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The Ocular Surface Jun 2024Human donor corneas are an essential control tissue for corneal research. We utilized whole mount immunofluorescence (WM-IF) to evaluate how the storage affects the...
PURPOSE
Human donor corneas are an essential control tissue for corneal research. We utilized whole mount immunofluorescence (WM-IF) to evaluate how the storage affects the tissue integrity and putative limbal stem cells in human and porcine corneas. Moreover, we compare this information with the marker expression patterns observed in human pluripotent stem cell (hPSC)-derived LSCs.
METHODS
The expression of putative LSC markers was analyzed with WM-IF and the fluorescence intensity was quantified in human donor corneas stored for 1-30 days, and in porcine corneas processed 0-6 hours after euthanasia. The results were compared with the staining of human and porcine corneal cryosections and with both primary and hPSC-derived LSC cultures.
RESULTS
WM-IF analyses emerged as a more effective method when compared to tissue sections for visualizing the expression of LSC markers within human and porcine corneas. Storage duration was a significant factor influencing the expression of LSC markers, as human tissues stored longer exhibited notable epithelial degeneration and lack of LSC markers. Porcine corneas replicated the expression patterns observed in fresh human tissue. We validated the diverse expression patterns of PAX6 in the limbal-corneal region, which aligned with findings from hPSC-LSC differentiation experiments.
CONCLUSIONS
WM-IF coupled with quantification of fluorescence intensities proved to be a valuable tool for investigating LSC marker expression in both human and porcine tissues ex vivo. Prolonged storage significantly influences the expression of LSC markers, underscoring the importance of fresh human or substitute control tissue when studying limbal stem cell biology.
PubMed: 38945477
DOI: 10.1016/j.jtos.2024.06.004 -
Journal of Theoretical Biology Jun 2024Across early childhood development, sleep behavior transitions from a biphasic pattern (a daytime nap and nighttime sleep) to a monophasic pattern (only nighttime...
Across early childhood development, sleep behavior transitions from a biphasic pattern (a daytime nap and nighttime sleep) to a monophasic pattern (only nighttime sleep). The transition to consolidated nighttime sleep, which occurs in most children between 2- and 5-years-old, is a major developmental milestone and reflects interactions between the developing homeostatic sleep drive and circadian system. Using a physiologically-based mathematical model of the sleep-wake regulatory network constrained by observational and experimental data from preschool-aged participants, we analyze how developmentally-mediated changes in the homeostatic sleep drive may contribute to the transition from napping to non-napping sleep patterns. We establish baseline behavior by identifying parameter sets that model typical 2-year-old napping behavior and 5-year-old non-napping behavior. Then we vary six model parameters associated with the dynamics of and sensitivity to the homeostatic sleep drive between the 2-year-old and 5-year-old parameter values to induce the transition from biphasic to monophasic sleep. We analyze the individual contributions of these parameters to sleep patterning by independently varying their age-dependent developmental trajectories. Parameters vary according to distinct evolution curves and produce bifurcation sequences representing various ages of transition onset, transition durations, and transitional sleep patterns. Finally, we consider the ability of napping and non-napping light schedules to reinforce napping or promote a transition to consolidated sleep, respectively. These modeling results provide insight into the role of the homeostatic sleep drive in promoting interindividual variability in developmentally-mediated transitions in sleep behavior and lay foundations for the identification of light- or behavior-based interventions that promote healthy sleep consolidation in early childhood.
PubMed: 38945471
DOI: 10.1016/j.jtbi.2024.111892 -
The Journal of Biological Chemistry Jun 2024The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness...
The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRp) of prototypic respiratory viruses. GS-646939 is the active 5'-triphosphate (TP) metabolite of a 4'-cyano modified C-adenosine analog phosphoramidate prodrug GS-7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 (EV-71) incorporate GS-646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase (h-mtRNAP) does not show significant incorporation of GS-646939. Once incorporated into the nascent RNA strand, GS-646939 acts as a chain-terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4'-modification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 1'-cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1'-cyano and 4'-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses.
PubMed: 38945449
DOI: 10.1016/j.jbc.2024.107514 -
The Journal of Pediatrics Jun 2024To demonstrate a high-yield molecular diagnostic workflow for lateralized overgrowth (LO), a congenital condition with abnormal enlargement of body parts, and to...
OBJECTIVE
To demonstrate a high-yield molecular diagnostic workflow for lateralized overgrowth (LO), a congenital condition with abnormal enlargement of body parts, and to classify it by molecular genetics. and STUDY DESIGN: We categorized 186 retrospective cases of LO diagnosed between 2003 and 2023 into suspected Beckwith-Wiedemann spectrum (BWSp), PIK3CA-Related Overgrowth Spectrum (PROS), vascular overgrowth (VO) , or isolated (ILO), based on initial clinical assessments, to determine the appropriate first-tier molecular tests and tissue for analysis. Patients underwent testing for 11p15 epigenetic abnormalities or somatic variants in genes related to PI3K/AKT/mTOR, vascular proliferation, and RAS-MAPK cascades using blood or skin DNA. For cases with negative initial tests, a sequential cascade molecular approach was employed to improve diagnostic yield.
RESULTS
This approach led to a molecular diagnosis in 54% of cases, 89% of cases consistent with initial clinical suspicions and 11% reclassified. BWSp was the most common cause, with 43% of cases exhibiting 11p15 abnormalities. PROS had the highest confirmation rate, with 74% of clinically diagnosed patients showing a PIK3CA variant. VO demonstrated significant clinical overlap with other syndromes. Molecular diagnosis of ILO proved challenging, with only 21% of cases classifiable into a specific condition.
CONCLUSION
Despite, LO is underdiagnosed from a molecular viewpoint and to date has had no diagnostic guidelines, which would be crucial for addressing potential cancer predisposition, enabling precision medicine treatments, or guiding management. This study sheds light on the molecular etiology of LO, highlighting the importance of tailored diagnostic approach and of selecting appropriate testing to achieve the highest diagnostic yield.
PubMed: 38945442
DOI: 10.1016/j.jpeds.2024.114177 -
International Journal of Infectious... Jun 2024The emergence of new SARS-CoV-2 variants has led to the development of Omicron-targeting bivalent mRNA vaccines. It is crucial to understand how bivalent vaccines may...
OBJECTIVES
The emergence of new SARS-CoV-2 variants has led to the development of Omicron-targeting bivalent mRNA vaccines. It is crucial to understand how bivalent vaccines may improve antibody responses against new variants.
METHODS
A total of 107 participants, who had three COVID-19 WT mRNA vaccine doses, were recruited, and given either a monovalent (WT) or a bivalent mRNA vaccination (Pfizer/BioNTech Bivalent (WT and BA.4/BA.5) or Moderna Bivalent (WT and BA.1)). Blood samples were taken before booster and at 28 days post-booster.
RESULTS
We found significantly lower fold change in serum binding IgA responses against BA.1, BA.5 and EG.5.1 spike in the bivalent booster group, compared with the monovalent (WT) booster group, following vaccination. However, this was only observed in individuals with prior infection. The relative fold change in serum binding IgA response was more skewed towards WT over variant (BA.1, BA.5 or EG.5.1) spike in previously infected bivalent-booster-vaccinees, as compared with previously infected monovalent-(WT)-booster-vaccinees.
CONCLUSION
The findings suggest imprinting of antibody responses that is shaped by the first vaccination (WT spike). Previous infection also affects the boosting effect of follow-up vaccination. Studies are needed to understand how to induce a robust and long-lasting IgA immunity for protection against COVID-19 infection.
PubMed: 38945433
DOI: 10.1016/j.ijid.2024.107147 -
International Journal of Infectious... Jun 2024Hospitalised neonates are vulnerable to infection and have high rates of antibiotic utilisation.
Prospective antimicrobial stewardship interventions by multidisciplinary teams to reduce neonatal antibiotic use in South Africa: the Neonatal Antimicrobial Stewardship (NeoAMS) study.
BACKGROUND
Hospitalised neonates are vulnerable to infection and have high rates of antibiotic utilisation.
METHODS
Fourteen South African neonatal units (seven public, seven private sector) assembled multidisciplinary teams involving neonatologists, microbiologists, pharmacists, and nurses to implement prospective audit and feedback neonatal antimicrobial stewardship (NeoAMS) interventions. The teams attended seven online training sessions. Pharmacists conducted weekday antibiotic prescription reviews in the neonatal intensive care unit and/or neonatal wards providing feedback to the clinical teams. Anonymised demographic and NeoAMS interventions data were aggregated for descriptive purposes and statistical analysis.
FINDINGS
During the 20-week NeoAMS intervention in 2022, 565 neonates were enrolled. Pharmacists evaluated seven hundred antibiotic prescription episodes; rule-out sepsis (180; 26%) and culture-negative sepsis (138; 20%) were the most frequent indications for antibiotic prescription. For infection episodes with an identified pathogen, only 51% (116/229) of empiric treatments provided adequate antimicrobial coverage. Pharmacists recommended 437 NeoAMS interventions (0·6 per antibiotic prescription episode), with antibiotic discontinuation (42%), therapeutic drug monitoring (17%), and dosing (15%) recommendations most frequent. Neonatal clinicians' acceptance rates for AMS recommendations were high (338; 77%). Mean antibiotic length of therapy decreased by 24% from 9·1 to 6·9 days (0·1 day decrease per intervention week; p=0·001), with the greatest decline in length of therapy for culture-negative sepsis (8·2 days (95%CI 5·7-11·7) to 5·9 days (95% CI 4·6-7·5); p=0·032).
INTERPRETATION
This neonatal AMS programme was successfully implemented in heterogenous and resource-limited settings. Pharmacist-recommended AMS interventions had high rates of clinician acceptance. The NeoAMS intervention significantly reduced neonatal antibiotic use, particularly for culture-negative sepsis.
FUNDING
A grant from Merck provided partial support.
PubMed: 38945432
DOI: 10.1016/j.ijid.2024.107158 -
International Journal of Infectious... Jun 2024Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory infection (ALRI) in young children. With substantial advances in RSV research, we aimed...
OBJECTIVES
Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory infection (ALRI) in young children. With substantial advances in RSV research, we aimed to conduct an updated systematic review of risk factors for RSV-ALRI in children under five years.
METHODS
We updated our previously published literature search to November 2022 among three English databases and additionally searched three Chinese databases (from January 1995) to identify all relevant publications. We performed random-effects meta-analyses to estimate the pooled odds ratio and 95% confidence interval (CI) for each risk factor and each outcome (RSV-ALRI in the community and RSV-ALRI hospitalisation).
RESULTS
A total of 47 studies were included (26 from the updated search). Indoor air pollution was identified as a possible risk factor for RSV-ALRI in the community (OR 1.45, 95% CI: 1.10-1.90). The identified risk factors for RSV-ALRI hospitalisation fall into four categories: demographic (male sex, Māori and Pacific ethnicities vs European or other ethnicities), pre- and post- neonatal (prematurity, low birth weight, small for gestational age, maternal smoking during pregnancy or lactation, maternal age <30 years vs 30-34 years, multiparity, caesarean section vs vaginal), household and environmental (having siblings, passive smoking, maternal asthma, daycare centre attendance), and health and medical conditions (any chronic diseases, bronchopulmonary dysplasia, HIV infections, congenital heart disease, Down syndrome, cystic fibrosis, previous asthma). The pooled ORs ranged from 1.14 to 4.55.
CONCLUSIONS
Our findings on the risk factors for RSV-ALRI help identify RSV high-risk groups, which has important implications for RSV prevention at both individual and population levels.
PubMed: 38945430
DOI: 10.1016/j.ijid.2024.107125 -
Biological Psychiatry Jun 2024Diverse antidepressants were recently described to bind to TrkB and drive a positive allosteric modulation of endogenous BDNF. Although neurotrophins such as BDNF can...
BACKGROUND
Diverse antidepressants were recently described to bind to TrkB and drive a positive allosteric modulation of endogenous BDNF. Although neurotrophins such as BDNF can bind to the p75 neurotrophin receptor (p75NTR), their precursors are the high affinity p75NTR ligands. While part of an unrelated receptor family capable of inducing completely opposite physiological changes, TrkB and p75NTR feature a cross-like conformation dimer and carry a cholesterol-recognition and alignment consensus in the transmembrane domain. Since such qualities were found crucial for antidepressants to bind to TrkB and drive behavioral and neuroplasticity effects, we hypothesized that their effects might also depend on p75NTR.
METHODS
ELISA-based binding assay and NMR spectroscopy were accomplished to assess whether antidepressants would bind to p75NTR. HEK293T cells and a variety of in vitro assays were used to address whether fluoxetine (FLX) or ketamine (KET) would trigger any α- and γ-secretase-dependent p75NTR proteolysis, and lead to p75NTR nuclear localization. Ocular dominance shift was performed with male and female p75KO mice to study the effects of KET and FLX on brain plasticity, in addition to pharmacological interventions to verifying how p75NTR signaling is important for the effects of KET and FLX in enhancing extinction memory in male WT mice and rats.
RESULTS
Antidepressants were found binding to p75NTR, FLX and KET triggered the p75NTR proteolytic pathway and induced p75NTR-dependent behavioral/neuroplasticity changes.
CONCLUSION
We thus hypothesize that antidepressants co-opt both BDNF/TrkB and proBDNF/p75NTR systems to induce a more efficient activity-dependent synaptic competition, thereby boosting the brain ability for remodeling.
PubMed: 38945387
DOI: 10.1016/j.biopsych.2024.06.021 -
The Journal of Pain Jun 2024The human brain is a dynamic system that shows frequency-specific features. Neuroimaging studies have shown that both healthy individuals and those with chronic pain...
The human brain is a dynamic system that shows frequency-specific features. Neuroimaging studies have shown that both healthy individuals and those with chronic pain disorders experience pain influenced by various processes that fluctuate over time. Primary dysmenorrhea is a chronic visceral pain that disrupts the coordinated activity of brain's functional network. However, it remains unclear whether the dynamic interactions across the whole-brain network over time and their associations with neurobehavioral symptoms are dependent on the frequency bands in patients with primary dysmenorrhea during the pain-free periovulation phase. In this study, we used an energy landscape analysis to examine the interactions over time across the large-scale network in a sample of 59 patients with primary dysmenorrhea and 57 healthy controls at different frequency bands. Compared to healthy controls, patients with primary dysmenorrhea exhibit aberrant brain dynamics, with more significant differences in the slow-4 frequency band. Patients with primary dysmenorrhea show more indirect neural transition times due to an unstable intermediate state, whereas neurotypical brain activity frequently transitions between two major states. This data-driven approach further revealed that the brains of individuals with primary dysmenorrhea have more abnormal brain dynamics than healthy controls. Our results suggested that unstable brain dynamics were associated with the strength of brain functional segregation and the Pain Catastrophizing Scale (PCS) score. Our findings provide preliminary evidence that atypical dynamics in the functional network may serve as a potential key feature and biological marker of patients with PDM during the pain-free phase. PERSPECTIVE: We applied energy landscape analysis on brain-imaging data to identify relatively stable and dominant brain activity patterns for patients with primary dysmenorrhea(PDM). More atypical brain dynamics were found in the slow-4 band and were related to the strength of functional segregation, providing new insights into the dysfunction brain dynamics.
PubMed: 38945381
DOI: 10.1016/j.jpain.2024.104618 -
Human Pathology Jun 2024MLH1 promoter hypermethylation (MPH) analysis is an essential step in the universal tumor testing algorithm for Lynch syndrome, the most common inherited predisposition...
MLH1 promoter hypermethylation (MPH) analysis is an essential step in the universal tumor testing algorithm for Lynch syndrome, the most common inherited predisposition to colorectal cancer (CRC). MPH usually indicates sporadic CRC. EPM2AIP1 gene shares the same promoter as MLH1, therefore MPH should also silence EPM2AIP1 transcription leading to loss of protein expression on immunohistochemistry (IHC). It has been previously reported that EPM2AIP1 IHC can be used as a surrogate for MPH in endometrial cancer. Our goal was to evaluate the feasibility of EPM2AIP1 IHC as a surrogate for MPH in CRC. 101 microsatellite instable CRC cases were selected, including 19 cases from whole tumor sections and 82 cases from tissue microarrays. 74 cases were with MPH and 27 without MPH. All 74 cases with MPH showed absent MLH1 by IHC, but only 47 (64%) exhibited loss of expression of EPM2AIP1. Of the 27 cases without MPH, 9 (33%) cases had unexpected loss of EPM2AIP1 expression. Of note, 10 cases were MLH1-mutated Lynch syndrome without MPH, 2 cases showed unexpected loss of EPM2AIP1 staining. Of the 6 cases with double somatic mutations of MLH1 gene (without MPH), only 4 cases demonstrated intact expression of EPM2AIP1 as expected. Taken together, EPM2AIP1 loss was 64% sensitive and 67% specific for MPH, with an accuracy of 64%. We conclude that, unless stain quality improves with different clones or platforms, EPM2AIP1 IHC will likely not be useful as a surrogate test for MPH in CRC.
PubMed: 38945374
DOI: 10.1016/j.humpath.2024.06.017