Influence of periodontitis and diabetes on structure and cytokine content of platelet-rich fibrin.

BACKGROUND: Platelet-rich fibrin (PRF) is a second-generation platelet concentrate with multiple applications in wound healing and regeneration in both periodontitis and diabetes. However, the three dimensional (3-D) structure and cytokine content of PRF might be altered in patients suffering from either/both of the chronic inflammatory conditions, ultimately influencing the efficacy of PRF as a biomaterial for regenerative medicine. AIM: The aim of the present study was hence to evaluate the effect of both these chronic inflammatory diseases on the 3-D structure of PRF membrane. An attempt was also made to compare the growth factor content between the plasma and RBC ends of the prepared PRF gel. MATERIALS & METHODS: L-PRF was prepared for twenty participants, healthy (5), periodontitis (5), T2DM (5) and T2DM with periodontitis (5). Porosity and fiber diameter of PRF membranes was visualized under FE-SEM and measured using ImageJ Software. PDGF-BB and TGF-ß1 levels in PRF gel were assessed by ELISA. RESULTS: The average diameter of fibrin fibers under FE-SEM was 0.15 to 0.30 micrometers. Porosity was higher at the plasma end (p = 0.042). Red blood cell (RBC) end of the membrane had thinner fibers arranged in a comparatively more dense and compact structure with smaller porosities. Healthy subjects had the least porous PRF compared to subjects with either/both of the chronic conditions. PDGF-BB levels were similar along all the four groups. TGF-ß1 levels were highest in healthy subjects. DISCUSSION: 3-D structure and growth factor content of PRF are influenced by a person's periodontal and/or diabetic status. The RBC end of the PRF membrane, as compared to the plasma end, has thinner fibers arranged in a comparatively more dense and compact structure with smaller porosities, and hence should be favored during periodontal regenerative procedures. CONCLUSION: Both periodontitis and diabetes have a significant influence on the 3-D structure and growth factor content of PRF produced.

COVID19-inhibitory activity of withanolides involves targeting of the host cell surface receptor ACE2: insights from computational and biochemical assays.

SARS-CoV-2 outbreak in China in December 2019 and its spread as worldwide pandemic has been a major global health crisis. Extremely high infection and mortality rate has severely affected all sectors of life and derailed the global economy. While drug and vaccine development have been prioritized and have made significant progression, use of phytochemicals and herbal constituents is deemed as a low-cost, safer and readily available alternative. We investigated therapeutic efficacy of eight withanolides (derived from Ashwagandha) against the angiotensin-converting enzyme 2 (ACE2) proteins, a target cell surface receptor for SARS-CoV-2 and report results on the (i) computational analyses including binding affinity and stable interactions with ACE2, occupancy of ACE2 residues in making polar and nonpolar interactions with different withanolides/ligands and (2) in vitro mRNA and protein analyses using human cancer (A549, MCF7 and HSC3) cells. We found that among all withanolides, Withaferin-A, Withanone, Withanoside-IV and Withanoside-V significantly inhibited the ACE2 expression. Analysis of withanolides-rich aqueous extracts derived from Ashwagandha leaves and stem showed a higher ACE2 inhibitory potency of stem-derived extracts. Taken together, we demonstrated the inhibitory potency of Ashwagandha withanolides and its aqueous extracts against ACE2.Communicated by Ramaswamy H. Sarma.

Experimental evidence and mechanism of action of some popular neuro-nutraceutical herbs.

Brain and neuronal circuits constitute the most complex organ networks in human body. They not only control and coordinate functions of all other organs, but also represent one of the most-affected systems with stress, lifestyle and age. With global increase in aging populations, these neuropathologies have emerged as major concern for maintaining quality of life. Recent era has witnessed a surge in nutritional remediation of brain dysfunctions primarily by "nutraceuticals" that refer to functional foods and supplements with pharmacological potential. Specific dietary patterns with a balanced intake of carbohydrates, fatty acids, vitamins and micronutrients have also been ascertained to promote brain health. Dietary herbs and their phytochemicals with wide range of biological and pharmacological activities and minimal adverse effects have gained remarkable attention as neuro-nutraceuticals. Neuro-nutraceutical potentials of herbs are often expressed as effects on cognitive response, circadian rhythm, neuromodulatory, antioxidant and anti-inflammatory activities that are mediated by effects on gene expression, epigenetics, protein synthesis along with their turnover and metabolic pathways. Epidemiological and experimental evidence have implicated enormous applications of herbal supplementation in neurodegenerative and psychiatric disorders. The present review highlights the identification, experimental evidence and applications of some herbs including Bacopa monniera, Withania somnifera, Curcuma longa, Helicteres angustifolia, Undaria pinnatifida, Haematococcus pluvialis, and Vitis vinifera, as neuro-nutraceuticals.

Molecular mechanism of anti-SARS-CoV2 activity of Ashwagandha-derived withanolides.

COVID-19 caused by SARS-CoV-2 corona virus has become a global pandemic. In the absence of drugs and vaccine, and premises of time, efforts and cost required for their development, natural resources such as herbs are anticipated to provide some help and may also offer a promising resource for drug development. Here, we have investigated the therapeutic prospective of Ashwagandha for the COVID-19 pandemic. Nine withanolides were tested in silico for their potential to target and inhibit (i) cell surface receptor protein (TMPRSS2) that is required for entry of virus to host cells and (ii) viral protein (the main protease Mpro) that is essential for virus replication. We report that the withanolides possess capacity to inhibit the activity of TMPRSS2 and Mpro. Furthermore, withanolide-treated cells showed downregulation of TMPRSS2 expression and inhibition of SARS-CoV-2 replication in vitro, suggesting that Ashwagandha may provide a useful resource for COVID-19 treatment.

Bioinformatics and Molecular Insights to Anti-Metastasis Activity of Triethylene Glycol Derivatives.

The anti-metastatic and anti-angiogenic activities of triethylene glycol derivatives have been reported. In this study, we investigated their molecular mechanism(s) using bioinformatics and experimental tools. By molecular dynamics analysis, we found that (i) triethylene glycol dimethacrylate (TD-10) and tetraethylene glycol dimethacrylate (TD-11) can act as inhibitors of the catalytic domain of matrix metalloproteinases (MMP-2, MMP-7 and MMP-9) by binding to the S1' pocket of MMP-2 and MMP-9 and the catalytic Zn ion binding site of MMP-7, and that (ii) TD-11 can cause local disruption of the secondary structure of vascular endothelial growth factor A (VEGFA) dimer and exhibit stable interaction at the binding interface of VEGFA receptor R1 complex. Cell-culture-based in vitro experiments showed anti-metastatic phenotypes as seen in migration and invasion assays in cancer cells by both TD-10 and TD-11. Underlying biochemical evidence revealed downregulation of VEGF and MMPs at the protein level; MMP-9 was also downregulated at the transcriptional level. By molecular analyses, we demonstrate that TD-10 and TD-11 target stress chaperone mortalin at the transcription and translational level, yielding decreased expression of vimentin, fibronectin and hnRNP-K, and increase in extracellular matrix (ECM) proteins (collagen IV and E-cadherin) endorsing reversal of epithelial-mesenchymal transition (EMT) signaling.

Anti-stress, Glial- and Neuro-differentiation Potential of Resveratrol: Characterization by Cellular, Biochemical and Imaging Assays.

Environmental stress, exhaustive industrialization and the use of chemicals in our daily lives contribute to increasing incidence of cancer and other pathologies. Although the cancer treatment has revolutionized in last 2-3 decades, shortcomings such as (i) extremely high cost of treatment, (ii) poor availability of drugs, (iii) severe side effects and (iv) emergence of drug resistance have prioritized the need of developing alternate natural, economic and welfare (NEW) therapeutics reagents. Identification and characterization of such anti-stress NEW drugs that not only limit the growth of cancer cells but also reprogram them to perform their specific functions are highly desired. We recruited rat glioma- and human neuroblastoma-based assays to explore such activities of resveratrol, a naturally occurring stilbenoid. We demonstrate that nontoxic doses of resveratrol protect cells against a variety of stresses that are largely involved in age-related brain pathologies. These included oxidative, DNA damage, metal toxicity, heat, hypoxia, and protein aggregation stresses. Furthermore, it caused differentiation of cells to functional astrocytes and neurons as characterized by the upregulation of their specific protein markers. These findings endorse multiple bioactivities of resveratrol and encourage them to be tested for their benefits in animal models and humans.

Stress-induced changes in CARF expression determine cell fate to death, survival, or malignant transformation.

CARF (Collaborator of ARF) was discovered as an ARF-interacting protein that activated ARF-p53-p21WAF1 signaling involved in cellular response to a variety of stresses, including oxidative, genotoxic, oncogenic, or telomere deprotection stresses, leading to senescence, growth arrest, or apoptosis. Of note, whereas suppression of CARF was lethal, its enrichment was associated with increased proliferation and malignant transformation of cells. These reports have predicted that CARF could serve as a multi-stress marker with a predictive value for cell fates. Here, we recruited various in vitro stress models and examined their effect on CARF expression using human normal fibroblasts. We demonstrate that CARF levels in stress and post-stress conditions could predict the fate of cells towards either death or enhanced proliferation and malignant transformation. We provide extensive molecular evidence that (i) CARF expression changes in response to stress, (ii) it modulates cell death or survival signaling and determines the fate of cells, and (iii) it may serve as a predictive measure of cellular response to stress and an important marker for biosafety.

Induction of Senescence in Cancer Cells by a Novel Combination of Cucurbitacin B and Withanone: Molecular Mechanism and Therapeutic Potential.

Cancer, an uncontrolled proliferation syndrome, is treated with synthetic chemotherapeutic drugs that are associated with severe adverse effects. Development and application of new natural compounds is warranted to deal with the exponentially increasing incidence of cancer worldwide. Keeping selective toxicity to cancer cells as a priority criterion, we developed a combination of Cucurbitacin B and Withanone, and analyzed its anticancer potential using non-small cell lung cancer cells. We demonstrate that the selective cytotoxicity of the combination, called CucWi-N, to cancer cells is mediated by induction of cellular senescence that was characterized by decrease in Lamin A/C, CDK2, CDK4, Cyclin D, Cyclin E, phosphorylated RB, mortalin and increase in p53 and CARF proteins. It compromised cancer cell migration that was mediated by decrease in mortalin, hnRNP-K, vascular endothelial growth factor, matrix metalloproteinase 2, and fibronectin. We provide in silico, molecular dynamics and experimental data to support that CucWi-N (i) possesses high capability to target mortalin-p53 interaction and hnRNP-K proteins, (ii) triggers replicative senescence and inhibits metastatic potential of the cancer cells, and (iii) inhibits tumor progression and metastasis in vivo. We propose that CucWi-N is a potential natural anticancer drug that warrants further mechanistic and clinical studies.

The Importance of Cell-Cell Interaction Dynamics in Bottom-Up Tissue Engineering: Concepts of Colloidal Self-Assembly in the Fabrication of Multicellular Architectures.

Building tissue from cells as the basic building block based on principles of self-assembly is a challenging and promising approach. Understanding how far principles of self-assembly and self-sorting known for colloidal particles apply to cells remains unanswered. In this study, we demonstrate that not just controlling the cell-cell interactions but also their dynamics is a crucial factor that determines the formed multicellular structure, using photoswitchable interactions between cells that are activated with blue light and reverse in the dark. Tuning dynamics of the cell-cell interactions by pulsed light activation results in multicellular architectures with different sizes and shapes. When the interactions between cells are dynamic, compact and round multicellular clusters under thermodynamic control form, while otherwise branched and loose aggregates under kinetic control assemble. These structures parallel what is known for colloidal assemblies under reaction- and diffusion-limited cluster aggregation, respectively. Similarly, dynamic interactions between cells are essential for cells to self-sort into distinct groups. Using four different cell types, which expressed two orthogonal cell-cell interaction pairs, the cells sorted into two separate assemblies. Bringing concepts of colloidal self-assembly to bottom-up tissue engineering provides a new theoretical framework and will help in the design of more predictable tissue-like structures.

Express ELISA for detection of mortalin.

Mortalin is a widely studied stress chaperone that plays a significant role in diseases such as cancer, diabetes mellitus, liver cirrhosis, neurodegeneration and generalized aging. Based on these, the level of mortalin expression has been predicted to be an important and valuable diagnostic and prognostic marker. Conventional methods of protein analyses, such as Western blotting, immunohistochemistry or ELISA with antibodies provide specific, sensitive and useful outcomes. However, they are limited by lengthy and time-consuming protocols. Here, we present an upgrade to the existing ELISA techniques. We have prepared a conjugate of anti-mortalin antibody and luciferase enzyme that can be recruited for rapid (∼3 h) and quantitative detection of mortalin expression in a given biological sample.

Marine Carotenoid Fucoxanthin Possesses Anti-Metastasis Activity: Molecular Evidence.

Fucoxanthin is commonly found in marine organisms; however, to date, it has been one of the scarcely explored natural compounds. We investigated its activities in human cancer cell culture-based viability, migration, and molecular assays, and found that it possesses strong anticancer and anti-metastatic activities that work irrespective of the p53 status of cancer cells. In our experiments, fucoxanthin caused the transcriptional suppression of mortalin. Cell phenotype-driven molecular analyses on control and treated cells demonstrated that fucoxanthin caused a decrease in hallmark proteins associated with cell proliferation, survival, and the metastatic spread of cancer cells at doses that were relatively safe to the normal cells. The data suggested that the cancer therapy regimen may benefit from the recruitment of fucoxanthin; hence, it warrants further attention for basic mechanistic studies as well as drug development.

Rat Glioma Cell-Based Functional Characterization of Anti-Stress and Protein Deaggregation Activities in the Marine Carotenoids, Astaxanthin and Fucoxanthin.

Stress, protein aggregation, and loss of functional properties of cells have been shown to contribute to several deleterious pathologies including cancer and neurodegeneration. The incidence of these pathologies has also been shown to increase with age and are often presented as evidence to the cumulative effect of stress and protein aggregation. Prevention or delay of onset of these diseases may prove to be unprecedentedly beneficial. In this study, we explored the anti-stress and differentiation-inducing potential of two marine bioactive carotenoids (astaxanthin and fucoxanthin) using rat glioma cells as a model. We found that the low (nontoxic) doses of both protected cells against UV-induced DNA damage, heavy metal, and heat-induced protein misfolding and aggregation of proteins. Their long-term treatment in glioma cells caused the induction of physiological differentiation into astrocytes. These phenotypes were supported by upregulation of proteins that regulate cell proliferation, DNA damage repair mechanism, and glial differentiation, suggesting their potential for prevention and treatment of stress, protein aggregation, and age-related pathologies.

Identification and Functional Characterization of Anti-metastasis and Anti-angiogenic Activities of Triethylene Glycol Derivatives.

We had previously reported anticancer activity in the water extract (WEX) of Ashwagandha leaves, and identified Triethylene glycol (TEG) as an active tumor suppressor component. In this study, we investigated anti-migratory and anti-angiogenesis activities of WEX and TEG. We conducted in vitro and in vivo experiments using TEG, and its two derivatives, Triethyleneglycol dimethacrylate (TD-10), and Tetraethyleneglycol dimethacrylate (TD-11). The data revealed strong anticancer and anti-metastasis potentials in the derivatives. Non-toxic, anti-migratory doses of the derivatives showed inhibition of canonical Wnt/ß-catenin axis and consequent downregulation of EMT-signaling proteins (Vimentin, MMPs and VEGF). These results endorse that the TD-10 and TD-11 have potential to safely put a check on the aggressiveness of the metastatic cells and therefore represent promising candidates for the treatment of metastatic cancers.

Integration of conventional cell viability assays for reliable and reproducible read-outs: experimental evidence.

OBJECTIVE: Short-term viability assays of cultured cells in 96-well plates are routinely used to determine the cytotoxicity or safety of drugs. These are often based on the formation of chromogen, generated selectively in viable cells. The innate problems of such short-term cell viability assays include (i) effect of drugs is determined by cell density (ii) some drugs have slow/gradual effect and hence may escape such assays, (iii) cell morphology that reveal significant hints to molecular signaling underlining the effect of drugs cannot be effectively captured, (iv) long-term effect on viability and clonogenic potential of cells cannot be determined and (v) herbal extracts often possess intrinsic color that interferes with spectrophotometer estimation. In light of the ease and importance of cell culture-based assessment of drug safety and cytotoxicity, we attempted to combine the conventional cell-based assays in a way that allows multiple readouts (quantitative and qualitative) from a single experiment, and avoids the drawbacks of color interference. RESULTS: We have established and validated (using 16 types of cultured mammalian cells) a Quantitative and Qualitative Cell Viability assay in 12-well cell culture plates. It overcomes several shortcomings as discussed above and allows long-term observations on cell morphology and clonogenicity.

CARF enrichment promotes epithelial-mesenchymal transition via Wnt/ß-catenin signaling: its clinical relevance and potential as a therapeutic target.

CARF (Collaborator of ARF)/CDKN2AIP was discovered as a novel ARF-binding protein. It has been established as an essential cell survival, p53-, and cell proliferation-regulatory protein. Although a moderate upregulation of CARF caused growth arrest and senescence, its excessively enriched levels were shown to facilitate aggressive proliferation and malignant transformation of cancer cells. Here, we examined the relevance of CARF levels in clinical tumors and found its amplification (both at gene and transcript levels) in a variety of invasive and metastatic malignancies. Consistent with the clinical readouts, enrichment of CARF in cancer cells promoted epithelial-mesenchymal transition (EMT). Cancer database and molecular analyses revealed that it activates Wnt/ß-catenin signaling axis, as evident by enhanced nuclear localization and function of ß-catenin marked by increased level of SNAIL1, SNAIL2, ZEB1, and TWIST1 and its downstream gene targets. Of note, targeted knockdown of CARF led to decrease in nuclear ß-catenin and its key downstream effectors, involved in EMT progression. Consistent with this, CARF targeting in vivo either by naked siRNA or CARF shRNA harboring adeno-oncolytic virus caused suppression of tumor progression and lung metastasis. Taken together, we report clinical and therapeutic relevance of CARF in EMT and cancer invasiveness/metastasis, and propose it as a potent therapeutic target of aggressive cancers.

Cucurbitacin B and cancer intervention: Chemistry, biology and mechanisms (Review).

Cancer is one of the most important healthcare matters, with the worst prognosis but the best possibilities for scientific development. It is likely to increase in the future and cause global havoc designating it as an epidemic. Cancer development requires urgent intervention. Past few decades have witnessed extensive research to challenge carcinogenesis. Treatment involving synthetic discipline is often associated with severe adverse effects, or even worsened prognosis. Accordingly, newer economic and patient friendly molecules are warranted. Many natural substances have proved their potential so far. Cucurbitacin B against cancer and other diseases has achieved towering popularity among the researchers around the world, as detailed in the below sections with summarized tables. In line with the fascinating role of cucurbitacin B against various types of cancers, through various molecular signaling pathways, it is justifiable to propose cucurbitacin B as a mainline chemotherapy before the onset and after the diagnosis of cancer.

Anti-Stress and Glial Differentiation Effects of a Novel Combination of Cucurbitacin B and Withanone (CucWi-N): Experimental Evidence.

BACKGROUND: Natural extracts and compounds used in traditional home medicine are known for their safety and a variety of health promoting and therapeutic potentials. In contrast to the single molecule mediated targets, the combinational therapies are preferred for their multi-functional and limited toxic regimens and may be useful for disease therapeutics as well as to increase the quality of life during a variety of environmental stresses. PURPOSE: We aimed to combine the active ingredients of Chinese (Helicteres angustifolia) and Indian (Withania somnifera) ginsengs to develop a natural, efficient, and welfare combinatorial mixture with high anti-stress and glial differentiation potentials. METHODS: Using cultured cells as a model system, we developed a combination of active ingredients of Chinese (Cucurbitacin B [Cuc]) and Indian (Withanone [Wi-N]) ginsengs. Eleven chemical models of environmental stresses were used. Cytotoxicity studies were performed using human skin fibroblast for anti-stress and rat glioma cells for glial differentiation effects. RESULTS: We demonstrate that the novel combination of Cuc and Wi-N, CucWi-N, was non-toxic to normal cells. It caused stress protection in assays using normal human fibroblasts subjected to a variety of stresses. Of note, cells showed remarkable protection against oxidative and UV stresses and marked by decrease in DNA damage and reactive oxygen species. We examined and found the glial differentiation potential of CucWi-N in rat glioblastoma cells. CucWi-N clearly induced differentiation phenotype, well-marked with upregulation of GAP43, MAP2, and GFAP, which have been shown to play a key role in glial differentiation. CONCLUSION: These data demonstrate anti-stress and glial differentiation potential of CucWi-N (a novel combination of Cuc and Wi-N) that could be recruited in nutraceutical and pharmaceutical avenues and hence warrant further evaluation and mechanistic studies.

Novel Methods to Generate Active Ingredients-Enriched Ashwagandha Leaves and Extracts.

Ashwagandha (Withania somnifera) is an Ayurvedic herb commonly used in world-renowned traditional Indian home medicine system. Roots of Ashwagandha have been traditionally known to possess a variety of therapeutic and health promoting potentials that have not been sufficiently supported by laboratory studies. Nevertheless, most, if not all, of the preventive and therapeutic potentials have been assigned to its bioactive components, steroidal alkaloids and lactones. In contrast to the traditional use of roots, we have been exploring bioactivities in leaves of Ashwagandha. Here, we report that the leaves possess higher content of active Withanolides, Withaferin-A (Wi-A) and Withanone (Wi-N), as compared to the roots. We also established, for the first time, hydroponic cultivation of Ashwagandha and investigated the effect of various cultivation conditions on the content of Wi-A and Wi-N by chemical analysis and bioassays. We report that the Withanone/Withaferin A-rich leaves could be obtained by manipulating light condition during hydroponic cultivation. Furthermore, we recruited cyclodextrins to prepare extracts with desired ratio of Wi-N and Wi-A. Hydroponically grown Ashwagandha and its extracts with high ratio of withanolides are valuable for cancer treatment.

Functional Characterisation of Anticancer Activity in the Aqueous Extract of Helicteres angustifolia L. Roots.

Helicteres angustifolia L. is a shrub that forms a common ingredient of several cancer treatment recipes in traditional medicine system both in China and Laos. In order to investigate molecular mechanisms of its anticancer activity, we prepared aqueous extract of Helicteres angustifolia L. Roots (AQHAR) and performed several in vitro assays using human normal fibroblasts (TIG-3) and osteosarcoma (U2OS). We found that AQHAR caused growth arrest/apoptosis of U2OS cells in a dose-dependent manner. It showed no cytotoxicity to TIG-3 cells at doses up to 50 µg/ml. Biochemical, imaging and cell cycle analyses revealed that it induces ROS signaling and DNA damage response selectively in cancer cells. The latter showed upregulation of p53, p21 and downregulation of Cyclin B1 and phospho-Rb. Furthermore, AQHAR-induced apoptosis was mediated by increase in pro-apoptotic proteins including cleaved PARP, caspases and Bax. Anti-apoptotic protein Bcl-2 showed decrease in AQHAR-treated U2OS cells. In vivo xenograft tumor assays in nude mice revealed dose-dependent suppression of tumor growth and lung metastasis with no toxicity to the animals suggesting that AQHAR could be a potent and safe natural drug for cancer treatment.

Ameliorative potential of pioglitazone and ceftriaxone alone and in combination in rat model of neuropathic pain: Targeting PPARγ and GLT-1 pathways.

BACKGROUND: The relation between glutamate homeostasis and PPAR gamma has got tremendous importance in nerve trauma and pain. Present study has been designed to elucidate the interaction between the GLT-1 activator (ceftriaxone) and PPAR gamma agonist (pioglitazone) in the spinal nerve ligation induced neuropathic pain. METHODS: Male SD rats were subjected to spinal nerve ligation to induce neuropathic pain. Pioglitazone, ceftriaxone and their combination treatments were given for 28 days. Various behavioral, biochemical, neuroinflammatory and apoptotic mediators were assessed subsequently. RESULTS: In the present study, ligation of L5 and L6 spinal nerves resulted in marked hyperalgesia and allodynia to different mechanical and thermal stimuli. In addition there is marked increase in oxidative-nitrosative stress parameters, inflammatory and apoptotic markers in spinal cord of spinal nerve ligated rats. Treatment with pioglitazone and ceftriaxone significantly prevented these behavioral, biochemical, mitochondrial and cellular alterations in rats. Further, combination of pioglitazone (10mg/kg, ip) with ceftriaxone (100mg/kg, ip) significantly potentiated the protective effects as compared to their effects per se. CONCLUSION: Based on these results we propose that possible interplay between the neuroprotective effects of pioglitazone and ceftriaxone exists in suppressing the behavioral, biochemical, mitochondrial, neuroinflammatory and apoptotic cascades in spinal nerve ligation induced neuropathic pain in rats.