Most recent advances and applications of extracellular vesicles in tackling neurological challenges.

Over the past few decades, there has been a notable increase in the global burden of central nervous system (CNS) diseases. Despite advances in technology and therapeutic options, neurological and neurodegenerative disorders persist as significant challenges in treatment and cure. Recently, there has been a remarkable surge of interest in extracellular vesicles (EVs) as pivotal mediators of intercellular communication. As carriers of molecular cargo, EVs demonstrate the ability to traverse the blood-brain barrier, enabling bidirectional communication. As a result, they have garnered attention as potential biomarkers and therapeutic agents, whether in their natural form or after being engineered for use in the CNS. This review article aims to provide a comprehensive introduction to EVs, encompassing various aspects such as their diverse isolation methods, characterization, handling, storage, and different routes for EV administration. Additionally, it underscores the recent advances in their potential applications in neurodegenerative disorder therapeutics. By exploring their unique capabilities, this study sheds light on the promising future of EVs in clinical research. It considers the inherent challenges and limitations of these emerging applications while incorporating the most recent updates in the field.

A substrate for a cell free in vitro assay system to screen drugs targeting trypsin like protease-based cleavage of SARS-CoV-2 spike glycoprotein and viral entry.

Host proteases trypsin and trypsin-like proteases have been reported to facilitate the entry of coronavirus SARS-CoV-2 in its host cells. These protease enzymes cleave the viral surface glycoprotein, spike, leading to successful cell surface receptor attachment, fusion and entry of the virus in its host cell. The spike protein has protease cleavage sites between the two domains S1 and S2. Since the cleavage site is recognized by the host proteases, it can be a potential antiviral therapeutic target. Trypsin-like proteases play an important role in virus infectivity and the property of spike protein cleavage by trypsin and trypsin-like proteases can be used to design assays for screening of antiviral candidates against spike protein cleavage. Here, we have documented the development of a proof-of-concept assay system for screening drugs against trypsin/trypsin-like proteases that cleave spike protein between its S1 and S2 domains. The assay system developed uses a fusion substrate protein containing a NanoLuc luciferase reporter protein, the protease cleavage site between S1 and S2 domains of SARS-CoV-2 spike protein and a cellulose binding domain. The substrate protein can be immobilized on cellulose via the cellulose binding domain of the substrate. When trypsin and trypsin-like proteases cleave the substrate, the cellulose binding domain remain bound to the cellulose and the reporter protein is dislodged. Reporter assay using the released reporter protein is the read out of the protease activity. We have demonstrated the proof-of-concept using multiple proteases like trypsin, TMPRSS2, furin, cathepsin B, human airway trypsin and cathepsin L. A significant increment in fold change was observed with increasing enzyme concentration and incubation time. Introduction of increasing amounts of enzyme inhibitors in the reaction reduced the luminescent signal, thus validating the assay. Furthermore, we used SDS-PAGE and immunoblot analyses to study the cleavage band pattern and re-confirm the cleavage for enzymes tested in the assay. Taken together, we have tested an in-vitro assay system using the proposed substrate for screening drugs against trypsin like protease-based cleavage of SARS-CoV-2 spike glycoprotein. The assay system can also be potentially used for antiviral drug screening against any other enzyme that might cleave the used cleavage site.

Complexity of Tumor Microenvironment: Therapeutic Role of Curcumin and Its Metabolites.

The tumor microenvironment (TME) is a complex network of cellular and non-cellular components surrounding the tumor. The cellular component includes fibroblasts, adipocytes, endothelial cells, and immune cells, while non-cellular components are tumor vasculature, extracellular matrix and signaling molecules. The tumor cells have constant close interaction with their surrounding TME components that facilitate their growth, survival, and metastasis. Targeting a complex TME network and its interaction with the tumor can offer a novel strategy to disrupt cancer cell progression. Curcumin, from turmeric rhizome, is recognized as a safe and effective natural therapeutic agent against multiple diseases including cancer. Here the effects of curcumin and its metabolites on tumor-TME interaction modulating ability have been described. Curcumin and its metabolites regulate TME by inhibiting the growth of its cellular components such as cancer-associated adipocytes, cancer-associated fibroblast, tumor endothelial cells, tumor-stimulating immune cells, and inducing anticancer immune cells. They also inhibit the interplay of tumor cells to TME by suppressing non-cellular components such as extracellular matrix, and associated tumor promoting signaling-pathways. In addition, curcumin inhibits the inflammatory environment, suppresses angiogenic factors, and increases antioxidant status in TME. Overall, curcumin has the capability to regulate TME components and their interaction with tumor cells.

KLF8 is activated by TGF-ß1 via Smad2 and contributes to ovarian cancer progression.

Krüppel-like factor 8 (KLF8) is a transcription factor expressed abnormally in various cancer types and promotes oncogenic transformation. However, the role of KLF8 in ovarian cancer (OC) progression remains unclear. This study reports that transforming growth factor-ß1 (TGF-ß1)/Smad2/KLF8 axis regulates epithelial-mesenchymal transition (EMT) and contributes to OC progression. We analyzed the KLF8 expression in OC cells and tissues, wherein a significant overexpression of KLF8 was observed. Increased KLF8 expressions were correlated with higher cell proliferation, EMT, migration, and invasion and conferred poor clinical outcomes in OC patients. Overexpressed KLF8 increases F-actin polymerization and induces cytoskeleton remodeling of OC cells. Furthermore, a dissection of the molecular mechanism defined that TGF-ß1 triggers KLF8 through the Smad2 pathway and regulates EMT. Pharmacological and genetic inhibition of Smad2 followed by TGF-ß1 treatment failed to activate KLF8 expression and induction of EMT. Using promoter-luciferase reporter assays, we defined that upon TGF-ß1 activation, phosphorylated Smad2 binds and promotes the KLF8 promoter activity, and knockdown of Smad2 inhibits KLF8 promoter activation. Together, these results demonstrate that TGF-ß1 activates KLF8 expression by the Smad2 pathway, and KLF8 contributes to OC progression and may serve as a potential therapeutic strategy for treating OC patients.

Anti-viral triterpenes: a review.

Triterpenes are naturally occurring derivatives biosynthesized following the isoprene rule of Ruzicka. The triterpenes have been reported to possess a wide range of therapeutic applications including anti-viral properties. In this review, the recent studies (2010-2020) concerning the anti-viral activities of triterpenes have been summarized. The structure activity relationship studies have been described as well as brief biosynthesis of these triterpenes is discussed.

DNA polymerase eta: A potential pharmacological target for cancer therapy.

In the last two decades, intensive research has been carried out to improve the survival rates of cancer patients. However, the development of chemoresistance that ultimately leads to tumor relapse poses a critical challenge for the successful treatment of cancer patients. Many cancer patients experience tumor relapse and ultimately die because of treatment failure associated with acquired drug resistance. Cancer cells utilize multiple lines of self-defense mechanisms to bypass chemotherapy and radiotherapy. One such mechanism employed by cancer cells is translesion DNA synthesis (TLS), in which specialized TLS polymerases bypass the DNA lesion with the help of monoubiquitinated proliferating cell nuclear antigen. Among all TLS polymerases (Pol η, Pol ι, Pol κ, REV1, Pol ζ, Pol µ, Pol λ, Pol ν, and Pol θ), DNA polymerase eta (Pol η) is well studied and majorly responsible for the bypass of cisplatin and UV-induced DNA damage. TLS polymerases contribute to chemotherapeutic drug-induced mutations as well as therapy resistance. Therefore, targeting these polymerases presents a novel therapeutic strategy to combat chemoresistance. Mounting evidence suggests that inhibition of Pol η may have multiple impacts on cancer therapy such as sensitizing cancer cells to chemotherapeutics, suppressing drug-induced mutagenesis, and inhibiting the development of secondary tumors. Herein, we provide a general introduction of Pol η and its clinical implications in blocking acquired drug resistance. In addition; this review addresses the existing gaps and challenges of Pol η mediated TLS mechanisms in human cells. A better understanding of the Pol η mediated TLS mechanism will not merely establish it as a potential pharmacological target but also open possibilities to identify novel drug targets for future therapy.

Neuroinvasion of SARS-CoV-2 may play a role in the breakdown of the respiratory center of the brain.

The recent outbreak of the novel coronavirus, SARS-CoV-2, has emerged to be highly pathogenic in nature. Although lungs are considered as the primary infected organs by SARS-CoV-2, some of the other organs, including the brain, have also been found to be affected. Here, we have discussed how SARS-CoV-2 might infect the brain. The infection of the respiratory center in the brainstem could be hypothesized to be responsible for the respiratory failure in many COVID-19 patients. The virus might gain entry through the olfactory bulb and invade various parts of the brain, including the brainstem. Alternatively, the entry might also occur from peripheral circulation into the central nervous system by compromising the blood-brain barrier. Finally, yet another possible entry route could be its dispersal from the lungs into the vagus nerve via the pulmonary stretch receptors, eventually reaching the brainstem. Therefore, screening neurological symptoms in COVID-19 patients, especially toward the breakdown of the respiratory center in the brainstem, might help us better understand this disease.

Land gradient and configuration effects on yield, irrigation amount and irrigation water productivity in rice-wheat and maize-wheat cropping systems in Eastern India.

Laser land levelling is expanding rapidly in the rice-wheat (RW) and maize-wheat (MW) systems of the Indo-Gangetic Plains of India and Pakistan. Current practice is to level to zero (0%) gradient, whereas a small gradient (e.g. 0.1%) is typically used in developed countries. Therefore, experiments were conducted in farmers' plots (~15 m x 40 m) in the Eastern Gangetic Plains to evaluate laser levelling with a 0.1% gradient in comparison with 0% and farmer levelling practice (FL). The study was conducted over two years in RW and MW systems. In the MW system, raised beds in plots lasered with 0% and 0.1% gradients were also evaluated. Laser levelling with 0% gradient significantly reduced irrigation amount and/or increased irrigation water productivity (WPi) in all crops/systems grown on the flat compared to FL except for wheat in the MW system. While there was a consistent trend for higher yield with a 0% gradient compared with FL, the differences were not significant in any crop/system. For the RW system, the results suggest no to marginal benefits in irrigation amount and WPi from levelling with a 0.1% gradient in comparison with 0% gradient. In that system, by far the bigger gains were from changing from FL to laser levelling with 0% gradient. This resulted in substantial reductions in irrigation amount, which greatly increased WPi in both crops (by ~40%), while yield was not affected. Rice grown with FL was not profitable, but lasering with 0% gradient significantly increased gross margin for rice, wheat and the total RW system. As for the RW system, levelling to 0% with a flat configuration significantly increased WPi of both crops in the MW system compared to FL, but by a lesser proportion. Raised beds significantly increased yield of maize by 8% (0.5 t ha-1), reduced irrigation amount by 20% (40 mm) and increased WPi by 34% (1.0 kg m-3) in comparison with the laser levelled flat plots. Gross margin of the MW system on beds was 17-20% higher than FL, and gross margin with beds on a 0.1% gradient was significantly higher than either gradient on the flat. The results suggest that the gains from levelling with a 0.1% gradient compared to 0% are marginal; however, this may change if the goal of consolidation of small farmer plots into larger fields becomes a reality provided there is a proportionate increase in irrigation flow rates, and ability to drain.

Use of Tourniquet During Knee Arthroplasty in Patients With Radiographic Arterial Calcifications.

BACKGROUND: Use of tourniquet during total knee arthroplasty (TKA) in patients with radiographic arterial calcifications is controversial. Intimal arterial calcifications are feared to be associated with ischemic complications such as delayed wound healing and arterial thrombosis, whereas medial calcifications stiffen the arterial wall, possibly leading to tourniquet failure and increased blood loss. METHODS: We conducted a prospective cohort study to determine the incidence of tourniquet failure (inflated up to 300 mm Hg), blood transfusions, wound healing, and ischemic complications in thighs with and without arterial calcifications on preoperative radiographs, in 2548 consecutive primary TKAs conducted in our unit over a 5-year period. Eighty-six thighs showed vascular calcifications: 58 medial and 28 intimal. RESULTS: Thighs with vascular calcifications had higher risk of tourniquet failure as compared to those without calcifications (P < .001), but with no significant increase in incidence of blood transfusions. All cases of tourniquet failure in the calcification group occurred in thighs with medial calcifications, whereas all cases of tourniquet failure in the control group occurred in obese patients. There was no difference in wound healing and ischemic complications in limbs with and without arterial calcifications. CONCLUSION: The presence of arterial calcifications on preoperative radiographs increases the risk of tourniquet failure at 300 mm Hg in patients undergoing TKA, with no significant increase in rate of blood transfusions, wound healing or ischemic complications.

Agricultural labor, COVID-19, and potential implications for food security and air quality in the breadbasket of India.

To contain the COVID-19 pandemic, India imposed a national lockdown at the end of March 2020, a decision that resulted in a massive reverse migration as many workers across economic sectors returned to their home regions. Migrants provide the foundations of the agricultural workforce in the 'breadbasket' states of Punjab and Haryana in Northwest India.There are mounting concerns that near and potentially longer-term reductions in labor availability may jeopardize agricultural production and consequently national food security. The timing of rice transplanting at the beginning of the summer monsoon season has a cascading influence on productivity of the entire rice-wheat cropping system. To assess the potential for COVID-related reductions in the agriculture workforce to disrupt production of the dominant rice-wheat cropping pattern in these states, we use a spatial ex ante modelling framework to evaluate four scenarios representing a range of plausible labor constraints on the timing of rice transplanting. Averaged over both states, results suggest that rice productivity losses under all delay scenarios would be low as compare to those for wheat, with total system productivity loss estimates ranging from 9%, to 21%, equivalent to economic losses of USD $674 m to $1.48 billion. Late rice transplanting and harvesting can also aggravate winter air pollution with concomitant health risks. Technological options such as direct seeded rice, staggered nursery transplanting, and crop diversification away from rice can help address these challenges but require new approaches to policy and incentives for change.

Inflammation-Stimulated Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Inflammation.

Extracellular vesicles (EVs) secreted by mesenchymal stromal cells (MSCs) have been proposed to be a key mechanistic link in the therapeutic efficacy of cells in response to cellular injuries through paracrine effects. We hypothesize that inflammatory stimulation of MSCs results in the release of EVs that have greater anti-inflammatory effects. The present study evaluates the immunomodulatory abilities of EVs derived from inflammation-stimulated and naive MSCs (MSCEv+ and MSCEv, respectively) isolated using a current Good Manufacturing Practice-compliant tangential flow filtration system. Detailed characterization of both EVs revealed differences in protein composition, cytokine profiles, and RNA content, despite similarities in size and expression of common surface markers. MSCEv+ further attenuated release of pro-inflammatory cytokines in vitro when compared to MSCEv, with a distinctly different pattern of EV-uptake by activated primary leukocyte subpopulations. The efficacy of EVs was partially attributed to COX2/PGE2 expression. The present study demonstrates that inflammatory stimulation of MSCs renders release of EVs that have enhanced anti-inflammatory properties partially due to COX2/PGE2 pathway alteration. Stem Cells 2018;36:79-90.

Teriflunomide Modulates Vascular Permeability and Microglial Activation after Experimental Traumatic Brain Injury.

Despite intensive research and clinical trials with numerous therapeutic treatments, traumatic brain injury (TBI) is a serious public health problem in the United States. There is no effective FDA-approved treatment to reduce morbidity and mortality associated with TBI. Inflammation plays a pivotal role in the pathogenesis of TBI. We looked to re-purpose existing drugs that reduce immune activation without broad immunosuppression. Teriflunomide, an FDA-approved drug, has been shown to modulate immunological responses outside of its ability to inhibit pyrimidine synthesis in rapidly proliferating cells. In this study, we tested the efficacy of teriflunomide to treat two different injury intensities in rat models of TBI. Our results show that teriflunomide restores blood-brain barrier integrity, decreases inflammation, and increases neurogenesis in the subgranular zone of the hippocampus. While we were unable to detect neurocognitive effects of treatment on memory and special learning abilities after treatment, a 2-week treatment following injury was sufficient to reduce neuroinflammation up to 120 days later.

Taking the climate risk out of transplanted and direct seeded rice: Insights from dynamic simulation in Eastern India.

Rice productivity in Eastern Indo-Gangetic plains (EIGP) is extremely low, in part due to the prevailing practice of cultivating long-duration transplanted rice under rainfed conditions which leads to water stress and significant yield losses in many seasons. Rice establishment alternatives such as direct seeded rice (DSR) require less water at planting but also are accompanied by climate risks that constrain adoption. For both conventional transplanted and DSR systems, successfully addressing climate-based production risks may provide a strong basis for sustainable rice intensification in EIGP. In this ex ante study of rice yield and yield variability, the APSIM cropping system model was used to evaluate the efficacy of risk-reducing management practices in both transplanted and DSR systems. Simulations were conducted with 44 years (1970-2013) of historical weather data from central Bihar, India. Results confirm that the prevailing farmer practice of transplanting long-duration cultivars under rainfed conditions (fTR) often results in delayed transplanting and the use of older seedlings, leading to low (median 1.6 t ha-1) and variable (Standard deviation (SD) 2.1 t ha-1) rice yields. To improve the fTR system, simulations suggest that adoption of medium-duration hybrid rice (3.2 t ha-1), provision of supplemental post-establishment irrigation (3.2 t ha-1), or transplanting appropriately aged seedlings (3.4 t ha -1) can double yields as single interventions while, in the case of supplemental irrigation, significantly reducing inter-annual production variability. Additional gains are achievable when interventions are layered: supplemental irrigation paired with medium-duration hybrids increased median rice yields to 4.6 t ha-1 with much lower variability (SD 1.0 t ha-1). In these improved systems where irrigation is used to transplant the crop, simulations revealed the importance of timely planting: high and stable yields are achievable for long-duration cultivars when transplanting is completed by 2 August with this window of opportunity extending to 16 August for medium-duration hybrids. In rainfed DSR systems, the potential pay-offs from single interventions were even higher with medium-duration hybrids resulting in a median yield of 4.5 t ha-1 against 1.8 t ha-1 with long-duration cultivars. For irrigated DSR systems, an optimum sowing window of early to mid-June was identified which resulted in higher and more stable yields with lower water requirements. Simulation results suggest several risk-reducing intensification pathways that can be selectively matched to farmer risk preferences and investment capabilities within the target region in EIGP.

Social inclusion increases with time for zero-tillage wheat in the Eastern Indo-Gangetic Plains.

Sustainable intensification (SI) approaches to agricultural development are urgently needed to meet the growing demand for crop staples while protecting ecosystem services and environmental quality. However, SI initiatives have been criticized for neglecting social welfare outcomes. A recent review found that better-off farmers benefitted disproportionately from SI and highlighted the dearth of studies assessing the equity of outcomes. In this study, we explore the social inclusiveness of zero-tillage (ZT) wheat adoption in Bihar, India. ZT is a proven SI technology for enhancing wheat productivity while boosting profitability and reducing greenhouse gas emissions from agricultural machinery in the densely populated Indo-Gangetic Plains. With an average landholding size of 0.39 ha, most farmers in Bihar depend on custom-hiring services to access the technology. While service provision models should foster inclusive growth by reducing financial barriers to technology adoption, early evidence suggested that smallholders remained at a disadvantage. Building on this previous research, we use a panel dataset from 961 wheat-growing households that spans a six-year period to analyze ZT adoption dynamics over time while accounting for the role of social networks and access to service provision. Using a heckprobit approach to correct for non-exposure bias, we compare determinants of ZT awareness and use in 2012 and 2015. We apply a multinomial logit model to identify determinants of early adoption, recent adoption, non-adoption, and dis-adoption. Furthermore, we explore the quality of ZT services as an additional dimension of socially-inclusive technology access. We find that the strong initial scale bias in ZT use declined substantially as awareness of the technology increased and the service economy expanded. Land fragmentation replaced total landholding size as a significant adoption determinant, which also affected the quality of ZT services received. Hence, farmers with small but contiguous landholdings appear to have gained a significant degree of access over time. We conclude that early-stage assessments of SI may be misleading, and that private sector-based service provision can contribute to socially inclusive development outcomes as markets mature.

Preclinical progenitor cell therapy in traumatic brain injury: a meta-analysis.

BACKGROUND: No treatment is available to reverse injury associated with traumatic brain injury (TBI). Progenitor cell therapies show promise in both preclinical and clinical studies. We conducted a meta-analysis of preclinical studies using progenitor cells to treat TBI. METHODS: EMBASE, MEDLINE, Cochrane Review, Biosis, and Google Scholar were searched for articles using prespecified search strategies. Studies meeting inclusion criteria underwent data extraction. Analysis was performed using Review Manager 5.3 according to a fixed-effects model, and all studies underwent quality scoring. RESULTS: Of 430 abstracts identified, 38 met inclusion criteria and underwent analysis. Average quality score was 4.32 of 8 possible points. No study achieved a perfect score. Lesion volume (LV) and neurologic severity score (NSS) outcomes favored cell treatment with standard mean difference (SMD) of 0.86 (95% CI: 0.64-1.09) and 1.36 (95% CI: 1.11-1.60), respectively. Rotarod and Morris water maze outcomes also favored treatment with improvements in SMD of 0.34 (95% CI: 0.02-0.65) and 0.46 (95% CI: 0.17-74), respectively. Although LV and NSS were robust to publication bias assessments, rotarod and Morris water maze tests were not. Heterogeneity (I2) ranged from 74%-85% among the analyses, indicating a high amount of heterogeneity among studies. Precision as a function of quality score showed a statistically significant increase in the size of the confidence interval as quality improved. CONCLUSIONS: Our meta-analysis study reveals an overall positive effect of progenitor cell therapies on LV and NSS with a trend toward improved motor function and spatial learning in different TBI animal models.

Mapping of PARK2 and PACRG overlapping regulatory region reveals LD structure and functional variants in association with leprosy in unrelated indian population groups.

Leprosy is a chronic infectious disease caused by Mycobacterium Leprae, where the host genetic background plays an important role toward the disease pathogenesis. Various studies have identified a number of human genes in association with leprosy or its clinical forms. However, non-replication of results has hinted at the heterogeneity among associations between different population groups, which could be due to differently evolved LD structures and differential frequencies of SNPs within the studied regions of the genome. A need for systematic and saturated mapping of the associated regions with the disease is warranted to unravel the observed heterogeneity in different populations. Mapping of the PARK2 and PACRG gene regulatory region with 96 SNPs, with a resolution of 1 SNP per 1 Kb for PARK2 gene regulatory region in a North Indian population, showed an involvement of 11 SNPs in determining the susceptibility towards leprosy. The association was replicated in a geographically distinct and unrelated population from Orissa in eastern India. In vitro reporter assays revealed that the two significantly associated SNPs, located 63.8 kb upstream of PARK2 gene and represented in a single BIN of 8 SNPs, influenced the gene expression. A comparison of BINs between Indian and Vietnamese populations revealed differences in the BIN structures, explaining the heterogeneity and also the reason for non-replication of the associated genomic region in different populations.

Clinical relevance of stem cell therapies in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS), characterized by the progressive loss of both upper and lower motor neurons, is a fatal neurodegenerative disorder. This disease is often accompanied by a tremendous physical and emotional burden not only for the patients, but also for their families and friends as well. There is no clinically relevant treatment available for ALS. To date, only one Food and Drug Administration (FDA)-approved drug, Riluzole, licensed 18 years ago, has been proven to marginally prolong patients' survival without improving the quality of their lives. Because of the lack of an effective drug treatment and the promising outcomes from several preclinical studies, researchers have highlighted this disease as a suitable candidate for stem cell therapy. This review article highlights the finding of key preclinical studies that present a rationale for the use of different types of stem cells for the treatment of ALS, and the most recent updates on the stem cell-based ALS clinical trials around the world.

Recent Advances in Genomic Studies of Gestational Duration and Preterm Birth.

Preterm birth (PTB) is the leading cause of infant mortality and morbidity. For several decades, extensive epidemiologic and genetic studies have highlighted the significant contribution of maternal and offspring genetic factors to PTB. This review discusses the challenges inherent in conventional genomic analyses of PTB and underscores the importance of adopting nonconventional approaches, such as analyzing the mother-child pair as a single analytical unit, to disentangle the intertwined maternal and fetal genetic influences. We elaborate on studies investigating PTB phenotypes through 3 levels of genetic analyses: single-variant, multi-variant, and genome-wide variants.

Recovery after human bone marrow mesenchymal stem cells (hBM-MSCs)-derived extracellular vesicles (EVs) treatment in post-MCAO rats requires repeated handling.

Rehabilitation is the only current intervention that improves sensorimotor function in ischemic stroke patients, similar to task-specific intensive training in animal models of stroke. Bone marrow mesenchymal stem cells (BM-MSCs)-derived extracellular vesicles (EVs) are promising in restoring brain damage and function in stroke models. Additionally, the non-invasive intranasal route allows EVs to reach the brain and target specific ischemic regions. Yet unclear is how handling might enhance recovery or influence other therapies such as EVs after stroke. We used the transient middle cerebral artery occlusion (MCAO) model of stroke in rats to assess how intensive handling alone, in the form of sensorimotor behavioral tests, or in combination with an intranasal treatment of EVs restored neurological function and ischemic damage. Handled rats were exposed to a battery of sensorimotor tests, including the modified Neurological Severity Score (mNSS), beam balance, corner, grid walking, forelimb placement, and cylinder tests, together with Magnetic Resonance Imaging (MRI) at 2, 7, 14, 21, and 28 days post-stroke (dps). Handled MCAO rats were also exposed to an intranasal multidose or single dose of EVs. Non-handled rats were evaluated only by mNSS and MRI at 2, 28, and 56 dps and were treated with a single intranasal dose of EVs. Our results showed that handling animals after MCAO is necessary for EVs to work at the tested dose and frequency, and that a single cumulative dose of EVs further improves the neurological function recovered during handling. These results show the importance of rehabilitation in combination with other treatments such as EVs, and highlight how extensive behavioral testing might influence functional recovery after stroke.

Heritability Estimation Approaches Utilizing Genome-Wide Data.

Prior to the development of genome-wide arrays and whole genome sequencing technologies, heritability estimation mainly relied on the study of related individuals. Over the past decade, various approaches have been developed to estimate SNP-based narrow-sense heritability ( h SNP 2 ${\rm{h}}_{{\rm{SNP}}}^2$ ) in unrelated individuals. These latter approaches use either individual-level genetic variations or summary results from genome-wide association studies (GWAS). Recently, several studies compared these approaches using extensive simulations and empirical datasets. However, sparse information on hands-on training necessitates revisiting these approaches from the perspective of a stepwise guide for practical applications. Here, we provide an overview of the commonly used SNP-heritability estimation approaches utilizing genome-wide array, imputed or whole genome data from unrelated individuals, or summary results. We not only discuss these approaches based on their statistical concepts, utility, advantages, and limitations, but also provide step-by-step protocols to apply these approaches. For illustration purposes, we estimate h SNP 2 ${\rm{h}}_{{\rm{SNP}}}^2$ of height and BMI utilizing individual-level data from The Northern Finland Birth Cohort (NFBC) and summary results from the Genetic Investigation of ANthropometric Traits (GIANT;) consortium. We present this review as a template for the researchers who estimate and use heritability in their studies and as a reference for geneticists who develop or extend heritability estimation approaches. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: GREML (GCTA) Alternate Protocol 1: Stratified GREML Basic Protocol 2: LDAK Alternate Protocol 2: Stratified LDAK Basic Protocol 3: Threshold GREML Basic Protocol 4: LD score (LDSC) regression Basic Protocol 5: SumHer.