Selective Late-Stage Functionalization of Tryptophan-Containing Peptides To Facilitate Bioorthogonal Tetrazine Ligation.

Site-selective modification of complex peptides and the functionalization of their C-H bonds hold great promise for expanding their use in therapeutics and biomedical research. Herein, we leverage the power of late-stage chemoenzymatic catalysis using an indole prenyltransferase (IPT) enzyme and alkyl diphosphates to specifically modify the indole ring of tryptophan in clinically relevant peptides. Furthermore, the installed handle enables bioorthogonal click chemistry through an inverse electron-demand Diels-Alder (IEDDA) reaction with a biotin-conjugated tetrazine probe.

Chemical Hypoxic Preconditioning Improves Survival and Proliferation of Mesenchymal Stem Cells.

Increasing evidence has demonstrated that mesenchymal stem cells (MSCs) have been linked to tissue regeneration both in vitro and in vivo. However, poor engraftment and low survival rate of transplanted MSCs are still a major concern. It has been found that the proliferation, survival, and migration of MSCs are all increased by hypoxic preconditioning. However, the molecular mechanism through which hypoxic preconditioning enhances these beneficial properties of MSCs remains to be fully investigated. Therefore, the present study is aimed to investigate the mechanism by which hypoxic preconditioning enhances the survival of MSCs. We used proteomic analysis to explore the molecules that may contribute to the survival and proliferation of hypoxic preconditioned (HP) MSCs. The analysis revealed a higher expression of prelamin A/C (Lmna), glutamate dehydrogenase 1(Glud1), Actin, cytoplasmic 1(Actb), Alpha-enolase (Eno1), Glucose-6-phosphate 1-dehydrogenase (G6pd), Protein disulfide-isomerase A3 (Pdia3), Malate dehydrogenase (Mdh1), Peroxiredoxin-6 (Prdx6), Superoxide dismutase (Sod1), and Annexin A2 (Anxa2) in HP-MSCs. These proteins are possibly involved in cellular survival and proliferation through various cellular pathways. This research could aid in understanding the processes involved in hypoxic preconditioning of MSCs and designing of cell-based therapeutic strategies for tissue regeneration.

Differential Protein Expression in Response to Varlitinib Treatment in Oral Cancer Cell Line: an In Vitro Therapeutic Approach.

Epidermal growth factor receptor (EGFR) is the most frequently overexpressed receptor histologically exhibited by oral squamous cell carcinoma (OSCC) patients. Aberrated EGFR signaling may lead to recurrence and metastasis, thus laying the foundation of targeted therapy. Deactivating EGFR is likely to prevent downstream signaling thus resulting in apoptosis. Tyrosine kinase inhibitors (TKIs) have come into play to revert aggressiveness of OSCC. We exploited comparative proteomic analyses based on anti-EGFR potential of varlitinib, using cellular proteomes from treated and untreated groups of oral cancer cells to identify protein players functional during oral carcinogenesis. Following separation by two-dimensional electrophoresis, differentially expressed cellular proteins (varlitinib-treated and untreated cells) were analyzed and later identified using QTOF mass spectrometer. In silico analysis for protein-protein interaction was carried out using STRING. Six differentially expressed proteins were identified as binding immunoglobulin protein (BiP), heat shock protein 7 C (HSP7C), protein disulfide isomerase 1 A (PDIA1), vimentin (VIME), keratin type I cytoskeletal 14 (K1C14), and ß-Actin (ACTB). Relative expression of five proteins was found to be downregulated upon varlitinib treatment, whereas only K1C14 was upregulated in treated cells compared to control. Protein network analysis depicts the interaction between BiP, PDIA1, VIME, etc. indicating their role in oral carcinogenesis. Oral cancer cells show proteome shift based on varlitinib treatment compared to corresponding controls. Our data suggest candidature of varlitinib as a potent therapeutic agent and BiP, PDIA1, HSP7C, VIME, and ß-Actin as complementary/prognostic markers of OSCC.

Development and validation of an ultrahigh-performance liquid chromatography-tandem mass spectrometry method to investigate the plasma pharmacokinetics of a KCa 2.2/KCa 2.3 positive allosteric modulator in mice.

RATIONALE: There is currently no treatment for spinocerebellar ataxias (SCAs), which are a group of genetic disorders that often cause a lack of coordination, difficulty walking, slurred speech, tremors, and eventually death. Activation of KCa 2.2/KCa 2.3 channels reportedly exerts beneficial effects in SCAs. Here, we report the development and validation of an analytical method for quantitating a recently developed positive allosteric modulator of KCa 2.2/KCa 2.3 channels (compound 2q) in mouse plasma. METHODS: Mouse plasma samples (10 µL) containing various concentrations of 2q were subjected to protein precipitation in the presence of a structurally similar internal standard (IS). Subsequently, the analytes were separated on a C18 ultrahigh-performance liquid chromatography column and detected by a tandem mass spectrometer. The method was validated using US Food and Drug Administration (FDA) guidelines. Finally, the validated assay was applied to the measurement of the plasma concentrations of 2q in plasma samples taken from mice after single intravenous doses of 2 mg/kg of 2q, and the pharmacokinetic parameters of 2q were determined. RESULTS: The calibration standards were linear (r2 ≥ 0.99) in the range of 1.56-200 nM of 2q with intra- and inter-run accuracy and precision values within the FDA guidelines. The lower limit of quantitation of the assay was 1.56 nM (0.258 pg on the column). The recoveries of 2q and IS from plasma were >94%, with no appreciable matrix effect. The assay showed no significant carryover, and the plasma samples stored at -80°C or the processed samples stored in the autosampler at 10°C were stable for at least 3 weeks and 36 h, respectively. After intravenous injection, 2q showed a bi-exponential decline pattern in the mouse plasma, with a clearance of 30 mL/min/kg, a terminal volume of distribution of 1.93 mL/kg, and a terminal half-life of 45 min. CONCLUSIONS: The developed assay is suitable for preclinical pharmacokinetic-pharmacodynamic studies of 2q as a potential drug candidate for ataxias.

Novel Synergistic Combination of Pamidronate and Temozolomide for Breast Cancer Therapeutics.

OBJECTIVE: Human breast cancer is among one major health concerns with high prevalence and mortality among women worldwide. Various cellular signaling pathways are implicated in carcinogenesis. One of the major pathways that affect the downstream cellular growth cascades is Mevalonate pathway (MVA). The inhibition of MVA is therapeutically beneficial for various cancers. Pamidronate (PAM) (MVA inhibitor), a nitrogen-containing bisphosphosphonate, is an antiresorptive FDAapproved drug. The objective of our study was to explore adjuvant therapy using a combination of PAM and an alkylating agent, Temozolomide (TMZ) against breast cancer. METHODS: We have examined the differential gene and protein expression in response to the combination treatment strategy. For gene expression analysis RT-qPCR and for proteomic study, twodimensional gel electrophoresis and mass spectrometry techniques were utilized. RESULTS: Combination treatment (PAM+TMZ) showed more pronounced cytotoxic effect as compared to single agent treatment. Our results indicate that MVA pathway regulatory genes (FDFT1, FDPS, KRAS) are significantly (p<0.05) downregulated in combination-treated breast cancer cells. The differential proteomic analysis showed lower expression of GFAP, PPA1 and TRIM68 proteins after synergistic treatment whereas, these proteins are found to be up-regulated in multiple cancers. CONCLUSION: The present study reveals that a combination of PAM and TMZ produces an effective anti-cancerous effect on breast cancer cells. Therefore, this novel therapeutic regimen is likely to provide a better treatment strategy for breast cancer.

Prenatal diagnosis of maternal serum from mothers carrying ß-thalassemic fetus.

BACKGROUND: This study focuses on the discovery of protein biomarkers from the maternal serum of ß-thalassemic trait mothers carrying the normal fetus and ß-thalassemic major fetus. METHODS: Serum samples from ß-thalassemic trait mothers carrying major (N = 5) and normal fetuses (N = 5) were studied. The IVS1-5 thalassemia mutation was common among ß-thalassemic trait mothers who were carrying a homozygous ß-thalassemic fetus (IVS1-5/ IVS1-5 mutation) or a normal fetus (no mutation). We employed two-dimensional gel electrophoresis and mass spectrometry analysis to explore differentially expressed maternal serum proteins from thalassemia carrier couples with the same ß-thalassemia mutation. Western blotting was performed for one of the identified proteins to validate our data. RESULTS: Ten proteins were identified in the maternal serum of ß-thalassemic trait mothers carrying the ß-thalassemic major fetus and normal fetus. Among these, serotransferrin, haptoglobin, α-1 anti-trypsin, apo-lipoprotein A1, and the fibrinogen-ß chain were found to be upregulated in mothers carrying major fetuses and are known to be associated with pregnancy-related disorders. The expression of α-1 anti-trypsin was validated through western blotting. CONCLUSIONS: Proteins identified in the current study from maternal serum are reported to contribute to hereditary disorders. We suggest that these can serve as putative screening markers for non-invasive prenatal diagnosis in ß-thalassemic pregnancies.

Serum HSP90-Alpha and Oral Squamous Cell Carcinoma: A Prospective Biomarker.

AIM: This 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 usually diagnosed late, which results in poor survival and high mortality. Identification of non-invasive prognostic biomarkers is of utmost importance for early diagnosis and proper management of the disease; hence we used a 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, up-regulation of heat shock protein alpha (HSP90α) from the serum of oral cancer patients was observed. We also observed elevated levels of Haptoglobin (HP) along with downregulation of Type II keratin cytoskeletal 1(KRT1) and serum albumin (ALB) in oral cancer patients. Gene expression studies on 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 a contribution of HSP90 alpha and other identified proteins in oral pathology as pro/anti-apoptotic modulators, thus considering their potential as predictive biomarkers.

Stability Evaluation of Extemporaneously Compounded Vancomycin Ophthalmic Drops: Effect of Solvents and Storage Conditions.

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.

An Update of the Virion Proteome of Kaposi Sarcoma-Associated Herpesvirus.

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.

Antiproliferative and apoptotic effects of proteins from black seeds (Nigella sativa) on human breast MCF-7 cancer cell line.

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.

Validation of an immunoassay for soluble Klotho protein: decreased levels in diabetes and increased levels in chronic kidney disease.

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.