Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis.

Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.

A spatiotemporally resolved single-cell atlas of the Plasmodium liver stage.

Malaria infection involves an obligatory, yet clinically silent liver stage1,2. Hepatocytes operate in repeating units termed lobules, exhibiting heterogeneous gene expression patterns along the lobule axis3, but the effects of hepatocyte zonation on parasite development at the molecular level remain unknown. Here we combine single-cell RNA sequencing4 and single-molecule transcript imaging5 to characterize the host and parasite temporal expression programmes in a zonally controlled manner for the rodent malaria parasite Plasmodium berghei ANKA. We identify differences in parasite gene expression in distinct zones, including potentially co-adaptive programmes related to iron and fatty acid metabolism. We find that parasites develop more rapidly in the pericentral lobule zones and identify a subpopulation of periportally biased hepatocytes that harbour abortive infections, reduced levels of Plasmodium transcripts and parasitophorous vacuole breakdown. These 'abortive hepatocytes', which appear predominantly with high parasite inoculum, upregulate immune recruitment and key signalling programmes. Our study provides a resource for understanding the liver stage of Plasmodium infection at high spatial resolution and highlights the heterogeneous behaviour of both the parasite and the host hepatocyte.

RNA-seq validation: software for selection of reference and variable candidate genes for RT-qPCR.

BACKGROUND: Real-time quantitative PCR (RT-qPCR) is one of the most widely used gene expression analyses for validating RNA-seq data. This technique requires reference genes that are stable and highly expressed, at least across the different biological conditions present in the transcriptome. Reference and variable candidate gene selection is often neglected, leading to misinterpretation of the results. RESULTS: We developed a software named "Gene Selector for Validation" (GSV), which identifies the best reference and variable candidate genes for validation within a quantitative transcriptome. This tool also filters the candidate genes concerning the RT-qPCR assay detection limit. GSV was compared with other software using synthetic datasets and performed better, removing stable low-expression genes from the reference candidate list and creating the variable-expression validation list. GSV software was used on a real case, an Aedes aegypti transcriptome. The top GSV reference candidate genes were selected for RT-qPCR analysis, confirming that eiF1A and eiF3j were the most stable genes tested. The tool also confirmed that traditional mosquito reference genes were less stable in the analyzed samples, highlighting the possibility of inappropriate choices. A meta-transcriptome dataset with more than ninety thousand genes was also processed successfully. CONCLUSION: The GSV tool is a time and cost-effective tool that can be used to select reference and validation candidate genes from the biological conditions present in transcriptomic data.

Effectiveness of Laser Acupuncture for Reducing Pain and Increasing Mouth Opening Range in Individuals with Temporomandibular Disorder: A Systematic Review and Network Meta-Analysis.

PURPOSE OF REVIEW: Laser acupuncture (LA) demonstrates promising results in the treatment of musculoskeletal disorders. However, its effects on temporomandibular disorder (TMD) are not yet fully understood. Thus, the aim of this systematic review and network meta-analysis was to assess the effectiveness of LA on pain intensity and maximum mouth opening range (MMO) related to TMD. A search was carried out in 11 electronic databases and references of included studies to locate randomized clinical trials (RCTs) that evaluated LA as a primary treatment for TMD. The risk of bias was assessed using the RoB 2 tool. Network meta-analysis was conducted on the MetaInsight platform, considering the pain intensity and counseling (C) as the outcome of reference. The GRADE system was used to assess the certainty of the evidence. RECENT FINDINGS: Five studies evaluated pain intensity, four with a high risk of bias and one with a low risk. Two studies evaluated pain intensity on palpation (one with high and one with low risk of bias), and one study with high risk of bias evaluated MMO. Laser parameters were: 690-810 nm, 40-150 mW, and 7.5-112.5 J/cm2. Occlusal splint (OS) [- 2.47; CI 95% - 3.64, - 1.30] and Physiotherapy (PT) [-2.64; CI 95% - 3.94, - 1.34] reduced pain intensity compared to C. The ranking of treatments in order of effectiveness was PT > OS > LA > C > CR (craniopuncture). The certainty of the evidence was very low or low. The data do not support the indication of LA for the treatment of TMDs and new placebo-controlled RCTs must be conducted to demonstrate its effectiveness more precisely.

Walk or be walked by the dog? The attachment role.

BACKGROUND: The human-animal bond has been recognized as having positive effects on the health and well-being of both humans and pets. The present study aims to explore the influence of attachment on physical activity (PA), lifestyle, and health outcomes of dog owners (DO), highlighting the mutual benefits resulting from the relationship between DO and dogs. METHODS: Thirty-eight DO and their dogs participated in this study. Socio-demographic data, the Self-Rated Health (SRH), FANTASTICO Lifestyle Scale, and the Lexington Attachment Pet Scale (LAPS) were assessed. PA was measured in both the DO and the dogs, using an ActiGraph GT3X accelerometer in the context of daily routine. Descriptive statistics and Spearman rank correlation analyses were performed to examine the associations between LAPS, PA levels, socio-demographic variables, lifestyle behaviors, and SRH. RESULTS: Significant correlations were found between the dog owners' light-level PA and the pets' vigorous level of PA (rho = 0.445, p = 0.01). Furthermore, the importance of the pets' health (rho = -0.785, p = 0.02) and the LAPS subscales, namely proximity (rho = 0.358, p = 0.03), and attachment (rho = 0.392, p = 0.01), were related to taking the pet for a walk. Regarding lifestyle, DO with a healthier lifestyle had a better self-assessment of their health using the SRH (rho = 0.39, p = 0.02). Moreover, DO with better lifestyles also exhibited greater concern for their pet's health (rho = 0.398, p = 0.01). CONCLUSIONS: This study emphasizes that individuals who adopt healthier habits tend to perceive themselves as healthier and exhibit greater concern for their pets' health. The attachment between DO and dogs is important in promoting healthy lifestyle behaviors and engagement in PA. Our results highlight that the presence of a dog is associated with a higher level of PA in DO, depending on the strength of the human-animal bond.

Longitudinal SARS-CoV-2 seroprevalence in Portugal and antibody maintenance 12 months after infection.

During the COVID-19 pandemic, Portugal has experienced three distinct SARS-CoV-2 infection waves. We previously documented the prevalence of SARS-CoV-2 immunity, measured by specific antibodies, in September 2020, 6 months after the initial moderate wave. Here, we show the seroprevalence changes 6 months later, up to the second week of March 2021, shortly following the third wave, which was one of the most severe in the world, and 2 months following the start of the vaccination campaign. A longitudinal epidemiological study was conducted, with a stratified quota sample of the Portuguese population. Serological testing was performed, including ELISA determination of antibody class and titers. The proportion of seropositives, which was 2.2% in September 2020, rose sharply to 17.3% (95% CI: 15.8-18.8%) in March 2021. Importantly, circulating IgG and IgA antibody levels were very stable 6 months after the initial determination and up to a year after initial infection, indicating long-lasting infection immunity against SARS-CoV-2. Moreover, vaccinated people had higher IgG levels from 3 weeks post-vaccination when compared with previously infected people at the same time post-infection.

Prospective cohort study of incidence and risk factors for catheter-associated urinary tract infections in 145 intensive care units of 9 Latin American countries: INICC findings.

PURPOSE: Identify urinary catheter (UC)-associated urinary tract infections (CAUTI) incidence and risk factors (RF) in Latin American Countries. METHODS: From 01/01/2014 to 02/10/2022, we conducted a prospective cohort study in 145 ICUs of 67 hospitals in 35 cities in nine Latin American countries: Argentina, Brazil, Colombia, Costa Rica, Dominican Republic, Ecuador, Mexico, Panama, and Peru. To estimate CAUTI incidence, we used the number of UC-days as the denominator, and the number of CAUTIs as numerator. To estimate CAUTI RFs, we analyzed the following 10 variables using multiple logistic regression: gender, age, length of stay (LOS) before CAUTI acquisition, UC-days before CAUTI acquisition, UC-device utilization (DU) ratio, UC-type, hospitalizationtype, ICU type, facility ownership, and time period. RESULTS: 31,631 patients, hospitalized for 214,669 patient-days, acquired 305 CAUTIs. The pooled CAUTI rate per 1000 UC-days was 2.58, for those using suprapubic catheters, it was 2.99, and for those with indwelling catheters, it was 2.21. The following variables were independently associated with CAUTI: age, rising risk 1% yearly (aOR = 1.01; 95% CI 1.01-1.02; p < 0.0001 female gender (aOR = 1.28; 95% CI 1.01-1.61; p = 0.04), LOS before CAUTI acquisition, rising risk 7% daily (aOR = 1.07; 95% CI 1.06-1.08; p < 0.0001, UC/DU ratio (aOR = 1.14; 95% CI 1.08-1.21; p < 0.0001, public facilities (aOR = 2.89; 95% CI 1.75-4.49; p < 0.0001. The periods 2014-2016 and 2017-2019 had significantly higher risks than the period 2020-2022. Suprapubic catheters showed similar risks as indwelling catheters. CONCLUSION: The following CAUTI RFs are unlikely to change: age, gender, hospitalization type, and facility ownership. Based on these findings, it is suggested to focus on reducing LOS, UC/DU ratio, and implementing evidence-based CAUTI prevention recommendations.

Nutrient sensing modulates malaria parasite virulence.

The lifestyle of intracellular pathogens, such as malaria parasites, is intimately connected to that of their host, primarily for nutrient supply. Nutrients act not only as primary sources of energy but also as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Canonical nutrient-sensing pathways are presumed to be absent from the causative agent of malaria, Plasmodium, thus raising the question of whether these parasites can sense and cope with fluctuations in host nutrient levels. Here we show that Plasmodium blood-stage parasites actively respond to host dietary calorie alterations through rearrangement of their transcriptome accompanied by substantial adjustment of their multiplication rate. A kinome analysis combined with chemical and genetic approaches identified KIN as a critical regulator that mediates sensing of nutrients and controls a transcriptional response to the host nutritional status. KIN shares homology with SNF1/AMPKα, and yeast complementation studies suggest that it is part of a functionally conserved cellular energy-sensing pathway. Overall, these findings reveal a key parasite nutrient-sensing mechanism that is critical for modulating parasite replication and virulence.

γδ-T cells promote IFN-γ-dependent Plasmodium pathogenesis upon liver-stage infection.

Cerebral malaria (CM) is a major cause of death due to Plasmodium infection. Both parasite and host factors contribute to the onset of CM, but the precise cellular and molecular mechanisms that contribute to its pathogenesis remain poorly characterized. Unlike conventional αß-T cells, previous studies on murine γδ-T cells failed to identify a nonredundant role for this T cell subset in experimental cerebral malaria (ECM). Here we show that mice lacking γδ-T cells are resistant to ECM when infected with Plasmodium berghei ANKA sporozoites, the liver-infective form of the parasite and the natural route of infection, in contrast with their susceptible phenotype if challenged with P. berghei ANKA-infected red blood cells that bypass the liver stage of infection. Strikingly, the presence of γδ-T cells enhanced the expression of Plasmodium immunogenic factors and exacerbated subsequent systemic and brain-infiltrating inflammatory αß-T cell responses. These phenomena were dependent on the proinflammatory cytokine IFN-γ, which was required during liver stage for modulation of the parasite transcriptome, as well as for downstream immune-mediated pathology. Our work reveals an unanticipated critical role of γδ-T cells in the development of ECM upon Plasmodium liver-stage infection.

Phenotypic and genotypic characterization of oxacillin-susceptible and mecA positive Staphylococcus aureus strains isolated in Uruguay.

The aim of this study was to characterize phenotypically and genotypically 27 mecA positive Staphylococcus aureus strains with oxacillin MICs of ≤2µg/ml by Vitek 2, isolated in different regions of Uruguay. Susceptibility to oxacillin and cefoxitin was studied by gradient diffusion, disk diffusion to cefoxitin, and Phoenix and MicroScan systems. PBP2a was determined. SCCmec typing was performed and the isolates were compared by PFGE. Twenty-six isolates were susceptible to oxacillin; one strain was susceptible to cefoxitin by disk diffusion and 3 strains by gradient diffusion. Phoenix and MicroScan panels detected methicillin resistance in 25 and 27 strains, respectively. Twenty-six strains tested positive for PBP2a. Twenty-six strains carried SCCmec V and 24 belonged to pulsotype A. One strain carried SCCmec IV and did not belong to pulsotype A. Cefoxitin disk diffusion test and PBP2a detection correctly identified 26 of these 27 strains as MRSA. PFGE results suggest the dissemination of a cluster of MRSA carrying SCCmec V.

Prevalence of SARS-CoV-2 Antibodies after First 6 Months of COVID-19 Pandemic, Portugal.

In September 2020, we tested 13,398 persons in Portugal for antibodies against severe acute respiratory syndrome coronavirus 2 by using a quota sample stratified by age and population density. We found a seroprevalence of 2.2%, 3-4 times larger than the official number of cases at the end of the first wave of the pandemic.

From malaria control to elimination within a decade: lessons learned from Timor Leste, a newly independent country.

BACKGROUND: Timor Leste has made remarkable progress from malaria control to malaria elimination in a span of 10 years during which organized malaria control efforts were instituted. The good practices and possible factors that have contributed to the remarkable transition from malaria control to elimination in a newly independent country devastated by civil unrest which left the entire administrative structure including the health sector in a disrupted non-functional state are highlighted. METHODS: Data from the National Malaria Control Programme were reviewed. A literature search was carried out using the key words "malaria", "Timor Leste", "East Timor", and "malaria control" and "malaria elimination". All relevant manuscripts and reports that were identified in the search were reviewed. Key personnel of the NMCP, WHO and the GFATM involved in the project were interviewed. RESULTS: With the setting up of the National Malaria Control Programme just after independence in 2003 with two officers, the programme expanded over the years and strategic malaria control activities in an organized manner commenced in 2009 with funding from the Global Fund to fight AIDS, Tuberculosis and Malaria. The incidence of malaria declined dramatically from 223,002 cases in 2006 with the last indigenous case being reported in June 2017. The decline in malaria was associated with strategic application of key evidence-based interventions taking into account the burden of disease, characteristics of vectors, and stratification of risk areas ensuring universal access to malaria prevention, and quality assured diagnostic tools and effective anti-malarial medicines at point of care, intensified surveillance, monitoring and evaluation, and capacity building, including training of staff, with adequate programme funding. The programme was provided with technical expertise and sustained political commitment that ensured uninterrupted implementation of interventions based on national strategic plans. The incorporation of the malaria control programme within an evolving health system helped the transition from malaria control to malaria elimination. CONCLUSION: Universal access to quality assured malaria diagnosis and treatment and focussed vector control, implemented throughout the country in an organized manner with adequate funding and political commitment were key to the successful interruption of malaria transmission in the country. All the practices or factors listed did not work in isolation but rather synergistically in an integrated manner. Malaria elimination is possible even in tropical areas of South and Southeast Asia.

Plasmodium berghei EXP-1 interacts with host Apolipoprotein H during Plasmodium liver-stage development.

The first, obligatory replication phase of malaria parasite infections is characterized by rapid expansion and differentiation of single parasites in liver cells, resulting in the formation and release of thousands of invasive merozoites into the bloodstream. Hepatic Plasmodium development occurs inside a specialized membranous compartment termed the parasitophorous vacuole (PV). Here, we show that, during the parasite's hepatic replication, the C-terminal region of the parasitic PV membrane protein exported protein 1 (EXP-1) binds to host Apolipoprotein H (ApoH) and that this molecular interaction plays a pivotal role for successful Plasmodium liver-stage development. Expression of a truncated EXP-1 protein, missing the specific ApoH interaction site, or down-regulation of ApoH expression in either hepatic cells or mouse livers by RNA interference resulted in impaired intrahepatic development. Furthermore, infection of mice with sporozoites expressing a truncated version of EXP-1 resulted in both a significant reduction of liver burden and delayed blood-stage patency, leading to a disease outcome different from that generally induced by infection with wild-type parasites. This study identifies a host-parasite protein interaction during the hepatic stage of infection by Plasmodium parasites. The identification of such vital interactions may hold potential toward the development of novel malaria prevention strategies.

Three new species of Anacanthorus Mizelle & Price, 1965 (Monogenea: Dactylogyridae) from Markiana nigripinnis Perugia (Actinopterygii: Characidae) in Pantanal wetlands, Brazil.

Three monogenean species, Anacanthorus luquei n. sp., A. scholzi n. sp. and A. cohenae n. sp. are described from the gills of the tetra fish Markiana nigripinnis (Perugia) (Characidae), collected in the Pantanal wetlands, State of Mato Grosso do Sul, Brazil. Among other differences, Anacanthorus luquei n. sp. differs from the most morphologically similar species, based on the structure of the accessory piece as follows: branches with smooth margins (vs with irregular margins in A. cuticulovaginus), without pointed projections at distal end (vs with projections in A. dipelecinus) and with 2 branches (vs 3 in A. quinqueramus). Anacanthorus scholzi n. sp. is most morphologically similar to A. luquei n. sp., differing from it because one of the branches of the accessory piece is bifurcated at the distal portion. Anacanthorus cohenae n. sp. can be differentiated from the congeners based on the combination of the following features: MCO cylindrical and robust with sclerotised flanges on the extremities, accessory piece V-shaped, bearing two branches similar in length and with blunt distal ends, and hooks with a proximal bulb. This is the first parasitological study on M. nigripinnis and, currently, Anacanthorus allocates 88 species infesting characiform fishes in the Neotropical region, including the three new species described here.

Plasmodium falciparum subtilisin-like ookinete protein SOPT plays an important and conserved role during ookinete infection of the Anopheles stephensi midgut.

Transmission of the malaria parasite Plasmodium falciparum involves infection of Anopheles mosquitoes. Here we characterize SOPT, a protein expressed in P. falciparum ookinetes that facilitates infection of the mosquito midgut. SOPT was identified on the basis that it contains a signal peptide, a PEXEL-like sequence and is expressed in asexual, ookinete and sporozoite stages, suggesting it is involved in infecting the human or mosquito host. SOPT is predicted to contain a subtilisin-like fold with a non-canonical catalytic triad and is orthologous to P. berghei PIMMS2. Localization studies reveal that SOPT is not exported to the erythrocyte but is expressed in ookinetes at the parasite periphery. SOPT-deficient parasites develop normally through the asexual and sexual stages and produce equivalent numbers of ookinetes to NF54 controls, however, they form fewer oocysts and sporozoites in mosquitoes. SOPT-deficient parasites were also unable to activate the immune-responsive midgut invasion marker SRPN6 after mosquito ingestion, suggesting they are defective for entry into the midgut. Disruption of SOPT in P. berghei (PIMMS2) did not affect other lifecycle stages or ookinete development but again resulted in fewer oocysts and sporozoites in mosquitoes. Collectively, this study shows that SOPT/PIMMS2 plays a conserved role in ookinetes of different Plasmodium species.

Synthesis and Novel Purification Process of PANI and PANI/AgNPs Composite.

In this work, polyaniline (PANI) is synthesized via oxidative polymerization of aniline and purified using organic solvents where the emeraldine phase is isolated by employing a phase separation system. The above contributes to the increase in the percentage yield compared to previous works and the possibility of being used as a single phase. In addition, the PANI/AgNPs composite is prepared in situ at the polymerization of aniline, adding silver nitrate and glycine to create the AgNPs inside the PANI matrix by controlling the pH, temperature, time of reaction and incorporating a new purification technique.

Fermentation at non-conventional conditions in food- and bio-sciences by the application of advanced processing technologies.

The interest in improving the yield and productivity values of relevant microbial fermentations is an increasingly important issue for the scientific community. Therefore, several strategies have been tested for the stimulation of microbial growth and manipulation of their metabolic behavior. One promising approach involves the performance of fermentative processes during non-conventional conditions, which includes high pressure (HP), electric fields (EF) and ultrasound (US). These advanced technologies are usually applied for microbial inactivation in the context of food processing. However, the approach described in this study focuses on the use of these technologies at sub-lethal levels, since the aim is microbial growth and fermentation under these stress conditions. During these sub-lethal conditions, microbial strains develop specific genetic, physiologic and metabolic stress responses, possibly leading to fermentation products and processes with novel characteristics. In some cases, these modifications can represent considerable improvements, such as increased yields, productivities and fermentation rates, lower accumulation of by-products and/or production of different compounds. Although several studies report the successful application of these technologies during the fermentation processes, information on this subject is still scarce and poorly understood. For that reason, the present review paper intends to assemble and discuss the main findings reported in the literature to date, and aims to stimulate interest and encourage further developments in this field.

Disease characteristics and treatment of patients with diabetes mellitus attending government health services in Indonesia, Peru, Romania and South Africa.

OBJECTIVE: To describe the characteristics and management of Diabetes mellitus (DM) patients from low- and middle-income countries (LMIC). METHODS: We systematically characterised consecutive DM patients attending public health services in urban settings in Indonesia, Peru, Romania and South Africa, collecting data on DM treatment history, complications, drug treatment, obesity, HbA1c and cardiovascular risk profile; and assessing treatment gaps against relevant national guidelines. RESULTS: Patients (median 59 years, 62.9% female) mostly had type 2 diabetes (96%), half for >5 years (48.6%). Obesity (45.5%) and central obesity (females 84.8%; males 62.7%) were common. The median HbA1c was 8.7% (72 mmol/mol), ranging from 7.7% (61 mmol/mol; Peru) to 10.4% (90 mmol/mol; South Africa). Antidiabetes treatment included metformin (62.6%), insulin (37.8%), and other oral glucose-lowering drugs (34.8%). Disease complications included eyesight problems (50.4%), EGFR <60 ml/min (18.9%), heart disease (16.5%) and proteinuria (14.7%). Many had an elevated cardiovascular risk with elevated blood pressure (36%), LDL (71.0%) and smoking (13%), but few were taking antihypertensive drugs (47.1%), statins (28.5%) and aspirin (30.0%) when indicated. Few patients on insulin (8.0%), statins (8.4%) and antihypertensives (39.5%) reached treatment targets according to national guidelines. There were large differences between countries in terms of disease profile and medication use. CONCLUSION: DM patients in government clinics in four LMIC with considerable growth of DM have insufficient glycaemic control, frequent macrovascular and other complications, and insufficient preventive measures for cardiovascular disease. These findings underline the need to identify treatment barriers and secure optimal DM care in such settings.

Colorimetric protein determination in microalgae (Chlorophyta): association of milling and SDS treatment for total protein extraction.

The use of colorimetric methods for protein quantification in microalgae is hindered by their elevated amounts of membrane-embedded intracellular proteins. In this work, the protein content of three species of microalgae was determined by the Lowry method after the cells were dried, ball-milled, and treated with the detergent sodium dodecyl sulfate (SDS). Results demonstrated that the association of milling and SDS treatment resulted in a 3- to 7-fold increase in protein quantification. Milling promoted microalgal disaggregation and cell wall disruption enabling access of the SDS detergent to the microalgal intracellular membrane proteins and their efficient solubilization and quantification.