in 0.4%. BVS IV was observed in 14.4% of A and in 38% of S. BVS V was detected in 6% of A and in 18.8% of S. Yeasts were associated to BVS I and II in 55.5% and 23.2% of A, respectively; and in 32.4% and 31% of S, respectively. Trichomonas were associated to BVS I in 50% of A, to IV in 44.4% of S and to V in 33.3% of S. BAVACO susceptibility to detect yeasts was 80.4% and 85.5% in A and S, respectively; 40% and 75% in A and S, respectively, to detect trichomonas and 100% in A and S to detect BV. BAVACO specificity was 100% for all pathogens in A and S. The study of BVS proved useful as a guide to evaluate vaginal dysfunction, regardless of symptomatology. Therefore, this study is recommended as prenatal control.

Effect of street trees shade on perceived thermal comfort in a south temperate climate: The sidewalks of Montevideo (Uruguay).

The use of vegetation in cities is one of the most promising strategies for urban climate change adaptation and mitigation. Tree shade influences heat storage from surfaces reducing long wave radiation emission which directly affects people. People 's heat perception depends more on insolation and the temperature of surrounding objects than on air temperature itself. There is a need for analyzes that include the combined effects of physical and human variables on thermal comfort, as well as location-based studies to address its climatic and social conditions. In order to compare the effect of the trees on microenvironmental temperature and perceived thermal comfort, we measured physical parameters and performed structured interviews on three downtown streets of Montevideo, Uruguay, which had sections with and without trees on four dates during the summer. Generally, people surveyed under both treatments stated they did not feel fully comfortable due to summer heat, but the proportion of people who stated feeling in thermal comfort under tree shade was more than double than the unshaded sections. The seasonal ARIMA analysis supported that the tree shade reduced the microenvironmental temperature by its effect on radiant temperature. By using a statistical decision tree methodology that combines all the variables in the same analysis, we found a greater impact of physical variables than personal variables on people's thermal comfort and thermal preferences. We also identified gender as a significant variable that affects people's thermal preferences, where 46.4 % of females preferred a slightly colder environment.

Antifungal Susceptibility and Candida sp. Biofilm Production in Clinical Isolates of HIV-Positive Brazilian Patients under HAART Therapy.

The aim of the present study was to characterize biofilms formed by Candida spp. clinical isolates (n = 19), isolated from the oral mucosa of HIV-positive patients. For characterizing the biofilms formed by several Candida sp. strains, isolated from HIV-positive patients, in terms of formed biomass, matrix composition and antifungal susceptibility profile, clinical isolates (n = 19) were collected from oral mucosa and identified. The biofilm of the samples was cultured with fluconazole (1250 mg/L), voriconazole (800 mg/L), anidulafungin (2 mg/L) or amphotericin B (2 mg/L). Afterwards, the quantification of the total biomass was performed using crystal violet assay, while the proteins and carbohydrates levels were quantified in the matrix. The results showed a predominance of C. albicans, followed by C. krusei. Around 58% of the Candida spp. biofilm had susceptibility to fluconazole and voriconazole (800 mg/L), 53% to anidulafungin and 74% to amphotericin B. C. krusei presented both the lowest and the highest biofilm matrix contents in polysaccharides and proteins. The low resistance to antifungal agents reported here was probably due to the fact that none of the participants had a prolonged exposure to these antifungals. A predominance of less virulent Candida spp. strains with low or no resistance to antifungals was observed. This can be attributed to a low fungal selective pressure. This most probably happened due to a low fungal selective pressure but also due to a good adherence to HAART therapy, which guarantees a stable and stronger immune patient response.

Spatial Relationships in the Tumor Microenvironment Demonstrate Association with Pathologic Response to Neoadjuvant Chemoimmunotherapy in Muscle-invasive Bladder Cancer.

BACKGROUND: Platinum-based neoadjuvant chemotherapy (NAC) is standard for patients with muscle-invasive bladder cancer (MIBC). Pathologic response (complete: ypT0N0 and partial: