Mediator kinase inhibition further activates super-enhancer-associated genes in AML.

Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA, IRF8, IRF1 and ETV6 (refs 6-8). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.

DNA extraction from individual Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) specimens: Which is the method with better results?

Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are a group of small insects of great concern for Public Health. These dipterous are intensely studied worldwide due to their involvement in the transmission of several pathogens, mainly Leishmania spp. parasites. Nowadays, the molecular tools have been included in Phlebotomine sand flies studies and has shown to be powerful tools in bioecology studies of these dipterous. Thereby, when molecular approaches are employed, there is a great concern regarding the amount and quality of the DNA obtained for analysis. Here, seven methods of DNA extraction, between commercial kits and in house extraction protocols were evaluated. We considered measure of DNA concentration and purity ratios using a spectrophotometer to check the performance of each protocol. In addition, the quality evaluation of the DNA extracted was performed by endogenous gene PCR on samples. The results of the seven evaluated DNA extraction protocols and their implications are discussed.

The effect of different grazing heights on carcass and meat quality characteristics of Nellore cattle fattened in pastures of Convert grass.

Carcass characteristic and meat quality from bulls and Nellore steers (n = 64 total) subjected to different grazing heights (15, 25, 35, and 45 cm) were evaluated isolatedly, under continuous grazing and variable load, in pastures of Convert grass. The experimental period was from May 2015 to June 2017, in an area of 16 ha, divided into 16 paddocks of 1 ha. The experimental design was randomized blocks with four replications. Each paddock was grazed by three animals and regulators, used to adjust grazing heights. The final slaughter weight, hot carcass, and crude protein in the meat of steers were higher when the pastures were managed at 42 cm. When the pasture was managed between 25 and 35 cm in height, greater fat thickness, marbling, muscle:bone and muscle+fat:bone ratio and lower color* of the meat and percentage of bone were found. For the steers, the height of 40 cm provided higher fat thickness and marbling in the meat. The loss during thawing in meat was greater at 28 cm in height. The heights of grazing alter the carcass characteristics and meat of bulls and steers.

CO2 Cystoscopy for Evaluation of Ureteral Patency.

Gynecologic surgery is associated with various perioperative complications, especially urinary tract injuries. Intraoperative cystoscopy plays an important role in allowing assessment of the bladder to ensure the absence of injuries. Verification of the urinary jets from the ureters is a fundamental step that is not always easy to accomplish. Dyes are frequently used, but these are not always available and are associated with adverse effects. The present study aimed to demonstrate the use of CO2 as a medium for distension during cystoscopy. A total of 47 patients underwent CO2 cystoscopy after laparoscopic hysterectomy (n = 26) or bladder endometriosis nodule resection (n = 21). In all patients, the ureteral jets were readily identified, leaving no doubt as to their patency. The median interval between the onset of cystoscopy and the view of jetting from both ureteral ostia was 145 seconds (range, 80-300 seconds). All cystoscopies were normal, and no patient had any signs of accidental urinary tract injury in the follow-up period. Two patients experienced mild urinary tract infection. This cystoscopy technique using CO2 is fast, easy, safe, and efficient. We recommend bladder distension with CO2 as a reasonable alternative technique when cystoscopy is required during gynecologic procedures.

Artificial Intelligence-Based Grading Quality of Bovine Blastocyst Digital Images: Direct Capture with Juxtaposed Lenses of Smartphone Camera and Stereomicroscope Ocular Lens.

In this study, we developed an online graphical and intuitive interface connected to a server aiming to facilitate professional access worldwide to those facing problems with bovine blastocysts classification. The interface Blasto3Q, where 3Q refers to the three qualities of the blastocyst grading, contains a description of 24 variables that were extracted from the image of the blastocyst and analyzed by three Artificial Neural Networks (ANNs) that classify the same loaded image. The same embryo (i.e., the biological specimen) was submitted to digital image capture by the control group (inverted microscope with 40× magnification) and the experimental group (stereomicroscope with maximum of magnification plus 4× zoom from the cell phone camera). The images obtained from the control and experimental groups were uploaded on Blasto3Q. Each image from both sources was evaluated for segmentation and submitted (only if it could be properly or partially segmented) for automatic quality grade classification by the three ANNs of the Blasto3Q program. Adjustments on the software program through the use of scaling algorithm software were performed to ensure the proper search and segmentation of the embryo in the raw images when they were captured by the smartphone, since this source produced small embryo images compared with those from the inverted microscope. With this new program, 77.8% of the images from smartphones were successfully segmented and from those, 85.7% were evaluated by the Blasto3Q in agreement with the control group.

The influenza virus neuraminidase protein transmembrane and head domains have coevolved.

UNLABELLED: Transmembrane domains (TMDs) from single-spanning membrane proteins are commonly viewed as membrane anchors for functional domains. Influenza virus neuraminidase (NA) exemplifies this concept, as it retains enzymatic function upon proteolytic release from the membrane. However, the subtype 1 NA TMDs have become increasingly more polar in human strains since 1918, which suggests that selection pressure exists on this domain. Here, we investigated the N1 TMD-head domain relationship by exchanging a prototypical "old" TMD (1933) with a "recent" (2009), more polar TMD and an engineered hydrophobic TMD. Each exchange altered the TMD association, decreased the NA folding efficiency, and significantly reduced viral budding and replication at 37°C compared to at 33°C, at which NA folds more efficiently. Passaging the chimera viruses at 37°C restored the NA folding efficiency, viral budding, and infectivity by selecting for NA TMD mutations that correspond with their polar or hydrophobic assembly properties. These results demonstrate that single-spanning membrane protein TMDs can influence distal domain folding, as well as membrane-related processes, and suggest the NA TMD in H1N1 viruses has become more polar to maintain compatibility with the evolving enzymatic head domain. IMPORTANCE: The neuraminidase (NA) protein from influenza A viruses (IAVs) functions to promote viral release and is one of the major surface antigens. The receptor-destroying activity in NA resides in the distal head domain that is linked to the viral membrane by an N-terminal hydrophobic transmembrane domain (TMD). Over the last century, the subtype 1 NA TMDs (N1) in human H1N1 viruses have become increasingly more polar, and the head domains have changed to alter their antigenicity. Here, we provide the first evidence that an "old" N1 head domain from 1933 is incompatible with a "recent" (2009), more polar N1 TMD sequence and that, during viral replication, the head domain drives the selection of TMD mutations. These mutations modify the intrinsic TMD assembly to restore the head domain folding compatibility and the resultant budding deficiency. This likely explains why the N1 TMDs have become more polar and suggests the N1 TMD and head domain have coevolved.

Polar residues and their positional context dictate the transmembrane domain interactions of influenza A neuraminidases.

Interactions that facilitate transmembrane domain (TMD) dimerization have been identified mainly using synthetic TMDs. Here, we investigated how inherent properties within natural TMDs modulate their interaction strength by exploiting the sequence variation in the nine neuraminidase subtypes (N1-N9) and the prior knowledge that a N1 TMD oligomerizes. Initially, consensus TMDs were created from the influenza A virus database, and their interaction strengths were measured in a biological membrane system. The TMD interactions increased with respect to decreasing hydrophobicity across the subtypes (N1-N9) and within the human N1 subtype where the N1 TMDs from the pandemic H1N1 strain of swine origin were found to be significantly less hydrophobic. The hydrophobicity correlation was attributed to the conserved amphipathicity within the TMDs as the interactions were abolished by mutating residues on the polar faces that are unfavorably positioned in the membrane. Similarly, local changes enhanced the interactions only when a larger polar residue existed on the appropriate face in an unfavorable membrane position. Together, the analysis of this unique natural TMD data set demonstrates how polar-mediated TMD interactions from bitopic proteins depend on which polar residues are involved and their positioning with respect to the helix and the membrane bilayer.

Assembly of subtype 1 influenza neuraminidase is driven by both the transmembrane and head domains.

Neuraminidase (NA) is one of the two major influenza surface antigens and the main influenza drug target. Although NA has been well characterized and thought to function as a tetramer, the role of the transmembrane domain (TMD) in promoting proper NA assembly has not been systematically studied. Here, we demonstrate that in the absence of the TMD, NA is synthesized and transported in a predominantly inactive state. Substantial activity was rescued by progressive truncations of the stalk domain, suggesting the TMD contributes to NA maturation by tethering the stalk to the membrane. To analyze how the TMD supports NA assembly, the TMD was examined by itself. The NA TMD formed a homotetramer and efficiently trafficked to the plasma membrane, indicating the TMD and enzymatic head domain drive assembly together through matching oligomeric states. In support of this, an unrelated strong oligomeric TMD rescued almost full NA activity, whereas the weak oligomeric mutant of this TMD restored only half of wild type activity. These data illustrate that a large soluble domain can force assembly with a poorly compatible TMD; however, optimal assembly requires coordinated oligomerization between the TMD and the soluble domain.

Peptidases from latex of Carica candamarcensis upregulate COX-2 and IL-1 mRNA transcripts against Salmonella enterica ser. Typhimurium-mediated inflammation.

The immunomodulatory properties of a mixture of cysteine peptidases (P1G10) obtained from the fruit lattice of Carica candamarcensis were investigated. P1G10 was obtained from fresh latex samples by chromatography in a Sephadex column and initially administered to Swiss mice (n = 5; 1 or 10 mg/kg) via i.p. After 30 min, the mice were injected with carrageenan (0.5 mg/mouse) or heat-killed S. Typhimurium (10(7) CFU/mL; 100°C/30 min) into the peritoneal cavity. Afterwards, two animal groups were i.p. administered with P1G10 (n = 6; 1, 5, or 10 mg/Kg) or PBS 24 hours prior to challenge with live S. Typhimurium (10(7) CFU/mL). P1G10 stimulated the proliferation of circulating neutrophils and lymphocytes, 6 h after injection of carrageenan or heat-killed bacteria, respectively. Furthermore, survival after infection was dose-dependent and reached 60% of the animal group. On the other hand, control mice died 1-3 days after infection. The examination of mRNA transcripts in liver cells 24 h after infection confirmed fold variation increases of 5.8 and 4.8 times on average for IL-1 and COX-2, respectively, in P1G10 pretreated mice but not for TNF-α, IL-10, γ-IFN and iNOS, for which the results were comparable to untreated animals. These data are discussed in light of previous reports.

Colchicine Poisoning: A Rare Case.

Intoxication by colchicine is rare, and its rapid recognition is crucial, as severe toxicity or death is reported in 10% of cases. Here, we present the case of a 50-year-old female admitted to the emergency department 24 hours after ingesting 10 mg of colchicine. Upon examination, she was conscious and hemodynamically stable. Analytically, she exhibited leukocytosis with neutrophilia and an elevation of lactate dehydrogenase (LDH). She was initiated on fluid therapy and transferred to the intermediate care unit of medicine. On the third day of hospitalization, she developed anterior chest pain, reduced breath sounds in the right hemithorax, and dullness on percussion. Arterial blood gas analysis showed partial respiratory failure, and chest X-rays and a computed tomography (CT) scan revealed a right-sided pleural effusion. The likely diagnosis was pleural effusion secondary to cardiac dysfunction due to colchicine intoxication. This case aims to describe the potential toxic effects of colchicine in cases of overdose and to reflect on ways to reduce its morbidity and mortality.

Modelling seasonal household variation in harvested rainwater availability: a case study in Siaya County, Kenya.

Rainwater harvesting reliability, the proportion of days annually when rainwater demand is fully met, is challenging to estimate from cross-sectional household surveys that underpin international monitoring. This study investigated the use of a modelling approach that integrates household surveys with gridded precipitation data to evaluate rainwater harvesting reliability, using two local-scale household surveys in rural Siaya County, Kenya as an illustrative case study. We interviewed 234 households, administering a standard questionnaire that also identified the source of household stored drinking water. Logistic mixed effects models estimated stored rainwater availability from household and climatological variables, with random effects accounting for unobserved heterogeneity. Household rainwater availability was significantly associated with seasonality, storage capacity, and access to alternative improved water sources. Most households (95.1%) that consumed rainwater faced insufficient supply of rainwater available for potable needs throughout the year, with intermittencies during the short rains for most households with alternative improved sources. Although not significant, stored rainwater lasts longer for households whose only improved water source was rainwater (301.8 ± 40.2 days) compared to those having multiple improved sources (144.4 ± 63.7 days). Such modelling analysis could enable rainwater harvesting reliability estimation, and thereby national/international monitoring and targeted follow-up fieldwork to support rainwater harvesting.

Molecular cloning and characterization of sea bass (Dicentrarchus labrax, L.) Tapasin.

Mammalian tapasin (TPN) is a key member of the major histocompatibility complex (MHC) class I antigen presentation pathway, being part of the multi-protein complex called the peptide loading complex (PLC). Several studies describe its important roles in stabilizing empty MHC class I complexes, facilitating peptide loading and editing the repertoire of bound peptides, with impact on CD8(+) T cell immune responses. In this work, the gene and cDNA of the sea bass (Dicentrarchus labrax) glycoprotein TPN have been isolated and characterized. The coding sequence has a 1329 bp ORF encoding a 442-residue precursor protein with a predicted 24-amino acid leader peptide, generating a 418-amino acid mature form that retains a conserved N-glycosylation site, three conserved mammalian tapasin motifs, two Ig superfamily domains, a transmembrane domain and an ER-retention di-lysine motif at the C-terminus, suggestive of a function similar to mammalian tapasins. Similar to the human counterpart, the sea bass TPN gene comprises 8 exons, some of which correspond to separate functional domains of the protein. A three-dimensional homology model of sea bass tapasin was calculated and is consistent with the structural features described for the human molecule. Together, these results support the concept that the basic structure of TPN has been maintained through evolution. Moreover, the present data provides information that will allow further studies on cell-mediated immunity and class I antigen presentation pathway in particular, in this important fish species.

Number Needed to Quarantine and Proportion of Prevented Infectious Days by Quarantine: Evaluating the Effectiveness of COVID-19 Contact Tracing.

OBJECTIVES: Information on the effectiveness of COVID-19 contact tracing is lacking. We proposed 2 measures for evaluating the effectiveness of contact tracing and applied them in a public health unit in northern Portugal. METHODS: This retrospective cohort study included the contacts of people with COVID-19 diagnosed July 1-September 15, 2020. We examined 2 measures: (1) number needed to quarantine (NNQ), as the number of quarantine person-days needed to prevent 1 potential infectious person-day; and (2) proportion of prevented infectious days by quarantine (PPID), as the number of potential infectious days prevented by quarantine divided by all infectious days. We assessed these measures by sociodemographic characteristics, types of contacts, and intervention timings (ie, time between diagnosis or symptom onset and intervention). We considered 3 scenarios for infectiousness periods: 10 days before to 10 days after symptom onset, 3 days before to 3 days after symptom onset, and 2 days before to 10 days after symptom onset. RESULTS: We found an NNQ of 19.8-41.8 person-days and a PPID of 19.7%-38.2%, depending on the infectiousness period scenario. Effectiveness was higher among cohabitants and symptomatic contacts than among social or asymptomatic contacts. NNQ and PPID changed by intervention timings: the effectiveness of contact tracing decreased with time from diagnosis to quarantine of contacts and with time from symptom onset of the index case to contacts' quarantine. CONCLUSIONS: These proposed measures of contact tracing effectiveness of communicable diseases can be important for decision making and prioritizing contact tracing when resources are scarce. They are also useful measures for communication with the general population, policy makers, and clinicians because they are easy to understand and use to assess the impact of health interventions.

Kinetics and mineralization fraction of organic matter from sewage sludge mixed with soil under controlled laboratory conditions.

The objective of this work was to evaluate the kinetics and mineralization fraction of organic matter from sewage sludge mixed with the soil under controlled laboratory conditions. For this, organic material samples accumulated in layers 0-5 cm, 5-10 cm and 10-15 cm in relation to the surface of a vertical flow constructed wetland system (VF-CW) used for treatment of septic tank sludge, in addition to samples of dewatered sludge from a septic tank and a UASB reactor and non-dewatered sludge from an anaerobic digester were mixed with material collected from the surface layer of a Red Yellow Argisol at rates equivalent to the applications, via organic residues, of 300 and 600 kg ha-1 year-1 of total nitrogen. It was found that the two-phase kinetic equation fit better to the mineralization data of labile and recalcitrant organic carbon. For the lowest nitrogen application rate in the mixtures, the mineralization fractions of the total organic carbon were higher than 73%, while at the highest dose there was a reduction in the mineralization of organic matter by 17% and 63%, respectively in samples collected in layer 10-15 cm from the VF-CW and in the septic tank sludge dewatered in the drying bed.

Acetabular Coverage Area Occupied by the Femoral Head as an Indicator of Hip Congruency.

Accurate radiographic screening evaluation is essential in the genetic control of canine HD, however, the qualitative assessment of hip congruency introduces some subjectivity, leading to excessive variability in scoring. The main objective of this work was to validate a method-Hip Congruency Index (HCI)-capable of objectively measuring the relationship between the acetabulum and the femoral head and associating it with the level of congruency proposed by the Fédération Cynologique Internationale (FCI), with the aim of incorporating it into a computer vision model that classifies HD autonomously. A total of 200 dogs (400 hips) were randomly selected for the study. All radiographs were scored in five categories by an experienced examiner according to FCI criteria. Two examiners performed HCI measurements on 25 hip radiographs to study intra- and inter-examiner reliability and agreement. Additionally, each examiner measured HCI on their half of the study sample (100 dogs), and the results were compared between FCI categories. The paired t-test and the intraclass correlation coefficient (ICC) showed no evidence of a systematic bias, and there was excellent reliability between the measurements of the two examiners and examiners' sessions. Hips that were assigned an FCI grade of A (n = 120), B (n = 157), C (n = 68), D (n = 38) and E (n = 17) had a mean HCI of 0.739 ± 0.044, 0.666 ± 0.052, 0.605 ± 0.055, 0.494 ± 0.070 and 0.374 ± 0.122, respectively (ANOVA, p < 0.01). Therefore, these results show that HCI is a parameter capable of estimating hip congruency and has the potential to enrich conventional HD scoring criteria if incorporated into an artificial intelligence algorithm competent in diagnosing HD.

Xenodiagnosis in four domestic cats naturally infected by Leishmania infantum.

Leishmaniasis is a neglected tropical disease that continues to pose a serious public health problem. Albeit dogs have long been held as the major reservoirs of Leishmania infantum, the involvement of domestic cats in the zoonotic cycle of visceral leishmaniasis has gained prominence. Here, 240 cats were evaluated by clinical signs and haematological/biochemical changes compatible with leishmaniasis and were diagnosed by serological, molecular, and parasitological techniques. Thus, four cats naturally infected by L. infantum were submitted to xenodiagnosis. A total of 203 females of Lutzomyia longipalpis were subjected to feeding on four cats, with all females completing the blood meal. Parasitological and molecular assays were carried out to evaluate the presence of L. infantum in the sand flies' midgut. Promastigotes were observed in 10 females (6.5%) that fed on one cat, and L. infantum DNA was detected in 17 (8.4%) females that fed on two cats. Our results strengthen the evidence that naturally infected cats are capable of transmitting L. infantum to sand flies.

A mixed methods study to evaluate participatory mapping for rural water safety planning in western Kenya.

Water safety planning is an approach to ensure safe drinking-water access through comprehensive risk assessment and water supply management from catchment to consumer. However, its uptake remains low in rural areas. Participatory mapping, the process of map creation for resource management by local communities, has yet to be used for rural water safety planning. In this mixed methods study, to evaluate the validity of participatory mapping outputs for rural water safety planning and assess community understanding of water safety, 140 community members in Siaya County, Kenya, attended ten village-level participatory mapping sessions. They mapped drinking-water sources, ranked their safety and mapped potential contamination hazards. Findings were triangulated against a questionnaire survey of 234 households, conducted in parallel. In contrast to source type ranking for international monitoring, workshop participants ranked rainwater's safety above piped water and identified source types such as broken pipes not explicitly recorded in water source typologies often used for formal monitoring. Participatory mapping also highlighted the overlap between livestock grazing areas and household water sources. These findings were corroborated by the household survey and subsequent participatory meetings. However, comparison with household survey data suggested participatory mapping outputs omitted some water sources and landscape-scale contamination hazards, such as open defecation areas or flood-prone areas. In follow-up visits, participant groups ranked remediation of rainwater harvesting systems as the most acceptable intervention to address hazards. We conclude that participatory mapping can complement other established approaches to rural water safety planning by capturing informally managed source use and facilitating community engagement.

Diagnostic accuracy of Panbio rapid antigen tests on oropharyngeal swabs for detection of SARS-CoV-2.

BACKGROUND: Antigen-detecting rapid diagnostic tests (Ag-RDTs) for the detection of SARS-CoV-2 offer new opportunities for testing in the context of the COVID-19 pandemic. Nasopharyngeal swabs (NPS) are the reference sample type, but oropharyngeal swabs (OPS) may be a more acceptable sample type in some patients. METHODS: We conducted a prospective study in a single screening center to assess the diagnostic performance of the Panbio™ COVID-19 Ag Rapid Test (Abbott) on OPS compared with reverse-transcription quantitative PCR (RT-qPCR) using NPS during the second pandemic wave in Switzerland. RESULTS: 402 outpatients were enrolled in a COVID-19 screening center, of whom 168 (41.8%) had a positive RT-qPCR test. The oropharyngeal Ag-RDT clinical sensitivity compared to nasopharyngeal RT-qPCR was 81% (95%CI: 74.2-86.6). Two false positives were noted out of the 234 RT-qPCR negative individuals, which resulted in a clinical specificity of 99.1% (95%CI: 96.9-99.9) for the Ag-RDT. For cycle threshold values ≤ 26.7 (≥ 1E6 SARS-CoV-2 genomes copies/mL, a presumed cut-off for infectious virus), 96.3% sensitivity (95%CI: 90.7-99.0%) was obtained with the Ag-RDT using OPS. INTERPRETATION: Based on our findings, the diagnostic performance of the Panbio™ Covid-19 RDT with OPS samples, if taken by a trained person and high requirements regarding quality of the specimen, meet the criteria required by the WHO for Ag-RDTs (sensitivity ≥80% and specificity ≥97%) in a high incidence setting in symptomatic individuals.

Effect of Inter-Observer Variation on the Association between Contamination Hazards and the Microbiological Quality of Water Sources: A Longitudinal Study.

Sanitary risk inspection protocols are often used to identify contamination hazards at water sources; however, different observers sometimes struggle to record hazards consistently. This study aimed to assess the effect of inter-observer variation in hazard observations on the strength of relationships between observed hazards and the bacterial contamination of water sources, particularly relationships with animal-related hazards. In a longitudinal study, five surveyors independently recorded hazards at 93 water sources used by 234 households in Siaya County, Kenya, in both wet and dry seasons. One surveyor collected samples from sources for subsequent Escherichia coli and intestinal enterococci testing. The relationship between each surveyor's hazard observations and high bacterial contamination was examined using logistic regression. After controlling for water source type and preceding rainfall; percentage scores for animal-related hazards were significantly related to high contamination with enterococci and E. coli for one surveyor (odds ratio 1.02; 95% confidence intervals 1.00-1.03 for both parameters), but not for the remaining four surveyors. The relationship between observed contamination hazards and the microbiological contamination of water sources is sensitive to variation in hazard recording between surveyors. Sanitary risk protocols should be designed to enable robust and consistent observation of hazards.