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The violence of incarceration creates greater responsibility for higher education administrators in supporting students who are in prison. Using focus group data with incarcerated students and formerly incarcerated alumni who participated in or are actively participating in Second Chance Pell, we explore their perceptions and understandings of the Pell Grant and eligibility for the Pell Grant, including lifetime eligibility used limits. Through a lens of Witnessing, we argue that deprivation and censorship of information negatively influence students' access to accurate and timely information about federal student aid and their ability to fully participate in the process. Accordingly, college and university staff have a fiduciary duty to incarcerated applicants and students who are subjected to the information deprivation and censorship that characterizes imprisonment in the U.S.

Abstract Osteosarcoma (OS) is the most frequent primary malignant bone tumour, whose heterogeneity represents a major challenge for common antitumour therapies. Inflammatory cytokines are known to be necessary for OS progression. Therefore, to optimise therapy, it is important to discover reliable biomarkers by identifying the mechanism generating OS and investigating the inflammatory pathways that support the undifferentiated state. In this work, we highlight the differences of epigenetic activities of IL-1β and TNFα, and the susceptibility of TET-1 enzymatic inhibition, in tumour progression of three different OS cell lines. Investigating DNA methylation of IL-6 promoter and determining its expression, we found that TET enzymatic inhibition influences proliferation induced by inflammatory cytokines in OS cell lines. Moreover, Bobcat 339 treatment blocks IL-1β epigenetic action on IL-6 promoter, while only partially those of TNFα as well as inhibits IL-1β-dependent epithelial–mesenchymal transition (EMT) process, but only partially those of TNFα. In conclusion, this work highlights that IL-1β and TNFα have different effects on DNA demethylation in OS cell lines, making DNA methylation a potential biomarker of disease. Specifically, in IL-1β treatment, TET-1 inhibition completely blocks tumour progression, while in TNFα actions, it is only partially effective. Given that these two inflammatory pathways can be therapeutic targets for treating these tumours, knowledge of their distinct epigenetic behaviours can be useful for developing precise and specific therapeutic strategies for this disease.

In implantology, among the key choices, to obtain predictable results, it is essential to establish, using cone beam computed tomography (CBCT), the bone site and where to insert the implants; during the surgical phase, these sites must be identified on the oral mucosa. Surgical templates are a valid aid, especially in complex cases which require the insertion of more than three or four implants. In cases of a single implant, on the other hand, surgical guides are rarely used, and the implant is often inserted freehand; therefore, the identification of the implant site on the oral mucosa (after choosing the location on the CBCT) is more difficult. For this reason, the clinician uses the teeth in the arch as a reference. This study evaluates the ability of the human eye to identify, on the oral mucosa, where the implant collars will be positioned, the position of which has previously been chosen on the CBCT, in cases where the hands-free surgical technique (without surgical guides) is used. The verification of this precision is carried out using particular thermo-printed templates which contain radiopaque metal spheres. The results show that, in the freehand technique, it is difficult to precisely identify the implant sites (chosen via X-ray) on the mucosa, especially when they are far from natural teeth adjacent to the edentulous area. In case of monoedentulism, the freehand implant technique seems to be applicable by expert implantologists with a reduced risk of error; in fact, clinical experience helps to find the correct correspondence between the implant site chosen on the CBCT and its identification on the mucosa. The level of experience is fundamental in the clinician’s decision about whether or not to use surgical guides; in fact, doctors with little experience should use surgical guides even in the simplest cases to reduce the risk of error.

Abstract Many plants have evolved nutrient rewards to attract pollinators to flowers, but most research has focused on the sugar content of floral nectar resources. Concentrations of sodium in floral nectar (a micronutrient in low concentrations in nectar) can vary substantially both among and within co‐occurring species. It is hypothesized that sodium concentrations in floral nectar might play an important and underappreciated role in plant–pollinator interactions, especially because many animals, including pollinators, are sodium limited in nature. Yet, the consequences of variation in sodium concentrations in floral nectar remain largely unexplored. Here, we investigate whether enriching floral nectar with sodium influences the composition, diversity, and frequency of plant–pollinator interactions. We experimentally enriched sodium concentrations in four plant species in a subalpine meadow in Colorado, USA. We found that flowers with sodium‐enriched nectar received more visits from a greater diversity of pollinators throughout the season. Different pollinator species foraged more frequently on flowers enriched with sodium and showed evidence of other changes to foraging behavior, including greater dietary evenness. These findings are consistent with the “salty nectar hypothesis,” providing evidence for the importance of sodium limitation in pollinators and suggesting that even small nectar constituents can shape plant–pollinator interactions.

Patient-reported outcomes (PROs) are essential for understanding the effects of MS and its treatments on patients’ lives; they play an important role in multiple sclerosis (MS) research and practice. We present the protocol for an observational study to prospectively assess the effect of cladribine tablets on PROs and their correlation to disability and physical activity in adults with highly active relapsing MS switching from a first disease modifying drug (DMD) to cladribine tablets in routine clinical practice at study sites in Italy. The primary objective will be to evaluate changes from baseline in the impact of highly active MS on self-assessed physical functioning 52 weeks after the switch to cladribine tablets using the Multiple Sclerosis Impact Scale-29 (MSIS-29). Secondary objectives will include self-assessed psychological impact of highly active MS in daily life and general health after the switch to cladribine tablets as well as changes in cognitive function, anxiety, and depression symptoms. Additional PRO measures will include the Hospital Anxiety and Depression Scale (HADS), the EuroQoL 5-Dimension 5-Level (EQ-5D-5L), the Work Productivity and Activity Impairment Questionnaire: Multiple Sclerosis (WPAI:MS), and the Patient-Reported Outcomes Measurement Information System (PROMIS). Wearable devices will acquire activity data (step counts, walking speed, time asleep, and energy expenditure). Additional clinical, radiological, and laboratory data will be collected when available during routine management. The findings will complement data from controlled trials by providing insight from daily clinical practice into the effect of cladribine tablets on the patient’s experience and self-assessed impact of treatment on daily life.

Plant-derived nanovesicles represent a novel approach in the field of plant-derived biomaterials, offering a sustainable and biocompatible option for various biomedical applications. The unique properties of these vesicles, such as their ability to encapsulate bioactive compounds, make them suitable for therapeutic, cosmetic, and nutraceutical purposes. In this study, we have, for the first time, successfully bio-fabricated vesicles derived from Opuntia ficus-indica (FicoVes) using an efficient and cost-effective method. Characterized by a size of approximately of 114 nm and a negative zeta potential of −20.9 mV, FicoVes exhibited excellent biocompatibility and hemocompatibility, showing no reduction in the viability of human and animal cells. Our results showed that FicoVes possess significant antioxidant properties as they reduced ROS generation in TBH-stimulated cells. FicoVes displayed anti-inflammatory properties by reducing the expression of pro-inflammatory cytokines (Il 1β, TNF α) and enhancing the expression of anti-inflammatory cytokines (IL4, IL10) following an inflammatory stimulus. Furthermore, FicoVes accelerated epithelial wound closure in L929 fibroblast monolayers in a dose-dependent manner, highlighting their potential role in tissue repair. This study establishes FicoVes as a promising candidate for nutrigenomic applications, particularly in the context of inflammation-related disorders and wound healing. Further research, including in vivo studies, is essential to validate these findings and fully explore their therapeutic potential.

Starting in 2026 Washington State building performance standards will come into effect that require commercial buildings larger than 50,000 sf to meet site energy use intensity targets. To support a state incentive program designed to encourage building owners to start complyingearly, we characterized the building stock energy use of 11 building types, analyzed 43 energy upgrade measures, and developed seven packages of energy upgrade measures using the ComStock energy analysis tool. Each energy upgrade package included from 4 to 17 energymeasures consisting of lighting, HVAC, and envelope upgrades. Package savings were calculated for four priority building types using a sample of 35,000 buildings characterized by four building area bins, three climate zones, and two county types (urban and rural). Of the 28package and building type combinations analyzed, 17 (61%) met or exceeded program energy savings targets and two packages met targets for all four building types.

Trimethyl-N-oxide (TMAO) has been linked to peripheral artery disease (PAD). TaurisoloⓇ is a natural, balanced phytocomplex containing resveratrol, quercetin, catechins, procianidins, gallic acid, and caffeic acid. Numerous studies have shown that TaurisoloⓇ reduces the damage of TMAO and exerts a protective effect on endothelial cells (ECs). The aim of this randomized, double-blind, single-center study was to evaluate the effects of TaurisoloⓇ on claudication in patients with PAD (Rutheford grade I, category II, Fontaine Classification: Stage IIA, American Medical Association Whole Person Impairment Classification: Class 0—WPI 0%) in two parallel groups of 31 patients. The primary outcomes were an increase in the pain-free walking distance and the ankle/brachial pressure index at the beginning and at the end of the treatment with Taurisolo. The secondary endpoint was the serum TMAO changes. The claudication distance improved by 14.1% in the Taurisolo group and by 2.0% in the placebo group, while the maximal distance increased by 15.8% and 0.6% only, respectively (both p < 0.05). The TMAO plasma levels decreased from 3.97 ± 2.13 micromole/L to 0.87 ± 0.48 (p < 0.0001) in the treated group. All these changes were highly significant both in univariate mixed models as well as in the adjusted model. Ultimately, TaurisoloⓇ might be an effective intervention to ameliorate intermittent claudication.

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, not always accessible and poorly compatible with many antigens. Here, we describe 'autonomous hypermutation yeast surface display' (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. By encoding antibody fragments on an error-prone orthogonal DNA replication system, surface-displayed antibody repertoires continuously mutate through simple cycles of yeast culturing and enrichment for antigen binding to produce high-affinity clones in as little as two weeks. We applied AHEAD to generate potent nanobodies against the SARS-CoV-2 S glycoprotein, a G-protein-coupled receptor and other targets, offering a template for streamlined antibody generation at large.

Abstract Low affinity is common for germline B cell receptors (BCR) seeding development of broadly neutralizing antibodies (bnAbs) that engage hypervariable viruses, including HIV. Antibody affinity selection is also non-homogenizing, insuring the survival of low affinity B cell clones. To explore whether this provides a natural window for expanding human B cell lineages against conserved vaccine targets, we deploy transgenic mice mimicking human antibody diversity and somatic hypermutation (SHM) and immunize with simple monomeric HIV glycoprotein envelope immunogens. We report an immunization regimen that focuses B cell memory upon the conserved CD4 binding site (CD4bs) through both conventional affinity maturation and reproducible expansion of low affinity BCR clones with public patterns in SHM. In the latter instance, SHM facilitates target acquisition by decreasing binding strength. This suggests that permissive B cell selection enables the discovery of antibody epitopes, in this case an HIV bnAb site.

IntroductionGoiter is a common problem in clinical practice, representing a large part of clinical evaluations for thyroid disease. It tends to grow slowly and progressively over several years, eventually occupying the thoracic inlet with its lower portion, defining the situation known as retrosternal goiter. Total thyroidectomy is a standardized procedure that represents the treatment of choice for all retrosternal goiters, but when is performed for such disease, a higher risk of postoperative morbidity is variously reported in the literature. The aims of our study were to compare the perioperative and postoperative outcomes in patients with cervical goiters and retrosternal goiters undergoing total thyroidectomy.MethodsIn our retrospective, multicentric evaluation we included 4,467 patients, divided into two groups based on the presence of retrosternal goiter (group A) or the presence of a classical cervical goiter (group B).ResultsWe found statistically significant differences in terms of transient hypoparathyroidism (19.9% in group A vs. 9.4% in group B, p

Shigellosis is one of the leading causes of diarrheal disease in low- and middle-income countries, particularly in young children, and is more often associated with antimicrobial resistance. Therefore, a preventive vaccine against shigellosis is an urgent medical need. We have proposed Generalised Modules for Membrane Antigens (GMMA) as an innovative delivery system for Shigella sonnei O-antigen, and an Alhydrogel formulation (1790GAHB) has been extensively tested in preclinical and clinical studies. Alhydrogel has been used as an adsorbent agent with the main purpose of reducing potential GMMA systemic reactogenicity. However, the immunogenicity and systemic reactogenicity of this GMMA-based vaccine formulated with or without Alhydrogel have never been compared. In this work, we investigated the potential adjuvant effect of aluminium salt-based adjuvants (Alhydrogel and AS37) on S. sonnei GMMA immunogenicity in mice and rabbits, and we found that S. sonnei GMMA alone resulted to be strongly immunogenic. The addition of neither Alhydrogel nor AS37 improved the magnitude or the functionality of vaccine-elicited antibodies. Interestingly, rabbits injected with either S. sonnei GMMA adsorbed on Alhydrogel or S. sonnei GMMA alone showed a limited and transient body temperature increase, returning to baseline values within 24 h after each vaccination. Overall, immunisation with unadsorbed GMMA did not raise any concern for animal health. We believe that these data support the clinical testing of GMMA formulated without Alhydrogel, which would allow for further simplification of GMMA-based vaccine manufacturing.

Shigellosis is leading bacterial cause of diarrhea with high prevalence in children younger than 5 years in low- and middle-income countries, and increasing number of reports of Shigella cases associated to anti-microbial resistance. No vaccines against Shigella are still licensed, but different candidates based on the O-antigen portion of lipopolysaccharides are in clinic. Generalized Modules for Membrane Antigens (GMMA) have been proposed as an alternative delivery system for the O-antigen, and a 4-component vaccine candidate (altSonflex1-2-3), containing GMMA from S. sonnei and S. flexneri 1b, 2a and 3a is being tested in a phase 1/2 clinical trial, with the aim to elicit broad protection against the most prevalent Shigella serotypes. Here, the 4-component GMMA vaccine candidate has been compared to a more traditional glycoconjugate formulation for the ability to induce functional antibodies in mice and rabbits. In mice, in the absence of Alhydrogel, GMMA induce higher IgG antibodies than glycoconjugates and stronger bactericidal titers against all Shigella serotypes. In the presence of Alhydrogel, GMMA induce O-antigen specific IgG levels similar to traditional glycoconjugates, but with a broader range of IgG subclasses, resulting in stronger bactericidal activity. In rabbits, GMMA elicit higher functional antibodies than glycoconjugates against S. sonnei, and similar responses to S. flexneri 1b, 2a and 3a, independently from the presence of Alhydrogel. Different O-antigen based vaccines against Shigella are now in clinical stage and it will be of particular interest to understand how the preclinical findings in the different animal models translate in humans.

We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.

Seasonal environmental conditions shape the behavior and life history of virtually all organisms. Climate change is modifying these seasonal environmental conditions, which threatens to disrupt population dynamics. It is conceivable that climatic changes may be beneficial in one season but result in detrimental conditions in another because life-history strategies vary between these time periods. We analyzed the temporal trends in seasonal survival of yellow-bellied marmots (Marmota flaviventer) and explored the environmental drivers using a 40-y dataset from the Colorado Rocky Mountains (USA). Trends in survival revealed divergent seasonal patterns, which were similar across age-classes. Marmot survival declined during winter but generally increased during summer. Interestingly, different environmental factors appeared to drive survival trends across age-classes. Winter survival was largely driven by conditions during the preceding summer and the effect of continued climate change was likely to be mainly negative, whereas the likely outcome of continued climate change on summer survival was generally positive. This study illustrates that seasonal demographic responses need disentangling to accurately forecast the impacts of climate change on animal population dynamics.

Abstract Development of SARS-CoV-2 vaccines that protect vulnerable populations is a public health priority. Here, we took a systematic and iterative approach by testing several adjuvants and SARS-CoV-2 antigens to identify a combination that elicits antibodies and protection in young and aged mice. While demonstrating superior immunogenicity to soluble receptor-binding domain (RBD), RBD displayed as a protein nanoparticle (RBD-NP) generated limited antibody responses. Comparison of multiple adjuvants including AddaVax, AddaS03, and AS01B in young and aged mice demonstrated that an oil-in-water emulsion containing carbohydrate fatty acid monosulphate derivative (CMS:O/W) most effectively enhanced RBD-NP-induced cross-neutralizing antibodies and protection across age groups. CMS:O/W enhanced antigen retention in the draining lymph node, induced injection site, and lymph node cytokines, with CMS inducing MyD88-dependent Th1 cytokine polarization. Furthermore, CMS and O/W synergistically induced chemokine production from human PBMCs. Overall, CMS:O/W adjuvant may enhance immunogenicity and protection of vulnerable populations against SARS-CoV-2 and other infectious pathogens.

Abstract The aim of the present work is the evaluation of biological effects of natural stilbenoids found in Vitis vinifera, with a focus on their activity as epigenetic modulators. In the present study, resveratrol, pterostilbene and for the first time their dimers (±)-trans-δ-viniferin, (±)-trans-pterostilbene dehydrodimer were evaluated in Caco-2 and HepG-2 cell lines as potential epigenetic modulators. Stilbenoids were added in a Caco-2 cell culture as a model of the intestinal epithelial barrier and in the HepG-2 as a model of hepatic environment, to verify their dose-dependent toxicity, ability to interact with DNA, and epigenomic action. Resveratrol, pterostilbene, and (±)-trans-pterostilbene dehydrodimer were found to have no toxic effects at tested concentration and were effective in reversing arsenic damage in Caco-2 cell lines. (±)-trans-δ-viniferin showed epigenomic activity, but further studies are needed to clarify its mode of action.

Ecological communities are maintained through species interactions, and the resilience of species interactions is critical to the persistence of natural communities. Keystone species play outsized roles in maintaining species interaction networks, and within plant-pollinator communities are high priorities for conservation. The loss of a keystone plant from a plant-pollinator network is expected to cause changes to network structure and composition of pollinator species, with the potential to cause secondary losses of plants and pollinators. To understand how the unmanipulated decline of a keystone plant affects the structure and composition of its network, we studied the plant-pollinator interactions of a Lake Michigan dune plant community where the population of the keystone plant, Cirsium pitcheri, is in rapid decline. The network prior to C. pitcheri decline (2016) was compared to the network as C. pitcheri continued to decline (2021 and 2022) in response to habitat loss. We find evidence that the loss of C. pitcheri altered network structure such that the community may be more sensitive to perturbations. Furthermore, changes in the composition of pollinators were explained by species turnover to a greater extent than by interaction rewiring, including the loss of bumblebees. Short-term negative consequences based on the changes to network structure and composition might lead to long-term effects on the persistence of the dune community. Our study exemplifies that the decline of a keystone plant can have negative implications for conservation of a plant-pollinator community. Using an interaction network framework to assess plant-pollinator communities has potential to develop strategies for best conservation and restoration practices in habitats vulnerable to habitat loss and disturbance.

The Mediterranean diet features plant-based foods renowned for their health benefits derived from bioactive compounds. This review aims to provide an overview of the bioactive molecules present in some representative Mediterranean diet plants, examining their human nutrigenomic effects and health benefits as well as the environmental advantages and sustainability derived from their cultivation. Additionally, it explores the facilitation of producing fortified foods aided by soil and plant microbiota properties. Well-studied examples, such as extra virgin olive oil and citrus fruits, have demonstrated significant health advantages, including anti-cancer, anti-inflammatory, and neuroprotective effects. Other less renowned plants are presented in the scientific literature with their beneficial traits on human health highlighted. Prickly pear’s indicaxanthin exhibits antioxidant properties and potential anticancer traits, while capers kaempferol and quercetin support cardiovascular health and prevent cancer. Oregano and thyme, containing terpenoids like carvacrol and γ-terpinene, exhibit antimicrobial effects. Besides their nutrigenomic effects, these plants thrive in arid environments, offering benefits associated with their cultivation. Their microbiota, particularly Plant Growth Promoting (PGP) microorganisms, enhance plant growth and stress tolerance, offering biotechnological opportunities for sustainable agriculture. In conclusion, leveraging plant microbiota could revolutionize agricultural practices and increase sustainability as climate change threatens biodiversity. These edible plant species may have crucial importance, not only as healthy products but also for increasing the sustainability of agricultural systems.

Given the recent evidence in the clinical application of regenerative medicine, mostly on integumentary systems, we focused our interests on recent bladder regeneration approaches based on mesenchymal stem cells (MSCs), platelet-rich plasma (PRP), and hyaluronic acid (HA) in the treatment of interstitial cystitis/bladder pain syndrome (IC/BPS) in humans. IC/BPS is a heterogeneous chronic disease with not-well-understood etiology, characterized by suprapubic pain related to bladder filling and urothelium dysfunction, in which the impairment of immunological processes seems to play an important role. The histopathological features of IC include ulceration of the mucosa, edema, denuded urothelium, and increased detection of mast cells and other inflammatory cells. A deeper understanding of the molecular mechanism underlying this disease is essential for the selection of the right therapeutic approach. In fact, although various therapeutic strategies exist, no efficient therapy for IC/BPS has been discovered yet. This review gives an overview of the clinical and pathological features of IC/BPS, with a particular focus on the molecular pathways involved and a special interest in the ongoing few investigational therapies in IC/BPS, which use new regenerative medicine approaches, and their synergetic combination. Good knowledge of the molecular aspects related to stem cell-, PRP-, and biomaterial-based treatments, as well as the understanding of the molecular mechanism of this pathology, will allow for the selection of the right and best use of regenerative approaches of structures involving connective tissue and epithelia, as well as in other diseases.

We report first-time reverberation-mapping results for 14 active galactic nuclei (AGNs) from the ongoing Monitoring AGNs with H β Asymmetry campaign (MAHA). These results utilize optical spectra obtained with the Long Slit Spectrograph on the Wyoming Infrared 2.3 m Telescope between 2017 November and 2023 May. MAHA combines long-duration monitoring with high cadence. We report results from multiple observing seasons for nine of the 14 objects. These results include H β time lags, supermassive black hole masses, and velocity-resolved time lags. The velocity-resolved lags allow us to investigate the kinematics of the broad-line region.

Osteoarthritis (OA) is a degenerative joint disease that is age-related and progressive. It causes the destruction of articular cartilage and underlying bone, often aggravated by inflammatory processes and oxidative stresses. This pathology impairs the quality of life of the elderly, causing pain, reduced mobility, and functional disabilities, especially in obese patients. Phytochemicals with anti-inflammatory and antioxidant activities may be used for long-term treatment of OA, either in combination with current anti-inflammatories and painkillers, or as an alternative to other products such as glucosamine and chondroitin, which improve cartilage structure and elasticity. The current systematic review provides a comprehensive understanding of the use of flavonoids. It highlights chondrocyte, cartilage, and subchondral bone activities, with a particular focus on their nutrigenomic effects. The molecular mechanisms of these molecules demonstrate how they can be used for the prevention and treatment of OA in the elderly population. However, clinical trials are still needed for effective use in clinical practice.

Summary: Immunogens that elicit broadly neutralizing antibodies targeting the conserved receptor-binding site (RBS) on influenza hemagglutinin may serve as candidates for a universal influenza vaccine. Here, we develop a computational model to interrogate antibody evolution by affinity maturation after immunization with two types of immunogens: a heterotrimeric “chimera” hemagglutinin that is enriched for the RBS epitope relative to other B cell epitopes and a cocktail composed of three non-epitope-enriched homotrimers of the monomers that comprise the chimera. Experiments in mice find that the chimera outperforms the cocktail for eliciting RBS-directed antibodies. We show that this result follows from an interplay between how B cells engage these antigens and interact with diverse helper T cells and requires T cell-mediated selection of germinal center B cells to be a stringent constraint. Our results shed light on antibody evolution and highlight how immunogen design and T cells modulate vaccination outcomes.

Abstract The genomic effects of circulating glucocorticoids are particularly relevant in cortico-limbic structures, which express a high concentration of steroid hormone receptors. To date, no studies have investigated genomic differences in hippocampal subregions, namely the dorsal (dHPC) and ventral (vHPC) hippocampus, in preclinical models treated with exogenous glucocorticoids. Chronic oral corticosterone (CORT) in mouse is a pharmacological approach that disrupts the activity of the hypothalamic-pituitary-adrenal axis, increases affective behavior, and induces genomic changes after stress in the HPC of wildtype (WT) mice and mice heterozygous for the gene coding for brain-derived neurotrophic factor Val66Met (hMet), a variant associated with genetic susceptibility to stress. Using RNA-sequencing, we investigated the genomic signatures of oral CORT in the dHPC and vHPC of WT and hMet male and female mice, and examined sex and genotype differences in response to oral CORT. Males under CORT showed lower glycemia and increased anxiety- and depression-like behavior compared to females that showed instead opposite affective behavior in response to CORT. Rank–rank-hypergeometric overlap (RRHO) was used to identify genes from a continuous gradient of significancy that were concordant across groups. RRHO showed that CORT-induced differentially expressed genes (DEGs) in WT mice and hMet mice converged in the dHPC of males and females, while in the vHPC, DEGs converged in males and diverged in females. The vHPC showed a higher number of DEGs compared to the dHPC and exhibited sex differences related to glucocorticoid receptor (GR)-binding genes and epigenetic modifiers. Methyl-DNA-immunoprecipitation in the vHPC revealed differential methylation of the exons 1C and 1F of the GR gene (Nr3c1) in hMet females. Together, we report behavioral and endocrinological sex differences in response to CORT, as well as epigenetic signatures that i) differ in the dHPC and vHPC,ii) are distinct in males and females, and iii) implicate differential methylation of Nr3c1 selectively in hMet females.

Background: Bone marrow (BM)-derived stem cells were implanted to induce angiogenesis in patients with no-option critical limb-threatening ischemia. Considering the potential for this therapy, conflicting results related to BM harvesting methods have been reported that could affect stem cell concentrations and quality. Methods: A total of 75 patients with no-option critical limb-threatening ischemia were treated with BM implantation. For 58 patients, BM was harvested using a BM aspirate concentrate system (Harvest Technologies; group HT) with a standard aspiration needle, followed by an automated centrifugation process, to produce BM aspirate concentrate. For 17 patients, BM was harvested using the Marrow Cellution system (Aspire Medical Innovation; group MC). CD34+ cells/mL, CD117+ cells/mL, CD133+ cells/mL, CD309+ cells/mL, hematocrit, and BM purity were compared between the two BM preparations. Results: The retrospective analysis of a subset group after adjustment for age shows that the quality of BM obtained using the Marrow Cellution system is better, in terms of purity, than the classic harvesting method before centrifugation. Harvested BM before centrifugation is characterized by a higher percentage of CD133+ cells compared with BM after centrifugation. In contrast, the MC aspirate had a larger amount of very small embryonic-like cells, as indicated by the higher percentage of CD133+, CD34+, and CD45− cells. These differences translated into an increased occurrence of leg amputations in group HT than in group MC and an increase in transcutaneous oxygen pressure in patients treated with BM aspirated using MC. Conclusions: BM manipulation, such as centrifugation, affects the quality and number of stem cells, with detrimental consequences on clinical outcomes, as reflected by the different amputation rates between the two groups. : Clinical Relevance: Critical limb-threatening ischemia is the most advanced form of peripheral arterial disease with major economic and social effects due to the high amputation rate and mortality. The problem is even greater for diabetic patients, for whom the expected incidence of amputation is ∼40% to 50%. Thus, the need for new therapeutic options is urgent. The present report highlights the striking effects of angiogenic therapy by bone marrow-derived stem cells obtained using a novel technology. We found that the choice of bone marrow harvesting method does influence the clinical outcome; however, further studies are needed. The present study presents a meaningful background for future development.