Restoring susceptibility to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus.

Infections by Staphylococcus aureus have been treated historically with ß-lactam antibiotics. However, these antibiotics have become obsolete in methicillin-resistant S. aureus by acquisition of the bla and mec operons. The presence of the ß-lactam antibiotic is detected by the sensor domains of BlaR and/or MecR, and the information is transmitted to the cytoplasm, resulting in derepression of the antibiotic-resistance genes. We hypothesized that inhibition of the sensor domain would shut down this response system, and ß-lactam susceptibility would be restored. An in silico search of 11 million compounds led to a benzimidazole-based hit and, ultimately, to the boronate 4. The X-ray structure of 4 is covalently engaged with the active-site serine of BlaR. Compound 4 potentiates by 16- to 4,096-fold the activities of oxacillin and of meropenem against methicillin-resistant S. aureus strains. The combination of 4 with oxacillin or meropenem shows efficacy in infected mice, validating the strategy.

The Ethnopharmacological Properties of Green-Engineered Metallic Nanoparticles against Metabolic Disorders.

Metabolic syndrome is a multifaceted pathophysiologic condition that is largely caused by an imbalance between caloric intake and energy expenditure. The pathogenesis of metabolic syndrome is determined by an individual's genetic/epigenetics and acquired factors. Natural compounds, notably plant extracts, have antioxidant, anti-inflammatory, and insulin-sensitizing properties and are considered to be a viable option for metabolic disorder treatment due to their low risk of side effects. However, the limited solubility, low bioavailability, and instability of these botanicals hinder their performance. These specific limitations have prompted the need for an efficient system that reduces drug degradation and loss, eliminates unwanted side effects, and boosts drug bioavailability, as well as the percentage of the drug deposited in the target areas. The quest for an enhanced (effective) drug delivery system has led to the formation of green-engineered nanoparticles, which has increased the bioavailability, biodistribution, solubility, and stability of plant-based products. The unification of plant extracts and metallic nanoparticles has helped in the development of new therapeutics against metabolic disorders such as obesity, diabetes mellitus, neurodegenerative disorders, non-alcoholic fatty liver, and cancer. The present review outlines the pathophysiology of metabolic diseases and their cures with plant-based nanomedicine.

Influence of Bone Substitutes on Mesenchymal Stromal Cells in an Inflammatory Microenvironment.

Bone regeneration is driven by mesenchymal stromal cells (MSCs) via their interactions with immune cells, such as macrophages (MPs). Bone substitutes, e.g., bi-calcium phosphates (BCPs), are commonly used to treat bone defects. However, little research has focused on MSC responses to BCPs in the context of inflammation. The objective of this study was to investigate whether BCPs influence MSC responses and MSC-MP interactions, at the gene and protein levels, in an inflammatory microenvironment. In setup A, human bone marrow MSCs combined with two different BCP granules (BCP 60/40 or BCP 20/80) were cultured with or without cytokine stimulation (IL1ß + TNFα) to mimic acute inflammation. In setup B, U937 cell-line-derived MPs were introduced via transwell cocultures to setup A. Monolayer MSCs with and without cytokine stimulation served as controls. After 72 h, the expressions of genes related to osteogenesis, healing, inflammation and remodeling were assessed in the MSCs via quantitative polymerase chain reactions. Additionally, MSC-secreted cytokines related to healing, inflammation and chemotaxis were assessed via multiplex immunoassays. Overall, the results indicate that, under both inflammatory and non-inflammatory conditions, the BCP granules significantly regulated the MSC gene expressions towards a pro-healing genotype but had relatively little effect on the MSC secretory profiles. In the presence of the MPs (coculture), the BCPs positively regulated both the gene expression and cytokine secretion of the MSCs. Overall, similar trends in MSC responses were observed with BCP 60/40 and BCP 20/80. In summary, within the limits of in vitro models, these findings suggest that the presence of BCP granules at a surgical site may not necessarily have a detrimental effect on MSC-mediated wound healing, even in the event of inflammation.

Clinical characteristic and epidemiological features of SARS CoV -2 disease patients from a COVID 19 designated Hospital in New Delhi.

SARS CoV -2 infection is rapidly evolving as a serious global pandemic. The present study describes the clinical characteristics of SARS CoV-2 infection patients. The Samples were subjected to RT - PCR or Rapid Antigen test for diagnosis of SARS CoV- 2. A cohort of 3745 patients with confirmed diagnosis of SARS CoV -2 infection in a tertiary care center in New Delhi, India were included in this study. Data was collected from offline and online medical records over a period of six months. Amongst 3745 SARS CoV -2 infected patients, 2245 (60%) were symptomatic and 1500 (40%) were asymptomatic. Most common presenting symptom was cough (49.3%) followed febrile episodes (47.1%), breathlessness (42.7%) and sore throat (35.1%). Cough along with breathlessness (24.1) was the most common combination of symptoms followed by fever with cough (22.7). The most common comorbidity found among symptomatic group was diabetes (42.5%) followed by hypertension (21.4%) and chronic kidney disease (18%). Comorbidities like diabetes mellitus, chronic diseases of lungs, heart and kidneys were found to be common in symptomatic group and this was found to be statistically significant (p<0.05). COVID-19 is an evolving disease and data from our study help in understanding the clinic-epidemiological profile of patients. This article is protected by copyright. All rights reserved.

Establishment of a novel cancer cell line derived from vulvar carcinoma associated with lichen sclerosus exhibiting a fibroblast-dependent tumorigenic potential.

Vulvar squamous cell carcinoma associated with lichen sclerosus (VLS-VSCC) are rare tumors but with higher recurrence and worse prognosis than other types of VSCC. Lack of experimental models has limited the search for better understanding of the biology and development of treatment modalities. In this study, we isolated and characterized primary cells from VSCC (n = 7) and normal vulvar tissue adjacent to tumor (n = 7). Detailed characterization of the novel spontaneously immortalized cell line, VCC1 revealed a characteristic epithelial morphology in vitro and a well-differentiated keratinizing SCC histology in vivo, closely resembling the tumor of origin. VCC1 expressed higher levels of epithelial-mesenchymal transition markers and higher clonogenic properties as compared to other established non VLS-VSCC cell lines. In vitro 3D organotypic assays and in vivo xenografts revealed a prominent role of cancer-associated fibroblasts in VCC1 invasion and tumor formation. In conclusion, VCC1 mirrored several major VLS-VSCC features and provided a robust experimental tool for further elucidation of VLS-related oncogenesis and drug testing.

Termite's potential in solid waste management in Himachal Pradesh: A mini review.

One of the major reasons for the degradation of Earth's setting is inappropriate disposal of solid waste. Mountains of solid waste are increasing in every country making solid waste management a challenge almost everywhere on Earth. It is vital to look for such municipal solid waste management solutions that are financially sustainable, technically possible, socially and legally acceptable and environmentally friendly. Currently vermicomposting is the only biological solid waste treatment process that uses multicellular organisms to biodegrade organic wastes. A few species of insects are capable of digesting lignin and cellulose. Of these, termites are the most numerous and play a decisive role as scavengers. Microflora which inhabit the termites' gut contribute to their waste degrading potential. Termites act as major soil ecosystem managers and are able to breakdown and recycle organic matter and composite. Although studies in the potential of termites to increase soil fertility are well-accounted for in the literature, the potential of termites for solid waste management still needs to be explored. This mini review presents the state of information on the use of termite species in solid waste degradation focused on the potential application in the Himachal Pradesh region, India. This review highlights different termite species found in Himachal Pradesh and the challenges that are needed to be conquered. The study also aims at encouraging competent authorities/researchers to work towards the improvement of the present system by further exploring the use of termites in solid waste management through suggestions and recommendations.

Liquid-Liquid Phase Separation of Histone Proteins in Cells: Role in Chromatin Organization.

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids has emerged as an important phenomenon in membraneless intracellular organization. We demonstrate that the linker histone H1 condenses into liquid-like droplets in the nuclei of HeLa cells. The droplets, observed during the interphase of the cell cycle, are colocalized with DNA-dense regions indicative of heterochromatin. In vitro, H1 readily undergoes LLPS with both DNA and nucleosomes of varying lengths but does not phase separate in the absence of DNA. The nucleosome core particle maintains its structural integrity inside the droplets, as demonstrated by FRET. Unexpectedly, H2A also forms droplets in the presence of DNA and nucleosomes in vitro, whereas the other core histones precipitate. The phase diagram of H1 with nucleosomes is invariant to the nucleosome length at physiological salt concentration, indicating that H1 is capable of partitioning large segments of DNA into liquid-like droplets. Of the proteins tested (H1, core histones, and the heterochromatin protein HP1α), this property is unique to H1. In addition, free nucleotides promote droplet formation of H1 nucleosome in a nucleotide-dependent manner, with droplet formation being most favorable with ATP. Although LLPS of HP1α is known to contribute to the organization of heterochromatin, our results indicate that H1 also plays a role. Based on our study, we propose that H1 and DNA act as scaffolds for phase-separated heterochromatin domains.

Bacillus cereus: public health burden associated with ready-to-eat foods in Himachal Pradesh, India.

The study determined incidence, enterotoxigenecity and antimicrobial susceptibility profiles of Bacillus cereus isolated from ready-to-eat (RTE) milk products (n = 80), RTE meat products (n = 40), beverages (n = 40) and water samples (n = 60, from food preparing and serving outlets/restaurants) collected from eight different tourist places of Himachal Pradesh. 11.4% (25/220) samples were contaminated with Bacillus and isolates were identified as B. cereus (76.0%, n = 19), B. alvei (12.0%, n = 3), B. polymyxa (8.0%, n = 2) and B. firmus (4.0%, n = 1) by conventional and molecular methods. B. cereus incidence was highest in cheese based foods (25.0%) followed by vegetable soups (16.7%), khoa based foods (14.0%), milk based beverages (10.5%), paneer based foods (8.6%), cream based foods (8.3%) and water (8.3%) samples. Multiplex polymerase chain reaction detected enterotoxigenic genes only in B. cereus isolates. nhe complex (encoding non-haemolytic enterotoxins, ABC) genes were detected only in B. cereus isolates. 57.6% (11/19), 36.8% (7/19) and 5.3% (1/19) harboured all three (nheA, nheB, nheC), two (nheB, nheC) and one (nheC) nhe gene, respectively. Among hbl complex genes (encoding haemolytic enterotoxins CAD), only hblC (36.8%, 7/19) was detected. Incidence B. cereus cytK (encoding cytotoxin enterotoxin) was 52.6% (10/19). Each B. cereus isolate harboured two or more enterotoxigenic genes. Seven isolates had at least one gene from haemolytic and non-haemolytic complexes along with cytK. High levels (> 50%) of antimicrobial resistance were recorded for penicillin, amoxicillin, ampicillin cefixime and ceftazidine in tested B. cereus isolates. Two isolates were identified as multidrug resistant isolates with resistance to ≥ 3 antibiotic classes.

Gynecological associated disorders and management.

BACKGROUND: Chronic pelvic pain syndrome is complex and involves multiple organ systems. The gynecological aspects of chronic pelvic pain syndrome can be divided into four different areas: intra-abdominal, vaginal, pelvic floor muscles and sexual pain. This article provides an overview of gynecological evaluation in patients with chronic pelvic pain and reviews the most common gynecological diagnoses and their management. METHODS: An extensive review of the literature including guidelines from the International Continence Society, the European Association of Urology, and the International Association for the Study of Pain was performed. RESULTS: Gynecological evaluation of patients with chronic pelvic pain begins with a thorough history and physical examination. Laboratory tests, imaging studies and diagnostic procedures can be used as adjuncts to make a diagnosis. Treatment modalities include physical therapy, medications, trigger points injections, and surgery. CONCLUSION: Common gynecological diagnoses of chronic pelvic pain include endometriosis, adenomyosis, vulvodynia, high tone pelvic floor dysfunction, and genitopelvic pain/penetration disorder. Gynecology is one of the many systems that can be associated with chronic pelvic pain. Managing patients with chronic pelvic pain requires a multimodal and multidisciplinary approach.

The fundamentals of chronic pelvic pain assessment, based on international continence society recommendations.

AIMS: Chronic pelvic pain (CPP) is defined as a noncyclical pain that has duration of at least 6 months and can lead to decreased quality of life and physical performance. The pain can be attributed to problems in the pelvic organs and/or problems in related systems, and possible psycho-social attributes may contribute to the manifestation. Due to the complex nature, CPP syndromes are multifactorial and the terminology needs to reflect the setting. METHODS: The current review is a synthesis of key aspects of the recent International Continence Society Standardization for Terminology in CPP Syndromes. RESULTS: Nine domains can be used for a detailed description of CPP. They include four domains specific to the pelvic organs (lower urinary tract, female genital, male genital, gastrointestinal), two related to other sources of pain which may be perceived in the pelvis (musculoskeletal, neurological) and three which may influence the response to the pain or its impact on the individual (psychological, sexual, and comorbidities). For an individual patient with CPP, each domain should be reviewed in terms of symptoms and signs, noting that positive findings could reflect either a primary cause or a secondary consequence. The findings will guide further evaluations and subsequent treatment. CONCLUSION: We present a synthesis of the standard for terminology in CPP syndromes in women and men, which serves as a systematic framework to consider possible sources of pain (pelvic organs or other sources) and the individual responses and impact.

Chemoenzymatic Synthesis, Nanotization, and Anti-Aspergillus Activity of Optically Enriched Fluconazole Analogues.

Despite recent advances in diagnostic and therapeutic methods in antifungal research, aspergillosis still remains a leading cause of morbidity and mortality. One strategy to address this problem is to enhance the activity spectrum of known antifungals, and we now report the first successful application of Candida antarctica lipase (CAL) for the preparation of optically enriched fluconazole analogues. Anti-Aspergillus activity was observed for an optically enriched derivative, (-)-S-2-(2',4'-difluorophenyl)-1-hexyl-amino-3-(1‴,2‴,4‴)triazol-1‴-yl-propan-2-ol, which exhibits MIC values of 15.6 µg/ml and 7.8 µg/disc in broth microdilution and disc diffusion assays, respectively. This compound is tolerated by mammalian erythrocytes and cell lines (A549 and U87) at concentrations of up to 1,000 µg/ml. When incorporated into dextran nanoparticles, the novel, optically enriched fluconazole analogue exhibited improved antifungal activity against Aspergillus fumigatus (MIC, 1.63 µg/ml). These results not only demonstrate the ability of biocatalytic approaches to yield novel, optically enriched fluconazole derivatives but also suggest that enantiomerically pure fluconazole derivatives, and their nanotized counterparts, exhibiting anti-Aspergillus activity may have reduced toxicity.

Molecular Modeling Evaluation of the Enantiomers of a Novel Adenylyl Cyclase 2 Inhibitor.

Adenylyl cyclase 2 (AC2) is one of nine membrane-bound isoforms of adenylyl cyclase that converts ATP into cyclic AMP (cAMP), an important second messenger molecule. Upregulation of AC2 is linked to cancers like pancreatic and small intestinal neuroendocrine tumors (NETs). The structures of the various isoforms of adenylyl cyclases are highly homologous, posing a significant challenge to drug discovery efforts for an effective, isoform-selective modulator of AC2. In a previous study, a screen identified a potential isoform-selective and noncompetitive inhibitor of AC2, SKF83566. In the present study, molecular modeling is used to explore the mode of inhibition of AC2 by SKF83566 and to investigate the active enantiomer of SKF83566. Homology models of hAC2 were built based on canine AC5-C1a and rat AC2-C2a templates. With these models, a combination of flexible docking, molecular dynamics simulations, and free energy calculations using the MM/GBSA methodology suggested an allosteric mechanism in which (S)-SKF83566 binds to an allosteric site near ATP and alters the protein conformation of the ATP binding site, potentially preventing the adenosine moiety of ATP from forming an archlike shape to form cAMP. The predicted binding preference for the (S)-SKF83566 enantiomer and the predicted free energy are consistent with the experimental data.

Enzymatic separation of epimeric 4-C-hydroxymethylated furanosugars: Synthesis of bicyclic nucleosides.

Conversion of D-glucose to 4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose, which is a key precursor for the synthesis of different types of bicyclic/spiro nucleosides, led to the formation of an inseparable 1:1 mixture of the desired product and 4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-xylofuranose. A convenient environment friendly Novozyme®-435 catalyzed selective acetylation methodology has been developed for the separation of an epimeric mixture of ribo- and xylotrihydroxyfuranosides in quantitative yields. The structure of both the monoacetylated epimers, i.e., 5-O-acetyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribo- and xylofuranose obtained by enzymatic acetylation, has been confirmed by an X-ray study on their corresponding 4-C-p-toluenesulfonyloxymethyl derivatives. Furthermore, the two separated epimers were used for the convergent synthesis of two different types of bicyclic nucleosides, which confirms their synthetic utility.

Biocatalytic Synthesis of Novel Partial Esters of a Bioactive Dihydroxy 4-Methylcoumarin by Rhizopus oryzae Lipase (ROL).

Highly regioselective acylation has been observed in 7,8-dihydroxy-4-methylcoumarin (DHMC) by the lipase from Rhizopus oryzae suspended in tetrahydrofuran (THF) at 45 °C using six different acid anhydrides as acylating agents. The acylation occurred regioselectively at one of the two hydroxy groups of the coumarin moiety resulting in the formation of 8-acyloxy-7-hydroxy-4-methylcoumarins, which are important bioactive molecules for studying biotansformations in animals, and are otherwise very difficult to obtain by only chemical steps. Six monoacylated, monohydroxy 4-methylcoumarins have been biocatalytically synthesised and identified on the basis of their spectral data and X-ray crystal analysis.

Effect of acyl chain length on selective biocatalytic deacylation on O-aryl glycosides and separation of anomers.

It has been demonstrated that Lipozyme® TL IM (Thermomyces lanuginosus lipase immobilised on silica) can selectively deacylate the ester function involving the C-5' hydroxyl group of α-anomers over the other acyl functions of anomeric mixture of peracylated O-aryl α,ß-D-ribofuranoside. The analysis of results of biocatalytic deacylation reaction revealed that the reaction time decreases with the increase in the acyl chain length from C1 to C4. The unique selectivity of Lipozyme® TL IM has been harnessed for the separation of anomeric mixture of peracylated O-aryl α,ß-D-ribofuranosides, The lipase mediated selective deacylation methodology has been used for the synthesis of O-aryl α-D-ribofuranosides and O-aryl ß-D-ribofuranosides in pure forms, which can be used as chromogenic substrate for the detection of pathogenic microbial parasites containing glycosidases.

Recent Developments and Future Perspectives of Purine Derivatives as a Promising Scaffold in Drug Discovery.

Numerous purine-containing compounds have undergone extensive investigation for their medical efficacy across various diseases. The swift progress in purine-based medicinal chemistry has brought to light the therapeutic capabilities of purine-derived compounds in addressing challenging medical conditions. Defined by a heterocyclic ring comprising a pyrimidine ring linked with an imidazole ring, purine exhibits a diverse array of therapeutic attributes. This review systematically addresses the multifaceted potential of purine derivatives in combating various diseases, including their roles as anticancer agents, antiviral compounds (anti-herpes, anti-HIV, and anti-influenzae), autoimmune and anti-inflammatory agents, antihyperuricemic and anti-gout solutions, antimicrobial agents, antitubercular compounds, anti-leishmanial agents, and anticonvulsants. Emphasis is placed on the remarkable progress made in developing purine-based compounds, elucidating their significant target sites. The article provides a comprehensive exploration of developments in both natural and synthetic purines, offering insights into their role in managing a diverse range of illnesses. Additionally, the discussion delves into the structure-activity relationships and biological activities of the most promising purine molecules. The intriguing capabilities revealed by these purine-based scaffolds unequivocally position them at the forefront of drug candidate development. As such, this review holds potential significance for researchers actively involved in synthesizing purine-based drug candidates, providing a roadmap for the continued advancement of this promising field.

Facial cutaneous phaeohyphomycosis in an immunocompetent individual.

ABSTRACT: Fungal infection is a rare condition in immunocompetent individuals, and it is associated with high rates of morbidity and mortality. We report on a case of cutaneous phaeohyphomycosis in healthy 25-year-old man. Based on the clinical findings, the case was first thought to be cervico-facial actinomycosis, but Alternaria was identified on the culture after debridement. Simple surgical excision resulted in the complete cure without administration of systemic antifungals.

Molecular detection of enterotoxins in multidrug resistant Aeromonas from ready to eat foods in North Western Himalayas: Public health significance.

Aeromonas spp. are normal inhabitants of aquatic environments and are emerging foodborne bacterial pathogens. Aeromonas spp. contamination is frequent in ready-to-eat (RTE) seafood and can also occur in products prepared from milk or meat. The study determined the enterotoxin and antimicrobial susceptibility profiles of Aeromonas spp. isolates recovered from RTE milk products (n = 105), RTE meat/fish products (n = 40) and drinking water (n = 60) samples collected from tourist places in Himachal Pradesh, India, in northwestern Himalayas. 7.3 % (16/220) samples were found contaminated with Aeromonas spp. These isolates were identified as A. hydrophila (31.3 %), A. schubertii (25.0 %), A. sobria (25.0 %) and A. veronii (18.8 %). Aeromonas spp. contamination was significantly higher (14.3 %, 15/105, p = 0.0001) in RTE milk products. The contamination levels for water samples were 1.7 % whereas none of the tested RTE meat or fish products yielded Aeromonas spp. Among RTE milk products, contamination was significantly higher in paneer (South Asian soft cheese) (26.1 %, p = 0.0027) and cream (25.0 %, p = 0.046) based RTE foods. All isolates carried alt (361 bp), encoding a cytotonic heat-labile enterotoxin. Ampicillin resistance was 100 % and high levels (>30 %) of resistance were recorded for amoxicillin/clavulanic acid, amikacin, cefotaxime and ceftazidime. Six (37.5 %) isolates were multi drug resistant (MDR), showing resistance to aminoglycosides, cephams and penicillins. Isolation of alt carrying MDR isolates from RTE foods indicates that Aeromonas spp. can be potential foodborne public health threat in northwestern Himalayas.

Phytofabrication, characterization of silver nanoparticles using Hippophae rhamnoides berries extract and their biological activities.

Introduction: Fabrication of plant-based metal nanoparticles has yielded promising results, establishing this approach as viable, sustainable, and non-toxic in the biomedical sector for targeted drug delivery, diagnostic imaging, biosensing, cancer therapy, and antimicrobial treatments. Methods: The present work demonstrates the suitability of Hippophae rhamnoides berries for the instant green synthesis of silver nanoparticles to check their antioxidant, lipid peroxidation, and antimicrobial potential. The preliminary characterization of Hippophae rhamnoides-mediated AgNPs was validated by monitoring the color shift in the solution from pale yellow to reddish brown, which was further confirmed by UV-vis spectroscopy and the plasmon peaks were observed at 450 nm. Field Emission Scanning Electron Microscopy (FESEM) and X-ray diffraction (XRD) were used to evaluate the surface topography and structure of AgNPs. Herein, the antioxidant potential of synthesized AgNPs was investigated using DPPH free radical assay and the antimicrobial efficacy of similar was checked against E. coli and S. aureus by following MIC (minimum inhibitory concentration) and MBC (Minimum bactericidal concentration) assay. Along with the inhibitory percentage of lipid peroxidation was analysed by following TBARS (Thiobarbituric acid reactive species) assay. Results & discussion: The results revealed that the AgNPs were spherical in shape with an average size distribution within the range of 23.5-28 nm and a crystalline structure. Negative zeta potential (-19.7 mV) revealed the physical stability of synthesized AgNPs as the repulsive force to prevent immediate aggregation. The bioactive functional moieties involved in reducing bulk AgNO3 into AgNPs were further validated by FTIR. TBARS was adapted to test lipid peroxidation, and Hippophae rhamnoides-mediated AgNPs showed a 79% inhibition in lipid peroxidation compared to Hippophae rhamnoides berries extract as 65%. Furthermore, the antibacterial tests showed 37 ± 0.01 mm and 35 ± 0.0132 mm, zones of inhibition against E. coli MTCC 1698 and S. aureus MTCC 3160 with MIC and MBC values of 1 mg/mL, respectively.

Osteogenic human MSC-derived extracellular vesicles regulate MSC activity and osteogenic differentiation and promote bone regeneration in a rat calvarial defect model.

BACKGROUND: There is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects. METHODS: EVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10 µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50 µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry. RESULTS: Although Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM. CONCLUSIONS: This study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.