Untargeted and temporal analysis of retinal lipidome in bacterial endophthalmitis.

Bacterial endophthalmitis is a blinding infectious disease typically acquired during ocular surgery. We previously reported significant alterations in retinal metabolism during Staphylococcus (S) aureus endophthalmitis. However, the changes in retinal lipid composition during endophthalmitis are unknown. Here, using a mouse model of S. aureus endophthalmitis and an untargeted lipidomic approach, we comprehensively analyzed temporal alterations in total lipids and oxylipin in retina. Our data showed a time-dependent increase in the levels of lipid classes, sphingolipids, glycerolipids, sterols, and non-esterified fatty acids, whereas levels of phospholipids decreased. Among lipid subclasses, phosphatidylcholine decreased over time. The oxylipin analysis revealed increased prostaglandin-E2, hydroxyeicosatetraenoic acids, docosahexaenoic acid, eicosapentaenoic acid, and α-linolenic acid. In-vitro studies using mouse bone marrow-derived macrophages showed increased lipid droplets and lipid-peroxide formation in response to S. aureus infection. Collectively, these findings suggest that S. aureus-infection alters the retinal lipid profile, which may contribute to the pathogenesis of bacterial endophthalmitis.

Synergistic Antibacterial Activity of Gallic Acid Based Chalcone Indl 2 by Inhibiting Efflux Pump Transporters.

As a part of novel discovery of drugs from natural resources, present study was undertaken to explore the antibacterial potential of chalcone Indl-2 in combination with different group of antibiotics. MIC of antibiotics was reduced up to eight folds against the different cultures of E. coli by both chalcones. Among the two compounds, the i. e. 1-(3', 4,'5'-trimethoxyphenyl)-3-(3-Indyl)-prop-2-enone (6, Indl-2), a chalcone derivative of gallic acid (Indl-2) was better along with tetracycline (TET) worked synergistically and was found to inhibit efflux transporters as obvious by ethidium bromide efflux confirmed by ATPase assays and docking studies. In combination, Indl-2 kills the MDREC-KG4 cells, post-antibiotic effect (PAE) of TET was prolonged and mutant prevention concentration (MPC) of TET was also decreased. In-vivo studies revealed that Indl-2 reduces the concentration of TNF-α. In acute oral toxicity study, Indl-2 was non-toxic and well tolerated up-to dose of 2000 mg/kg. Perhaps, the study is going to report gallic acid derived chalcone as synergistic agent acting via inhibiting the primary efflux pumps.

Case 314: Cerebral Proliferative Angiopathy.

HISTORY: An 18-year-old man presented to the neurosurgery outpatient department with recurrent episodes of epistaxis for the past 8 years and altered behavior for the past month. Epistaxis was scanty in amount, intermittent, spontaneous, and not associated with any trauma or nasal obstruction or breathing difficulties. Bleeding used to stop spontaneously after some time. There was no history of associated headache, seizures, vomiting, fever, or loss of consciousness. On physical examination, the patient was afebrile, with normal vital signs and normal Glasgow Coma Scale score (15 of 15) at the time of presentation. Multiple dilated engorged veins were visible on the forehead; however, there was no evidence of abnormal skin pigmentation. Neurologic examination yielded findings that were within normal limits. Laboratory examinations revealed a hemoglobin level of 11 g/dL (normal range, 13.2-16.6 g/dL), with the rest of the parameters within normal limits. This patient underwent unenhanced CT of the brain and paranasal sinuses followed by contrast-enhanced MRI of the brain for further evaluation.

Case 314.

HISTORY: An 18-year-old man presented to the neurosurgery outpatient department with recurrent episodes of epistaxis for the past 8 years and altered behavior for the past month. Epistaxis was scanty in amount, intermittent, spontaneous, and not associated with any trauma or nasal obstruction or breathing difficulties. Bleeding used to stop spontaneously after some time. There was no history of associated headache, seizures, vomiting, fever, or loss of consciousness. On physical examination, the patient was afebrile, with normal vital signs and normal Glasgow Coma Scale score (15 of 15) at the time of presentation. Multiple dilated engorged veins were visible on the forehead; however, there was no evidence of abnormal skin pigmentation. Neurologic examination yielded findings that were within normal limits. Laboratory examinations revealed a hemoglobin level of 11 g/dL (normal range, 13.2-16.6 g/dL), with the rest of the parameters within normal limits. This patient underwent unenhanced CT of the brain and paranasal sinuses followed by contrast-enhanced MRI of the brain for further evaluation (Figs 1-3).

Whole exome sequencing identifies novel variants of PIK3CA and validation of hotspot mutation by droplet digital PCR in breast cancer among Indian population.

BACKGROUND: Breast cancer (BC) is the most common malignancy with very high incidence and relatively high mortality in women. The PIK3CA gene plays a pivotal role in the pathogenicity of breast cancer. Despite this, the mutational status of all exons except exons 9 and 20 still remains unknown. METHODS: This study uses the whole exome sequencing (WES) based approach to identify somatic PIK3CA mutations in Indian BC cohorts. The resultant hotspot mutations were validated by droplet digital PCR (ddPCR). Further, molecular dynamics (MD) simulation was applied to elucidate the conformational and functional effects of hotspot position on PIK3CA protein. RESULTS: In our cohort, PIK3CA showed a 44.4% somatic mutation rate and was among the top mutated genes. The mutations of PIK3CA were confined in Exons 5, 9, 11, 18, and 20, whereas the maximum number of mutations lies within exons 9 and 20. A total of 9 variants were found in our study, of which 2 were novel mutations observed on exons 9 (p.H554L) and 11 (p.S629P). However, H1047R was the hotspot mutation at exon 20 (20%). In tumor tissues, there was a considerable difference between copy number of wild-type and H1047R mutant was detected by ddPCR. Significant structural and conformational changes were observed during MD simulation, induced due to point mutation at H1047R/L position. CONCLUSIONS: The current study provides a comprehensive view of novel as well as reported single nucleotide variants (SNVs) in PIK3CA gene associated with Indian breast cancer cases. The mutation status of H1047R/L could serve as a prognostic value in terms of selecting targeted therapy in BC.

A double blind randomized controlled trial to assess efficacy of nutritional therapy for prevention of recurrence of hepatic encephalopathy in patients with cirrhosis.

BACKGROUND AND AIM: Overt hepatic encephalopathy (OHE) has high risk of recurrence and is associated with poor survival. The role of nutrition therapy is well documented in cirrhosis, but its efficacy in preventing the recurrence of OHE has not been studied. METHODS: In double blind RCT, we randomly assigned 150 patients with liver cirrhosis, with history of OHE in recent past to receive nutrition therapy (group I) or no nutrition therapy (group II) and followed up for 6 months. The primary efficacy end points were occurrence of breakthrough episodes and time to breakthrough episode of OHE. Secondary end points were OHE related hospitalizations and time to hospitalization involving OHE. Other parameters included anthropometry, changes in serum cytokines (IL-1, IL-6, IL-10, and TNF-α), endotoxin and myostatin. RESULTS: There was significant reduction in occurrence of breakthrough episodes of OHE in group I [10 vs 36, hazard ratio 0.20; P < 0.001], OHE-related hospitalization [8 vs 24, hazard ratio 0.27; P < 0.001)]. Times to breakthrough episode of OHE and OHE-related hospitalization were longer in group I. At the end of 6 months, inflammatory and anthropometry parameters showed significant improvement in group I compared with worsening of serum albumin, anthropometric parameters, IL-6, IL-10 and TNF-α in group II. At the end of 6 months, ascites (50 vs 66, P = 0.01), gastrointestinal bleed (2 vs 11, P = 0.007), and jaundice (16 vs 41, P < 0.001) were lower in group I. CONCLUSIONS: Treatment with nutrition therapy prevented recurrence of OHE and decreased OHE-related hospitalizations as compared with no nutrition therapy.

Nutritional therapy for persistent cognitive impairment after resolution of overt hepatic encephalopathy in patients with cirrhosis: A double-blind randomized controlled trial.

BACKGROUND AND AIM: Minimal hepatic encephalopathy (MHE) reflects cognitive impairment in patients with liver cirrhosis and is associated with poor prognosis. We assessed the effects of nutritional therapy on cognitive functions, health-related quality of life (HRQOL), anthropometry, endotoxins, and inflammatory markers in cirrhotic patients with MHE. METHODS: In a double-blind randomized controlled trial, cirrhotic patients with MHE were randomized to nutritional therapy (group I: 30-35 kcal/kg/day and 1.0-1.5 g of protein/kg/day) and no nutritional therapy (group II: diet as patients were taking before) for 6 months. MHE was diagnosed based on psychometric hepatic encephalopathy score (PHES). Anthropometry, ammonia, endotoxins, inflammatory markers, myostatin, and HRQOL were assessed at baseline and after 6 months. Primary endpoints were improvement or worsening in MHE and HRQOL. RESULTS: A total of 150 patients were randomized to group I (n = 75, age 46.3 ± 12.5 years, 58 men) and group II (n = 75, age 45.2 ± 9.3 years, 56 men). Baseline PHES (-8.16 ± 1.42 vs -8.24 ± 1.43; P = 0.54) was comparable in both groups. Reversal of MHE was higher in group I (73.2% vs 21.4%; P = 0.001) than group II. Improvement in PHES (Δ PHES 4.0 ± 0.60 vs -4.18 ± 0.40; P = 0.001), HRQOL (Δ Sickness Impact Profile 3.24 ± 3.63 vs 0.54 ± 3.58; P = 0.001), anthropometry, ammonia, endotoxins, cytokines, and myostatin levels was also significantly higher in group I than group II. Overt hepatic encephalopathy developed in 6 patients in group I and 13 in group II (P = 0.04). CONCLUSIONS: Nutritional therapy is effective in treatment of MHE and associated with improvement in nutritional status, HRQOL, ammonia, endotoxins, inflammatory markers, and myostatin levels.

Effective corrosion inhibition of mild steel using novel 1,3,4-oxadiazole-pyridine hybrids: Synthesis, electrochemical, morphological, and computational insights.

An easy synthesis of two 1,3,4-oxadiazole derivatives, namely, 2-phenyl-5-(pyridin-3-yl)-1,3,4-oxadiazole (POX) and 2-(4-methoxyphenyl)-5-(pyridin-3-yl)-1,3,4-oxadiazole (4-PMOX), and their corrosion-inhibition efficacy against mild steel corrosion in 1 N HCl, is evaluated using weight loss from 303 to 323 K, Electrochemical Impedance Spectroscopy (EIS), Potentiodynamic Polarization (PDP), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray (EDX), UV-Vis spectroscopy, along with theoretical evaluation. Both POX and 4-PMOX exhibit excellent inhibition efficiency, with values reaching 97.83% and 98% at 500 ppm, respectively. The PDP analysis reveals that both derivatives act as mixed-type inhibitors. The Langmuir adsorption isotherm provides insights into the adsorption phenomena, demonstrating that 4-PMOX exhibits superior adsorption behavior on the mild steel surface compared to POX. This finding is further supported by SEM, DFT, RDF, and MSD analyses. Quantum mechanical parameters, including EHOMO, ELUMO, dipole moment (µ), energy gap (ΔE), etc., are in good agreement with the effectiveness of inhibition performance revealing ΔE values of 3.10 and 2.75 for POX and 4-PMOX, respectively. The results obtained from this study hold significant implications for researchers aiming to design more efficient organic inhibitors to combat metal corrosion.

Tear Film MicroRNAs as Potential Biomarkers: A Review.

MicroRNAs are non-coding RNAs that serve as regulatory molecules in a variety of pathways such as inflammation, metabolism, homeostasis, cell machinery, and development. With the progression of sequencing methods and modern bioinformatics tools, novel roles of microRNAs in regulatory mechanisms and pathophysiological states continue to expand. Advances in detection methods have further enabled larger adoption of studies utilizing minimal sample volumes, allowing the analysis of microRNAs in low-volume biofluids, such as the aqueous humor and tear fluid. The reported abundance of extracellular microRNAs in these biofluids has prompted studies to explore their biomarker potential. This review compiles the current literature reporting microRNAs in human tear fluid and their association with ocular diseases including dry eye disease, Sjögren's syndrome, keratitis, vernal keratoconjunctivitis, glaucoma, diabetic macular edema, and diabetic retinopathy, as well as non-ocular diseases, including Alzheimer's and breast cancer. We also summarize the known roles of these microRNAs and shed light on the future progression of this field.

Treatment of pharmaceutical wastewater through activated sludge process-a critical review.

The occurrence of pharmaceutical compounds in water is a rising issue in the environment. These drugs in the waste may be toxic to aquatic organisms and humans as they disrupt the endocrine system, cause genotoxicity, etc. Several techniques were used for the treatment of pharmaceutical wastewater, such as physical, chemical, physiochemical, and biological processes like adsorption, chemical coagulation, and activated sludge processes, but these techniques possess several merits and demerits, such as higher installation and operation costs. This technique is used to remove color and turbidity; reduce biochemical oxygen demand (BOD), chemical oxygen demand (COD), and total suspended solids (TSS) to permissible limits for reuse of effluent; and prevent diseases caused by pharmaceutical wastewater. This review focuses on the treatment of pharmaceutical wastewater containing drugs like antibiotics, depressants, and hormones, with the activated sludge process having several advantages like good quality effluent and low installation costs.

Role of Procalcitonin as a Biomarker in Early Identification of Adverse Events Following Esophageal Atresia Surgery.

Introduction: Surgical complication following esophageal atresia repair is one of the several factors known to influence the final outcomes. Early identification of such complications may help in timely institution of therapeutic measures and translate into improved prognosis. Objective: The objective of this study was to evaluate the role of procalcitonin in early prediction of the adverse events after surgery in patients of esophageal atresia and the temporal relationship with clinical manifestations and other inflammatory biomarkers such as C-reactive protein (CRP). Materials and Methods: This was a prospective study on consecutive patients of esophageal atresia (n = 23). Serum procalcitonin and CRP levels were assessed at baseline (prior to surgery) and on postoperative days (POD) 1, 3, 5, 7, and 14. The trends in the biomarker values and temporal relationships of deviation in trend with the clinical and conventional laboratory parameters and patient outcomes were analyzed. Results: Baseline serum procalcitonin was elevated (n = 23; 1.7 ng/ml: min: 0.07 ng/ml-max: 24.36 ng/ml) in 18/23 (78.3%) patients. Procalcitonin nearly doubled on POD-1 (n = 22; 3.28 ng/ml: min: 0.64 ng/ml-max: 16.51 ng/ml) followed by a gradual decline. CRP was also elevated on POD-1 (three times the baseline) and depicted a delayed peak at POD-3. POD-1 procalcitonin and CRP levels correlated with survival. POD-1 procalcitonin cutoff at 3.28 ng/ml predicted mortality with a sensitivity and specificity of 100% and 57.9% (P = 0.05). Serum procalcitonin and CRP were higher for patients who sustained complications, so was the time required for hemodynamic stabilization. Procalcitonin (baseline and POD-5) and CRP (POD-3 and POD-5) values correlated with the clinical course after surgery. Baseline procalcitonin cutoff at 2.91 ng/ml predicted the possibility of a major complication with a sensitivity of 71.4% and a specificity of 93.3%. POD-5 procalcitonin cutoff at 1.38 ng/ml predicted the possibility of a major complication with a sensitivity of 83.3% and a specificity of 93.3%. Patients who sustained major complications depicted a change in serum procalcitonin trend 24-48 h ahead of clinical manifestation of an adverse event. Conclusions: Procalcitonin is a good indicator to identify the adverse events in neonates after surgery for esophageal atresia. The procalcitonin levels in patients who sustained a major complication depicted a reversal in trend 24-48 h of clinical manifestation. POD-1 procalcitonin correlated with survival while the baseline and POD-5 serum procalcitonin predicted the clinical course.

MicroRNA cargo of extracellular vesicles released by skeletal muscle fibro-adipogenic progenitor cells is significantly altered with disuse atrophy and IL-1ß deficiency.

Fibro-adipogenic progenitor cells (FAPs) are a population of stem cells in skeletal muscle that play multiple roles in muscle repair and regeneration through their complex secretome; however, it is not well understood how the FAP secretome is altered with muscle disuse atrophy. Previous work suggests that the inflammatory cytokine IL-1ß is increased in FAPs with disuse and denervation. Inflammasome activation and IL-1ß secretion are also known to stimulate the release of extracellular vesicles (EVs). Here, we examined the microRNA (miRNA) cargo of FAP-derived, platelet-derived growth factor receptor A (PDGFRα+) EVs from hindlimb muscles of wild-type and IL-1ß KO mice after 14 days of single-hindlimb immobilization. Hindlimb muscles were isolated from mice following the immobilization period, and PDGFRα+ extracellular vesicles were isolated using size-exclusion chromatography and immunoprecipitation. Microarrays were performed to detect changes in miRNAs with unloading and IL-1ß deficiency. Results indicate that the PDGFRα+, FAP-derived EVs show a significant increase in miRNAs, such as miR-let-7c, miR-let-7b, miR-181a, and miR-124. These miRNAs have previously been demonstrated to play important roles in cellular senescence and muscle atrophy. Furthermore, the expression of these same miRNAs was not significantly altered in FAP-derived EVs isolated from the immobilized IL-1ß KO. These data suggest that disuse-related activation of IL-1ß can mediate the miRNA cargo of FAP-derived EVs, contributing directly to the release of senescence- and atrophy-related miRNAs. Therapies targeting FAPs in settings associated with muscle disuse atrophy may therefore have the potential to preserve muscle function and enhance muscle recovery.

Molecular mechanism(s) of regulation(s) of c-MET/HGF signaling in head and neck cancer.

Head and neck cancer is the sixth most common cancer across the globe. This is generally associated with tobacco and alcohol consumption. Cancer in the pharynx majorly arises through human papillomavirus (HPV) infection, thus classifying head and neck squamous cell carcinoma (HNSCC) into HPV-positive and HPV-negative HNSCCs. Aberrant, mesenchymal-epithelial transition factor (c-MET) signal transduction favors HNSCC progression by stimulating proliferation, motility, invasiveness, morphogenesis, and angiogenesis. c-MET upregulation can be found in the majority of head and neck squamous cell carcinomas. c-MET pathway acts on several downstream effectors including phospholipase C gamma (PLCγ), cellular Src kinase (c-Src), phosphotidylinsitol-3-OH kinase (PI3K), alpha serine/threonine-protein kinase (Akt), mitogen-activated protein kinase (MAPK), and wingless-related integration site (Wnt) pathways. c-MET also establishes a crosstalk pathway with epidermal growth factor receptor (EGFR) and contributes towards chemoresistance in HNSCC. In recent years, the signaling communications of c-MET/HGF in metabolic dysregulation, tumor-microenvironment and immune modulation in HNSCC have emerged. Several clinical trials have been established against c-MET/ hepatocyte growth factor (HGF) signaling network to bring up targeted and effective therapeutic strategies against HNSCC. In this review, we discuss the molecular mechanism(s) and current understanding of c-MET/HGF signaling and its effect on HNSCC.

Impact of noncoding RNAs on cancer directed immune therapies: Now then and forever.

Accumulating evidence demonstrates that the host genome's epigenetic modifications are essential for living organisms to adapt to extreme conditions. DNA methylation, covalent modifications of histone and interassociation of noncoding RNAs facilitate the cellular manifestation of epigenetic changes in the genome. Out of various factors involved in the epigenetic programming of the host, noncoding RNAs (ncRNAs) such as microRNA (miRNA), long noncoding RNA (lncRNA), circular RNA, snoRNA and piRNA are new generation noncoding molecules that influence a variety of cellular processes like immunity, cellular differentiation and tumor development. During tumor development, temporal changes in miRNA/lncRNA rheostat influence sterile inflammatory responses accompanied by the changes in the carcinogenic signaling in the host. At the cellular level, this is manifested by the upregulation of inflammasome and inflammatory pathways, which promotes cancer-related inflammation. Given this, we discuss the potential of lncRNAs, miRNAs, circular RNA, snoRNA and piRNA in regulating inflammation and tumor development in the host.

Dietary interventions and molecular mechanisms for healthy musculoskeletal aging.

Over the past decade, extensive efforts have focused on understanding age-associated diseases and how to prolong a healthy lifespan. The induction of dietary protocols such as caloric restriction (CR) and protein restriction (PR) has positively affected a healthy lifespan. These intervention ideas (nutritional protocols) have been the subject of human cohort studies and clinical trials to evaluate their effectiveness in alleviating age-related diseases (such as type II diabetes, cardiovascular disease, obesity, and musculoskeletal fragility) and promoting human longevity. This study summarizes the literature on the nutritional protocols, emphasizing their impacts on bone and muscle biology. In addition, we analyzed several CR studies using Gene Expression Omnibus (GEO) database and identified common transcriptome changes to understand the signaling pathway involved in musculoskeletal tissue. We identified nine novel common genes, out of which five were upregulated (Emc3, Fam134b, Fbxo30, Pip5k1a, and Retsat), and four were downregulated (Gstm2, Per2, Fam78a, and Sel1l3) with CR in muscles. Gene Ontology enrichment analysis revealed that CR regulates several signaling pathways (e.g., circadian gene regulation and rhythm, energy reserve metabolic process, thermogenesis) involved in energy metabolism. In conclusion, this study summarizes the beneficiary role of CR and identifies novel genes and signaling pathways involved in musculoskeletal biology.

Preclinical pharmacokinetics, CYP phenotyping, and tissue distribution study of novel anti-breast cancer candidate S-011-1559.

S-011-1559 is a tyrosine-derived novel benzoxazine CDRI molecule targeted to the oestrogen-related receptor (ER-α/ß) modulator in breast cancer. To explore the pharmacokinetics of S-011-1559, a selective and sensitive bioanalytical method using LC-MS/MS was established and validated in different biological matrices of female rats.Blood-to-plasma ratio and plasma protein binding (PPB) of S-011-1559 were found to be <1 and >97% in both rats and humans, respectively. The human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG) binding was found in the range of > 68 to 45% and >14% respectively. Half-life and intrinsic clearance by microsomal stability study were found to be 28.83 min and 0.05 mL/min/mg in rats, 78.35 min and 0.036 mL/min/mg in humans, respectively. The IC50 value of S-011-1559 against CYP isoforms was revealed to moderately inhibit CYP2D6 by a reversible non-competitive mechanism.Tissue distribution of S-011-1559 on single intravenous injection at 2 mg/kg was found in the order of C lungs > C mammary gland > C spleen > C heart > C kidney > C liver > C brain.The data from the present study provides crucial information about S-011-1559 for further development as a novel potential drug candidate in modulating ER-α/ß receptors of lung and breast neoplasia.

Role of cardiovascular magnetic resonance imaging in COVID-19 recovered patients: A short-term follow-up study.

OBJECTIVE: Cardiac involvement in recovered COVID-19 patients assessed by cardiac magnetic resonance imaging (MRI). METHODS: Subjects recently recovered from COVID-19 and with an abnormal left ventricular global longitudinal strain were enrolled. Cardiac MRI in all the enrolled subjects was done at baseline (within 30-90 days following recovery from COVID-19) with a follow-up scan at 6 months in individuals with an abnormal baseline scan. Additionally, 20 age-and sex-matched individuals were enrolled as healthy controls (HCs). RESULTS: All the 30 enrolled subjects were symptomatic during active COVID-19 disease and were categorized as mild: 11 (36.7%), moderate: 6 (20%), and severe: 13 (43.3%). Of the 30 patients, 16 (53.3%) had abnormal CMR findings. Myocardial edema was reported in 12 (40%) patients while 10 (33.3%) had late gadolinium enhancement (LGE). No difference was observed in terms of conventional left ventricular (LV) parameters; however, COVID-19-recovered patients had significantly lower right ventricular (RV) ejection fraction, RV stroke volume, and RV cardiac index compared to HCs. Follow-up scan was abnormal in 4/16 (25%) with LGE persisting in three patients (who had severe COVID-19 [3/4;75%]). Subjects with severe COVID-19 had a greater frequency of LGE (53.8%) and myocardial edema (61.5%) as compared to mild and moderate cases. Myocardial T1 (1284 ± 43.8 ms vs. 1147.6 ± 68.4 ms; p < .0001) and T2 values (50.8 ± 16.7 ms vs. 42.6 ± 3.6 ms; p = .04) were significantly higher in post COVID-19 subjects compared to HCs. Similarly, T1 and T2 values of severe COVID-19 patients were significantly higher compared to mild and moderate cases. CONCLUSIONS: An abnormal CMR was seen in half of the recovered patients with persistent abnormality in one-fourth at 6 months. Our study suggests a need for closer follow-up among recovered subjects in order to evaluate for long-term cardiovascular sequelae. COVID-19 causes structural changes in the myocardium in a small segment of patients with partial spontaneous resolution.

Comparison of Different Mass Spectrometry Workflows for the Proteomic Analysis of Tear Fluid.

The tear film is a multi-layer fluid that covers the corneal and conjunctival epithelia of the eye and provides lubrication, nutrients, and protection from the outside environment. Tear fluid contains a high concentration of proteins and has thus been recognized as a potential source of biomarkers for ocular disorders due to its proximity to disease sites on the ocular surface and the non-invasive nature of its collection. This is particularly true in the case of dry eye disease, which directly impacts the tear film and its components. Proteomic analysis of tear fluid is challenging mainly due to the wide dynamic range of proteins and the small sample volumes. However, recent advancements in mass spectrometry have revolutionized the field of proteomics enabling unprecedented depth, speed, and accuracy, even with small sample volumes. In this study using the Orbitrap Fusion Tribrid mass spectrometer, we compared four different mass spectrometry workflows for the proteomic analysis of tear fluid collected via Schirmer strips. We were able to establish a method of in-strip protein digestion that identified >3000 proteins in human tear samples from 11 healthy subjects. Our method offers a significant improvement in the number of proteins identified compared to previously reported methods without pooling samples.

Generalizability of Homeopathic Prognostic Factor Research Outcome in COVID-19 Treatment: Comparison of Data.

BACKGROUND/OBJECTIVE: During the coronavirus disease 2019 (COVID-19) pandemic, several homeopathic prognostic factor research (PFR) projects have been undertaken. We found two projects with comparable outcomes to assess consistency and possible flaws. METHODS: Two comparisons were made. (1) Outcome of a PFR data collection from the Liga Medicorum Homoeopathica Internationalis (LMHI) by about 100 doctors with 541 cases was compared with a previous analysis of 161 cases in the same database. (2) The updated LMHI database was also compared with a data collection carried out in India by four doctors with a total of 1,445 cases. Differences that resulted in conflicting outcomes (indication in one, contraindication in the other) were examined for possible causes. RESULTS: There was only a single outcome in the updated LMHI database that conflicted with the previous dataset, and this could have been due to statistical variation. The Indian data contained many cases, from few doctors, while the LMHI database had few cases per doctor, but many doctors. The overlap between the projects (individual cases entered in both) was between zero and 22%. In 72 comparisons we found six (8.3%) conflicting outcomes. Possible causes were statistical error due to small numbers of cases and/or observers, confirmation bias, and keynote prescribing if this resulted in symptoms being inadequately checked. CONCLUSION: There was little conflict between the outcomes of the two versions of one project and between the two different PFR projects. Differences could mostly be explained by causes that can be managed. This consistency should primarily be interpreted as showing a strong overall consensus between homeopathic practitioners worldwide, but with variation of consensus between small groups of practitioners.

Impact of Bias in Data Collection of COVID-19 Cases.

BACKGROUND: Prognostic factor research (PFR), prevalence of symptoms and likelihood ratio (LR) play an important role in identifying prescribing indications of useful homeopathic remedies. It involves meticulous unbiased collection and analysis of data collected during clinical practice. This paper is an attempt to identify causes of bias and suggests ways to mitigate them for improving the accuracy in prescribing for better clinical outcomes and execution of randomized controlled studies. METHODS: A prospective, open label, observational study was performed from April 2020 to December 2020 at two COVID Health Centers. A custom-made Excel spreadsheet containing 71 fields covering a spectrum of COVID-19 symptoms was shared with doctors for regular reporting. Cases suitable for PFR were selected. LR was calculated for commonly occurring symptoms. Outlier values with LR ≥5 were identified and variance of LRs was calculated. RESULTS: Out of 1,889 treated cases of confirmed COVID-19, 1,445 cases were selected for pre-specified reasons. Nine medicines, Arsenicum album, Bryonia alba, Gelsemium sempervirens, Pulsatilla nigricans, Hepar sulphuricus, Magnesia muriaticum, Phosphorus, Nux vomica and Belladonna, were most frequently prescribed. Outlier values and large variance for Hepar sulphuricus and Magnesia muriaticum were noticed as indication of bias. Confirmation bias leading to lowering of symptom threshold, keynote prescribing, and deficiency in checking of all symptoms in each case were identified as the most important sources of bias. CONCLUSION: Careful identification of biases and remedial steps such as training of doctors, regular monitoring of data, checking of all pre-defined symptoms, and multicenter data collection are important steps to mitigate biases.