Efficacy and Safety of Stem Cell Therapy for Orthopedic Conditions, Including Osteoarthritis and Bone Defects.

INTRODUCTION: Orthopedic conditions like osteoarthritis and bone defects pose significant challenges due to their impact on individuals' quality of life. Traditional treatments often provide only symptomatic relief, necessitating alternative therapies for long-term management. Stem cell therapy has grabbed attention for its regenerative and immunomodulatory properties, offering potential for tissue repair and functional restoration. OBJECTIVE: This study aims to assess the efficacy and safety of stem cell therapy for orthopedic conditions, specifically osteoarthritis and bone defects. MATERIALS AND METHODS: A retrospective cross-sectional study analyzed data from patients who underwent stem cell therapy for osteoarthritis or bone defects between January and September 2023. Outcome measures focused on pain and function improvements using tools such as Visual Analog Scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), alongside radiographic assessments. Adverse events, range of motion, quality of life, and demographic factors were also examined. Data were collected from electronic medical records while maintaining patient confidentiality. Descriptive statistics using SPSS (IBM Corp., Armonk, NY, USA) were employed to analyze patient characteristics, treatment variables, and outcomes, with statistical significance determined using Chi-square test and Independent t-test. RESULTS: Out of 50 individuals, the majority, i.e., 35 (or 70%), were diagnosed with osteoarthritis, while the remaining 15 (30%) had bone defects. Treatment outcomes showed significant improvements in pain and function, with a decrease in mean VAS and WOMAC scores at the six-month follow-up. Seven participants (28%) reported adverse events, and two participants (8%) experienced serious adverse events. CONCLUSION: Stem cell therapy shows promise for treating orthopedic conditions like osteoarthritis and bone defects. While demonstrating efficacy in pain management and functional improvement, safety considerations warrant further investigation and optimization of treatment protocols. Future research should focus on refining stem cell therapy techniques and addressing safety concerns to maximize its therapeutic potential in orthopedic practice.

Students' perceptions of and satisfaction with their Orthopaedic posting learning environment by using the Healthcare Education Micro-Learning Environment Measure (HEMLEM) questionnaire.

BACKGROUND: The learning environment in medical education is crucial for student development, encompassing social, psychological, and physical aspects that significantly affect learning. This study aimed to assess undergraduate medical students' perception of the orthopaedic ward's learning environment and examine the factors influencing their overall satisfaction during clinical rotation. METHODS: This cross sectional quantitative study was conducted in a private medical college in Pakistan. Data was collected through a pre-validated questionnaire, "The Healthcare Education Micro-Learning Environment Measure (HEMLEM)." Data analysis was done using SPSS version 23 software. RESULTS: A total of 205/300 students (response rate 68.33%) [103 (50.2%) males and 102(49.85) females] participated in this survey. Notably, 116 (56.6%) appreciated the ward's welcoming, friendly, and open atmosphere, and 114(55.6%) of the respondents appreciated the ward culture where they felt free to ask questions or comment. Additionally, 111(54.7%) appreciated the faculty's enthusiasm for teaching. A comparison between male and female students showed significantly higher satisfaction among males regarding staff attitudes and behaviours (p < .019). CONCLUSION: Undergraduate students held a predominantly positive view of the orthopaedic ward's learning environment, with differences observed based on gender and year of study. The study highlights the importance of both staff attitude and teaching quality in shaping the educational experience. It suggests that medical institutions should focus on enhancing teaching skills among clinicians to improve learning experiences and ultimately benefit patient care and the healthcare system.

Bringing back Galium aparine L. from forgotten corners of traditional wound treatment procedures: an antimicrobial, antioxidant, and in-vitro wound healing assay along with HPTLC fingerprinting study.

BACKGROUND: The wound healing process, restoring the functionality of the damaged tissue, can be accelerated by various compounds. The recent experimental analysis highlights the beneficial effects of phytochemicals in improving skin regeneration and wound healing. In traditional medicine, one of the widespread plants used for treating different injuries or skin afflictions is Galium aparine L. (GA). Besides, previously reported chemical compounds of GA suggested its therapeutic effects for the wound healing process, yet its regulatory effects on the cellular and molecular stages of the wound healing process have not been investigated. METHODS: In the present study, the phytochemical profile of the GA extract was analyzed using HPTLC fingerprinting, and further scientific evaluation of its phytochemicals was done. The wound-healing effects of GA extract were explored at the cellular and molecular levels while accounting for cell toxicity. The wound closure enhancing effect, antibacterial activity, and antioxidant activity were assessed. RESULTS: The HPTLC fingerprinting of the GA extract proved its previously reported phytochemical profile including phenols, flavonoids, tannins, plant acids, ergot alkaloids, flavonoids, anthraquinones, terpenoids, sterols, salicin, lipophilic compounds, saponins, iridoids, and heterocyclic nitrogen compounds. Antimicrobial assessment, of the extract, indicated the more susceptibility of S. aureus to the inhibitory effects of GA rather than E. coli and S. epidermidis. DPPH test results revealed the antioxidant property of GA extract, which was comparable to ascorbic acid. The results of the viability assay showed no cytotoxicity effects on human umbilical endothelial cell (HUVEC) and normal human dermal fibroblast (NHDF) cell lines treated with different concentrations of whole plant extract and cell viability increased in a dose-dependent manner. The results of the scratch assay showed improved cell migration and wound closure. CONCLUSIONS: This study shows the anti-oxidant, anti-microbial, and in vitro wound healing wound-healing effects of GA hydroalcoholic extract, which aligns with its use in traditional medicine. No cytotoxicity effects were shown. The results from this study can be the basis for further investigations such as animal models and phytochemical studies. Further evaluation of its effects on mechanisms and signaling pathways involved in the wound healing processes such as angiogenesis and cell proliferation can provide novel insights into the potential therapeutic effects of the GA extract.

Mitigating drought-induced oxidative stress in wheat (Triticum aestivum L.) through foliar application of sulfhydryl thiourea.

Drought stress is a major abiotic stress affecting the performance of wheat (Triticum aestivum L.). The current study evaluated the effects of drought on wheat phenology, physiology, and biochemistry; and assessed the effectiveness of foliar-applied sulfhydryl thiourea to mitigate drought-induced oxidative stress. The treatments were: wheat varieties; V1 = Punjab-2011, V2 = Galaxy-2013, V3 = Ujala-2016, and V4 = Anaaj-2017, drought stress; D1 = control (80% field capacity [FC]) and D2 = drought stress (40% FC), at  the reproductive stage, and sulfhydryl thiourea (S) applications; S0 = control-no thiourea and S1 = foliar thiourea application @ 500 mg L-1. Results of this study indicated that growth parameters, including height, dry weight, leaf area index (LAI), leaf area duration (LAD), crop growth rate (CGR), net assimilation rate (NAR) were decreased under drought stress-40% FC, as compared to control-80% FC. Drought stress reduced the photosynthetic efficiency, water potential, transpiration rates, stomatal conductances, and relative water contents by 18, 17, 26, 29, and 55% in wheat varieties as compared to control. In addition, foliar chlorophyll a, and b contents were also lowered under drought stress in all wheat varieties due to an increase in malondialdehyde and electrolyte leakage. Interestingly, thiourea applications restored wheat growth and yield attributes by improving the production and activities of proline, antioxidants, and osmolytes under normal and drought stress as compared to control. Thiourea applications improved the osmolyte defense in wheat varieties as peroxidase, superoxide dismutase, catalase, proline, glycine betaine, and total phenolic were increased by 13, 20, 12, 17, 23, and 52%; while reducing the electrolyte leakage and malondialdehyde content by 49 and 32% as compared to control. Among the wheat varieties, Anaaj-2017 showed better resilience towards drought stress and also gave better response towards thiourea application based on morpho-physiological, biochemical, and yield attributes as compared to Punjab-2011, Galaxy-2013, and Ujala-2016. Eta-square values showed that thiourea applications, drought stress, and wheat varieties were key contributors to most of the parameters measured. In conclusion, the sulfhydryl thiourea applications improved the morpho-physiology, biochemical, and yield attributes of wheat varieties, thereby mitigating the adverse effects of drought.  Moving forward, detailed studies pertaining to the molecular and genetic mechanisms under sulfhydryl thiourea-induced drought stress tolerance are warranted.

Nexus among the perceived infrastructural, social, economic, and environmental impact of CPEC: A case of Pakistan.

Despite its ambitious "economic sustainability" objectives, the China-Pakistan Economic Corridor (CPEC) has been the subject of growing environmental anxiety. Considering the CPEC developments, it is clear that Pakistan is ready to fully embrace this new industrial chapter and take advantage of its major benefits to solve social, energy, infrastructure, and economic problems. However, it should also seriously commit to undertaking proper environmental impact assessments and upgrading system resilience. Data was collected from 400 respondents from Pakistan, and structural equation modeling was applied with the help of AMOS. Factor analysis and structural equation modeling techniques were used to estimate the results and test the study's hypothesis. The results indicate a strong socio-economic impact across perceived economic, infrastructure, social, and total impacts, but they identify a negative association between infrastructure innovation and environmental sustainability. Moreover, results revealed that infrastructure supports social and economic growth, but it might have a substantial negative impact on biodiversity. According to findings, Pakistan may be more vulnerable to climate change due to three potential environmental issues: coal-fired power plants, CO2 concentration along the CPEC route, and increased traffic on the Karakorum Highway. Furthermore, future international trade will be significantly impacted by the corridor. It may, however, also accelerate the destruction of the ecosystem over time due to the industrial revolution.

Formulation and Optimization of Effective Oil Spill Dispersants Composed of Surface-Active Ionic Liquids and Nonionic Surfactants.

The use of chemical dispersants to remove oil spills in aquatic environments raises serious concerns, including heightened toxicity and limited biodegradability, which diminish their effectiveness. This study aimed to develop an environmentally friendly formulation by combining two nonionic surfactants (Tween 80, Span 80) with two surface-active ionic liquids (SAILs): 1-butyl-3-methylimidazolium lauroyl sarcosinate [Bmim][Lausar] and choline myristate [Cho][Mys], to remediate crude oil spill. The performance of the formulation was evaluated by its emulsion stability, surface tension, interfacial tension (IFT), and effectiveness. The toxicity and biodegradability of the formulation were also assessed to ensure their safe application in aquatic environments. The formulation (F9) exhibited the most stable emulsion, maintaining stability even after 5 h with a critical micelle concentration (CMC) of 3.52 mM. The efficiency of the formulation in dispersing various crude oils (Arab, Ratawi, and Doba) ranged from 70.12 to 93.72%. Acute toxicity tests conducted on zebrafish demonstrated that the formulation, with an LC50 value of 450 mg L-1, exhibited practically nontoxicity after 96 h. The formulation showed rapid biodegradability, exceeding 60% within a 28-day testing period. This research presents a promising approach for synthesizing the green formulation which can contribute to mitigating the environmental impacts of oil spills and enhancing the efficiency of cleanup operations.

Nanoparticle-Mediated Mucosal Vaccination: Harnessing Nucleic Acids for Immune Enhancement.

Recent advancements in in vitro transcribed mRNA (IVT-mRNA) vaccine manufacturing have attracted considerable interest as advanced methods for combating viral infections. The respiratory mucosa is a primary target for pathogen attack, but traditional intramuscular vaccines are not effective in generating protective ion mucosal surfaces. Mucosal immunization can induce both systemic and mucosal immunity by effectively eliminating microorganisms before their growth and development. However, there are several biological and physical obstacles to the administration of genetic payloads, such as IVT-mRNA and DNA, to the pulmonary and nasal mucosa. Nucleic acid vaccine nanocarriers should effectively protect and load genetic payloads to overcome barriers i.e., biological and physical, at the mucosal sites. This may aid in the transfection of specific antigens, epithelial cells, and incorporation of adjuvants. In this review, we address strategies for delivering genetic payloads, such as nucleic acid vaccines, that have been studied in the past and their potential applications.

Osteoprotegerin (OPG): A potential biomarker for adverse cardiovascular events in stable coronary artery disease.

Background: Osteoprotegerin (OPG) is a secretory glycoprotein known for its involvement in bone metabolism and immune regulation. Research has extended OPG's significance in cardiovascular diseases (CVDs). Elevated OPG levels have been associated with increased cardiovascular risks, prompting interest in its role as a potential biomarker. Main Body: This study summarizes several studies that investigated the relationship between OPG levels and the incidence of CVD. The studies indicate that higher plasma levels of OPG are associated with an increased incidence of all-cause death, cardiovascular death, and heart failure, even after adjusting for clinical confounders. Moreover, the findings suggest that OPG has the potential to serve as a predictive biomarker for adverse cardiovascular events in the patient population studied. The findings suggest that OPG could aid in risk stratification, allowing clinicians to identify high-risk patients who might benefit from intensified preventive measures or tailored therapeutic interventions. Therefore, early identification of individuals at risk for adverse cardiovascular events could lead to improved patient outcomes and reduced disease burden. Conclusions: OPG's role in bone health and immune regulation has expanded to potential use as a biomarker for adverse cardiovascular events in stable coronary artery disease (CAD) patients. Despite limitations, its association with cardiovascular risks highlights its importance in risk assessment and personalized interventions.

Early prognostication of overall survival for pediatric diffuse midline gliomas using MRI radiomics and machine learning: A two-center study.

Background: Diffuse midline gliomas (DMG) are aggressive pediatric brain tumors that are diagnosed and monitored through MRI. We developed an automatic pipeline to segment subregions of DMG and select radiomic features that predict patient overall survival (OS). Methods: We acquired diagnostic and post-radiation therapy (RT) multisequence MRI (T1, T1ce, T2, and T2 FLAIR) and manual segmentations from 2 centers: 53 from 1 center formed the internal cohort and 16 from the other center formed the external cohort. We pretrained a deep learning model on a public adult brain tumor data set (BraTS 2021), and finetuned it to automatically segment tumor core (TC) and whole tumor (WT) volumes. PyRadiomics and sequential feature selection were used for feature extraction and selection based on the segmented volumes. Two machine learning models were trained on our internal cohort to predict patient 12-month survival from diagnosis. One model used only data obtained at diagnosis prior to any therapy (baseline study) and the other used data at both diagnosis and post-RT (post-RT study). Results: Overall survival prediction accuracy was 77% and 81% for the baseline study, and 85% and 78% for the post-RT study, for internal and external cohorts, respectively. Homogeneous WT intensity in baseline T2 FLAIR and larger post-RT TC/WT volume ratio indicate shorter OS. Conclusions: Machine learning analysis of MRI radiomics has potential to accurately and noninvasively predict which pediatric patients with DMG will survive less than 12 months from the time of diagnosis to provide patient stratification and guide therapy.

Neuroprotective potential of topiramate, pregabalin and lacosamide combination in a rat model of acute SE and intractable epilepsy: Perspectives from electroencephalographic, neurobehavioral and regional degenerative analysis.

The lithium-pilocarpine model is commonly used to recapitulate characteristics of human intractable focal epilepsy. In the current study, we explored the impact of topiramate (TPM) alone and in combination with pregabalin and lacosamide administration for 6 weeks on the evolution of spontaneous recurrent seizures (SRS) and disease-modifying potential on associated neuropsychiatric comorbidities. In addition, redox impairments and neurodegeneration in hippocampus regions vulnerable to temporal lobe epilepsy (TLE) were assessed by cresyl violet staining. Results revealed that acute electrophysiological (EEG) profiling of the ASD cocktail markedly halted sharp ictogenic spikes as well as altered dynamics of brain wave oscillations thus validating the need for polytherapy vs. monotherapy. In TLE animals, pharmacological intervention for 6 weeks with topiramate 10 mg/kg in combination with PREG and LAC at the dose of 20 mg/kg exhibited marked protection from SRS incidence, improved body weight, offensive aggression, anxiety-like behavior, cognitive impairments, and depressive-like behavior (p < 0.05). Moreover, combination therapy impeded redox impairments as evidenced by decreased MDA and AchE levels and increased activity of antioxidant SOD, GSH enzymes. Furthermore, polytherapy rescued animals from SE-induced neurodegeneration with increased neuronal density in CA1, CA3c, CA3ab, hilus, and granular cell layer (GCL) of the dentate gyrus. In conclusion, early polytherapy with topiramate in combination with pregabalin and lacosamide prompted synergy and prevented epileptogenesis with associated psychological and neuropathologic alterations.

Bayesian sample size determination for coefficient of variation of normal distribution.

Sample size determination is an active area of research in statistics. Generally, Bayesian methods provide relatively smaller sample sizes than the classical techniques, particularly average length criterion is more conventional and gives relatively small sample sizes under the given constraints. The objective of this study is to utilize major Bayesian sample size determination techniques for the coefficient of variation of normal distribution and assess their performance by comparing the results with the freqentist approach. To this end, we noticed that the average coverage criterion is the one that provides relatively smaller sample sizes than the worst outcome criterion. By comparing with the existing frequentist studies, we show that a smaller sample size is required in Bayesian methods to achieve the same efficiency.

FDA-Approved Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) Inhibitors for Managing Rheumatoid Arthritis: A Narrative Review of the Literature.

Rheumatoid arthritis (RA) is a complex autoimmune disease causing chronic joint inflammation and, in more serious cases, organ involvement. RA typically affects people between the ages of 35 and 60; however, it can also afflict children younger than the age of 16 years and can also demonstrate a pattern of remission later in the disease course. Non-steroidal anti-inflammatory drugs, glucocorticoids, exercise, and patient education are all used in the management of RA, which is divided into symptomatic management and disease-modifying management (disease-modifying antirheumatic drugs) to reduce pain and inflammation, thereby preserving joint function. Janus kinase inhibitors (JAKis) have led to a substantial improvement in the management of RA. By specifically targeting the JAK-signal transducer and activator of transcription pathway, which is essential for immunological modulation, these inhibitors also demonstrate promise in treating various autoimmune illnesses, including inflammatory bowel diseases, giant cell arteritis, ankylosing spondylitis, and psoriatic arthritis. Tofacitinib, baricitinib, upadacitinib, peficitinib, delgocitinib, and filgotinib are examples of FDA-approved JAKis that have distinct properties and indications for treating a range of autoimmune illnesses. JAKis demonstrate a promising treatment approach for managing RA and other autoimmune diseases while enhancing patient outcomes and quality of life. However, due to major safety concerns and the need for long-term success, meticulous patient monitoring is essential.

Evaluation of agricultural non-point source pollution using an in-situ and automated photochemical flow analysis system.

Off-line leachate collection from agricultural landscapes cannot guarantee precise evaluation of agricultural non-point source (ANPS) due to geospatial variations, time, and transportation from the field to the laboratory. Implementing an in-situ nitrogen and phosphorous monitoring system with a robust photochemical flow analysis is imperative for precision agriculture, enabling real-time intervention to minimize non-point source pollution and overcome the limitations posed by conventional analysis in laboratory. A reliable, robust and in-situ approach was proposed to monitor nitrogen and phosphorous for determining ANPS pollution. In this study, a home-made porous ceramic probe and the frequency domain reflectometer (FDR) based water content sensors were strategically placed at different soil depths to facilitate the collection of leachates. These solutions were subsequently analyzed by in-situ photochemical flow analysis monitoring system built across the field to estimate the concentrations of phosphorus and nitrogen. After applying both natural and artificial irrigation to the agricultural landscape, at least 10 mL of soil leachates was consistently collected using the porous ceramic probe within 20 min, regardless of the depth of the soil layers when the volumetric soil water contents are greater than 19%. The experimental results showed that under different weather conditions and irrigation conditions, the soil water content of 50 cm and 90 cm below the soil surface was 19.58% and 26.08%, respectively. The average concentrations of NH4+-N, NO3--N, PO43- are 0.584 mg/L, 15.7 mg/L, 0.844 mg/L, and 0.562 mg/L, 16.828 mg/L and 0.878 mg/L at depths of 50 cm and 90 cm below the soil surface, respectively. Moreover, the comparison with conventional laboratory spectroscopic analysis confirmed R2 values of 0.9951, 0.9943, 0.9947 average concentration ranges of NH4+-N, NO3--N, and PO43-, showcasing the accuracy and reliability of robust photochemical flow analysis in-situ monitoring system. The suggested monitoring system can be helpful in the assessment of soil nutrition for precision agriculture.

Assessment of physical literacy in 8- to 12-year-old Pakistani school children: reliability and cross-validation of the Canadian assessment of physical literacy-2 (CAPL-2) in South Punjab, Pakistan.

BACKGROUND: The increasing prevalence of physical inactivity, declining fitness, and rising childhood obesity highlight the importance of physical literacy (PL), as a foundational component for fostering lifelong health and active lifestyle. This recognition necessitates the development of effective tools for PL assessment that are applicable across diverse cultural landscapes. AIM: This study aimed to translate the Canadian Assessment of Physical Literacy-2 (CAPL-2) into Urdu and adapt it for the Pakistani cultural context, to assess PL among children aged 8-12 years in Pakistan. METHOD: The Urdu version of CAPL-2 was administered among 1,360 children aged 8-12 from 87 higher secondary schools across three divisions in South Punjab province, Pakistan. Statistical analysis includes test-retest reliability and construct validity, employing confirmatory factor analysis to evaluate the tool's performance both overall and within specific subdomains. RESULTS: The Urdu version of CAPL-2 demonstrated strong content validity, with a Content Validity Ratio of 0.89. Confirmatory factor analysis supported the four-factor structure proposed by the original developers, evidenced by excellent model fit indices (GFI = 0.984, CFI = 0.979, TLI = 0.969, RMSEA = 0.041). High internal consistency was observed across all domains (α = 0.988 to 0.995), with significant correlations among most, excluding the Knowledge and Understanding domains. Notably, gender and age significantly influenced performance, with boys generally scoring higher than girls, with few exceptions. CONCLUSION: This study marks a significant step in the cross-cultural adaptation of PL assessment tools, successfully validating the CAPL-2 Urdu version for the Pakistani context for the first time. The findings affirm the tool's suitability for assessing PL among Pakistani children, evidencing its validity and reliability across the Pakistani population.

Sero-epidemiology of bluetongue in ruminants raised on private holdings in North-Western Pakistan.

This study investigated the sero-epidemiology of bluetongue in ruminants in North-Western Pakistan. A total of 3,173 serum samples were collected from small (n = 1,651) and large (n = 1,522) ruminants being reared by farmers in 14 districts. Antibodies to bluetongue virus (BTV) were detected using competitive ELISA. The overall prevalence of BTV antibodies was 65%. A significant association (P < 0.05) between the prevalence of BTV antibodies and the risk factors including sex, species, age, area, husbandry practices and breed was shown by univariate analysis. In multivariate analysis, the seroprevalence was 6.5 (95% CL = 3.7-11.4), 5.9 (95% CL = 3.8-9.4) and 2.4 (95% CL = 1.5-3.7) times higher in buffaloes, cattle and goats than sheep, respectively. The seroprevalence was 1.4 (95% CL = 1.1-1.7) times higher in local breeds than in cross/exotic breeds. The seroprevalence was 1.6 (95% CL = 1.1 to 2.3) times higher in sedentary animals than in nomadic animals. The seroprevalence was significantly associated with age. Further work is required to determine the BTV serotypes prevalent in the study area for effective control of the disease.

Spontaneous Left Main Coronary Artery Dissection in a Male.

Spontaneous coronary artery dissection (SCAD) is one of the causes of acute coronary syndrome (ACS) that is increasingly recognized in young to middle-aged women without typical coronary risk factors. This case report describes a 46-year-old male with a rare presentation of SCAD involving the left main (LM) coronary artery. The patient underwent an emergency coronary angiogram for high-risk ACS and had percutaneous coronary intervention (PCI) of LM due to active ischemia and hemodynamic instability. The extension of intramural hematoma after the LM coronary artery stent confirmed the initial suspicion of SCAD. The diagnosis of SCAD is crucial, as its management differs from other causes of ACS. Coronary angiography is the gold standard for diagnosing SCAD, with adjunctive imaging using optical coherence tomography (OCT) and intravascular ultrasound (IVUS). In this patient, his physical examination findings and further imaging raised a suspicion for systemic connective tissue disease. Genetic analysis was executed, but no reportable variants in any of the 29 genes studied were identified. This case highlights the importance of recognizing SCAD as a potential cause of ACS even in men and emphasizes the findings during coronary angiography that can aid in an accurate diagnosis and appropriate management.

Problems in Commercial Kits of 25-Hydroxy Vitamin D and the Development of Simple, Robust and Faster HPLC Method.

Vitamin D is a lipid-soluble compound that plays a key role in bone mineral metabolism. The commercial current kits and several published assay methods (High-performance liquid chromatography (HPLC) and Immunoassay) are complicated due to the use of multiple reagents, larger sample volume, high backpressure, longer extraction time, evaporation under nitrogen after extraction, significant interference and antibody cross-reactivity. Here we report a new HPLC method for the determination of 25-hydroxyvitamin D2 (25-OHD2) and 25-hydroxyvitamin D3 (25-OHD3) that is simple (no evaporation), rapid (10-minute run time) and robust. Serum sample (300 µl) is mixed with 300 µl acetonitrile containing lauraphenone as internal standard. After vortexing and centrifugation, the supernatant was loaded into C18 extraction cartridges, washed with 70% methanol and then eluted with 200 µl of a mixture of 70% ethanol and 30% isopropyl alcohol (IPA). The eluent was mixed with 50 µl of water and injected into the HPLC-UV system for analysis. The method proved to be linear in the range of 10-750 nmol/L of 25-OHD2 and 25-OHD3. The intra- and inter-assay precision was less than 10 for both compounds at four different concentrations. The method was compared with (LC-MS/MS) and the correlation coefficients (R2) were 0.9454 and 0.9673 for 25-OHD2 and 25-OHD3 respectively. The proposed HPLC method is simple, rapid, robust and free from the most common problems encountered with commercial kits. It can be used in a high-volume laboratory that uses the HPLC method for the simultaneous determination of 25-OHD2 and 25-OHD3 in serum samples.

Neuroprotective Effect of Brivaracetam and Perampanel Combination on Electrographic Seizures and Behavior Anomalies in Pentylenetetrazole-Kindled Mice.

Pentylenetetrazole (PTZ)-induced kindling is a broadly used experimental model to study the anticonvulsive potential of new and existing chemical moieties with the aim of discovering drugs hindering seizure progression and associated neurological comorbidities. In the present study, the impact of brivaracetam (BRV) (10 and 20 mg/kg) as monotherapy as well as in combination with 0.25 mg/kg of perampanel (PRP) was investigated on seizure progression with simultaneous electroencephalographic changes in PTZ kindling mouse model. Subsequently, mice were experimentally analyzed for anxiety, cognition, and depression after which their brains were biochemically evaluated for oxidative stress. The outcomes demonstrated that BRV alone delayed the kindling process, but BRV + PRP combination significantly (p < 0.0001) protected the mice from seizures of higher severity and demonstrated an antikindling effect. The PTZ-kindled mice exhibited anxiety, memory impairment, and depression in behavioral tests, which were remarkably less (p < 0.001) in animals treated with drug combination (in a dose-dependent manner) as these mice explored central, illuminated, and exposed zones of open-field test, light/dark box, and elevated plus maze. Moreover, memory impairment was demonstrated by kindled mice, which was significantly (p < 0.001) protected by BRV + PRP as animal's spontaneous alteration, object discrimination, and step-through latencies were increased in various tests employed for the assessment of cognitive abilities. The brains of PTZ-kindled mice had increased malondialdehyde and reduced antioxidant enzymes while treatment with BRV + PRP combination prevented kindling-induced elevation in oxidative markers. The outcomes of this study demonstrate that combining the PRP at low dose augmented the antiseizure properties of BRV as both drugs when administered simultaneously hindered the process of kindling by reducing PTZ-induced excessive electrical activity and oxidative stress in the brain.