Investigating the Impact of Community Volunteerism on the Mental Health of Medical Students.

OBJECTIVE: This study aimed to analyze the impact of community service on the mental health of medical students through their perception of stress. METHODS: The 10-item Perceived Stress Scale was used to measure the stress levels of 82 medical students over a 3-month period. Additional survey questions gauged students' weekly volunteer experiences in clinical and nonclinical settings and their perceived effects on stress and quality of life. RESULTS: Results found an inverse relationship between the number of clinical volunteer hours and perceived stress (P = .0497). Nonclinical and total volunteer hours were correlated with both reduced perceived stress levels (nonclinical P = .0095, total P = .0052) and better quality of life (nonclinical P = .0301, total P = .0136). All individual perceived stress scores fell into the low or moderate stress ranges of the Perceived Stress Scale per the week-to-week analysis. CONCLUSION: The preliminary results raised important research questions about the impact of volunteering on medical student perceived stress. As medical students face higher levels of stress in comparison to the general population, it is exceedingly important to determine methods to decrease their risk of compromising their mental health. This study may aid in decision-making and research in favor of or against offering community service opportunities as part of the core medical education curriculum.

Effect of Single-Residue Mutations on CTCF Binding to DNA: Insights from Molecular Dynamics Simulations.

In humans and other eukaryotes, DNA is condensed into chromatin fibers that are further wound into chromosomes. This organization allows regulatory elements in the genome, often distant from each other in the linear DNA, to interact and facilitate gene expression through regions known as topologically associating domains (TADs). CCCTC-binding factor (CTCF) is one of the major components of TAD formation and is responsible for recruiting a partner protein, cohesin, to perform loop extrusion and facilitate proper gene expression within TADs. Because single-residue CTCF mutations have been linked to the development of a variety of cancers in humans, we aim to better understand how these mutations affect the CTCF structure and its interaction with DNA. To this end, we compare all-atom molecular dynamics simulations of a wildtype CTCF-DNA complex to those of eight different cancer-linked CTCF mutant sequences. We find that most mutants have lower binding energies compared to the wildtype protein, leading to the formation of less stable complexes. Depending on the type and position of the mutation, this loss of stability can be attributed to major changes in the electrostatic potential, loss of hydrogen bonds between the CTCF and DNA, and/or destabilization of specific zinc fingers. Interestingly, certain mutations in specific fingers can affect the interaction with the DNA of other fingers, explaining why mere single mutations can impair CTCF function. Overall, these results shed mechanistic insights into experimental observations and further underscore CTCF's importance in the regulation of chromatin architecture and gene expression.

Overcoming Poor Transgene Expression in the Wild-Type Chlamydomonas Chloroplast: Creation of Highly Mosquitocidal Strains of Chlamydomonas reinhardtii.

High-level expression of transgenes in the chloroplast of wild-type Chlamydomonas reinhardtii (C. reinhardtii) remains challenging for many genes (e.g., the cry toxin genes from Bacillus thuringiensis israelensis). The bottleneck is presumed to be post-transcriptional and mediated by the 5' element and the coding region. Using 5' elements from highly expressed photosynthesis genes such as atpA did not improve the outcome with cry11A regardless of the promoter. However, when we employed the 5' UTR from mature rps4 mRNA with clean fusions to promoters, production of the rCry11A protein became largely promoter-dependent. The best results were obtained with the native 16S rrn promoter (−91 to −1). When it was fused to the mature 5' rps4 UTR, rCry11A protein levels were ~50% higher than was obtained with the inducible system, or ~0.6% of total protein. This level was sufficient to visualize the 73-kDa rCry11A protein on Coomassie-stained gels of total algal protein. In addition, analysis of the expression of these transgenes by RT-PCR indicated that RNA levels roughly correlated with protein production. Live cell bioassays using the best strains as food for 3rd instar Aedes aegypti larvae showed that most larvae were killed even when the cell concentration was as low as 2 × 104 cells/mL. Finally, the results indicate that these highly toxic strains are also quite stable, and thus represent a key milestone in using C. reinhardtii for mosquito control.

Affordable IgY-based antiviral prophylaxis for resource-limited settings to address epidemic and pandemic risks.

Background: The COVID-19 pandemic caused by SARS-CoV-2 exposed a global problem, as highly effective vaccines are challenging to produce and distribute, particularly in regions with limited resources and funding. As an alternative, immunoglobulins produced in eggs of immunized hens (IgY) can be a simple and inexpensive source for a topical and temporary prophylaxis. Here, we developed a method to extract and purify IgY antibodies from egg yolks of hens immunized against viral pathogen-derived proteins using low-cost, readily available materials, for use in resource-limited settings. Methods: Existing protocols for IgY purification and equipment were modified, including extraction from yolks and separation of water-soluble IgY using common household reagents and tools. A replacement for a commercial centrifuge was developed, using a home food processor equipped with a 3D printed adapter to enable IgY precipitation. IgY purification was verified using standard gel electrophoresis and Western blot analyses. Results: We developed a step-by-step protocol for IgY purification for two settings in low- and middle-income countries (LMIC): a local laboratory, where commercial centrifuges are available, or a more rural setting, where an alternative for expensive centrifuges can be used. Gel electrophoresis and Western blot analyses confirmed that the method produced highly enriched IgY preparation; each commercial egg produced ~ 90 mg of IgY. We also designed a kit for IgY production in these two settings and provided a cost estimate of the kit. Conclusion: IgY purified from eggs of immunized local hens can offer a fast and affordable prophylaxis, provided that purification can be performed in a resource-limited setting. Here, we created a low-cost method that can be used anywhere where electricity is available using inexpensive, readily available materials in place of costly, specialized laboratory equipment and chemicals. This procedure can readily be used now to make an anti-SARS-CoV-2 prophylaxis in areas where vaccines are unavailable, and can be modified to combat future threats from viral epidemics and pandemics.

Hypoxia-induced macropinocytosis represents a metabolic route for liver cancer.

Hepatocellular carcinoma (HCC) invariably exhibits inadequate O2 (hypoxia) and nutrient supply. Hypoxia-inducible factor (HIF) mediates cascades of molecular events that enable cancer cells to adapt and propagate. Macropinocytosis is an endocytic process initiated by membrane ruffling, causing the engulfment of extracellular fluids (proteins), protein digestion and subsequent incorporation into the biomass. We show that macropinocytosis occurs universally in HCC under hypoxia. HIF-1 activates the transcription of a membrane ruffling protein, EH domain-containing protein 2 (EHD2), to initiate macropinocytosis. Knockout of HIF-1 or EHD2 represses hypoxia-induced macropinocytosis and prevents hypoxic HCC cells from scavenging protein that support cell growth. Germline or somatic deletion of Ehd2 suppresses macropinocytosis and HCC development in mice. Intriguingly, EHD2 is overexpressed in HCC. Consistently, HIF-1 or macropinocytosis inhibitor suppresses macropinocytosis and HCC development. Thus, we show that hypoxia induces macropinocytosis through the HIF/EHD2 pathway in HCC cells, harnessing extracellular protein as a nutrient to survive.

Prefrontal cortex PACAP signaling: organization and role in stress regulation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neuromodulatory peptide strongly implicated in nervous stress processing. Human polymorphism of the primary PACAP receptor (PAC1) is linked to psychiatric disorders, including posttraumatic stress disorder (PTSD). Prefrontal cortex PACAP signaling is associated with processing of traumatic stress and fear learning, suggesting a potential role in PTSD-related deficits. We used RNAscope to define the cellular location of PACAP and PAC1 in the infralimbic cortex (IL). Subsequent experiments used a pharmacological approach to assess the impact of IL PACAP infusion on behavioral and physiological stress response and fear memory. Adult male Sprague-Dawley rats were bilaterally microinjected with PACAP (1 ug) or vehicle into the IL, 30 minutes prior to forced swim test (FST). Blood was sampled at 15, 30, 60, and 120 minutes for analysis of hypothalamic pituitary adrenal (HPA) axis reactivity. Five days after, animals were tested in a 3-day passive avoidance paradigm with subsequent testing of fear retention two weeks later. We observed that PACAP is highly expressed in putative pyramidal neurons (identified by VGlut1 expression), while PAC1 is enriched in interneurons (identified by GAD). Pretreatment with PACAP increased active coping style in the FST, despite higher levels of ACTH and corticosterone. The treatment was also sufficient to cause an increase in anxiety-like behavior in a dark/light crossover test and enhanced retention of passive avoidance. Our data suggest that IL PACAP plays a role in driving stress responses and in processing of fear memories, likely mediated by inhibition of cortical drive.

Long-term impact of chronic variable stress in adolescence versus adulthood.

Adolescence is a period of active development of stress regulatory neurocircuitry. As a consequence, mechanisms that control the responses to stress are not fully matured during this developmental period, which may result in vulnerability to chronic stress. We hypothesized that adolescent chronic stress would have negative consequences on stress adaptation later in life. Male Wistar rats (PND40) were subjected to chronic variable stress (CVS) for 2 weeks, with 2 daily stressors randomly presented and overnight social stressors twice a week. After five weeks, animals were evaluated during adulthood, using the elevated plus maze (EPM) and the forced swim test (FST). The hypothalamic-pituitary adrenal (HPA) axis response to a 30-min restraint was also assessed. Results are compared to those of adult rats tested 5 weeks following CVS cessation. Our results demonstrate that the long-term effects of CVS are specific to the age of application of the stress regime. We show how behavior and HPA axis response as well as hypothalamic paraventricular nucleus activation can differ with age, resulting in differential behavioral adaptations for animals stressed in adolescence and dysregulation of the HPA axis in the animals stressed in adulthood, These data underscore the importance of the adolescent period in determining resilience of the HPA axis and programming behavioral responses later in life.