Extracellular Vesicle ASC: A Novel Mediator for Lung-Brain Axis in Preterm Brain Injury.

Bronchopulmonary dysplasia (BPD) and neurodevelopmental impairment (NDI) are among the most common morbidities affecting preterm infants. Although BPD is a predictor of poor NDI, it is currently uncertain how BPD contributes to brain injury in preterm infants. Extracellular vesicles (EVs) are involved in inter-organ communication in diverse pathological processes. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly and activation of inflammatory response. We assessed expression profiles of alveolar macrophage (AM) markers, CD11b, CD11c, and CD206, and ASC in EVs isolated from the plasma of preterm infants at risk for BPD at 1 week of age. We found that infants on higher fraction inspired oxygen (FiO2) therapy (HO2, ≥30%) had increased levels of AM-derived EV-ASC compared with infants on lower FiO2 (LO2, <30%). To assess the function of these EVs, we performed adoptive transfer experiments by injecting them into the circulation of newborn mice. We discovered that mice that received EVs from infants on HO2 had increased lung inflammation, decreased alveolarization, and disrupted vascular development, the hallmarks of BPD. Importantly, these EVs crossed the blood-brain barrier and the EVs from infants on HO2 caused inflammation, reduced cell survival, and increased cell death with features of pyroptosis and necroptosis in the hippocampus. These results highlight a novel role for AM-derived EV-ASC in mediating the lung-to-brain crosstalk that is critical in the pathogenesis of BPD and brain injury and identify potential novel targets for preventing and treating BPD and brain injury in preterm infants.

IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice.

BACKGROUND: Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation. METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR. RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2. CONCLUSION: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

Chorioamnionitis accelerates granule cell and oligodendrocyte maturation in the cerebellum of preterm nonhuman primates.

BACKGROUND: Preterm birth is often associated with chorioamnionitis and leads to increased risk of neurodevelopmental disorders, such as autism. Preterm birth can lead to cerebellar underdevelopment, but the mechanisms of disrupted cerebellar development in preterm infants are not well understood. The cerebellum is consistently affected in people with autism spectrum disorders, showing reduction of Purkinje cells, decreased cerebellar grey matter, and altered connectivity. METHODS: Preterm rhesus macaque fetuses were exposed to intra-amniotic LPS (1 mg, E. coli O55:B5) at 127 days (80%) gestation and delivered by c-section 5 days after injections. Maternal and fetal plasma were sampled for cytokine measurements. Chorio-decidua was analyzed for immune cell populations by flow cytometry. Fetal cerebellum was sampled for histology and molecular analysis by single-nuclei RNA-sequencing (snRNA-seq) on a 10× chromium platform. snRNA-seq data were analyzed for differences in cell populations, cell-type specific gene expression, and inferred cellular communications. RESULTS: We leveraged snRNA-seq of the cerebellum in a clinically relevant rhesus macaque model of chorioamnionitis and preterm birth, to show that chorioamnionitis leads to Purkinje cell loss and disrupted maturation of granule cells and oligodendrocytes in the fetal cerebellum at late gestation. Purkinje cell loss is accompanied by decreased sonic hedgehog signaling from Purkinje cells to granule cells, which show an accelerated maturation, and to oligodendrocytes, which show accelerated maturation from pre-oligodendrocytes into myelinating oligodendrocytes. CONCLUSION: These findings suggest a role of chorioamnionitis on disrupted cerebellar maturation associated with preterm birth and on the pathogenesis of neurodevelopmental disorders among preterm infants.

AGHmatrix: genetic relationship matrices in R.

MOTIVATION: The resemble between relatives computed from pedigree and genomic data is an important resource for geneticists and ecologists, who are interested in understanding how genes influence phenotypic variation, fitness adaptation, and population dynamics. RESULTS: The AGHmatrix software is an R package focused on the construction of pedigree (A matrix) and/or molecular markers (G matrix), with the possibility of building a combined matrix of pedigree corrected by molecular markers (H matrix). Designed to estimate the relationships for any ploidy level, the software also includes auxiliary functions related to filtering molecular markers, and checks pedigree errors in large data sets. After computing the relationship matrices, results from the AGHmatrix can be used in different contexts, including on prediction of (genomic) estimated breeding values and genome-wide association studies. AVAILABILITY AND IMPLEMENTATION: AGHmatrix v2.1.0 is available under GPL-3 license in CRAN at https://cran.r-project.org/web/packages/AGHmatrix/index.html and also in GitHub at https://github.com/rramadeu/AGHmatrix. It has a comprehensive tutorial, and it follows with real data examples.

Enterotoxigenic Staphylococcus aureus in Brazilian artisanal cheeses: Occurrence, counts, phenotypic and genotypic profiles.

The present study aimed to assess the occurrence and counts of Staphylococcus aureus in Brazilian artisanal cheeses (BAC) produced in five regions of Brazil: Coalho and Manteiga (Northeast region); Colonial and Serrano (South); Caipira (Central-West); Marajó (North); and Minas Artisanal cheeses, from Araxá, Campos das Vertentes, Cerrado, Serro and Canastra microregions (Southeast). The resistance to chlorine-based sanitizers, ability to attach to stainless steel surfaces, and antibiogram profile of a large set of S. aureus strains (n = 585) were assessed. Further, a total of 42 isolates were evaluated for the presence of enterotoxigenic genes (sea, seb, sec, sed, see, seg, sei, sej, and ser) and submitted to typing using pulsed-field gel electrophoresis (PFGE). BAC presented high counts of S. aureus (3.4-6.4 log CFU/g), varying from 25 to 62.5%. From the S. aureus strains (n = 585) assessed, 16% could resist 200 ppm of sodium hypochlorite, whereas 87.6% produced strong ability to attach to stainless steel surfaces, corroborating with S. aureus ability to persist and spread in the environment. Furthermore, the relatively high frequency (80.5%) of multidrug-resistant S. aureus and the presence of enterotoxin genes in 92.6% of the strains is of utmost attention. It reveals the lurking threat of SFP that can survive when conditions are favorable. The presence of enterotoxigenic and antimicrobial-resistant strains of S. aureus in cheese constitutes a potential risk to public health. This result calls for better control of cheese contamination sources, and taking hygienic measures is necessary for food safety. More attention should be paid to animal welfare and hygiene practices in some dairy farms during manufacturing to enhance the microbiological quality of traditional cheese products.

GSDMD gene knockout alleviates hyperoxia-induced hippocampal brain injury in neonatal mice.

BACKGROUND: Neonatal hyperoxia exposure is associated with brain injury and poor neurodevelopment outcomes in preterm infants. Our previous studies in neonatal rodent models have shown that hyperoxia stimulates the brain's inflammasome pathway, leading to the activation of gasdermin D (GSDMD), a key executor of pyroptotic inflammatory cell death. Moreover, we found pharmacological inhibition of caspase-1, which blocks GSDMD activation, attenuates hyperoxia-induced brain injury in neonatal mice. We hypothesized that GSDMD plays a pathogenic role in hyperoxia-induced neonatal brain injury and that GSDMD gene knockout (KO) will alleviate hyperoxia-induced brain injury. METHODS: Newborn GSDMD knockout mice and their wildtype (WT) littermates were randomized within 24 h after birth to be exposed to room air or hyperoxia (85% O2) from postnatal days 1 to 14. Hippocampal brain inflammatory injury was assessed in brain sections by immunohistology for allograft inflammatory factor 1 (AIF1) and CD68, markers of microglial activation. Cell proliferation was evaluated by Ki-67 staining, and cell death was determined by TUNEL assay. RNA sequencing of the hippocampus was performed to identify the transcriptional effects of hyperoxia and GSDMD-KO, and qRT-PCR was performed to confirm some of the significantly regulated genes. RESULTS: Hyperoxia-exposed WT mice had increased microglia consistent with activation, which was associated with decreased cell proliferation and increased cell death in the hippocampal area. Conversely, hyperoxia-exposed GSDMD-KO mice exhibited considerable resistance to hyperoxia as O2 exposure did not increase AIF1 + , CD68 + , or TUNEL + cell numbers or decrease cell proliferation. Hyperoxia exposure differentially regulated 258 genes in WT and only 16 in GSDMD-KO mice compared to room air-exposed WT and GSDMD-KO, respectively. Gene set enrichment analysis showed that in the WT brain, hyperoxia differentially regulated genes associated with neuronal and vascular development and differentiation, axonogenesis, glial cell differentiation, hypoxia-induced factor 1 pathway, and neuronal growth factor pathways. These changes were prevented by GSDMD-KO. CONCLUSIONS: GSDMD-KO alleviates hyperoxia-induced inflammatory injury, cell survival and death, and alterations of transcriptional gene expression of pathways involved in neuronal growth, development, and differentiation in the hippocampus of neonatal mice. This suggests that GSDMD plays a pathogenic role in preterm brain injury, and targeting GSDMD may be beneficial in preventing and treating brain injury and poor neurodevelopmental outcomes in preterm infants.

Extubation in the Operating Room After Coronary Artery Bypass Graft Surgery Reduces Hospital Stay.

OBJECTIVES: The aim of this analysis was to compare the effect of extubating in the operating room (OR) versus and the intensive care unit (ICU) among patients undergoing coronary artery bypass grafting (CABG). DESIGN: A retrospective cohort analysis. SETTING: Ten cardiac referral hospitals in Latin America; participants of the São Paulo Registry of Cardiovascular Surgery II (REPLICCAR II). PARTICIPANTS: The database included a total of 4,015 patients who underwent primary and isolated CABG surgery and were ≥18 years old, of whom 205 patients were extubated in the OR. INTERVENTIONS: The analysis was made after a propensity score matching (PSM) adjustment in the population sample of patients extubated in the OR and ICU by the following variables: sex, age, body mass index, smoking, type of surgery, chronic obstructive pulmonary disease, preoperative atrial fibrillation, cardiopulmonary bypass time, preoperative creatinine, and preoperative left ventricular ejection fraction. MEASUREMENTS AND MAIN RESULTS: This study focused on the analysis of the ICU and hospital length of stay, need for reintubation, morbidity, and mortality. After PSM, 402 patients were analyzed. Both groups had similar baseline characteristics, such as age (p = 0.132), sex (p = 1.00), and estimated risk of prolonged ventilation (>24 hours, p = 0.168); however, the median ventilation time was significantly shorter in the group extubated in the OR compared to the ICU group (5.67 hours v 17.55 hours, p < 0.001). The group of patients extubated in the ICU had a longer postoperative stay (7.54 ± 3.40 days v 6.41 ± 2.91 days, p < 0.001) and longer total hospitalization time (11.49 ± 5.70 days v 10.36 ± 5.72, p = 0.013) compared to those extubated in the OR. The authors did not observe a significant difference in the need for reintubation, morbidity, or mortality rates among the evaluated groups. CONCLUSIONS: In the REPLICCAR II database, extubation performed in the OR was associated with a reduced length of postoperative and total hospital stays compared to extubation in the ICU.

Patients and Parents' Satisfaction and Self-Reported Evaluation After Single-Event Multilevel Surgery in Cerebral Palsy.

PURPOSE: The aim of this study was to analyze the results of single-event multilevel surgery (SEMLS) in cerebral palsy (CP) based on objective gait outcomes and self-reported evaluations. METHODS: In total, 258 patients with spastic diplegic CP, GMFCS I-III, who underwent SEMLS and with preoperative and postoperative gait analyses, were included in the SEMLS group (SG). The same database was used to compose the control group (CG) formed of 88 subjects who had performed at least 2 gait analyses and did not undergo surgical intervention between tests. Demographic data, Gait Deviation Index (GDI), and a self-reported questionnaire were analyzed, and results were compared between groups. RESULTS: The GDI decreased from 59.6 to 57.9 in the CG and increased from 51.3 to 58.4 in the SG ( P <0.001). There was no change in patients' walking ability in the CG. The number of patients who walk community distances increased after SEMLS in the group that had a GDI improvement >5 points (from 12.3% to 24.7%, P =0.008) and in patients GMFCS I and II (from 9.2% to 20.4%, P =0.028). According to patient and parental responses on satisfaction, the most significant improvements were reported in self-esteem, mobility, body image, and independence. In total, 51.1% of the patients were extremely satisfied or satisfied, while 3.9% were unsatisfied or extremely unsatisfied with the treatment results. Of the parents surveyed, 60.1% were extremely satisfied or satisfied, whereas just 5.0% were unsatisfied or extremely unsatisfied. CONCLUSIONS: In the studied group, an improvement in outdoor walking for community distances after SEMLS, as reported in questionnaires, was observed only when GDI increase was >5 points and in GMFCS I and II. LEVEL OF EVIDENCE: Level III.

Continuous but not pulsed low-dose fetal betamethasone exposures extend the durability of antenatal steroid therapy.

Antenatal steroid (ANS) therapy is the standard care for women at imminent risk of preterm labor. Despite extensive and long-standing use, 40%-50% of babies exposed antenatally to steroids do not derive benefit; remaining undelivered 7 days or more after ANS treatment is associated with a lack of treatment benefit and increased risk of harm. We used a pregnant sheep model to evaluate the impact of continuous versus pulsed ANS treatments on fetal lung maturation at an extended, 8-day treatment to delivery interval. Continuous low-dose ANS treatments for more than 72 h in duration improved fetal lung maturation at 8 days after treatment initiation. If fetal ANS exposure was interrupted, the beneficial ANS effect was lost. Truncated treatments, including that simulating the current clinical treatment regimen, did not improve lung function. Variable fetal lung maturation was correlated to the amount of saturated phosphatidylcholine present in the lung fluid. These data demonstrate that 1) the durability of ANS therapy may be enhanced by employing an extended, low-dose treatment regimen by reducing total dose and 2) interrupting the continuity of fetal exposure by allowing it to fall below a minimal threshold was associated with comparably poor functional maturation of the preterm ovine lung.

Shedding Light on the Enigmatic TcO2 ⋠xH2 O Structure with Density Functional Theory and EXAFS Spectroscopy.

The ß-emitting 99 Tc isotope is a high-yield fission product in 235 U and 239 Pu nuclear reactors, raising special concern in nuclear waste management due to its long half-life and the high mobility of pertechnetate (TcO4 - ). Under the conditions of deep nuclear waste repositories, Tc is retained through biotic and abiotic reduction of TcO4 - to compounds like amorphous TcO2 ⋅ xH2 O precipitates. It is generally accepted that these precipitates have linear (Tc(µ-O)2 (H2 O)2 )n chains, with trans H2 O. Although corresponding Tc-Tc and Tc-O distances have been obtained from extended X-ray absorption fine structure (EXAFS) spectroscopy, this structure is largely based on analogy with other compounds. Here, we combine density-functional theory with EXAFS measurements of fresh and aged samples to show that, instead, TcO2 ⋅ xH2 O forms zigzag chains that undergo a slow aging process whereby they combine to form longer chains and, later, a tridimensional structure that might lead to a new TcO2 polymorph.

The complex challenge of antenatal steroid therapy nonresponsiveness.

Antenatal steroid therapy is standard care for women at imminent risk of preterm delivery. When deliveries occur within 7 days of treatment, antenatal steroid therapy reduces the risk of neonatal death and improves preterm outcomes by exerting diverse developmental effects on the fetal organs, in particular the preterm lung and cardiovascular system. There is, however, sizable variability in antenatal steroid treatment efficacy, and an important percentage of fetuses exposed to antenatal steroid therapy do not respond sufficiently to derive benefit. Respiratory distress syndrome, for example, is a central metric of clinical trials to assess antenatal steroid outcomes. In the present analysis, we addressed the concept of antenatal steroid nonresponsiveness, and defined a failed or suboptimal response to antenatal steroids as death or a diagnosis of respiratory distress syndrome following treatment. For deliveries at 24 to 35 weeks' gestation, the number needed to treat to prevent 1 case of respiratory distress syndrome was 19 (95% confidence interval, 14-28). Reflecting gestation-dependent risk, for deliveries at >34 weeks' gestation the number needed to treat was 55 (95% confidence interval, 30-304), whereas for elective surgical deliveries at term this number was 106 (95% confidence interval, 61-421). We reviewed data from clinical and animal studies investigating antenatal steroid therapy to highlight the significant incidence of antenatal steroid therapy nonresponsiveness (ie, residual mortality or respiratory distress syndrome after treatment), and the potential mechanisms underpinning this outcome variability. The origins of this variability may be related to both the manner in which the therapy is applied (ie, the treatment regimen itself) and factors specific to the individual (ie, genetic variation, stress, infection). The primary aims of this review were: (1) to emphasize to the obstetrical and neonatal communities the extent of antenatal steroid response variability and its potential impact; (2) to propose approaches by which antenatal steroid therapy may be better applied to improve overall benefit; and (3) to stimulate further research toward the empirical optimization of this important antenatal therapy.

Betamethasone phosphate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights when administered to pregnant sheep in combination with betamethasone acetate.

BACKGROUND: Antenatal corticosteroid therapy is a standard of care for women at imminent risk of preterm labor. However, the optimal (maximum benefit and minimal risk of side effects) antenatal corticosteroid dosing strategy remains unclear. Although conveying overall benefit when given to the right patient at the right time, antenatal corticosteroid treatment efficacy is highly variable and is not risk-free. Building on earlier findings, we hypothesized that when administered in combination with slow-release betamethasone acetate, betamethasone phosphate and the high maternal-fetal betamethasone concentrations it generates are redundant for fetal lung maturation. OBJECTIVE: Using an established sheep model of prematurity and postnatal ventilation of the preterm lamb, we aimed to compare the pharmacodynamic effects of low-dosage treatment with betamethasone acetate only against a standard dosage of betamethasone phosphate and betamethasone acetate as recommended by the American College of Obstetricians and Gynecologists for women at risk of imminent preterm delivery between 24 0/7 and 35 6/7 weeks' gestation. STUDY DESIGN: Ewes carrying a single fetus at 122±1 days' gestation (term=150 days) were randomized to receive either (1) maternal intramuscular injections of sterile saline (the saline negative control group, n=12), (2) 2 maternal intramuscular injections of 0.25 mg/kg betamethasone phosphate+betamethasone acetate administered at 24-hour dosing intervals (the betamethasone phosphate+betamethasone acetate group, n=12); or (3) 2 maternal intramuscular injections of 0.125 mg/kg betamethasone acetate administered at 24-hour dosing intervals (the betamethasone acetate group, n=11). The fetuses were surgically delivered 48 hours after treatment initiation and ventilated for 30 minutes to determine functional lung maturation. The fetuses were euthanized after ventilation, and the lungs were collected for analysis using quantitative polymerase chain reaction and Western blot assays. Fetal plasma adrenocorticotropic hormone levels were measured in the cord blood samples taken at delivery. RESULTS: Preterm lambs were defined as either antenatal corticosteroid treatment responders or nonresponders using an arbitrary cutoff, being a PaCO2 level at 30 minutes of ventilation being more extreme than 2 standard deviations from the mean value of the normally distributed saline control group values. Compared with the animals in the saline control group, the animals in the antenatal corticosteroid treatment groups showed significantly improved lung physiological responses (blood gas and ventilation data) and had a biochemical signature (messenger RNA and surfactant protein assays) consistent with functional maturation. However, the betamethasone acetate group had a significantly higher treatment response rate than the betamethasone phosphate+betamethasone acetate group. These physiological results were strongly correlated to the amount of surfactant protein A. Birthweight was lower in the betamethasone phosphate+betamethasone acetate group and the fetal hypothalamic-pituitary-adrenal axis was suppressed to a greater extent in the betamethasone phosphate+betamethasone acetate group. CONCLUSION: Low-dosage antenatal corticosteroid therapy solely employing betamethasone acetate was sufficient for fetal lung maturation. The elevated maternal-fetal betamethasone concentrations associated with the coadministration of betamethasone phosphate did not in addition improve lung maturation but were associated with greater fetal hypothalamic-pituitary-adrenal axis suppression, a lower antenatal corticosteroid treatment response rate, and lower birthweight-outcomes not desirable in a clinical setting. These data warranted a clinical investigation of sustained low-dosage antenatal corticosteroid treatments that avoid high maternal-fetal betamethasone exposures.

A novel surgical toxicological-free model of diaphragmatic hernia in fetal rats.

BACKGROUND: Teratogen-induced congenital diaphragmatic hernia (CDH) rat models are commonly used to study the pathophysiology. We have created a new and reliable surgically induced diaphragmatic hernia (DH) model to obtain a purely mechanical DH rat model, and avoid the confounding teratogen-induced effects on the lung development. METHODS: Fetal DH was surgically created on fetuses at E18.5 and harvested at E21.5 in rats. Four groups were evaluated (n = 16): control (CONT), control exposed to Nitrofen (CONT NIT), DH surgically created (DH SURG), and CDH Nitrofen (CDH NIT). Body weight, total lung weights, and their ratio (BW, TLW, and TLBR) were compared. Air space (AS), parenchyma (PA), total protein, and DNA contents were measured to verify lung hypoplasia. Medial wall thickness (MWT) of pulmonary arterioles was also analyzed. RESULTS: DH SURG showed significant hypoplasia (decreased in total protein and DNA) vs CONT (p < 0.05); DH SURG vs CDH NIT were similar in TLW and TLBR. DH SURG has less AS than CONT (p < 0.05) and similar PA compared to CONT NIT and CDH NIT, MWT were similarly increased in CONT NIT, DH SURG, and CDH NIT. CONCLUSIONS: This novel surgical model generates fetal lung hypoplasia contributing to the study of the mechanical compression effect on fetal lung development in DH. IMPACT: There is a critical need to develop a surgical model in rat to complement the findings of the well-known Nitrofen-induced CDH model. This experimental study is pioneer and can help to understand better the CDH pathophysiological changes caused by herniated abdominal viscera compression against the lung during the final stage of gestation in CDH fetuses, and also to develop more efficient treatments in near future.

Transit-time flow measurement parameters after protamine infusion in CABG surgeries.

OBJECTIVE: The objective of this study is to evaluate protamine sulfate effects on graft's blood flow by comparing transit-time flow measurement (TTFM) values before and after protamine administration. METHODS: This is an observational study with data collected between years 2018 and 2020. Immediate graft patency was evaluated using TTFM. Only patients with TTFM parameters registered before and after protamine infusion were included. The main three parameters studied were:  mean graft flow  (MGF),  pulsatility index (PI), and  diastolic flow (DF). In the first analysis, all conduits were evaluated regardless of the surgical technique used. In a second analysis, on-pump and off-pump groups were compared. Evaluated grafts were left internal thoracic artery, saphenous vein graft (SVG), radial artery, and right internal thoracic artery. Since SVG was numerically the most used graft, an exclusive analysis was created. RESULTS: Our study included 575 patients, resulting in a total of 1686 grafts, mean 2.93 grafts/patient. Off-pump surgery was performed in 158 patients. Before protamine infusion, inadequate TTFM parameters were observed in 3.8% of grafts. Overall, after protamine administration, MGF decreased in all grafts, but its reduction was not statistically significant. PI values increased in the SVG and DF values reduced in LIMA grafts. SVG group analysis showed that after protamine PI values were higher in OM1 and RCA. DF values increased in RCA. The comparison between off and on-pump surgeries, showed that in off-pump cases TTFM measures did not present statistically significant differences. CONCLUSION: Significant variations were observed in TTFM values before and after protamine administration. Although different, those values remained within the normal reference ranges. We recommend that flow measurement should be performed before protamine infusion.

Correlating blast vibrations and geomechanical properties to determine damage profiles and improve wall conditions in open pit mining.

Slope stability is one of the biggest concerns for mining practices and to consider the rock mass response over blasting is fundamental to achieve pit geometry. This study consists in developing a methodology which connects the dynamic behavior of one lithological domain to blast designs applied at a copper mine. The central element of this study was the construction of vibration attenuation and seed wave model which, in conjunction with geomechanical properties, has allowed the characterization of this particular rock mass and the vibration attenuation phenomena. The new blast design was developed from the model simulations, once it was possible to recognize which parameters of the blast design affect most of the damage induced by blasting. To guarantee model representativeness, two blast tests were conducted: one with the usual blast design and another using the new one. Furthermore, holes were drilled behind the blasts, which were inspected before and after each blast to compare the produced fracturing with the fracturing expected by the model. The results obtained in these blast tests show a strong correlation between the modeled and the real. The modeling proved to be a useful tool providing manners to stablish a blast design, which generates stable walls.

Helicobacter pylori Urease: Potential Contributions to Alzheimer's Disease.

Alzheimer's disease (AD) causes dementia and memory loss in the elderly. Deposits of beta-amyloid peptide and hyperphosphorylated tau protein are present in a brain with AD. A filtrate of Helicobacter pylori's culture was previously found to induce hyperphosphorylation of tau in vivo, suggesting that bacterial exotoxins could permeate the blood-brain barrier and directly induce tau's phosphorylation. H. pylori, which infects ~60% of the world population and causes gastritis and gastric cancer, produces a pro-inflammatory urease (HPU). Here, the neurotoxic potential of HPU was investigated in cultured cells and in rats. SH-SY5Y neuroblastoma cells exposed to HPU (50-300 nM) produced reactive oxygen species (ROS) and had an increased [Ca2+]i. HPU-treated BV-2 microglial cells produced ROS, cytokines IL-1ß and TNF-α, and showed reduced viability. Rats received daily i.p., HPU (5 µg) for 7 days. Hyperphosphorylation of tau at Ser199, Thr205 and Ser396 sites, with no alterations in total tau or GSK-3ß levels, and overexpression of Iba1, a marker of microglial activation, were seen in hippocampal homogenates. HPU was not detected in the brain homogenates. Behavioral tests were performed to assess cognitive impairments. Our findings support previous data suggesting an association between infection by H. pylori and tauopathies such as AD, possibly mediated by its urease.

Circulating extracellular vesicles activate the pyroptosis pathway in the brain following ventilation-induced lung injury.

BACKGROUND: Mechanical ventilation of preterm newborns causes lung injury and is associated with poor neurodevelopmental outcomes. However, the mechanistic links between ventilation-induced lung injury (VILI) and brain injury is not well defined. Since circulating extracellular vesicles (EVs) are known to link distant organs by transferring their cargos, we hypothesized that EVs mediate inflammatory brain injury associated with VILI. METHODS: Neonatal rats were mechanically ventilated with low (10 mL/kg) or high (25 mL/kg) tidal volume for 1 h on post-natal day 7 followed by recovery for 2 weeks. Exosomes were isolated from the plasma of these rats and adoptively transferred into normal newborn rats. We assessed the effect of mechanical ventilation or exosome transfer on brain inflammation and activation of the pyroptosis pathway by western blot and histology. RESULTS: Injurious mechanical ventilation induced similar markers of inflammation and pyroptosis, such as increased IL-1ß and activated caspase-1/gasdermin D (GSDMD) in both lung and brain, in addition to inducing microglial activation and cell death in the brain. Isolated EVs were enriched for the exosomal markers CD9 and CD81, suggesting enrichment for exosomes. EVs isolated from neonatal rats with VILI had increased caspase-1 but not GSDMD. Adoptive transfer of these EVs led to neuroinflammation with microglial activation and activation of caspase-1 and GSDMD in the brain similar to that observed in neonatal rats that were mechanically ventilated. CONCLUSIONS: These findings suggest that circulating EVs can contribute to the brain injury and poor neurodevelopmental outcomes in preterm infants with VILI through activation of GSDMD.

Evaluation of Cardiotonic Steroid Modulation of Cellular Cholesterol and Phospholipid.

We have previously shown that 21-benzylidene digoxin (21-BD) increases the total cholesterol and phospholipid content on the membrane of HeLa cells. Lipid modulation caused by cardiotonic steroids (CTS) is still unexplored. Therefore, the aim of the present study was to evaluate the cholesterol and phospholipid modulation of the cell membrane caused by ouabain and 21-BD and the possible involvement of the caveolae on this modulation. For this, one cell line containing caveolae (HeLa) and other not containing (Caco-2) were used. The modulation of the lipid profile was evaluated by total cholesterol and phospholipids measurements, and identification of membrane phospholipids by HPTLC. The cholesterol distribution was evaluated by filipin staining. The caveolin-1 expression was evaluated by Western Blotting. Ouabain had no effect on the total membrane lipid content in both cell lines. However, 21-BD increased total membrane phospholipid content and had no effect on the membrane cholesterol content in Caco-2 cells. CTS were not able to alter the specific phospholipids content. In the filipin experiments, 21-BD provoked a remarkable redistribution of cholesterol to the perinuclear region of HeLa cells. In Caco-2 cells, it was observed only a slight increase in cholesterol, especially as intracellular vesicles. The caveolin-1 expression was not altered by any of the compounds. Our data mainly show different effects of two cardiotonic steroids. Ouabain had no effect on the lipid profile of cells, whereas 21-BD causes important changes in cholesterol and phospholipid content. Therefore, the modulation of cholesterol content in the plasma membrane of HeLa cells is not correlated with the expression of caveolin-1.

Compensated hypogonadism in men with sickle cell disease.

OBJECTIVE: To evaluate the prevalence of hypogonadism in a population of men with SCD and characterize its aetiology. Sickle cell disease (SCD) is associated with the development of hypogonadism, but there is still controversy regarding its aetiology and clinical implications. METHODS: We performed a cross-sectional study of 34 men with SCD aged > 18 years. Sociodemographic and clinical data, including anthropometric measurements (weight, height and BMI), were obtained. Early morning, blood samples were collected and total testosterone (TT), free testosterone (FT), luteinizing hormone (LH), follicle stimulating hormone (FSH), a complete blood count and haemoglobin electrophoresis were measured. Eugonadism was defined as T ≥300 ng/dL and LH ≤9.4 mUI/mL; primary hypogonadism as T < 300 ng/dL and LH > 9.4 mUI/mL; secondary hypogonadism as T < 300 ng/dL and LH ≤ 9.4 mUI/mL; and compensated hypogonadism as T ≥ 300 ng/dL and LH > 9.4 mUI/mL. RESULTS: Median age was 33 (26-41) years, and SS genotype was the most frequent (73.5%). The prevalence of eugonadism, compensated hypogonadism and secondary hypogonadism was 67.5%, 26.4% and 5.88%, respectively. No men with primary hypogonadism were identified in our sample. Those with compensated hypogonadism had also higher FSH levels (>7.8 mUI/mL, P < .0001). CONCLUSION: In our study population of men with SCD, a high prevalence of compensated hypogonadism was identified, which is a controversial and distinct clinical entity that warrants monitoring and further research.

Is bioelectrical impedance vector analysis a good indicator of nutritional status in children and adolescents?

OBJECTIVE: The study objective is to propose bioelectrical impedance vector analysis (BIVA) ellipses for the population and investigate its use to assess nutritional status through the BMI/age ratio and the cardiovascular risk through waist circumference (WC). DESIGN: Age, weight, height and WC were recorded, along with the values of resistance (R), reactance (Xc) and phase angle (PA) by impedance. Student's t test and ANOVA were applied to ascertain the significance between means and a specific programme was applied to investigate the significance between ellipses. SETTING: Fortaleza, Brazil. PARTICIPANTS: A total of 467 students from public schools participated in the study: 120 children and 347 adolescents were evaluated, with respective means of age, weight and height of 8·2 years, 27·6 kg and 1·29 m and 12·7 years, 48·6 kg and 1·53 m, respectively. RESULTS: The mean values of R/H, Xc/H and PA were 569·0 and 424·7 Ohm/m, 59·1 and 50·4 Ohm/m, and 5·9º and 6·8º for children and adolescents, respectively. The mean vectors for R/H and Xc/H show significant differences between for both age ranges and are inversely proportional to the BMI/A. As for WC, individuals without cardiovascular risk had higher R/H and lower Xc/H, but Xc and R in participants without cardiovascular risk were greater than with cardiovascular risk. CONCLUSIONS: It was possible to propose confidence and tolerance BIVA ellipses for children and adolescent's clinical evaluation. The method was also suitable to identify cardiovascular risk ellipses in these age groups, but it was not possible to draw nutritional classifications ellipses by BMI/age data.