Assessment of Salivary MMP-8 and IL-1ß for the Diagnosis of Periodontal Diseases in Pakistani Population.

OBJECTIVE: Clinical methods use the subjective diagnosis of periodontal diseases by visual observation that could result in differences and variability of diagnosis. The addition of specific markers could aid in the accurate diagnosis of the local population. The objective of the study was to target two of the major proteins for possible significance in such an approach. MATERIALS AND METHODS: Unstimulated saliva samples were collected from 60 participants aged between 18 and 70 years. Three groups each with twenty participants were recruited into periodontitis, gingivitis, and healthy control. STATISTICAL ANALYSIS: The samples were analyzed using human enzyme-linked immunosorbent assay kits for matrix metalloproteinase-8 (MMP-8) and interleukin-1ß (IL-1ß). RESULTS: SPSS version 20 was used to analyze the result. Posthoc analysis by Tukey's test revealed that MMP-8 levels were higher in gingivitis and periodontitis groups as compared with healthy controls. The test also revealed that IL-1ß levels were higher in the periodontitis group compared with the healthy control and gingivitis group. Additionally, one-way analysis of variance analysis showed a significant effect on probing depth in gingivitis and periodontitis patients. The mean age of periodontitis group was significantly higher than other groups. CONCLUSION: Salivary biomarkers may provide useful diagnostic information and could be utilized as tests for periodontal disease screening, prognosis, and prediction.

Design and Immunoinformatic Assessment of Candidate Multivariant mRNA Vaccine Construct against Immune Escape Variants of SARS-CoV-2.

To effectively counter the evolving threat of SARS-CoV-2 variants, modifications and/or redesigning of mRNA vaccine construct are essentially required. Herein, the design and immunoinformatic assessment of a candidate novel mRNA vaccine construct, DOW-21, are discussed. Briefly, immunologically important domains, N-terminal domain (NTD) and receptor binding domain (RBD), of the spike protein of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) were assessed for sequence, structure, and epitope variations. Based on the assessment, a novel hypothetical NTD (h-NTD) and RBD (h-RBD) were designed to hold all overlapping immune escape variations. The construct sequence was then developed, where h-NTD and h-RBD were intervened by 10-mer gly-ala repeat and the terminals were flanked by regulatory sequences for better intracellular transportation and expression of the coding regions. The protein encoded by the construct holds structural attributes (RMSD NTD: 0.42 Å; RMSD RBD: 0.15 Å) found in the respective domains of SARS-CoV-2 immune escape variants. In addition, it provides coverage to the immunogenic sites of the respective domains found in SARS-CoV-2 variants. Later, the nucleotide sequence of the construct was optimized for GC ratio (56%) and microRNA binding sites to ensure smooth translation. Post-injection antibody titer was also predicted (~12000 AU) to be robust. In summary, the construct proposed in this study could potentially provide broad spectrum coverage in relation to SARS-CoV-2 immune escape variants.

Association of salivary Cathepsin B in different histological grades among patients presenting with oral squamous cell carcinoma.

BACKGROUND: Oral cancer is considered a major public health problem due to its high mortality and morbidity rates. Survival rate of OSCC can be significantly improved by using non-invasive tool such as salivary biomarkers for detection of OSCC which is considered a promising approach. Cathepsin B is a lysosomal cysteine protease, present in abundant quantities in lysosome of cells, tissues and different biological fluids. Increased expression of Cathepsin B was observed in many malignancies including oral cancer. The present study was designed to determine the salivary levels of Cathepsin B in different histological grades of OSCC. METHOD: In this study, total no. of 80 research participants were enrolled which were divided into four groups. Each group comprised 20 participants, group 1 comprised 20 patients of OSCC (well differentiated), group 2 comprised 20 patients of OSCC (moderately differentiated), group 3 comprised 20 patients of OSCC (poorly differentiated) and group 4 comprised 20 healthy controls. Saliva sample was collected from all the four study groups and salivary Cathepsin B levels were analyzed by ELISA sandwich technique in duplicate. RESULTS: Salivary levels of Cathepsin B were significantly increased with p value (< 0.001) in patients of OSCC as compared to control group according to both histological grades and tumor size. Highest mean Cathepsin B levels in well differentiated OSCC followed by poorly differentiated OSCC and moderately differentiated OSCC were observed. CONCLUSION: Results of the present study suggests that Cathepsin B has a great value as a salivary biomarker for diagnosis and monitoring of OSCC in different histological grades. This will further lead to increase survival rate and improve the prognosis of OSCC.

Differentially Expressed Salivary Proteins in Dental Caries Patients.

Dental caries is a multifactorial disease mainly caused by cariogenic bacteria commonly found in the oral cavity. Dental caries may cause demineralization of the tooth, cavitation, hypersensitivity, pulp inflammation, and even tooth loss if left untreated. Saliva secreted in the oral cavity can serve as a tool for identification of biomarkers for early detection of diseases. In the present study, differential expression of salivary proteins from 33 dental caries patients was compared with 10 control subjects. The unstimulated saliva was analyzed by 12% SDS-PAGE and two-dimensional gel electrophoresis. Gelatin and casein zymography was performed to check for protease activity. Also, salivary IgAs from both groups were compared by sandwich ELISA technique. Dental caries patient's saliva showed decreased caseinolytic and increased gelatinolytic activity probably due to metalloproteases and cathepsins. Mean salivary levels of sIgA were also significantly higher (p < 0.018) in dental caries saliva samples. The 2D electrophoresis profile of both the groups showed regions on gel with visually detectable alterations in protein expression. The present study is among the few initial studies in the locality for identification of protein differences in saliva from dental caries patients and has demonstrated a good potential to identify alterations. However, a large population-based analysis is required to validate these findings to be translated as a tool for indicative applications.

Purification and Characterization of a Nonspecific Lipid Transfer Protein 1 (nsLTP1) from Ajwain (Trachyspermum ammi) Seeds.

Ajwain (Trachyspermum ammi) belongs to the family Umbelliferae, is commonly used in traditional, and folk medicine due to its carminative, stimulant, antiseptic, diuretic, antihypertensive, and hepatoprotective activities. Non-specific lipid transfer proteins (nsLTPs) reported from various plants are known to be involved in transferring lipids between membranes and in plants defense response. Here, we describe the complete primary structure of a monomeric non-specific lipid transfer protein 1 (nsLTP1), with molecular weight of 9.66 kDa, from ajwain seeds. The nsLTP1 has been purified by combination of chromatographic techniques, and further characterized by mass spectrometry, and Edman degradation. The ajwain nsLTP1 is comprised of 91 amino acids, with eight conserved cysteine residues. The amino acid sequence based predicted three dimensional (3D) structure is composed of four α-helices stabilized by four disulfide bonds, and a long C-terminal tail. The predicted model was verified by using different computational tools; i.e. ERRAT, verify 3D web server, and PROCHECK. The docking of ajwain nsLTP1 with ligands; myristic acid (MYR), and oleic acid (OLE) was performed, and molecular dynamics (MD) simulation was used to validate the docking results. The findings suggested that amino acids; Leu11, Leu12, Ala55, Ala56, Val15, Tyr59, and Leu62 are pivotal for the binding of lipid molecules with ajwain nsLTP1.

Snake Venom: From Deadly Toxins to Life-saving Therapeutics.

Snakes are fascinating creatures and have been residents of this planet well before ancient humans dwelled the earth. Venomous snakes have been a figure of fear, and cause notable mortality throughout the world. The venom constitutes families of proteins and peptides with various isoforms that make it a cocktail of diverse molecules. These biomolecules are responsible for the disturbance in fundamental physiological systems of the envenomed victim, leading to morbidity which can lead to death if left untreated. Researchers have turned these life-threatening toxins into life-saving therapeutics via technological advancements. Since the development of captopril, the first drug that was derived from bradykininpotentiating peptide of Bothrops jararaca, to the disintegrins that have potent activity against certain types of cancers, snake venom components have shown great potential for the development of lead compounds for new drugs. There is a continuous development of new drugs from snake venom for coagulopathy and hemostasis to anti-cancer agents. In this review, we have focused on different snake venom proteins / peptides derived drugs that are in clinical use or in developmental stages till to date. Also, some commonly used snake venom derived diagnostic tools along with the recent updates in this exciting field are discussed.

The Primary Structure of ß(I)-Chain of Hemoglobin from Snake Sindhi Krait (Bungarus sindanus sindanus).

The amino acid sequence of ß(I)-globin chain from Sindhi Krait (Bungarus sindanus sindanus) was determined to study the molecular evolution among snakes. The hemoglobin was isolated from the red blood cells and was analyzed by ion-exchange chromatography (IEX). The crude globin was subjected to reversed phased-high performance liquid chromatography (RP-HPLC) using C4 column. The N-terminal sequences of intact globin chains and tryptic peptides were determined by Edman degradation in a pulsed liquid gas phase sequencer using an online Phenylthiohydantoin analyzer. Sindhi Krait is expected to express three hemoglobin components that are composed of ß(II), ß(I), α(D) and α(A)-globin chains, as apparent by IEX, RP-HPLC and N-terminal sequence analyses. Sequence alignment and phylogenetic analyses of ß(I) globin chain from Sindhi Krait showed closest relationship with ß(I) globin chain from Rattlesnake, Water snake and Indigo snake. Interestingly, comparison of primary sequence of ß(I) globin chain of Sindhi Krait with human ß chain revealed 63 % similarity along with the retention of all heme contact points. Variations among the two sequences were prominent at αß contact points and in regions directly not important for function.