Herb Extracellular Vesicle-Chitosan-PEGylated Graphene Oxide Conjugate Delivers Estrogen Receptor α Targeting siRNA to Breast Cancer Cells.

Herb-based extracellular vesicles (EV), inherently replete with bioactive proteins, RNA, lipids, and other medicinal compounds, are noncytotoxic and uniquely capable of cellular delivery to meet the ever-stringent challenges of ongoing clinical applications. EVs are abundant in nature, affordable, and scalable, but they are also incredibly fragile and stuffed with many biomolecules. To address the low drug binding abilities and poor stability of EVs, we demonstrated herb-based EVs (isolated from neem, mint, and curry leaves) conjugated with chitosan (CS) and PEGylated graphene oxide (GP) that led to their transformation into robust and efficient vectors. The designed conjugates successfully delivered estrogen receptor α (ERα1)-targeting siRNA to breast cancer MCF7 cells. Our data revealed that neem-based EV-CS-GP conjugates were most efficient in cellular siRNA delivery, which could be attributed to hyaluronic acid-mediated recognition of neem EVs by MCF7 cells via CD44 receptors. Our approach shows a futuristic direction in designing clinically viable, sustainable, nontoxic EV-based vehicles that can deliver a variety of functional siRNA cargos.

Discovery of Small Molecule Glycolytic Stimulants for Enhanced ApoE Lipidation in Alzheimer's Disease Cell Model.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by pathophysiological deposits of extracellular amyloid beta (Aß) peptides and intracellular neurofibrillary tangles of tau. The central role of Aß in AD pathology is well-established, with its increased deposition attributed mainly to its decreased cerebral clearance. Here, it is noteworthy that apolipoprotein E (ApoE), the most significant risk factor for AD, has been shown to play an isoform-specific role in clearing Aß deposits (ApoE2 > ApoE3 > ApoE4), owing mainly to its lipidation status. In addition to the pathophysiological Aß deposits, AD is also characterized by abnormal glucose metabolism, which is a distinct event preceding Aß deposition. The present study established, for the first time, a possible link between these two major AD etiologies, with glucose metabolism directly influencing ApoE lipidation and its secretion by astrocytes expressing human ApoE4. Specifically, glucose dose-dependently activated liver X receptor (LXR), leading to elevated ABCA1 and ABCG1 protein levels and enhanced ApoE lipidation. Moreover, co-treatment with a glycolytic inhibitor significantly inhibited this LXR activation and subsequent ApoE lipidation, further supporting a central role of glucose metabolism in LXR activation leading to enhanced ApoE lipidation, which may help against AD through potential Aß clearance. Therefore, we hypothesized that pharmacological agents that can target cellular energy metabolism, specifically aerobic glycolysis, may hold significant therapeutic potential against AD. In this context, the present study also led to the discovery of novel, small-molecule stimulants of astrocytic glucose metabolism, leading to significantly enhanced lipidation status of ApoE4 in astrocytic cells. Three such newly discovered compounds (lonidamine, phenformin, and berberine), owing to their promising cellular effect on the glycolysis-ApoE nexus, warrant further investigation in suitable in vivo models of AD.

Development and Validation of a Questionnaire to Measure a Medical Student's Interest in the Subject of Community Medicine.

Background: Lack of interest has been cited by many studies as the predominant cause for students undervaluing the subject of Community Medicine. However, there are few valid and reliable tools that could measure this interest. To develop and validate a questionnaire to measure a medical student's interest in the subject of Community Medicine. Material and Methods: Cross-sectional study conducted at MTMC Jamshedpur. The Community Medicine Interest Questionnaire (CMIQ) was developed in two phases: item generation and item reduction. Items were generated through a review of the literature, focused group discussions, and in-depth interviews. In the item reduction phase, the content and construct validity of the questionnaire were ascertained. Content validity was carried out by a group of experts based on three parameters: the interrater agreement on the representativeness of the item, the interrater agreement on the clarity of the items, and the content validity index. The construct validity was ascertained through pilot testing of 480 responses from undergraduate medical students. Exploratory factor analysis through principal axis factoring and Promax rotation. Results: Twenty-five items were generated. Three of these items were removed following expert validation. Furthermore, three items were removed after pilot testing. The resulting CMIQ consisted of 19 items distributed over three dimensions: feeling, value, and predisposition to reengage toward the subject. The internal consistency of each of the subscales was ascertained. Conclusions: CMIQ is a valid and reliable tool that can be used to measure such interest for providing educational interventions.

Inactive-enriched machine-learning models exploiting patent data improve structure-based virtual screening for PDL1 dimerizers.

INTRODUCTION: Small-molecule Programmable Cell Death Protein 1/Programmable Death-Ligand 1 (PD1/PDL1) inhibition via PDL1 dimerization has the potential to lead to inexpensive drugs with better cancer patient outcomes and milder side effects. However, this therapeutic approach has proven challenging, with only one PDL1 dimerizer reaching early clinical trials so far. There is hence a need for fast and accurate methods to develop alternative PDL1 dimerizers. OBJECTIVES: We aim to show that structure-based virtual screening (SBVS) based on PDL1-specific machine-learning (ML) scoring functions (SFs) is a powerful drug design tool for detecting PD1/PDL1 inhibitors via PDL1 dimerization. METHODS: By incorporating the latest MLSF advances, we generated and evaluated PDL1-specific MLSFs (classifiers and inactive-enriched regressors) on two demanding test sets. RESULTS: 60 PDL1-specific MLSFs (30 classifiers and 30 regressors) were generated. Our large-scale analysis provides highly predictive PDL1-specific MLSFs that benefitted from training with large volumes of docked inactives and enabling inactive-enriched regression. CONCLUSION: PDL1-specific MLSFs strongly outperformed generic SFs of various types on this target and are released here without restrictions.

A brief review on application of design of experiment for the analysis of pharmaceuticals using HPLC.

The quality pioneer Dr. Joseph M. Juran first proposed the idea of quality by design. According to him, pharmaceutical quality by design is an organised approach to product development that starts with predetermined goals and places an emphasis on product, process understanding, control based on reliable science and quality risk management. The quality of a product or process can typically be affected by a number of input elements. Design of experiments has been employed widely recently to understand the impacts of multidimensional and interactions of input parameters on the output responses of analytical procedures and pharmaceutical goods. Depending on the design of experiments objectives, screening, characterization, or optimization of the process and formulation, a variety of designs, such as factorial or mixture, can be used. The most popular designs used in the stage of screening or factor selection are the 2-Level Factorial and Plackett-Burman designs, both of which have two levels for each factor (k), both economical and effective, and in optimization widely used designs in this step are full factorial at three levels, central composite, Box-Behnken design. The analysis of variance, regression significance, and lack of fit of the regression model were some of the key topics covered in the discussion of the main components of multiple regression model adjustment. Design of experiments is thus the primary element of the formulation and analytical quality by design. The details about design of experiments used for the analysis of pharmaceutical formulation using HPLC.

Exploring Small Molecules Targeting Protein-Protein Interactions (PPIs): Advancements and Future Prospects.

This Special Issue of Pharmaceuticals is dedicated to the clinically relevant, intricate realm of "Small Molecules Targeting Protein-Protein Interactions (PPIs): Current Strategies for the Development of New Drugs" [...].

Grossing and reporting of radical prostatectomy specimens: An evidence-based approach.

Radical prostatectomy (RP) constitutes the primary treatment option for patients with clinically localized, biopsy-proven prostate cancer that requires local treatment with curative intent. Accurate reporting of radical prostatectomy specimens is required to guide further risk stratification and management of patients. Hence, for the handling and reporting of RP specimens, a standardized protocol should be followed. Many general pathologists may not be well-versed with the guidelines for the handling of radical prostatectomy specimens. This article discusses a detailed approach to grossing techniques, including specimen description, fixation requirements, gross cut-up, and reporting of the grade and stage of RP specimens. This will enable the pathologist to aid in multidisciplinary management.

Acute Herpes Simplex Virus Laryngitis Presenting as Airway Obstruction Post Influenza: A Viral Pneumonia.

Herpes simplex virus (HSV) typically presents with mucocutaneous or genital ulcerations but can also manifest with central nervous system involvement and occasionally other visceral or mucosal sites. However, laryngeal involvement almost exclusively presents in infants and children. Very few confirmed adult cases have been reported. Adults present with a broad spectrum of symptoms, usually in the context of significant immunocompromise. Diagnosis is difficult given a wide spectrum of nonspecific presenting symptoms and usually requires tissue biopsy. Frequently, patients have severe laryngeal edema that threatens to compromise the airway and requires tracheostomy. We present a case of HSV laryngitis in a 71-year-old female who presented with septic shock, acute renal failure, and acute hypoxic respiratory failure secondary to Influenza A and bacterial pneumonia for which she required intubation. The hospitalization course included extubation failures due to stridor, a positive cuff leak test resulting in an open tracheostomy, and a laryngeal biopsy confirming HSV infection, which was successfully treated with acyclovir.

Identification of hits as anti-obesity agents against human pancreatic lipase via docking, drug-likeness, in-silico ADME(T), pharmacophore, DFT, molecular dynamics, and MM/PB(GB)SA analysis.

Obesity, characterized by excessive fat accumulation, is a major health concern. Inhibition of human pancreatic lipase, an enzyme involved in fat digestion, offers a potential strategy for weight loss and obesity treatment. This study aimed to identify polyphenols capable of forming stable complexes with human pancreatic lipase to block its activity. Molecular docking, density functional theory (DFT), molecular dynamics (MD) simulations, and MMPBGBSA calculations were employed to evaluate ligand binding, stability, and energy profiles. Pharmacophore modeling was also performed to identify key structural features for effective inhibition. Virtual screening identified ZINC000015120539, ZINC000000899200, ZINC000001531702, and ZINC000013340267 as potential candidates, exhibiting favorable binding and stable interactions over 100 ns MD simulations. These findings provide insights into the inhibitory potential of selected polyphenols on human pancreatic lipase and support further experimental investigations for obesity treatment.Communicated by Ramaswamy H. Sarma.

Acute left knee prosthetic joint infection by Francisella tularensis with literature review.

Tularemia is a severe zoonotic disease caused by gram-negative bacillus Francisella tularensis. F. tularensis species account for most cases in the United States of America (USA). Apart from the six classical clinical presentations that include glandular, ulceroglandular, oculoglandular, pharyngeal, typhoidal, and pneumonic, skeletal disease is uncommon. Rare clinical manifestations include primary and secondary skin rashes, erythema nodosum, and erythema multiforme. Infrequent skeletal manifestations have presented as osteomyelitis and prosthetic joint infections. Prosthetic joint infection by F. tularensis is a rarity. PubMed literature review revealed a total of five prosthetic joint infection cases. Here we report the sixth and the third case in the USA in a 73-year-old white male with an acute left knee prosthetic joint infection (occurring after a recent episode of left lower extremity cellulitis with septic shock) successfully treated with 14 days of doxycycline.

A case of acute non-typeable Hemophilus influenza infective endocarditis in a patient with hypocomplementemia.

Hemophilus influenzae is a gram-negative bacteria responsible for significant cases of invasive infections, especially in the pediatric population and in immunosuppressed adult patients. Before vaccination, most cases were frequently caused by capsulated or typeable variants. Due to the absence of effective vaccination against the nontypeable variant, it is now responsible for most invasive infections. Predisposing risk factors in adults include asplenia, hypocomplementemia, cancer, human immunodeficiency virus infection, and chronic cardiopulmonary disease. Immunity to the nontypeable variants causing disease is perplexing and not yet wholly described as they are genetically diverse. Infective endocarditis (IE) is a cardiac infection with devastating consequences if not detected earlier and treated appropriately. Gram-positive bacteria are the primary cause of IE overall, followed by gram-negative bacteria. Hemophilus species belong to the HACEK group of gram-negative bacteria responsible for causing IE in the pediatric population more than in adults. Hemophilus species, especially the nontypeable variant, is a rare cause of IE in adults. Here we present a case of IE due to Nontypeable Hemophilus influenzae in a 49-year-old caucasian male with hypocomplementemia.

A case of spontaneous pneumothorax due to paragonimiasis in North America with literature review.

The species, Paragonimus kellicotti , causes human paragonimiasis in North America. As a foodborne disease, human infection with P. kellicotti occurs after eating raw or undercooked crayfish containing metacercariae. Many risk factors have been described in the literature, including young adult age, male, alcohol consumption, outdoor activities involving rivers within Missouri, and ingesting raw or partially cooked crayfish. Here, we report a case of a 41-year-old male with a 5-year history of cough who presented with acute shortness of breath. Further workup showed mild eosinophilia and spontaneous pneumothorax. A definitive diagnosis was made with a lung biopsy, which showed P. kellicotti eggs. Further questioning revealed that the patient took a hunting and river rafting trip on a river in Missouri 5 years ago, though the history was negative for any crayfish consumption. Paragonimiasis should be considered in those with associated clinical features, including cough and eosinophilia, with a history of a river raft float trip in Missouri, even if the history is negative for crayfish ingestion or travel.

Discovery of small-molecule PD-1/PD-L1 antagonists through combined virtual screening and experimental validation.

Inhibition of the interaction between the PD-1 protein on activated lymphocytes and the PD-L1 protein on tumors represents a novel therapeutic approach for selective activation of the innate immune response against a variety of cancers. Therefore, the present study utilized a combined virtual and experimental screening approach to screen databases of both lead-like and larger molecules for identification of novel inhibitors of PD-1/PD-L1 interaction. First, high-throughput virtual screening of ∼3.7 million lead-like molecules using a rigid-receptor docking approach against both human PD-1 and PD-L1 proteins revealed possible small-molecule tractability of PD-1, but not PD-L1, binding interface. The subsequent work, therefore, involved screening of the National Cancer Institute (NCI) compound database against the PD-1 pocket. Several NCI compounds were identified with potential to bind to the PD-1 pocket and in turn inhibit the PD-1/PD-L1 interaction. The dynamic binding behavior of these molecules was further investigated using long 100 ns molecular dynamics (MD) stimulation revealing NSC631535 to be a potentially stable binder at PD-1 interface pocket. In support of these MD data, the experimental testing of NSC631535 exhibited 50% inhibition at ∼15 µM test concentration. The observed activity of this compound is promising as despite its relatively low molecular weight (415.5 g/mol) it is still capable of inhibiting the PD-1/PD-L1 interaction having a large interface area (∼1970 Å2). In summary, our integrated computational and experimental screening led to identification of a novel PD-1 antagonist that may serve as a starting point for further optimization into more potent small-molecule PD-1/PD-L1 inhibitors for cancer immunotherapy.

Percent positivity and phylogenetic analysis of Mycoplasma gallisepticum and Mycoplasma synoviae in commercial poultry from the different States of India.

Background and Aim: The Indian and global poultry industries suffer significant economic losses due to Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) infections, which adversely affect egg production, hatchability, weight gain, and feed efficiency in farms, thus decreasing the overall production efficiency. This study aimed to determine the percent positivity and phylogenetic analysis of MG, MS, and co-infection of both mycoplasmas in commercial poultry farms across different states of India from 2017 to 2021. Materials and Methods: A total of 3620 tracheal or choacal swabs were collected from breeder and layer farms showing clinical signs of avian mycoplasma infections from commercial poultry farms across India, and the percent positivity for MG, MS, and co-infection of both mycoplasmas were determined by Polymerase chain reaction using the 16S rRNA and vlhA genes amplification, respectively. Phylogenetic analysis was carried out by sequencing the mgc2 and vlhA genes of 2 samples of MG and 24 samples of M. synoviae to gain insight into the genetic variability of Indian strains. The data were then compared with other Indian strains, vaccines strains, and strains from other countries. Results: Our data shows the percent positivity of MG, MS, and co-infection of both MG and MS was 6.43%, 23.61%, and 15.49%, respectively. The phylogenetic relationship between MG and MS was determined using the vlhA and mgc2 genes, revealing two samples of MG and 24 samples of MS clustered with other Indian strains. M. synoviae MSM22 and previously studied M. synoviae MGS 482 clustered with vaccine strain M. synoviae MS-H. Conclusion: Mycoplasma synoviae infections in breeder, layer, and in both is predominant compared to MG across the states investigated in India. Sequenced samples of MG and MS showed evolutionary relationships with the previously studied Indian strains of MG and MS. These findings support our view that monitoring chickens for avian mycoplasma infections are of paramount significance. It further lends credence to the contention that such information will pave the way for the development of a home-grown vaccination control program and thus safeguard the poultry sector against mycoplasma infections.

Combinatorial delivery of CPI444 and vatalanib loaded on PEGylated graphene oxide as an effective nanoformulation to target glioblastoma multiforme: In vitro evaluation.

Glioblastoma multiforme (GBM) is known as the primary malignant and most devastating form of tumor found in the central nervous system of the adult population. The active pharmaceutical component in current chemotherapy regimens is mostly hydrophobic and poorly water-soluble, which hampers clinical implications. Nanodrug formulations using nanocarriers loaded with such drugs assisted in water dispersibility, improved cellular permeability, and drug efficacy at a low dose, thus adding to the overall practical value. Here, we successfully developed a water-dispersible and biocompatible nanocargo (GO-PEG) based on covalently modified graphene oxide (GO) with a 6-armed poly(ethylene glycol) amine dendrimer for effective loading of the two hydrophobic anticancer drug molecules, CPI444 and vatalanib. These drug molecules target adenosine receptor (A2AR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and type III stem cell receptor tyrosine kinase (c-KIT), which plays a crucial role in cancers. The effective cellular delivery of the drugs when loaded on GO-PEG is attributed to the increased permeability of the drug-nanoconjugate formulation. We observed that this combinatorial drug treatment with nanocargo resulted in a significant reduction in the overall cell survival as supported by reduced calcium levels and stem cell markers such as Oct4 and Nanog, which are two of the prime factors for GBM stem cell proliferation. Furthermore, reduced expression of CD24 upon treatment with nanoformulation impeded cellular migration. Cellular assays confirmed inhibition of cell proliferation, migration, and angiogenic potential of GBM treated with GO-PEG-Drug conjugates. Ultimately, GBM U87 cells assumed programmed cell death at a very low concentration due to nanocarrier-mediated drug delivery along with the chosen combination of drugs. Together, this study demonstrated the advantage of GO-PEG mediated combined delivery of CPI444 and vatalanib drugs with increased permeability, a three-pronged combinatorial strategy toward effective GBM treatment.

Acute disseminated actinomycosis presenting as pneumonia with bilateral pulmonary nodules and pelvic osteomyelitis in an immunocompetent patient.

Actinomycosis is an indolent human infectious disease caused by gram-positive anaerobic filamentous bacteria Actinomyces. Despite its sluggish growth, clinical manifestations can be acute or chronic. Over the last five decades, a significant incidence decline in the western world is due to the discovery of effective antimicrobials and improved oral hygiene. Actinomycosis is now rarely encountered and often misdiagnosed as its manifestations mimic malignancy and other infectious diseases. Due to prior use of antimicrobials, laboratory diagnostic processes often fail to isolate the organism making it arduous to establish the diagnosis. Clinical classification is based on the geographical distribution of the disease as oro-cervicofacial, thoracic, abdominopelvic, neurologic, musculoskeletal, and disseminated. Disseminated and pulmonary actinomycosis in an immunocompetent individual is extremely rare. Here we present a 53-year-old healthy male presenting with acute disseminated actinomycosis with bilateral pulmonary nodules, right upper lobe pneumonia, and pelvic osteomyelitis from Actinomyces odontolyticus infection.

A fast, single column high-performance anion exchange chromatography with pulsed amperometric detection method for the determination of saccharides in atmospheric aerosol samples.

Saccharides, especially anhydro sugars present in atmospheric aerosols, can be used as tracers to track sources of atmospheric aerosols. High-performance anion-exchange chromatography with pulsed amperometric detection is a commonly used technique for determining these saccharides, but the reported methods suffer from three drawbacks. First, to achieve separation of the complete set of atmospheric saccharides, run times are very long, typically longer than 60 minutes. Second, some methods require two columns to achieve the desired separation. Finally, in an era when electrolytic eluent preparation allows for excellent precision and accuracy, these methods require manually prepared eluents, which can lead to separation inconsistency for closely eluting analytes. These drawbacks make existing methods difficult to automate. To address this issue, we developed a fast method that uses only a single column for separation and electrolytically generated eluent that resolves 12 key atmospheric aerosol saccharides in 20 minutes. The resolved saccharides include anhydro sugars (levoglucosan, galactosan, and mannosan), sugar alcohols (erythritol, xylitol, and mannitol), and mono-/disaccharides (arabinose, galactose, glucose, mannose, fructose, and sucrose). To our knowledge, this report is the first instance of achieving such a significant reduction in run time with good resolution for this set of saccharides.

Dendrimer-Functionalized Nanodiamonds as Safe and Efficient Drug Carriers for Cancer Therapy: Nucleus Penetrating Nanoparticles.

Nanodiamonds (NDs) are increasingly being assessed as potential candidates for drug delivery in cancer cells and they hold great promise in overcoming the side effects of traditional chemotherapeutics. In the current work, carboxylic acid functionalized nanodiamonds (ND-COOH) were covalently modified with poly(amidoamine) dendrimer (PAMAM) to form amine-terminated nanodiamonds (NP). Unlike ND-COOH, the chemically modified nanodiamond platform NP revealed a pH-independent aqueous dispersion stability, enhancing its potential as an effective carrier. Physical encapsulation of poorly water soluble cabazitaxel (CTX) drug on NP formed ND-PAMAM-CTX (NPC) nanoconjugates and substantially reduced the size of CTX from micrometer to nanometer. CTX was localized within the pores of nanoparticle aggregates and the cavities of the PAMAM dendrimer, thus facilitating the loaded drug's controlled and sustained release. NPC's cumulative CTX release efficiency was determined to be ∼95% at pH 4 after 96 h. A high cellular uptake of NPC both within the cytoplasm and nucleus of U87 cells is confirmed, accounting for a reduced IC50 value (1 nM). Both the cell cycle and Western blot analyses confirmed enhanced cell death and suppressed tubulin protein expression in NPC-treated cells. A significantly high inhibition to cell division with early apoptosis and reduced metastasis demonstrates the effective loading of CTX dosages on the nanocarrier. The present work highlights the potential of a newly designed nanocarrier NP as an efficient nanocargo for cellular delivery applications and may provide future insights to treat one of the most aggressive tumors in neuro-oncological research, glioblastoma multiforme (GBM).

Symptomatic Pancreaticojejunal Anastomotic Stricture After Pancreaticoduodenectomy Managed by Longitudinal Pancreaticogastrostomy: A Report of Two Cases.

Symptomatic pancreaticojejunal anastomotic stricture is a rare complication following pancreaticoduodenectomy. Literature for the management of pancreaticojejunal anastomotic strictures is limited. Revision of pancreaticojejunostomy anastomosis, endoscopic dilation, stenting of pancreaticojejunal stricture, and modified Puestow procedure have all been described with variable outcomes. We present a report of two patients who developed symptomatic pancreaticojejunal anastomotic stricture following a pancreaticoduodenectomy, managed by longitudinal pancreaticogastrostomy with no complications, and resolution of symptoms with an average follow-up interval of 45 months.

Machine-Learning Guided Discovery of Bioactive Inhibitors of PD1-PDL1 Interaction.

The selective activation of the innate immune system through blockade of immune checkpoint PD1-PDL1 interaction has proven effective against a variety of cancers. In contrast to six antibody therapies approved and several under clinical investigation, the development of small-molecule PD1-PDL1 inhibitors is still in its infancy with no such drugs approved yet. Nevertheless, a promising series of small molecules inducing PDL1 dimerization has revealed important spatio-chemical features required for effective PD1-PDL1 inhibition through PDL1 sequestration. In the present study, we utilized these features for developing machine-learning (ML) classifiers by fitting Random Forest models to six 2D fingerprint descriptors. A focused database of ~16 K bioactive molecules, including approved and experimental drugs, was screened using these ML models, leading to classification of 361 molecules as potentially active. These ML hits were subjected to molecular docking studies to further shortlist them based on their binding interactions within the PDL1 dimer pocket. The top 20 molecules with favorable interactions were experimentally tested using HTRF human PD1-PDL1 binding assays, leading to the identification of two active molecules, CRT5 and P053, with the IC50 values of 22.35 and 33.65 µM, respectively. Owing to their bioactive nature, our newly discovered molecules may prove suitable for further medicinal chemistry optimization, leading to more potent and selective PD1-PDL1 inhibitors. Finally, our ML models and the integrated screening protocol may prove useful for screening larger libraries for novel PD1-PDL1 inhibitors.