Vaccine for Diabetes-Where Do We Stand?

Diabetes is an endocrinological disorder with a rapidly increasing number of patients globally. Over the last few years, the alarming status of diabetes has become a pivotal factor pertaining to morbidity and mortality among the youth as well as middle-aged people. Current developments in our understanding related to autoimmune responses leading to diabetes have developed a cause for concern in the prospective usage of immunomodulatory agents to prevent diabetes. The mechanism of action of vaccines varies greatly, such as removing autoreactive T cells and inhibiting the interactions between immune cells. Currently, most developed diabetes vaccines have been tested in animal models, while only a few human trials have been completed with positive outcomes. In this review, we investigate the undergoing clinical trial studies for the development of a prototype diabetes vaccine.

Selenium Biofortification: Roles, Mechanisms, Responses and Prospects.

The trace element selenium (Se) is a crucial element for many living organisms, including soil microorganisms, plants and animals, including humans. Generally, in Nature Se is taken up in the living cells of microorganisms, plants, animals and humans in several inorganic forms such as selenate, selenite, elemental Se and selenide. These forms are converted to organic forms by biological process, mostly as the two selenoamino acids selenocysteine (SeCys) and selenomethionine (SeMet). The biological systems of plants, animals and humans can fix these amino acids into Se-containing proteins by a modest replacement of methionine with SeMet. While the form SeCys is usually present in the active site of enzymes, which is essential for catalytic activity. Within human cells, organic forms of Se are significant for the accurate functioning of the immune and reproductive systems, the thyroid and the brain, and to enzyme activity within cells. Humans ingest Se through plant and animal foods rich in the element. The concentration of Se in foodstuffs depends on the presence of available forms of Se in soils and its uptake and accumulation by plants and herbivorous animals. Therefore, improving the availability of Se to plants is, therefore, a potential pathway to overcoming human Se deficiencies. Among these prospective pathways, the Se-biofortification of plants has already been established as a pioneering approach for producing Se-enriched agricultural products. To achieve this desirable aim of Se-biofortification, molecular breeding and genetic engineering in combination with novel agronomic and edaphic management approaches should be combined. This current review summarizes the roles, responses, prospects and mechanisms of Se in human nutrition. It also elaborates how biofortification is a plausible approach to resolving Se-deficiency in humans and other animals.

Staphylococcus aureus Biofilm Infection Compromises Wound Healing by Causing Deficiencies in Granulation Tissue Collagen.

OBJECTIVE: The objective of this work was to causatively link biofilm properties of bacterial infection to specific pathogenic mechanisms in wound healing. BACKGROUND: Staphylococcus aureus is one of the four most prevalent bacterial species identified in chronic wounds. Causatively linking wound pathology to biofilm properties of bacterial infection is challenging. Thus, isogenic mutant stains of S. aureus with varying degree of biofilm formation ability was studied in an established preclinical porcine model of wound biofilm infection. METHODS: Isogenic mutant strains of S. aureus with varying degree (ΔrexB > USA300 > ΔsarA) of biofilm-forming ability were used to infect full-thickness porcine cutaneous wounds. RESULTS: Compared with that of ΔsarA infection, wound biofilm burden was significantly higher in response to ΔrexB or USA300 infection. Biofilm infection caused degradation of cutaneous collagen, specifically collagen 1 (Col1), with ΔrexB being most pathogenic in that regard. Biofilm infection of the wound repressed wound-edge miR-143 causing upregulation of its downstream target gene matrix metalloproteinase-2. Pathogenic rise of collagenolytic matrix metalloproteinase-2 in biofilm-infected wound-edge tissue sharply decreased collagen 1/collagen 3 ratio compromising the biomechanical properties of the repaired skin. Tensile strength of the biofilm infected skin was compromised supporting the notion that healed wounds with a history of biofilm infection are likely to recur. CONCLUSION: This study provides maiden evidence that chronic S. aureus biofilm infection in wounds results in impaired granulation tissue collagen leading to compromised wound tissue biomechanics. Clinically, such compromise in tissue repair is likely to increase wound recidivism.

Wound healing activity of a collagen-derived cryptic peptide.

Wound healing involves a well-controlled series of interactions among cells and several mediators leading to the restoration of damaged tissue. Degradation of the extracellular matrix (ECM) protein collagen during remodelling of wound tissue leads to the release of bioactive peptides that can possibly influence the healing process. The RGD-containing, antioxidative collagen peptide E1 isolated in an earlier work was screened in this study for its ability to influence multiple steps of the wound healing process. E1 was assayed for and found to be chemotactic. Excision and incision wounds were created on separate groups of rats and E1 was administered topically. The wound tissues were isolated on the 4th and 8th days post-wound and subjected to biochemical and biophysical analysis. A significant decrease in lipid peroxides in the treatment group confirmed the in vivo antioxidant capacity of E1. The treatment group also displayed significant increase in total protein, collagen and amino sugar synthesis indicating faster ECM formation. The significantly increased rate of wound contraction and reepithelialisation along with higher tensile strength of the wound tissue corroborated the results of biochemical analysis. The results confirm the significant role played by collagen peptides in accelerating the healing process and justify their possible use as a pharmaceutical agent.

Screening for novel cell adhesive regions in bovine Achilles tendon collagen peptides.

Collagen, a major structural protein of the ECM, is known for its high cell adherence capacity. This study was conducted to identify regions in collagen that harbour such bioactivity. Collagen from tendon was hydrolysed and the peptides fractionated using ion-exchange chromatography (IEC). Isolated peptide fractions were coated onto disposable dishes and screened for cell adherence and proliferative abilities. Active IEC fractions were further purified by chromatography, and two peptides, C2 and E1 with cell adhesion ability, were isolated. A cell adhesion assay done with different amounts of C2 coated onto disposable dishes revealed the maximum adhesion to be 94.6%, compared with 80% for collagen coated dishes and an optimum peptide coating density of 0.507 nmoles per cm(2) area of the dish. Growth of cells on C2, collagen, and E1 revealed a similar pattern and a reduction in the doubling time compared with cells grown on uncoated dishes. C2 had a mass of 2.046 kDa with 22 residues, and sequence analysis revealed a higher percentage occurrence of hydrophilic residues compared with other regions in collagen. Docking studies revealed GDDGEA in C2 as the probable site of interaction with integrins α2ß1 and α1ß1, and stability studies proved C2 to be mostly protease-resistant.

Nutritional Aspects, Chemistry Profile, Extraction Techniques of Lemongrass Essential Oil and It's Physiological Benefits.

Lemongrass contains a variety of substances that are known to have antioxidant and disease-preventing properties, including essential oils, compounds, minerals, and vitamins. Lemongrass (Cymbopogon Spp.) essential oil (LGEO) has been demonstrated to ameliorate diabetes and accelerate wound healing. A member of the Poaceae family, Lemongrass, a fragrant plant, is cultivated for the extraction of essential oils including myrcene and a mixture of geranial and neral isomers of citral monoterpenes. Active constituents in lemongrass essential oil are myrcene, followed by limonene and citral along with geraniol, citronellol, geranyl acetate, neral, and nerol, which are beneficial to human health. A large part of lemongrass' expansion is driven by the plant's huge industrial potential in the food, cosmetics, and medicinal sectors. A great deal of experimental and modeling study was conducted on the extraction of essential oils. Using Google Scholar and PubMed databases, a systematic review of the literature covering the period from 1996 to 2022 was conducted, in accordance with the PRISMA declaration. There were articles on chemistry, biosynthesis, extraction techniques and worldwide demand of lemongrass oil. We compared the effectiveness of several methods of extracting lemongrass essential oil, including solvent extraction, supercritical CO2 extraction, steam distillation, hydrodistillation (HD), and microwave aided hydrodistillation (MAHD). Moreover, essential oils found in lemongrass and its bioactivities have a significant impact on human health. This manuscript demonstrates the different extraction techniques of lemongrass essential oil and its physiological benefits on diabetic wound healing, tissue repair and regeneration, as well as its immense contribution in ameliorating arthritis and joint pain.Key teaching pointsThe international market demand prediction and the pharmacological benefits of the Lemongrass essential oil have been thoroughly reported here.This article points out that different extraction techniques yield different percentages of citral and other secondary metabolites from lemon grass, for example, microwave assisted hydrodistillation and supercritical carbon dioxide extraction process yields more citral.This article highlights the concept and application of lemongrass oil in aromatherapy, joint-pain, and arthritis.Moreover, this manuscript includes a discussion about the effect of lemongrass oil on diabetic wound healing and tissue regeneration - that paves the way for further research.

Collagenase-based wound debridement agent induces extracellular matrix supporting phenotype in macrophages.

Macrophages assume diverse phenotypes and functions in response to cues from the microenvironment. Earlier we reported an anti-inflammatory effect of Collagenase Santyl® Ointment (CSO) and the active constituent of CSO (CS-API) on wound macrophages in resolving wound inflammation indicating roles beyond debridement in wound healing. Building upon our prior finding, this study aimed to understand the phenotypes and subsets of macrophages following treatment with CS-API. scRNA-sequencing was performed on human blood monocyte-derived macrophages (MDM) following treatment with CS-API for 24 h. Unbiased data analysis resulted in the identification of discrete macrophage subsets based on their gene expression profiles. Following CS-API treatment, clusters 3 and 4 displayed enrichment of macrophages with high expression of genes supporting extracellular matrix (ECM) function. IPA analysis identified the TGFß-1 pathway as a key hub for the CS-API-mediated ECM-supportive phenotype of macrophages. Earlier we reported the physiological conversion of wound-site macrophages to fibroblasts in granulation tissue and impairment of such response in diabetic wounds, leading to compromised ECM and tensile strength. The findings that CSO can augment the physiological conversion of macrophages to fibroblast-like cells carry significant clinical implications. This existing clinical intervention, already employed for wound care, can be readily repurposed to improve the ECM response in chronic wounds.

Larvicidal activity of ß-Citral: An In-vitro and In-silico study to understand its potential against mosquito.

Tropical and subtropical regions face millions of deaths from mosquito-borne illnesses yearly. Insecticides prevent transmission but pose health risks like dermatitis and allergies. The primary objective was to mitigate the recurring dependence on synthetic insecticides, thereby curbing the development of mosquito resistance. Leaves of Cymbopogon flexuosus (lemongrass) was collected from Mayurbhanj, India, processed, then extracted by steam distillation for essential oils & analyzed spectroscopically. Larvicidal assays were performed across varying concentrations, revealing the significant mortality induced by the Cymbopogon flexuosus extract against Anopheles stephensi larvae. 3D structure was modelled by using G protein-coupled receptors (GPCR) sequence and structural stability was also validated. After docking the binding free energy was determined from GPCR protein with ß-citral complex. Molecular dynamics (MD) study was conducted on the docked pose that displayed an optimal interactome profile. The larvicidal assay at the 12th and 24th hour revealed the highest LC50 (lethal concentration) of 23.493 ppm and 19.664 ppm . ß-Citral has a high binding affinity and an identifiable binding site, which suggests that it may play a larvicidal role in regulating the receptor's function by creating stable complexes with it. ß-Citral from lemongrass oils has potential larvicidal activity and effective against GPCR family 1 of mosquito and highly effective repellents against mosquito-borne diseases.

Hydrolyzed Collagen Powder Dressing Improves Wound Inflammation, Perfusion, and Breaking Strength of Repaired Tissue.

Objective: Hydrolyzed collagen-based matrices are widely used as wound care dressings. Information on the mechanism of action of such dressings is scanty. The objective of this study was to test the effect of a specific hydrolyzed collagen powder (HCP), which is extensively used for wound care management in the United States. Approach: The effects of HCP on resolution of wound inflammation, perfusion, closure, and breaking strength of the repaired skin were studied in an experimental murine model. Results: In early (day 7) inflammatory phase of wound macrophages, HCP treatment boosted phagocytosis and efferocytosis of wound-site macrophages. In these cells, inducible reactive oxygen species were also higher on day (d) 7. HCP treatment potentiated the expression of anti-inflammatory interleukin (IL)-10 cytokine and proangiogenic vascular endothelial growth factor (VEGF) production. Excisional wounds dressed with HCP showed complete closure on day 21, while the control wounds remained open. HCP treatment also demonstrated improved quality of wound healing as marked by the improved breaking strength of the closed wound tissue/repaired skin. Innovation: These data represent first evidence on the mechanism of action of clinically used HCP. Conclusion: HCP dressing favorably influenced both wound inflammation and vascularization. Improved breaking strength of HCP-treated repaired skin lays the rationale for future studies testing the hypothesis that HCP-treated closed wounds would show fewer recurrences.

A novel peptide isolated from Catla skin collagen acts as a self-assembling scaffold promoting nucleation of calcium-deficient hydroxyapatite nanocrystals.

Catla collagen hydrolysate (CH) was fractionated by chromatography and each fraction was subjected to HA nucleation, with the resultant HA-fraction composites being scored based on the structural and functional group of the HA formed. The process was repeated till a single peptide with augmented HA nucleation capacity was obtained. The peptide (4.6 kDa), exhibited high solubility, existed in polyproline-II conformation and displayed a dynamic yet stable hierarchical self-assembling property. The 3D modelling of the peptide revealed multiple calcium and phosphate binding sites and a high propensity to self-assemble. Structural analysis of the peptide-HA crystals revealed characteristic diffraction planes of HA with mineralization following the (002) plane, retention of the self-assembled hierarchy of the peptide and intense ionic interactions between carboxyl groups and calcium. The peptide-HA composite crystals were mostly of 25-40 nm dimensions and displayed 79% mineralization, 92% crystallinity, 39.25% porosity, 12GPa Young's modulus and enhanced stability in physiological pH. Cells grown on peptide-HA depicted faster proliferation rates and higher levels of osteogenic markers. It was concluded that the prerequisite for HA nucleation by a peptide included: a conserved sequence with a unique charge topology allowing calcium chelation and its ability to form a dynamic self-assembled hierarchy for crystal propagation.

Formation of hierarchical assemblies by collagen peptides derived from fish skin and bladder and their subsequent application as antiperoxide agents in lipid-rich food.

This study attempts to identify the significant role played by the secondary and tertiary structure of collagen-derived peptides that are involved in lipid peroxide quenching in food products. Fish collagen hydrolysate (CH) was extracted with an efficiency of 70%. The constituent peptides of CH (8.2-9.7 kDa) existed in a polyproline-II (PP-II) conformation and at a minimum concentration of 1 mg ml-1 and pH range 7 to 8, assembled into a stable, hierarchical, quasi-fibrillar (QF) network. The peroxide quenching activity of this QF-CH increased with increasing ionic stability of the assembly and decreased upon proteolytic dismantling. Upon being used as an additive, the QF-CH reduced peroxide formation by 84.5% to 98.9% in both plant and fish-based oil and increased the shelf life of soya oil by a factor of 5 after 6 months of storage. The addition of QF-CH to cultured cells quenched peroxide ions generated in situ and decreased stressor activity by a factor of 12.16 abundant peptides were identified from the CH. The reason behind the high efficacy displayed by CH was attributed to its unique charge distribution, prevalence of proton-donating amino acid residues and proximal charge delocalization by the QF network, making fish derived CH a suitable substitute for antiperoxide agents in lipid-rich food.

Efficacy of a Proprietary Fenugreek Seed Extract (Trigonella foenum-graecum, Furocyst®) in Women with Polycystic Ovary Syndrome (PCOS): a Randomized, Double-Blind, Placebo-Controlled Study.

INTRODUCTION: Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenemia, a quite common heterogenous endocrine/hormonal disorder, and accompanied by elevated androgen level, menstrual irregularity, and hirsutism. The consequences include infertility or miscarriage. It is a challenging problem to the physicians. In a one-arm, non-randomized preliminary investigation in fifty premenopausal women, we demonstrated the efficacy of Furocyst®, a patented, standardized Trigonella foenum-graecum extract, in ameliorating the symptoms of PCOS over a period of 90 consecutive days. OBJECTIVE: In the present study, a double-blind, two-arm, single-center, randomized, comparative study was conducted to assess the efficacy of Furocyst® (2 capsules of 500 mg/day) in 208 pre-menopausal women diagnosed with PCOS. METHODS: Ethical committee approval was obtained. A total of 208 subjects (placebo = 95; Furocyst® = 113; age:18-45 years, BMI < 42 kg/m2) completed the investigation. The comparative efficacy of placebo and Furocyst® was assessed on the number of cysts, ovarian volume, hirsutism, LH:FSH ratio, titer of TSH, SHBG, prolactin and free testosterone. Key clinical parameters such as fasting blood glucose levels, HOMA Index, cholesterol, LDL, and triglyceride levels, as well as total blood chemistry were also investigated. RESULTS: Furocyst® supplementation significantly reduced the number of cysts, ovarian volume, and hirsutism levels, as well as normalized the menstrual cycle in Furocyst®-treated subjects as compared to placebo group. Furocyst® significantly reduced luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels, and thyroid stimulating hormone (TSH) levels, and reduced the prolactin and SHBG levels. Furocyst® significantly reduced the fasting blood glucose levels, HOMA Index, cholesterol, LDL, and triglyceride levels as compared to the placebo group, while the free testosterone levels were significantly decreased in the Furocyst® group. CONCLUSION: The studies collectively demonstrated the efficacy of Furocyst® as a safe, natural phytochemical-based formulation to alleviate the symptoms of PCOS. No significant adverse events were observed.

Hypoglycaemic Molecules for the Management of Diabetes Mellitus from Marine Sources.

Diabetes mellitus (DM) is a chronic metabolic disorder recognized as a major health problem globally. A defective insulin activity contributes to the prevalence and expansion of DM. Treatment of DM is often hampered by limited options of conventional therapies and adverse effects associated with existing procedures. This has led to a spike in the exploration for potential therapeutic agents from various natural resources for clinical applications. The marine environment is a huge store of unexplored diversity of chemicals produced by a multitude of organisms. To date, marine microorganisms, microalgae, macroalgae, corals, sponges, and fishes have been evaluated for their anti-diabetic properties. The structural diversity of bioactive metabolites discovered has shown promising hypoglycaemic potential through in vitro and in vivo screenings via various mechanisms of action, such as PTP1B, α-glucosidase, α-amylase, ß-glucosidase, and aldose reductase inhibition as well as PPAR alpha/gamma dual agonists activities. On the other hand, hypoglycaemic effect is also shown to be exerted through the balance of antioxidants and free radicals. This review highlights marine-derived chemicals with hypoglycaemic effects and their respective mechanisms of action in the management of DM in humans.

A Modified Collagen Dressing Induces Transition of Inflammatory to Reparative Phenotype of Wound Macrophages.

Collagen containing wound-care dressings are extensively used. However, the mechanism of action of these dressings remain unclear. Earlier studies utilizing a modified collagen gel (MCG) dressing demonstrated improved vascularization of ischemic wounds and better healing outcomes. Wound macrophages are pivotal in facilitating wound angiogenesis and timely healing. The current study was designed to investigate the effect of MCG on wound macrophage phenotype and function. MCG augmented recruitment of macrophage at the wound-site, attenuated pro-inflammatory and promoted anti-inflammatory macrophage polarization. Additionally, MCG increased anti-inflammatory IL-10, IL-4 and pro-angiogenic VEGF production, indicating a direct role of MCG in resolving wound inflammation and improving angiogenesis. At the wound-site, impairment in clearance of apoptotic cell bioburden enables chronic inflammation. Engulfment of apoptotic cells by macrophages (efferocytosis) resolves inflammation via a miR-21-PDCD4-IL-10 pathway. MCG-treated wound macrophages exhibited a significantly bolstered efferocytosis index. Such favorable outcome significantly induced miR-21 expression. MCG-mediated IL-10 production was dampened under conditions of miR-21 knockdown pointing towards miR-21 as a causative factor. Pharmacological inhibition of JNK attenuated IL-10 production by MCG, implicating miR-21-JNK pathway in MCG-mediated IL-10 production by macrophages. This work provides direct evidence demonstrating that a collagen-based wound-care dressing may influence wound macrophage function and therefore modify wound inflammation outcomes.

Cryptic Peptides from Collagen: A Critical Review.

Collagen, a predominant structural protein in extracellular matrix (ECM), is now considered to have probable roles in many biological activities and hence, in different forms have found application as nutraceutical or pharmaceutical therapy option. Many of the biological properties are believed to be due to small hidden peptide residues in the collagen molecules, which come into play after the biodegradation or biosorption of the parent molecule. These peptide regions are called cryptic peptides or by some, as cryptides. The proteolytic hydrolysis of the ECM protein releases the cryptic peptides with many novel biological activities not exhibited directly by the parental protein which include angiogenic, antimicrobial, mitogenic and chemotactic properties. The research for understanding the role of these cryptic peptide regions and making use of them in medical field is very active. Such an understanding could lead to the development of peptide supplements for many biomedical applications. The prolific research in this area is reviewed in this paper.

Bio-mimetic mineralization potential of collagen hydrolysate obtained from chromium tanned leather waste.

Hydroxyapatite (HA) ceramics serve as an alternative to autogenous-free bone grafting by virtue of their excellent biocompatibility. However, chemically synthesized HA lacks the strong load-bearing capacity as required by bone. The bio-mimetic growth of HA crystals on collagen surface provides a feasible solution for synthesizing bone substitutes with the desired properties. This study deals with the utilization of the collagen hydrolysate recovered from leather waste as a substrate for promoting HA crystal growth. Bio-mimetic growth of HA was induced by subjecting the hydrolysate to various mineralization conditions. Parameters that would have a direct effect on crystal growth were varied to determine the optimal conditions necessary. Maximum mineralization was achieved with a combination of 10mM of CaCl2, 5mM of Na2HPO4, 100mM of NaCl and 0.575% glutaraldehyde at a pH of 7.4. The metal-protein interactions leading to formation of HA were identified through Fourier-transform infrared (FTIR) spectroscopy and x-ray diffraction (XRD) studies. The crystal dimensions were determined to be in the nanoscale range by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The size and crystallinity of bio-mimetically grown HA indicate that hydrolysate from leather waste can be used as an ideal alternative substrate for bone growth.

Investigation into the cyto-protective and wound healing properties of cryptic peptides from bovine Achilles tendon collagen.

Many proteins have concealed regions in their amino acid sequences that when liberated or exposed by conformational changes can exhibit bioactivity. Two such cryptic bioactive peptides, C2 (with cell adhesive properties) and E1 (with cell adhesive and antioxidant properties) have been isolated from bovine tendon collagen. This investigation deals with the efficacy of these peptides in countering externally generated stress and imparting cyto-protection in mammalian cell systems. The cell survival activity was studied with two cell lines, viz., HeLa and Vero, with varying concentrations of five oxidative stress-generating agents. The activities of the peptides in supporting cell adhesion and countering stress were determined in their coated and dissolved forms. C2 and E1 coated dishes registered 8 times (p<0.01) higher rate of cell survival against oxidative stress than collagen coated dishes. E1 increased stress tolerance levels by >100 times in dissolved form and C2, by 8 times in coated form. The peptides supported faster wound closure than collagen under normal as well as stressed condition. Maximum stress tolerance was observed on C2 coated dishes in the presence of E1 in the medium suggesting that both enhanced cell adhesion and antioxidative activities significantly contribute to the cell survival during stress. The present study emphasizes that collagen peptides, apart from providing a suitable surface for cell adhesion, also confer protection to cells against oxidative stress.

Isolation and identification of cryptic bioactive regions in bovine achilles tendon collagen.

Several proteins are known to host specific regions within their sequence, that when exposed or excised out proteolytically can display a range of physiological activities quite different from that of the parent protein. Collagen, a class of structural biopolymers and an important component of the extracellular matrix, is now known to harbor several such bioactive peptides which can act as physiological regulators. This study was undertaken to identify such cryptic sites from bovine Achilles tendon collagen and an antioxidative assay was used to screen for bioactivity. Bacterial crude protease was used to hydrolyze collagen and the hydrolysate was subjected to separation through ion-exchange column chromatography. Fractions were screened using conventional antioxidative assays and further purified by gel permeation chromatography. Two biologically active cryptic peptides were obtained displaying high antioxidative properties, E1 and F3. At low concentrations, both peptides displayed higher chelating ability than EDTA and were able to reduce the auto-oxidation of unsaturated fatty acid. The molecular weights of the peptides were found out through column chromatography and Tricine SDS PAGE; both displayed molecular mass below 4 kDa. Overall E1 displayed a comparatively better antioxidative ability than the others and was further characterized by circular dichroism studies and sequencing. A BLAST search of the active peptide sequence revealed that an almost similar peptide also resides in human collagen Type I.