The Klotho gene has been identified as an aging suppressor gene that encodes a transmembrane protein, which is expressed primarily in renal tubules. There are 2 forms of Klotho, membrane and secreted. However, there is a paucity of data on levels of soluble Klotho in diseases like diabetes and kidney disease. We validated an enzyme-linked immunosorbent assay for Klotho and quantitated Klotho levels separately in patients with diabetes and also in patients with chronic kidney disease (CKD). The Klotho assay showed good precision and was linear down to 19 ng/mL. There were no significant effects on Klotho levels with the addition of common interferents such as ascorbate, triglycerides, or hemolysis; only bilirubin (250 mg/L) significantly reduced Klotho levels (P < .05). There was a significant reduction in Klotho levels in samples with glycated hemoglobin (HbA(1c)) levels of 6.5% or more compared with control samples (HbA(1c) < 6.5%; P < .001). We also documented significantly higher levels of Klotho with CKD. Thus, we validated an assay for Klotho and made the novel observation that levels are decreased in diabetes and increased in CKD.

The virion proteins of Kaposi sarcoma-associated herpesvirus (KSHV) were initially characterized in 2005 in two separate studies that combined the detection of 24 viral proteins and a few cellular components via LC-MS/MS or MALDI-TOF. Despite considerable advances in the sensitivity and specificity of mass spectrometry instrumentation in recent years, leading to significantly higher yields in detections, the KSHV virion proteome has not been revisited. In this study, we have re-examined the protein composition of purified KSHV virions via ultra-high resolution Qq time-of-flight mass spectrometry (UHR-QqTOF). Our results confirm the detection of all previously reported virion proteins, in addition to 17 other viral proteins, some of which have been characterized as virion-associated using other methods, and 10 novel proteins identified as virion-associated for the first time in this study. These results add KSHV ORF9, ORF23, ORF35, ORF48, ORF58, ORF72/vCyclin, K3, K9/vIRF1, K10/vIRF4, and K10.5/vIRF3 to the list of KSHV proteins that can be incorporated into virions. The addition of these proteins to the KSHV virion proteome provides novel and important insight into early events in KSHV infection mediated by virion-associated proteins. Data are available via ProteomeXchange with identifier PXD022626.

Background Nigella sativa (NS), a member of family Ranunculaceae is commonly known as black seed or kalonji. It has been well studied for its therapeutic role in various diseases, particularly cancer. Literature is full of bioactive compounds from NS seed. However, fewer studies have been reported on the pharmacological activity of proteins. The current study was designed to evaluate the anticancer property of NS seed proteins on the MCF-7 cell line. Methods NS seed extract was prepared in phosphate-buffered saline (PBS), and proteins were precipitated using 80% ammonium sulfate. The crude seed proteins were partially purified using gel filtration chromatography, and peaks were resolved by SDS-PAGE. MTT assay was used to screen the crude proteins and peaks for their cytotoxic effects on MCF-7 cell line. Active Peaks (P1 and P4) were further studied for their role in modulating the expression of genes associated with apoptosis by real-time reverse transcription PCR. For protein identification, proteins were digested, separated, and analyzed with LC-MS/MS. Data analysis was performed using online Mascot, ExPASy ProtParam, and UniProt Knowledgebase (UniProtKB) gene ontology (GO) bioinformatics tools. Results Gel filtration chromatography separated seed proteins into seven peaks, and SDS-PAGE profile revealed the presence of multiple protein bands. Among all test samples, P1 and P4 depicted potent dose-dependent inhibitory effect on MCF-7 cells exhibiting IC50 values of 14.25 ± 0.84 and 8.05 ± 0.22 μg/ml, respectively. Gene expression analysis demonstrated apoptosis as a possible cell killing mechanism. A total of 11 and 24 proteins were identified in P1 and P4, respectively. The majority of the proteins identified are located in the cytosol, associate with biological metabolic processes, and their molecular functions are binding and catalysis. Hydropathicity values were mostly in the hydrophilic range. Conclusion Our findings suggest NS seed proteins as a potential therapeutic agent for cancer. To our knowledge, it is the first study to report the anticancer property of NS seed proteins.

Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus keratitis and other ocular infections. Vancomycin ophthalmic drops are not commercially available and require compounding. The present study was designed to investigate the stability of vancomycin ophthalmic drops in normal saline, phosphate-buffered saline (PBS), and balanced salt solution (BSS) while stored at room temperature or under refrigeration. Vancomycin ophthalmic drops (50 mg/mL) were aseptically prepared from commercially available intravenous powder using PBS, BSS, and saline. Solutions were stored at room temperature and in a refrigerator for 28 days. The vancomycin stability was tested by a microbiology assay and high-performance liquid chromatography HPLC analysis immediately after formulation and at days 7, 14, and 28 after storage at room temperature or under refrigeration. The pH, turbidity was also tested. Vancomycin formulations in PBS, BSS and normal saline had initial pH of 5; 5.5; 3 respectively. The formulation in PBS developed turbidity and a slight decrease in pH upon storage. Microbiological assay did not show any change in zone of inhibition with any of the formulation upon storage either at room temperature or under refrigeration. HPLC analysis did not detect any decrease in vancomycin concentration or the accumulation of degraded products in any of the formulations upon storage either at room temperature or under refrigeration. Vancomycin ophthalmic drops prepared using PBS, BSS, and normal saline were stable up to the tested time point of 28 days, irrespective of their storage temperature.

Aim: Current study aims to perform differential protein expression analysis of serum samples from Oral squamous cell carcinoma (OSCC) patients and healthy controls in search of potential diagnostic and/or prognostic biomarker(s). Objective: OSCC is diagnosed late, resulting in poor survival and high mortality. Identification of non-invasive prognostic biomarker is of utmost importance for early diagnosis and proper management of the disease; hence we used proteomic approach to identify potential biomarkers from serum. Methods: Serum samples (OSCC n=45 and control n=30) were depleted and proteins were separated using 2-D gel electrophoresis followed by identification by mass spectrometric analysis. Gene expression analysis of identified proteins in malignant and normal tissue was also performed to complement proteomics studies. Results: Among differentially expressed proteins, a noteworthy observation was up regulation of Heat shock protein alpha (HSP90α) from serum of oral cancer patients. We also observed elevated levels of Haptoglobin (HP) along with down regulation of Type II keratin cytoskeletal 1(KRT1) and serum Albumin (ALB) in oral cancer patients. Gene expression studies of identified proteins in malignant and normal tissue revealed a similar pattern with the exception of KRT1. We believe that elevated levels of serum HSP90 alpha might be used as a potential biomarker. Conclusion: Our findings suggest the contribution of HSP90 alpha and other identified proteins in oral pathology as pro/anti apoptotic modulators, thus they are being considered as predictive biomarkers.