Nanofiber-microwell cell culture system for spatially patterned differentiation of pluripotent stem cells in 3D.

The intricate interplay between biochemical and physical cues dictates pluripotent stem cell (PSC) differentiation to form various tissues. While biochemical modulation has been extensively studied, the role of biophysical microenvironments in early lineage commitment remains elusive. Here, we introduce a novel 3D cell culture system combining electrospun nanofibers with microfabricated polydimethylsiloxane (PDMS) patterns. This system enables the controlled formation of semispherical human induced pluripotent stem cell (hiPSC) colonies, facilitating the investigation of local mechanical stem cell niches on mechano-responsive signaling and lineage specification. Our system unveiled spatially organized RhoA activity coupled with actin-myosin cable formation, suggesting mechano-dependent hiPSC behaviors. Nodal network analysis of RNA-seq data revealed RhoA downstream regulation of YAP signaling, DNA histone modifications, and patterned germ layer specification. Notably, altering colony morphology through controlled PDMS microwell shaping effectively modulated the spatial distribution of mechano-sensitive mediators and subsequent differentiation. This study provides a cell culture platform to decipher the role of biophysical cues in early embryogenesis, offering valuable insights for material design in tissue engineering and regenerative medicine applications.

Dissecting TGF-ß-induced glioblastoma invasion with engineered hyaluronic acid hydrogels.

Glioma stem cells (GSCs) contribute to rapid cellular invasion in glioblastoma (GBM). Transforming growth factor-ß (TGF-ß) has been strongly implicated in supporting key GSC functions, including stemness, immunosuppression, and resistance. Although TGF-ß is well-known as a driver of cancer invasion, how TGF-ß supports the invasion of GSCs is not well understood. Progress in understanding mechanisms of TGF-ß-driven invasion in GSC-derived tumors has been limited by an absence of three-dimensional (3D) culture systems that support TGF-ß-stimulated invasion. Here, we show that 3D hyaluronic acid (HA) matrices can address this need. We perform bioinformatic analysis of human glioma datasets, which reveals progressive enrichment of TGF-ß-related gene expression with increasingly aggressive glioma grade and GBM subtype. We then experimentally screen the invasion of a panel of human GSC spheroids through a set of 3D matrix systems, including collagen I, Matrigel, and HA, and find that only HA recapitulates TGF-ß-induced invasion. We then show that GSCs differ in their ability to invade HA in a way that can be predicted from TGF-ß receptor 2 expression and SMAD2 phosphorylation. GSC spheroid invasion depends strongly on the presence of RGD peptides on the HA backbone but is surprisingly independent of matrix metalloprotease degradability. Finally, we demonstrate that TGF-ß stimulates invasion through SMAD-dependent signaling, consistent with recent observations that TGF-ß/SMAD signals drive tumor microtube formation and invasion. Our work supports further development of HA as a matrix platform for dissecting contributions of TGF-ß and other cytokines to GBM invasion and screening of cytokine-dependent invasion in human tumors.

Pre- and post-surgical evaluation of the impact on Hansen disease sequealae patients' quality of life submitted to orthopedic surgery for closure of chronic plantar ulcers.

INTRODUCTION: Leprosy is a chronic infectious disease that still persists as a public health problem in Brazil. Plantar ulcers are serious complications due to leprosy neuropathy and intensify the isolation and stigma of these individuals. The difficulty in closing these lesions associated with the fetid odor negatively impact the quality of life of people with these lesions. OBJECTIVE: To evaluate the clinical, socioeconomic conditions, degree of satisfaction and quality of life (QoL) of patients after healing of chronic ulcers on feet submitted to orthopedic surgery. METHODOLOGY: This is a qualitative, exploratory, descriptive and observational study carried out with 92 people after surgical treatment of chronic leprosy plantar ulcers. These patients were submitted to a semi-structured questionnaire raising questions of an epidemiological, socioeconomic and perception of quality-of-life order, comparing before and after the surgical procedure. RESULTS: Decrease in indicators - alcohol consumption, tobacco consumption, average monthly cost of analgesic medications, fetid wound odor, foot pain and number of dressings performed weekly; Recurrence of lesions in 55.4 % of cases, related to irregular use or lack of shoes and insoles; Improvement in self-perception of Quality of Life (QoL) in 89.1 % of patients after surgery. CONCLUSION: Orthopedic surgical treatment with resection of plantar bony prominences and skin grafting is an effective therapeutic method for closing chronic plantar ulcers in leprosy, resulting in a decrease in the financial costs employed and in an important improvement in the Quality-of-Life parameters of the individuals undergoing to this procedure. The availability and regular use of shoes and insoles is crucial to prevent recurrence of these injuries.

Chronic Chagas Cardiomyopathy in the Brazilian Amazon region: clinical characteristics and regional distinctiveness.

Objectives: This study aims to provide a comprehensive analysis of clinical and epidemiological data related to Chronic Chagas Cardiomyopathy (CCC) in the Amazon region of Brazil. Methods: A review of observational, retrospective, and cross-sectional studies related to Chagas Disease in the Amazon region of Brazil was conducted, and a case series addressing CCC in patients treated at the FMT-HVD outpatient clinic, a reference center for Chagas disease in Brazil, was carried out. Results: Clinical characteristics of 55 patients from the Amazon region with CCC were described. The most common electrocardiographic alteration observed was abnormal ventricular repolarization (AVR), present in 40% of cases. The most common echocardiographic finding was left ventricular systolic dysfunction (49%), followed by akinesia or hypokinesia of the inferior and/or inferolateral walls (38.1%) and the presence of an apical aneurysm (32.7%). Conclusions: Overall, this study demonstrates that CCC in the Amazon region presents clinical characteristics and severity that are similar to those observed in other regions. However, certain peculiarities, such as the frequency of right bundle branch block (RBBB) and anterior and septal involvement during the acute phase, require additional investigation to better comprehend the disease in the region. Overall, the study provides crucial clinical insights for the diagnosis and treatment of CCC in the Amazon region.

Decreased level of TREM like Transcript 1 (TLT-1) is associated with prematurity and promotes the in-utero inflammatory response to maternal lipopolysaccharide (LPS) exposure.

PROBLEM: The occurrence of preterm birth is associated with multiple factors including bleeding, infection and inflammation. Platelets are mediators of hemostasis and can modulate inflammation through interactions with leukocytes. TREM like Transcript 1 (TLT-1) is a type 1 single Ig domain receptor on activated platelets. In adults, it plays a protective role by dampening the inflammatory response and facilitating platelet aggregation at sites of vascular injury. TLT-1 is expressed in human placenta and found in cord blood. We thus hypothesized that TLT-1 deficiency is associated with prematurity and fetal inflammation. METHOD OF STUDY: To test this hypothesis, we examined cord blood levels of soluble TLT-1 (sTLT) in premature and term infants and compared the inflammatory response in C57BL/6 (WT) and TLT-1-/- (treml1-/- , KO) mice given intraperitoneal LPS mid-gestation RESULTS: The preterm infant cord blood level of sTLT was significantly lower than that found at term. On exposure to LPS, histology of KO (as compared to WT) placenta and decidua showed increased hemorrhage, and KO decidual RNA expression of IL-10 was significantly lower. KO fetal interface tissues (placenta, membranes, amniotic fluid) over time showed increased expression of inflammatory cytokines such as IL-6, IFN-γ, and TNF, but not MCP-1. However, fetal organs showed similar levels. CONCLUSION: There is a potential association between insufficient TLT-1 expression and increased fetal inflammatory responses in the setting of prematurity. The data support further study of TLT-1 in the mechanistic link between bleeding, inflammation and preterm birth, and perhaps as a biomarker in human pregnancy.

Acute Chagas disease associated with ingestion of contaminated food in Brazilian western Amazon.

OBJECTIVE: To describe clinical, epidemiological and management information on cases of acute Chagas disease (ACD) by oral transmission in the state of Amazonas in western Amazon. METHODS: Manual and electronic medical records of patients diagnosed with ACD at the Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD) were included. RESULTS: There were 147 cases of acute CD registered from 10 outbreaks that occurred in the state of Amazonas between 2004 and 2022. The transmission pathway was through oral route, with probable contaminated palm fruit juice (açaí and/or papatuá), and involved people from the same family, friends or neighbours. Of 147 identified cases, 87 (59%) were males; cases were aged 10 months to 82 years. The most common symptom was the febrile syndrome (123/147; 91.8%); cardiac alterations were present in 33/100 (33%), (2/147; 1.4%) had severe ACD with meningoencephalitis, and 12 (8.2%) were asymptomatic. Most cases were diagnosed through thick blood smear (132/147; 89.8%), a few (14/147; 9.5%) were diagnosed by serology and (1/147; 0.7%) by polymerase chain reaction (PCR) and blood culture. In all these outbreaks, 74.1% of the patients were analysed by PCR, and Trypanosoma cruzi TcIV was detected in all of them. No deaths were recorded. The incidence of these foci coincided with the fruit harvest period in the state of Amazonas. CONCLUSION: The occurrence of ACD outbreaks in the Amazon affected individuals of both sexes, young adults, living in rural and peri-urban areas and related to the consumption of regional foods. Early diagnosis is an important factor in surveillance. There was a low frequency of cardiac alterations. Continuous follow-up of most patients was not carried out due to difficulty in getting to specialised centres; therefore, little is known about post-treatment.

Early Diagnosis of Oral Cancer and Lesions in Fanconi Anemia Patients: A Prospective and Longitudinal Study Using Saliva and Plasma.

Fanconi anemia (FA) patients display an exacerbated risk of oral squamous cell carcinoma (OSCC) and oral potentially malignant lesions (OPMLs) at early ages. As patients have defects in their DNA repair mechanisms, standard-of-care treatments for OSCC such as radiotherapy and chemotherapy, give rise to severe toxicities. New methods for early diagnosis are urgently needed to allow for treatment in early disease stages and achieve better clinical outcomes. We conducted a prospective, longitudinal study wherein liquid biopsies from sixteen patients with no clinical diagnoses of OPML and/or OSCC were analyzed for the presence of mutations in cancer genes. The DNA from saliva and plasma were sequentially collected and deep-sequenced, and the clinical evaluation followed over a median time of approximately 2 years. In 9/16 FA patients, we detected mutations in cancer genes (mainly TP53) with minor allele frequencies (MAF) of down to 0.07%. Importantly, all patients that had mutations and clinical follow-up data after mutation detection (n = 6) developed oral precursor lesions or OSCC. The lead-time between mutation detection and tumor diagnosis ranged from 23 to 630 days. Strikingly, FA patients without mutations displayed a significantly lower risk of developing precursor lesions or OSCCs. Therefore, our diagnostic approach could help to stratify FA patients into risk groups, which would allow for closer surveillance for OSCCs or precursor lesions.

The Assessment of Identity Development in Adolescence (AIDA) Questionnaire: First Psychometric Evaluation in Two North American Samples of Young People.

The Assessment of Identity Development in Adolescence (AIDA) is a recently developed measure of identity diffusion in young people. Originally validated in Switzerland, the English version has not yet been validated. Our aim was to evaluate the AIDA's internal factor structure, internal consistency, incremental validity, and relations to other variables in adolescents and young adults and derive clinical cutoffs for use in clinical settings. Study 1, involving 2,119 undergraduate students between the ages of 18-25 years, confirmed the expected bi-factor structure for the AIDA. Study 2 involved the recruitment of 122 adolescent inpatients (ages 12-17), of whom 36 met full DSM-based criteria for Borderline Personality Disorder, in addition to 164 adolescents of the same age, recruited from public schools (total 286 adolescents). Results of both studies demonstrated strong internal consistency and correlations of the expected magnitude and direction with other self-report measures of personality pathology. In addition, Study 2 demonstrated that the AIDA could successfully discriminate between adolescent inpatients with and without BPD, as well as healthy controls, and established preliminary clinical cutoffs that should be replicated in future studies. In all, these studies provide support for further validation and use of the AIDA in young people.

Hope and mental health problems among orphans and vulnerable children in South Africa.

ABSTRACTThere are over three million orphaned and vulnerable children (OVC) currently living in South Africa. OVC are at high risk for a number of negative outcomes, including poor mental health. Hope has been associated with well-being among youth, including youth in South Africa. However, the relationships between hope and mental health in high-adversity populations such as OVC has not been adequately described. The present study sought to address this research gap by evaluating the relationship between hope and mental health, controlling for gender, age, and orphan status, among OVC. This study includes 8- to 12-year-old OVC (N = 61) in Manguang, Free State, South Africa. Hope was assessed using the Children's Hope Scale (CHS) and mental health outcomes were assessed using the Strengths and Difficulties Questionnaire (SDQ). Hope was significantly, inversely associated with mental health outcomes after controlling for other variables in linear regression analysis. In contrast to previous research, this study found that increased hope scores were associated with adverse mental health outcomes among OVC in South Africa. Hope may be contextualized differently in this population due to resource scarcity and high rates of adversity including HIV-AIDS related stigma and poverty.

Development of a mouse model for spontaneous oral squamous cell carcinoma in Fanconi anemia.

Fanconi anemia (FA) patients frequently develop oral squamous cell carcinoma (OSCC). This cancer in FA patients is diagnosed within the first 3-4 decades of life, very often preceded by lesions that suffer a malignant transformation. In addition, they respond poorly to current treatments due to toxicity or multiple recurrences. Translational research on new chemopreventive agents and therapeutic strategies has been unsuccessful partly due to scarcity of disease models or failure to fully reproduce the disease. Here we report that Fanca gene knockout mice (Fanca-/-) frequently display pre-malignant lesions in the oral cavity. Moreover, when these animals were crossed with animals having conditional deletion of Trp53 gene in oral mucosa (K14cre;Trp53F2-10/F2-10), they spontaneously developed OSCC with high penetrance and a median latency of less than ten months. Tumors were well differentiated and expressed markers of squamous differentiation, such as keratins K5 and K10. In conclusion, Fanca and Trp53 genes cooperate to suppress oral cancer in mice, and Fanca-/-;K14cre;Trp53F2-10/F2-10 mice constitute the first animal model of spontaneous OSCC in FA.

Acute micro-outbreak of Chagas disease in the southeastern Amazon: a report of five cases.

BACKGROUND: Chagas disease is gaining importance in the Brazilian Amazon region as a differential diagnosis of febrile syndrome. The most recent microoutbreak occurred in Ipixuna, in Amazonas state. METHODS: An epidemiological survey was conducted using parasitological and serological tests, and electrocardiographic analysis. RESULTS: The patients belonged to one family and had ingested açaí acquired from Ipixuna. All patients reported fever and initially a thick blood smear test was done to identify Trypanosoma cruzi. Benznidazole treatment was administered to all patients. CONCLUSIONS: Knowledge of the epidemiological dynamics of Chagas disease allows us to improve control and management measures for this disease.

Does coding internal working models of attachment have to be so hard?

The Child Attachment Interview (CAI) has demonstrated promise in youth, yet widespread use is thwarted by the need for interview transcription, face-to-face training, and reliability certification. The present study sought to examine the empirical basis for these barriers. Thirty-five archival CAIs were re-coded by: (1) expert coders (i.e., trained and reliable) without access to transcripts, (2) trained coders who had not completed reliability training, and (3) novice coders who had no formal training. Agreement with consensus classifications was computed with the expectation of moderate agreement. Results supported coding by experts without transcription of the interview. Near-moderate agreement preliminarily supported the use of trained coders who have not attempted reliability certification with appropriate caveats. While moderate agreement was not achieved for novice raters, findings suggest that self-paced training options for the CAI may hold future promise. These contributions erode a number of significant barriers to the current use of the CAI.

Myocardial Injury in Patients With Acute and Subacute Chagas Disease in the Brazilian Amazon Using Cardiovascular Magnetic Resonance.

Background Chagas disease is a neglected tropical disease that is still considered a global health emergency. In the Amazon region, most of the reports are of acute cases that are associated with oral transmission. This study aimed to evaluate myocardial injury in patients with acute Chagas disease before and after treatment. Methods and Results We evaluated 23 patients with acute Chagas disease in 3 different stages of progression. Group 1 had 12 patients evaluated during the acute phase, at the time of diagnosis, and 1 year after treatment, and Group 2 had 11 patients in the late postacute phase who were evaluated 5.2 years on average after diagnosis and treatment. ECGs with the Selvester score, 24-hour Holter exam, and cardiovascular magnetic resonance imaging were performed. The mean age of the 23 patients was 44.3±18.9 years, and they were mostly men (15/65.24%) from Amazonas state (22/95.6%). In 69.6% (n=16) of the patients, some ECG alterations were found, the most frequent being left anterior fascicular block and ventricular repolarization. In Group 1, the 24-hour Holter exam showed atrial tachycardia in 3 (25%) patients and ventricular extrasystoles in 2 (16.7%) patients. In Group 2, 1 patient had ventricular extrasystoles. Myocardial injury was observed in 7 patients (58.3%) at the acute phase and in 5 (50%) patients at the 1-year follow-up in Group 1 and in 2 (18.2%) patients in Group 2. Conclusions This article describes, for the first time, myocardial injury shown by cardiovascular magnetic resonance imaging in a group of patients with acute Chagas disease and reveals the importance of early detection and follow-up of the cardiac impairment in these patients.

Prevalence, Mechanism, Genetic Diversity, and Cross-Resistance Patterns of Methicillin-Resistant Staphylococcus Isolated from Companion Animal Clinical Samples Submitted to a Veterinary Diagnostic Laboratory in the Midwestern United States.

Methicillin-resistant Staphylococcus (MRS) is a leading cause of skin and soft tissue infections in companion animals, with limited treatment options available due to the frequent cross-resistance of MRS to other antibiotics. In this study, we report the prevalence, species distribution, genetic diversity, resistance mechanism and cross-resistance patterns of MRS isolated from companion animal (mostly dog and cat) clinical cases submitted to Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) between 2012 and 2019. The majority of isolates were identified as Staphylococcus pseudintermedius (68.3%; 2379/3482) and coagulase-negative Staphylococcus (CoNS) (24.6%; 857/3482), of which 23.9% and 40.5% were phenotypically resistant to methicillin, respectively. Cross resistance to other ß-lactams (and to a lesser extent to non-ß-lactams) was common in both methicillin-resistant S. pseudintermedius (MRSP) and CoNS (MRCoNS), especially when oxacillin MIC was ≥4 µg/mL (vs. ≥0.5−<4 µg/mL). The PBP2a protein was detected by agglutination in 94.6% (521/551) MRSP and 64.3% (146/227) MRCoNS. A further analysis of 31 PBP2a-negative MRS isolates (all but one MRCoNS) indicated that 11 were mecA gene-positive while 20 were negative for mecA and other mec genes by PCR. The resistance to last-resort anti-staphylococcal human drugs (e.g., tigecycline, linezolid, vancomycin) among the MRS tested was none to very low. Even though genotyping indicated an overall high level of genetic diversity (87 unique PFGE patterns and 20 MLST types) among a subset of MRSP isolates tested (n = 106), certain genotypes were detected from epidemiologically connected cases at the same or different time points, suggesting persistence and/or nosocomial transmission. These results indicate a relatively high prevalence of MRS from companion animals in the Midwestern US; therefore, it is important to perform routine susceptibility testing of Staphylococcus in veterinary clinical settings for the selection of appropriate antimicrobial therapy.

Clinical and molecular characterization of Costello syndrome in unrelated Mexican patients.

This study intends to describe for the first time a cohort of Mexican patients with Costello syndrome. The five exons of the HRAS gene were amplified in DNA samples from 13 patients with a clinical suspicion of Costello syndrome. PCR products were sequenced using the Ready Reaction Big Dye Terminator v.3.0 Kit and an ABI PRISM 310 sequencer. Only five patients (38%) showed causal variant in codon 12 of the HRAS gene (four with the p.Gly12Ser and one with the p.Gly12Ala variant). Three patients showed silent polymorphic variants (p.His27His and p.Leu159Leu). Clinical features in patients carrying the causal variant were variable. The alternative diagnosis of cardio-facio-cutaneous syndrome was considered in patients who did not have a causative variant in HRAS.

Acute micro-outbreak of Chagas disease in the southeastern Amazon: a report of five cases

ABSTRACT Background: Chagas disease is gaining importance in the Brazilian Amazon region as a differential diagnosis of febrile syndrome. The most recent microoutbreak occurred in Ipixuna, in Amazonas state. Methods: An epidemiological survey was conducted using parasitological and serological tests, and electrocardiographic analysis. Results: The patients belonged to one family and had ingested açaí acquired from Ipixuna. All patients reported fever and initially a thick blood smear test was done to identify Trypanosoma cruzi. Benznidazole treatment was administered to all patients. Conclusions: Knowledge of the epidemiological dynamics of Chagas disease allows us to improve control and management measures for this disease.

High anti-SARS-CoV-2 antibody seroconversion rates before the second wave in Manaus, Brazil, and the protective effect of social behaviour measures: results from the prospective DETECTCoV-19 cohort.

BACKGROUND: The city of Manaus, Brazil, has seen two collapses of the health system due to the COVID-19 pandemic. We report anti-SARS-CoV-2 nucleocapsid IgG antibody seroconversion rates and associated risk factors in Manaus residents before the second wave of the epidemic in Brazil. METHODS: A convenience sample of adult (aged ≥18 years) residents of Manaus was recruited through online and university website advertising into the DETECTCoV-19 study cohort. The current analysis of seroconversion included a subgroup of DETECTCoV-19 participants who had at least two serum sample collections separated by at least 4 weeks between Aug 19 and Oct 2, 2020 (visit 1), and Oct 19 and Nov 27, 2020 (visit 2). Those who reported (or had no data on) having a COVID-19 diagnosis before visit 1, and who were positive for anti-SARS-CoV-2 nucleocapsid IgG antibodies at visit 1 were excluded. Using an in-house ELISA, the reactivity index (RI; calculated as the optical density ratio of the sample to the negative control) for serum anti-SARS-CoV-2 nucleocapsid IgG antibodies was measured at both visits. We calculated the incidence of seroconversion (defined as RI values ≤1·5 at visit 1 and ≥1·5 at visit 2, and a ratio >2 between the visit 2 and visit 1 RI values) during the study period, as well as incidence rate ratios (IRRs) through cluster-corrected and adjusted Poisson regression models to analyse associations between seroconversion and variables related to sociodemographic characteristics, health access, comorbidities, COVID-19 exposure, protective behaviours, and symptoms. FINDINGS: 2496 DETECTCoV-19 cohort participants returned for a follow-up visit between Oct 19 and Nov 27, 2020, of whom 204 reported having COVID-19 before the first visit and 24 had no data regarding previous disease status. 559 participants were seropositive for anti-SARS-CoV-2 nucleocapsid IgG antibodies at baseline. Of the remaining 1709 participants who were seronegative at baseline, 71 did not meet the criteria for seroconversion and were excluded from the analyses. Among the remaining 1638 participants who were seronegative at baseline, 214 showed seroconversion at visit 2. The seroconversion incidence was 13·06% (95% CI 11·52-14·79) overall and 6·78% (5·61-8·10) for symptomatic seroconversion, over a median follow-up period of 57 days (IQR 54-61). 48·1% of seroconversion events were estimated to be asymptomatic. The sample had higher proportions of affluent and higher-educated people than those reported for the Manaus city population. In the fully adjusted and corrected model, risk factors for seroconversion before visit 2 were having a COVID-19 case in the household (IRR 1·49 [95% CI 1·21-1·83]), not wearing a mask during contact with a person with COVID-19 (1·25 [1·09-1·45]), relaxation of physical distancing (1·31 [1·05-1·64]), and having flu-like symptoms (1·79 [1·23-2·59]) or a COVID-19 diagnosis (3·57 [2·27-5·63]) between the first and second visits, whereas working remotely was associated with lower incidence (0·74 [0·56-0·97]). INTERPRETATION: An intense infection transmission period preceded the second wave of COVID-19 in Manaus. Several modifiable behaviours increased the risk of seroconversion, including non-compliance with non-pharmaceutical interventions measures such as not wearing a mask during contact, relaxation of protective measures, and non-remote working. Increased testing in high-transmission areas is needed to provide timely information about ongoing transmission and aid appropriate implementation of transmission mitigation measures. FUNDING: Ministry of Education, Brazil; Fundação de Amparo à Pesquisa do Estado do Amazonas; Pan American Health Organization (PAHO)/WHO.