Neutrophils cultured ex vivo from CD34+ stem cells are immature and genetically tractable.

BACKGROUND: Neutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency granulopoiesis leads to release of immature neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development. METHODS: We optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors. RESULTS: We obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis. CONCLUSION: Ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.

Why Foot-and-Mouth Disease-Free with Vaccination Should Be Equivalent to Foot-and-Mouth Disease-Free without Vaccination.

Foot-and-Mouth Disease (FMD) is still one of the most relevant animal diseases and remains of global concern. The World Organization for Animal Health (WOAH) has specified two sanitary statuses that assure freedom from FMD: a country or zone can be free from FMD either with or without vaccination. To obtain either of the two statuses, absence of virus circulation must be shown. The standards set by WOAH are used for trade negotiations. During recent decades, different tools and approaches were developed to control FMD, including vaccines, diagnostics, and the Progressive Control Pathway for FMD. These tools improved over time, and nowadays high-quality, reliable vaccines and specific diagnostics are available to efficiently control and detect the infection, even in vaccinated populations. Due to these improvements, it is no longer justifiable to treat the two FMD-free statuses differently. The distinction between the statuses provides wrong incentives and tempts countries to take increased risks by stopping vaccination to improve their trade conditions, which can have potentially devastating consequences. The decision to stop vaccination should only be made on the basis of a careful and comprehensive analysis of the local and regional epidemiological situation. This paper presents the perspective that member countries and WOAH should recognize the two FMD-free statuses as equivalent.

Complete absence of GLUT1 does not impair human terminal erythroid differentiation.

The Glucose transporter 1 (GLUT1) is one of the most abundant proteins within the erythrocyte membrane and is required for glucose and dehydroascorbic acid (Vitamin C precursor) transport. It is widely recognized as a key protein for red cell structure, function, and metabolism. Previous reports highlighted the importance of GLUT1 activity within these uniquely glycolysis-dependent cells, in particular for increasing antioxidant capacity needed to avoid irreversible damage from oxidative stress in humans. However, studies of glucose transporter roles in erythroid cells are complicated by species-specific differences between humans and mice. Here, using CRISPR-mediated gene editing of immortalized erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate committed human erythroid cells completely deficient in expression of GLUT1. We show that absence of GLUT1 does not impede human erythroblast proliferation, differentiation, or enucleation. This work demonstrates for the first-time generation of enucleated human reticulocytes lacking GLUT1. The GLUT1-deficient reticulocytes possess no tangible alterations to membrane composition or deformability in reticulocytes. Metabolomic analyses of GLUT1-deficient reticulocytes reveal hallmarks of reduced glucose import, downregulated metabolic processes and upregulated AMPK-signalling, alongside alterations in antioxidant metabolism, resulting in increased osmotic fragility and metabolic shifts indicative of higher oxidant stress. Despite detectable metabolic changes in GLUT1 deficient reticulocytes, the absence of developmental phenotype, detectable proteomic compensation or impaired deformability comprehensively alters our understanding of the role of GLUT1 in red blood cell structure, function and metabolism. It also provides cell biological evidence supporting clinical consensus that reduced GLUT1 expression does not cause anaemia in GLUT1 deficiency syndrome.

Ammonia Detection by Electronic Noses for a Safer Work Environment.

Providing employees with proper work conditions should be one of the main concerns of any employer. Even so, in many cases, work shifts chronically expose the workers to a wide range of potentially harmful compounds, such as ammonia. Ammonia has been present in the composition of products commonly used in a wide range of industries, namely production in lines, and also laboratories, schools, hospitals, and others. Chronic exposure to ammonia can yield several diseases, such as irritation and pruritus, as well as inflammation of ocular, cutaneous, and respiratory tissues. In more extreme cases, exposure to ammonia is also related to dyspnea, progressive cyanosis, and pulmonary edema. As such, the use of ammonia needs to be properly regulated and monitored to ensure safer work environments. The Occupational Safety and Health Administration and the European Agency for Safety and Health at Work have already commissioned regulations on the acceptable limits of exposure to ammonia. Nevertheless, the monitoring of ammonia gas is still not normalized because appropriate sensors can be difficult to find as commercially available products. To help promote promising methods of developing ammonia sensors, this work will compile and compare the results published so far.

Early transfusion patterns improve the Molecular International Prognostic Scoring System (IPSS-M) prediction in myelodysplastic syndromes.

BACKGROUND: The Molecular International Prognostic Scoring System (IPSS-M) is the new gold standard for diagnostic outcome prediction in patients with myelodysplastic syndromes (MDS). This study was designed to assess the additive prognostic impact of dynamic transfusion parameters during early follow-up. METHODS: We retrieved complete transfusion data from 677 adult Swedish MDS patients included in the IPSS-M cohort. Time-dependent erythrocyte transfusion dependency (E-TD) was added to IPSS-M features and analyzed regarding overall survival and leukemic transformation (acute myeloid leukemia). A multistate Markov model was applied to assess the prognostic value of early changes in transfusion patterns. RESULTS: Specific clinical and genetic features were predicted for diagnostic and time-dependent transfusion patterns. Importantly, transfusion state both at diagnosis and within the first year strongly predicts outcomes in both lower (LR) and higher-risk (HR) MDSs. In multivariable analysis, 8-month landmark E-TD predicted shorter survival independently of IPSS-M (p < 0.001). A predictive model based on IPSS-M and 8-month landmark E-TD performed significantly better than a model including only IPSS-M. Similar trends were observed in an independent validation cohort (n = 218). Early transfusion patterns impacted both future transfusion requirements and outcomes in a multistate Markov model. CONCLUSION: The transfusion requirement is a robust and available clinical parameter incorporating the effects of first-line management. In MDS, it provides dynamic risk information independently of diagnostic IPSS-M and, in particular, clinical guidance to LR MDS patients eligible for potentially curative therapeutic intervention.

The Impact of Diabetes Education on Continuous Glucose Monitoring in SUS-Dependent Patients in a Northeastern Brazilian City.

BACKGROUND: Diabetes Mellitus (DM) is an important chronic disease that occurs worldwide. AIMS: This study aims to investigate how the use of the FreeStyle® Libre system in Unified Health System (SUS) patients impacts diabetes parameters in patients who receive education on proper insulin administration and the use of the continuous monitoring device, as well as how this affects patients without any concomitant multidisciplinary support in Sergipe, Brazil. METHODS: We conducted a prospective randomized study in a diabetes clinic in Sergipe, Brazil, using the flash method FreeStyle® Libre (Abbott). The participants were divided into two groups: one receiving diabetes education on CGM (continuous glucose monitoring), while the other did not. Before the intervention, the patient's treatment motivation and quality of life were assessed using a questionnaire, and baseline levels of glycated hemoglobin were measured using high-performance liquid chromatography (HPLC) and the point of care AlereTM Afinion with boronate fixation. We compared first- and second-phase data with respect to glycated hemoglobin, mean interstitial blood glucose, time on and above target for hypoglycemic and hyperglycemic events, and mean hypoglycemic duration. RESULTS: In group A, which received the diabetes education intervention, there was a significant reduction in average HbA1c levels from 8.6% to 7.9% after 3 months (p = 0.001). However, there was no significant difference in average glycemic values. Time above target decreased significantly from 50.62% to 29.43% (p = 0.0001), while time below target decreased from 22.90% to 20.21% (p = 0.002). There was no significant change in the number of hypoglycemic events, but the duration of hypoglycemia decreased significantly from 130.35 min to 121.18 min after 3 months (p = 0.0001). In Group B, there was no significant difference in mean HbA1c levels before (7.07%) and after (7.28%) sensor installation. This group maintained lower HbA1c levels compared to the other group. Average blood glucose levels also remained similar before (148.37 mg/dL) and after (154.65 mg/dL) the intervention. Although the time above the target glucose level increased significantly from 35.94% to 48.17%, the time at target decreased from 50.40% to 37.97%. No significant changes were observed in the time below target, the number of hypoglycemic events, or the duration of hypoglycemia. CONCLUSIONS: Our findings indicate that utilizing continuous glucose monitoring technology can enhance glycemic control, particularly in motivated, educated, low-income patients dependent on the SUS. To achieve positive results with FreeStyle Libre, it is imperative to allocate resources for multidisciplinary support.

COVID-19 Crisis in Brazil: Post-Vaccination Seroprevalence and Infection in More and Less Vaccinated Municipalities in a Northeastern State.

BACKGROUND: Given the impact of the pandemic in Brazil, vaccination is essential to prevent illness and death. Thus, this study sought to compare, after vaccination, the circulation of SARS-CoV-2 and the response to vaccination in the least and most vaccinated municipalities of a Brazilian state during the height of the pandemic when the Omicron variant was dominant. METHODS: We tested for the SARS-CoV-2 antigen and confirmed infection using an RT-qPCR and measured IgM and IgG antibodies in fully vaccinated participants from municipalities with higher and lower vaccination rates. RESULTS: We showed that participants from the least vaccinated municipalities were more likely to have detectable IgM antibodies and a positive antigen/RT-qPCR result for SARS-CoV-2 than participants from the most vaccinated municipalities. There were no differences between the vaccines used (BNT162b2, Ad26.COV2.S, AZD1222, and CoronaVac) and antibody production. CONCLUSIONS: Our study evaluated municipal vaccination coverage and its effects on mortality, infections, and anti-SARS-CoV-2 antibodies during a critical phase of the pandemic. The results suggest that higher vaccination coverage reduces acute cases and confers higher memory antibody levels against SARS-CoV-2. Even with a full vaccination schedule, individuals living in places with low vaccination rates are more susceptible to infection.

Comparison of the gut microbiome composition between men with erectile dysfunction and a matched cohort: a pilot study.

BACKGROUND: To-date there have been minimal studies to investigate an association between the gut microbiome and erectile dysfunction. There have been many inflammatory diseases linked to gut microbiome dysbiosis; such as cardiovascular disease and metabolic syndrome. These same inflammatory diseases have been heavily linked to erectile dysfunction. Given the correlations between both conditions and cardiovascular disease and the metabolic syndrome, we believe that it is worthwhile to investigate a link between the two. OBJECTIVE: To investigate the potential association between the gut microbiome and erectile dysfunction. METHODS: Stool samples were collected from 28 participants with erectile dysfunction and 32 age-matched controls. Metatranscriptome sequencing was used to analyze the samples. RESULTS: No significant differences were found in the gut microbiome characteristics, including Kyoto Encyclopedia of Genes and Genomes richness (p = 0.117), Kyoto Encyclopedia of Genes and Genomes diversity (p = 0.323), species richness (p = 0.364), and species diversity (p = 0.300), between the erectile dysfunction and control groups. DISCUSSION: The association of gut microbiome dysbiosis and pro-inflammatory conditions has been well studied and further literature continues to add to this evidence. Our main limitation for this study was our small-sample size due to recruitment issues. We believe that a study with a larger population size may find an association between the gut microbiome and erectile dysfunction. CONCLUSIONS: The results of this study do not support a significant association between the gut microbiome and erectile dysfunction. Further research is needed to fully understand the relationship between these two conditions.

Breath biomarkers in Non-Carcinogenic diseases.

The analysis of volatile organic compounds (VOCs) from human matrices like breath, perspiration, and urine has received increasing attention from academic and medical researchers worldwide. These biological-borne VOCs molecules have characteristics that can be directly related to physiologic and pathophysiologic metabolic processes. In this work, gathers a total of 292 analytes that have been identified as potential biomarkers for the diagnosis of various non-carcinogenic diseases. Herein we review the advances in VOCs with a focus on breath biomarkers and their potential role as minimally invasive tools to improve diagnosis prognosis and therapeutic monitoring.

Erythroid Differentiation Enhances RNA Mis-Splicing in SF3B1-Mutant Myelodysplastic Syndromes with Ring Sideroblasts.

Myelodysplastic syndromes with ring sideroblasts (MDS-RS) commonly develop from hematopoietic stem cells (HSC) bearing mutations in the splicing factor SF3B1 (SF3B1mt). Direct studies into MDS-RS pathobiology have been limited by a lack of model systems that fully recapitulate erythroid biology and RS development and the inability to isolate viable human RS. Here, we combined successful direct RS isolation from patient samples, high-throughput multiomics analysis of cells encompassing the SF3B1mt stem-erythroid continuum, and functional assays to investigate the impact of SF3B1mt on erythropoiesis and RS accumulation. The isolated RS differentiated, egressed into the blood, escaped traditional nonsense-mediated decay (NMD) mechanisms, and leveraged stress-survival pathways that hinder wild-type hematopoiesis through pathogenic GDF15 overexpression. Importantly, RS constituted a contaminant of magnetically enriched CD34+ cells, skewing bulk transcriptomic data. Mis-splicing in SF3B1mt cells was intensified by erythroid differentiation through accelerated RNA splicing and decreased NMD activity, and SF3B1mt led to truncations in several MDS-implicated genes. Finally, RNA mis-splicing induced an uncoupling of RNA and protein expression, leading to critical abnormalities in proapoptotic p53 pathway genes. Overall, this characterization of erythropoiesis in SF3B1mt RS provides a resource for studying MDS-RS and uncovers insights into the unexpectedly active biology of the "dead-end" RS. SIGNIFICANCE: Ring sideroblast isolation combined with state-of-the-art multiomics identifies survival mechanisms underlying SF3B1-mutant erythropoiesis and establishes an active role for erythroid differentiation and ring sideroblasts themselves in SF3B1-mutant myelodysplastic syndrome pathogenesis.

Adverse events leading to intensive care unit admission in a low-and-middle-income-country: A prospective cohort study and a systematic review.

INTRODUCTION: Adverse events (AE) are frequent in critical care and could be even more prevalent in LMIC due to a shortage of ICU beds and Human resources. There is limited data on how relevant AE are among the reasons for ICU admission, being all of which published by High-Income-Countries services. Our main goal is to describe the rate of adverse events-related ICU admissions and their preventability in a LMIC scenario, comparing our results with previous data. METHODS: This was a prospective cohort study, during a one-year period, in two general ICUs from a tertiary public academic hospital. Our exposure of interest was ICU admission related to an AE in adult patients, we further characterized their preventability and clinical outcomes. We also performed a systematic review to identify and compare previous published data on ICU admissions due to AE. RESULTS: Among all ICU admissions, 12.1% were related to an AE (9.8% caused by an AE, 2.3% related but not directly caused by an AE). These ICU admissions were not associated with a higher risk of death, but most of them were potentially preventable (70.9% of preventability rate, representing 8.6% of all ICU admissions). The meta-analysis resulted in a proportion of ICU admissions due to AE of 11% (95% CI 6%-16%), with a preventability rate of 54% (95% CI 42%-66%). CONCLUSIONS: In this prospective cohort, adverse events were a relevant reason for ICU admission. This result is consistent with data retrieved from non-LMIC as shown in our meta-analysis. The high preventability rate described reinforces that quality and safety programs could work as a tool to optimize scarce resources.

Evaluating the OH-EpiCap tool using the Danish integrated surveillance program for AMU and AMR as a case study.

Antimicrobial resistance (AMR) is considered a One Health (OH) challenge, ideally demanding concerted efforts from the animal, human and environmental side. DANMAP, the Danish Integrated Antimicrobial Resistance Monitoring and Research Program, is monitoring AMR and antimicrobial use in animals and humans. OH-EpiCap is an evaluation tool, developed to address essential elements in OH surveillance systems, such as the dimensions of the organization, operational activities and the impact of the surveillance activities. We aimed to evaluate DANMAP using OH-EpiCap and hereby assessed the suitability of OH-EpiCap to evaluate integrated AMR surveillance systems. During the evaluation, the strengths and weaknesses of DANMAP concerning the "OH-ness" of the program were discussed. Furthermore, possible adaptations of the standard operating procedures and governance structure were addressed. Attention was paid to the ability and easiness of DANMAP to cope with current and future challenges connected to integrated AMR surveillance. It was concluded that DANMAP has a strong OH approach covering relevant aspects for humans and animals, whereas environmental aspects are missing. OH-EpiCap proved to be straightforward to use and provided valuable insights. The authors recommend OH-EpiCap to be used by health authorities and stakeholders. It is not suitable for the technical evaluation of a surveillance program.

Diagnosis of Carcinogenic Pathologies through Breath Biomarkers: Present and Future Trends.

The assessment of volatile breath biomarkers has been targeted with a lot of interest by the scientific and medical communities during the past decades due to their suitability for an accurate, painless, non-invasive, and rapid diagnosis of health states and pathological conditions. This paper reviews the most relevant bibliographic sources aiming to gather the most pertinent volatile organic compounds (VOCs) already identified as putative cancer biomarkers. Here, a total of 265 VOCs and the respective bibliographic sources are addressed regarding their scientifically proven suitability to diagnose a total of six carcinogenic diseases, namely lung, breast, gastric, colorectal, prostate, and squamous cell (oesophageal and laryngeal) cancers. In addition, future trends in the identification of five other forms of cancer, such as bladder, liver, ovarian, pancreatic, and thyroid cancer, through perspective volatile breath biomarkers are equally presented and discussed. All the results already achieved in the detection, identification, and quantification of endogenous metabolites produced by all kinds of normal and abnormal processes in the human body denote a promising and auspicious future for this alternative diagnostic tool, whose future passes by the development and employment of newer and more accurate collection and analysis techniques, and the certification for utilisation in real clinical scenarios.

The State of the Art on Graphene-Based Sensors for Human Health Monitoring through Breath Biomarkers.

The field of organic-borne biomarkers has been gaining relevance due to its suitability for diagnosing pathologies and health conditions in a rapid, accurate, non-invasive, painless and low-cost way. Due to the lack of analytical techniques with features capable of analysing such a complex matrix as the human breath, the academic community has focused on developing electronic noses based on arrays of gas sensors. These sensors are assembled considering the excitability, sensitivity and sensing capacities of a specific nanocomposite, graphene. In this way, graphene-based sensors can be employed for a vast range of applications that vary from environmental to medical applications. This review work aims to gather the most relevant published papers under the scope of "Graphene sensors" and "Biomarkers" in order to assess the state of the art in the field of graphene sensors for the purposes of biomarker identification. During the bibliographic search, a total of six pathologies were identified as the focus of the work. They were lung cancer, gastric cancer, chronic kidney diseases, respiratory diseases that involve inflammatory processes of the airways, like asthma and chronic obstructive pulmonary disease, sleep apnoea and diabetes. The achieved results, current development of the sensing sensors, and main limitations or challenges of the field of graphene sensors are discussed throughout the paper, as well as the features of the experiments addressed.

COVID-19 Vaccination and Serological Profile of a Brazilian University Population.

BACKGROUND: COVID-19 led to the suspension academic activities worldwide, affecting millions of students and staff. METHODS: In this study, we evaluated the presence of IgM and IgG anti-SARS-CoV-2 antibodies in an academic population during the return to classes after a one-year suspension. The study took place over five months at a Brazilian university and included 942 participants. RESULTS: We found that most participants had reactive IgG and non-reactive IgM. All received at least one dose, and 940 received two or more doses, of different COVID-19 vaccines. We obtained a higher average of memory antibodies (IgG) in participants who received the CoronaVac/ChAdOx1 combination. IgG was consistently distributed for each vaccine group, but individuals who completed the vaccination schedule had higher levels. There were no differences between antibodies and gender, presence of symptoms, and previous COVID-19 infection, but older participants (>53 years) and contacts of infected individuals had higher IgM levels. CONCLUSION: This study makes significant contributions to the assessment of antibodies in the academic environment, allowing us to infer that most participants had memory immunity and low indications of recent infection when returning to face-to-face classes, as well as demonstrating the need to monitor immunity and update vaccinations.

Defining the proteomic landscape of cultured macrophages and their polarization continuum.

Macrophages have previously been characterized based on phenotypical and functional differences into suggested simplified subtypes of MØ, M1, M2a and M2c. These macrophage subtypes can be generated in a well-established primary monocyte culture model that produces cells expressing accepted subtype surface markers. To determine how these subtypes retain functional similarities and better understand their formation, we generated all four subtypes from the same donors. Comparative whole-cell proteomics confirmed that four distinct macrophage subtypes could be induced from the same donor material, with > 50% of 5435 identified proteins being significantly altered in abundance between subtypes. Functional assessment highlighted that these distinct protein expression profiles are primed to enable specific cell functions, indicating that this shifting proteome is predictive of meaningful changes in cell characteristics. Importantly, the 2552 proteins remained consistent in abundance across all macrophage subtypes examined, demonstrating maintenance of a stable core proteome that likely enables swift polarity changes. We next explored the cross-polarization capabilities of preactivated M1 macrophages treated with dexamethasone. Importantly, these treated cells undergo a partial repolarization toward the M2c surface markers but still retain the M1 functional phenotype. Our investigation of polarized macrophage subtypes therefore provides evidence of a sliding scale of macrophage functionality, with these data sets providing a valuable benchmark resource for further studies of macrophage polarity, with relevance for cell therapy development and drug discovery.

Suitability of Short- and Long-Term Storage of Volatile Organic Compounds Samples in Syringe-Based Containers: A Comparison Study.

The employment of advanced analytical techniques and instrumentation enables the tracing of volatile organic compounds (VOCs) in vestigial concentrations (ppbv-pptv range) for several emerging applications, such as the research of disease biomarkers in exhaled air, the detection of metabolites in several biological processes, and the detection of pollutants for air quality control. In this scope, the storage of gaseous samples is crucial for preserving the integrity and stability of the collected set of analytes. This study aims to assess the suitability of three commercially available syringes as air containers (AC) that are commonly used for the collection, storage, isolation, and transportation of samples: glass syringes with glass plungers (AC1), and two plastic syringes, one with plastic plungers (AC2), and one with rubbered plungers (AC3). For this purpose, 99 air samples with different times of storage (from 10 min to 24 h) were analyzed using a Gas Chromatography-Ion Mobility Spectrometry device and the degradation of the samples was properly assessed by comparing the changes in the VOCs' emission profiles. The quality of the method was assured by via the measurement of the blank's spectra before each experimental run, as well as by the consecutive measurement of the three replicates for each sample. A statistical analysis of the changes in the VOCs' emission patterns was performed using principal component analysis (PCA). The results, with a total explained variance of 93.61%, indicate that AC3 is the most suitable option for the long-term storage of air samples. Thus, AC3 containers demonstrated a higher capacity to preserve the stability and integrity of the analytes compared to AC1 and AC2. The findings of the short-term effects analysis, up to 1 h, confirm the suitability of all analyzed syringe-based containers for sample-transferring purposes in onsite analysis.