Role of three-dimensional cell culture in therapeutics and diagnostics: an updated review.

Drug development and testing are a tedious and expensive process with a high degree of uncertainty in the clinical success and preclinical validation of manufactured therapeutic agents. Currently, to understand the drug action, disease mechanism, and drug testing, most therapeutic drug manufacturers use 2D cell culture models to validate the drug action. However, there are many uncertainties and limitations with the conventional use of 2D (monolayer) cell culture models for drug testing that are primarily attributed due to poor mimicking of cellular mechanisms, disturbance in environmental interaction, and changes in structural morphology. To overcome such odds and difficulties in the preclinical validation of therapeutic medications, newer in vivo drug testing cell culture models with higher screening efficiencies are required. One such promising and advanced cell culture model reported recently is the "three-dimensional cell culture model." The 3D cell culture models are reported to show evident benefits over conventional 2D cell models. This review article outlines and describes the current advancement in cell culture models, their types, significance in high-throughput screening, limitations, applications in drug toxicity screening, and preclinical testing methodologies to predict in vivo efficacy.

Quantitation of mHLA-DR and nCD64 by Flow Cytometry to Study Dysregulated Host Response: The Use of QuantiBRITE™ PE Beads and Its Stability.

Quantitation of mHLA-DR and nCD64 is useful in understanding the dysregulated host response. The down regulation of HLA-DR expression on the circulating monocytes (mHLA-DR) is associated with anti-inflammatory response, and an increased expression of CD64 on neutrophil surface (nCD64) is associated with pro-inflammatory response. Quantitation of these antigen expression using beads (QuantiBRITE™ PE) is a precision technique. These beads are reported to be stable for 24 h after reconstitution. We report the results of our investigation examining the stability of QuantiBRITE PE beads over a period of 4-week post-reconstitution. The data suggest that reconstituted QuantiBRITE PE beads, if stored in dark at 2-8 °C, can be effectively used for up to 2 weeks for determining nCD64 and mHLA-DR antibody bound per cell (ABC) values.

HPTLC and GC-MS finger-printing of two potential multifunctional siddha tailams: Mathan and maha megarajanga tailam.

The Siddha system of medicine is an ancient medical lineage that is practiced primarily in the southern part of India. Siddha system of medicine has been in practice for thousands of years with documented evidence dating back to the 6th century BCE. According to siddha system of medicine's basic fundamental principle, the human body is made up of 96 thathuvam (primary components), which encompass physical, physiological, psychological, and intellectual aspects. Medicine (marunthu) is classified as a wide range of internal and external medicines. The major components of its medical formulations include plant parts, minerals and animal products. Various methods were carried out for the purification process to eliminate the toxins. Choornam, Guligai, Tailam, Parpam, Chendooram, Kattu, Pasai and Poochu are the most common medicines used in Siddha system of medicine for the treatment of various diseases. The pathophysiological classification of diseases is elaborated in detail in the classical Siddha literature. Siddha system of medicine plays an important role in protecting people from diseases such as COVID-19 by providing immune-protecting and immune-boosting medicines in today's world. Mathan tailam and maha megarajanga tailam are the two unique preparations used widely for various skin diseases including chronic wounds and burns. Scientific validation of both medicines will help in understanding their effectiveness against a typical wound condition. In the present study physio-chemical and phytochemical, HPTLC, and GC-MS analyses were carried out and discussed in detail on the multifunctional properties exhibited in the patient communities.

Monoclonal Antibody Functionalized, and L-lysine α-Oxidase Loaded PEGylated-Chitosan Nanoparticle for HER2/Neu Targeted Breast Cancer Therapy.

Herein, we designed a nanocarrier to deliver the LO specifically to HER2+ breast cancer (BC) cells, where functionalization of mAb (anti-HER2+) with PEGylated chitosan enabled it to target the HER2+ BC cells. Taking advantage of overexpression of HER2+ in cancer cells, our nanocarrier (CS-LO-PEG-HER NPs) exhibited promising potency and selectivity against HER2+ BC cells (BT474). The CS-LO-PEG-HER NPs demonstrated the cytotoxicity in BT474 cells by promoting reactive oxygen species, mitochondrial membrane potential loss, and nucleus damage. The biocompatibility of CS-LO-PEG-HER NPs was evidenced by the hemolysis assay and H & E staining of major organs. The CS-LO-PEG-HER NPs showed anticancer potency against the BT474-xenograft tumor-bearing mice, as evident by the reduction of tumor size and cell density. These results indicate that CS-LO-PEG-HER NPs are biocompatible with mice while inhibiting tumor growth through alter the oxidative stress. Overall, this work provides a promising approach for the delivery of LO for good therapeutic effect in combination with mAb.

Photothermally responsive chitosan-coated iron oxide nanoparticles for enhanced eradication of bacterial biofilms.

This work developed a pH/NIR responsive antibacterial agent (CS-FeNPs) composed of chitosan (CS) and Fe3O4 nanoparticles (FeNPs). CS triggers bacterial attraction through surface charge, while Fe acts as a photothermal agent (PTA). The CS-Fe NPs exhibited antibacterial and antibiofilm activity against both bacteria (G+/G-). However, higher activity was observed against bacteria (G-) due to electrostatic interactions. The CS-FeNPs bind with the bacterial membrane through electrostatic interactions and disturb bacterial cells. Later, in an acidic environment, CS-FeNPs bind with bacterial membrane, and NIR irradiation leads the antibacterial activity. CS-FeNPs exhibited a potential photothermal conversion efficiency (η) of 21.53 %. Thus, it converts NIR irradiation into heat to kill the bacterial pathogen. The CS-FeNPs were found to be less cytotoxic with great antibacterial efficiency on planktonic bacteria and their biofilm, which indicates that they deserve to develop potential and safe treatment strategies for the treatment of bacterial infections.

Application of hyaluronic acid in tissue engineering, regenerative medicine, and nanomedicine: A review.

Hyaluronic acid (HA) plays a vital role in cellular processes and its contribution to physical and immunological barriers is considered to be an important property for the formulation of modern therapeutics. With the increasing demand for non-toxic and targeted therapy, HA-based materials could be utilized for biomedical applications due to their tendency to bio-mimic the hosts. Moreover, HA is a versatile compound in the fabrication of HA-based products such as hydrogels, nanofibers, and 3D materials. These have been implemented in various medical fields, such as bone and tissue regeneration, topical gels for wound healing, and cancer treatment via HA-loaded drug delivery approaches. Herein, we have discussed the characteristics of HA and its significance in drug delivery in addition to synergistic effects with other therapeutic compounds in the fields of nanomedicine, tissue engineering, and regenerative medicine.

Unraveling the hazardous impact of diverse contaminants in the marine environment: Detection and remedial approach through nanomaterials and nano-biosensors.

Marine pollution is one of the most underlooked forms of pollution as it affects most aquatic lives and public health in the coastal area. The diverse form of the hazardous pollutant in the marine ecosystem leads the serious genetic level disorders and diseases which include cancer, diabetes, arthritis, reproductive, and neurological diseases such as Parkinson's, Alzheimer's, and several microbial infections. Therefore, a recent alarming study on these pollutants, the microplastics have been voiced out in many countries worldwide, it was even found to be in the human placenta. In recent times, nanomaterials have demonstrated their potential in the detection and remediation of sensitive contaminants. In this review, we presented a comprehensive overview of the source, and distribution of diverse marine pollution on both aquatic and human health by summarizing the concentration of diverse pollutions (heavy metals, pesticides, microbial toxins, and micro/nano plastics) in marine samples such as soil, water, and seafood. Followed by emphasizing its ecotoxicological impact on aquatic animal life and coastal public health. Also discussed are the applicability and advancements of nanomaterials and nano-based biosensors in the detection, prevention, and remediation of diverse pollution in the marine ecosystem.

Polysaccharide-Drug Conjugates: A Tool for Enhanced Cancer Therapy.

Cancer is one of the most widespread deadly diseases, following cardiovascular disease, worldwide. Chemotherapy is widely used in combination with surgery, hormone and radiation therapy to treat various cancers. However, chemotherapeutic drugs can cause severe side effects due to non-specific targeting, poor bioavailability, low therapeutic indices, and high dose requirements. Several drug carriers successfully overcome these issues and deliver drugs to the desired sites, reducing the side effects. Among various drug delivery systems, polysaccharide-based carriers that target only the cancer cells have been developed to overcome the toxicity of chemotherapeutics. Polysaccharides are non-toxic, biodegradable, hydrophilic biopolymers that can be easily modified chemically to improve the bioavailability and stability for delivering therapeutics into cancer tissues. Different polysaccharides, such as chitosan, alginates, cyclodextrin, pullulan, hyaluronic acid, dextran, guar gum, pectin, and cellulose, have been used in anti-cancer drug delivery systems. This review highlights the recent progress made in polysaccharides-based drug carriers in anti-cancer therapy.

Anticancer and immunomodulatory effect of rhaponticin on Benzo(a)Pyrene-induced lung carcinogenesis and induction of apoptosis in A549 cells.

In worldwide, one of the most important cancer-related death is lung cancer. Also has the highest mortality rate between various cancer types. The count of lung cancer occurrence is increasing with an increased frequency by smoking. Proficient chemoprevention approaches are needed to prevent the occurrence of lung cancer. Therefore, the aim of this exploration is to determine the therapeutic impact on the immune modulatory effect of rhaponticin on lung tumorigenesis in vivo and in vitro cytotoxicity effect in A549 cells of human lung cancer. Lung cancer tumorigenesis in mice was challenged with benzo(a)pyrene (BaP) with 50 mg/kg bodyweight (b.wt) as oral administration for 6 weeks (two times/week). Rhaponticin were given orally 30 mg/kg b.wt (two times/week) in BaP induced mice from 12 weeks to 18 weeks. After treatment completes, the body weight was measured and then blood, lung tissue was collected for various parameters detection. The results evidenced that BaP induced mice decreased the bodyweight, increased lung weight, increased tumor markers (AHH, CEA and LDH), and increased the proinflammatory cytokines. The enzyme catalase, superoxide dismutase activity was decreased and increased lipid peroxidation in immune comprising cells compared with the control cells. Moreover, rhaponticin treatment improves in chemical assays and also the histopathological alteration of lung tissues. The present findings provide evidence about the therapeutic potentials of rhaponticin against BaP triggered lung tumorigenesis.

Anti-Apoptotic Effect of Flavokawain A on Ochratoxin-A-Induced Endothelial Cell Injury by Attenuation of Oxidative Stress via PI3K/AKT-Mediated Nrf2 Signaling Cascade.

This study investigates the endothelial protective activity of flavokawain A (FKA) against oxidative stress induced by ochratoxin A (OTA), which acts as a mycotoxin, and its primary mechanisms in in vitro models. Reactive oxygen species, in general, regulate oxidative stress that significantly contributes to the pathophysiology of endothelial dysfunctions. OTA exerts toxicity through inflammation and the accumulation of ROS. This research is aimed at exploring the defensive function of FKA against the endothelial injury triggered by OTA through the Nrf2 pathway regulated by PI3K/AKT. OTA exposure significantly increased the nuclear translocation of NFκB, whereas we found a reduction in inflammation via NFκB inhibition with FKA treatment. FKA increased the PI3K and AKT phosphorylation, which may lead to the stimulation of antioxidative and antiapoptotic signaling in HUVECs. It also upregulated the phosphorylation of Nrf2 and a concomitant expression of antioxidant genes, such as HO-1, NQO-1, and γGCLC, depending on the dose under the oxidative stress triggered by OTA. Knockdown of Nrf2 through small interfering RNA (siRNA) impedes the protective role of FKA against the endothelial toxicity induced by OTA. In addition, FKA enhanced Bcl2 activation while suppressing apoptosis marker proteins. Therefore, FKA is regarded as a potential agent against endothelial oxidative stress caused by the deterioration of the endothelium. The research findings showed that FKA plays a key role in activating the p-PI3K/p-AKT and Nrf2 signaling pathways, while suppressing caspase-dependent apoptosis.

Nobiletin promotes osteogenic differentiation of human osteoblastic cell line (MG-63) through activating the BMP-2/RUNX-2 signaling pathway.

Nobiletin (NOB) is polymethoxy flavonoids, which plentifully there in Citrus depressa and they demonstrate numerous pharmacological effects. NOB has an anti-proliferative effect, attenuates ovalbumin-treated eosinophilic airway inflammation and Type II collagen treated arthritis. NOB noticeably inhibits bone resorption and renovates bone loss in mice model, but role of NOB in bone metabolism is unclear. Human bone is a important organ that sustains its homeostasis among bone resorpting osteoclasts and bone developing osteoblasts. The balances of among these two kind of cell outcomes are implicated in bone remodeling. The current study designed to explore possessions of NOB on differentiation and proliferation of MG-63 cells and contribution of morphogenetic protein signaling. Cell proliferation was analyzed by MTT, mineralization analysis by alizarin red staining and morphogenetic signaling protein by RT-PCR. No stimulus outcome of NOB on cell proliferation was found at days of 1, 3 and 7. Accumulation of calcium was augmented after that treatment of NOB. The mRNA expression of BMP-2, COL-I, ALP, OCN, RUNX2 and COL1A1 augmented markedly with NOB supplement. Hence, NOB can stimulate osteogenic differentiation of MG-63, almost certainly by promoting RUNX2 and BMP-2 signaling and this result might provide to its action on stimulation of osteoblast development, differentiation and augments of bone mass.

Identification of aspirin and diclofenac binding proteins in the red complex pathogens.

Red complex organisms are a group of organisms (Porphyromonas gingivalis ATCC 33277, Treponema denticola ATCC 35405, Tannerella forsythia ATCC 43037) that have been identified for the causation of periodontal diseases. Aspirin and diclofenac have been used as regular analgesics. Therefore, it is of interest to document the identification of aspirin and diclofenac binding proteins in the red complex pathogens using the STITCH v.5 pipeline. The virulence properties of these proteins were analyzed using VICMPred and VirulentPred software. Thus, we document 000 number of proteins having optimal binding features with the known analgesics.

Non-classical monocytes and its potential in diagnosing sepsis post cardiac surgery.

BACKGROUND: Sepsis is caused by a dysregulation of immune response to infection that results in very high mortality. Current laboratory tests and clinical criteria are inadequate to diagnose sepsis due to limited sensitivity and specificity. Circulating monocytes are important players in immune homeostasis and their altered HLA-DR expression indicate immune dysregulation. HLA-DR is an MHC Class II cell-surface receptor that can present foreign antigens to helper T cells and mount an inflammatory response. Therefore, we analyzed the variations in HLA-DR expression and the concentration of monocyte subsets for diagnosing post-surgical sepsis. METHODS: In this double-blinded prospective cohort study, we adopted immunophenotyping and quantification of antigen expression by flowcytometry to detect the changes in circulating monocyte subsets in patients undergoing cardiac surgery. Statistical analysis was performed to identify significant changes and based on the predictive potential of measured variables ROC curve analysis was done. ROC curve permitted the choice of appropriate cut-off values using which a diagnostic protocol was developed. RESULTS: We observed that the monocyte subset concentrations in circulation varied differently after surgery. There was a significant downregulation of monocytic HLA-DR on both intermediate (p = 0.0477) and non-classical monocytes (p = 0.0333) at 48 h post-surgery. The monocyte subset analysis clearly showed that the patients with reduced pre-surgical non-classical monocyte count (p = 0.0430) coupled with post-surgical down-regulation of HLA-DR expression on the same subset had a higher incidence of developing sepsis after cardiac surgery. CONCLUSIONS: Here we are reporting for the first time, the significant influence of non-classical monocytes in inducing dysregulated host response and sepsis after cardiac surgery. Using multiple biomarkers associated with this monocyte subset, we established an algorithm for the diagnosis of sepsis at 48 h post cardiac surgery with 100% sensitivity and 69.23% specificity.

Comparison of Immunological Profiles of SARS-CoV-2 Variants in the COVID-19 Pandemic Trends: An Immunoinformatics Approach.

The current dynamics of the COVID-19 pandemic have become a serious concern with the emergence of a series of mutant variants of the SARS-CoV-2 virus. Unlike the previous strain, it is reported that the descendants are associated with increased risk of transmission yet causing less impact in terms of hospital admission, the severity of illness, or mortality. Moreover, the vaccine efficacy is also not believed to vary among the population depending on the variants of the virus and ethnicity. It has been determined that the mutations recorded in the spike gene and protein of the newly evolved viruses are specificallyresponsible for this transformation in the behavior of the virus and its disease condition. Hence, this study aimed to compare the immunogenic profiles of the spike protein from the latest variants of the SARS-CoV-2 virus concerning the probability of COVID-19 severity. Genome sequences of the latest SARS-CoV-2 variants were obtained from GISAID and NCBI repositories. The translated protein sequences were run against T-cell and B-cell epitope prediction tools. Subsequently, antigenicity, immunogenicity, allergenicity, toxicity, and conservancy of the identified epitopes were ascertained using various prediction servers. Only the non-allergic and non-toxic potential epitopes were matched for population relevance by using the Human Leucocyte Antigen population registry in IEDB. Finally, the selected epitopes were validated by docking and simulation studies. The evaluated immunological parameters would concurrently reveal the severity of COVID-19, determining the infection rate of the pathogen. Our immunoinformatics approach disclosed that spike protein of the five variants was capable of forming potential T and B-cell epitopes with varying immune responses. Although the Wuhan strain showed a high number of epitope/HLA combinations, relatively less antigenicity and higher immunogenicity results in poor neutralizing capacity, which could be associated with increased disease severity. Our data demonstrate that increased viral antigenicity with moderate to high immunogenicity, and several potential epitope/HLA combinations in England strain, the USA, India, and South Africa variants, could possess a high neutralizing ability. Therefore, our findings reinforce that the newly circulating variants of SARS-CoV-2 might be associated with more infectiousness and less severe disease condition despite their greater viremia, as reported in the recent COVID-19 cases, whichconsequently determine their increased epidemiological fitness.

Molecular docking data of E3 ubiquitin-protein ligase WWP1 with compounds from a medicinal plant Justicia adhatoda L.

It is known that E3 ubiquitin-protein ligase WWP1 is linked to oral cancer. Therefore, it is of interest to document molecular docking data of E3 ubiquitin-protein ligase WWP1 with compounds ((Stigmasterol, Pyrazinamide, Vasicinone and Ethambutol)) from a medicinal plant Justicia adhatoda L for further consideration.

Toothbrushes as a Source of DNA for Gender and Human Identification-A Systematic Review.

BACKGROUND: Few studies have reported the use of toothbrushes as a reliable source of DNA for human or gender identification. The present systematic review with the available information was conducted to answer the focus question "Is a toothbrush a reliable source of DNA for human or gender identification?". METHODS: The keyword combination "Toothbrush" and "DNA" was used to search databases including MEDLINE, Scopus, and Web of Science along with a manual search of reference lists of relevant articles. Duplicates and irrelevant articles were excluded, and the remaining articles were fully read for the final selection of articles. The risk of bias of the included studies was evaluated using the Appraisal tool for Cross-Sectional Studies (AXIS tool). RESULTS: Of the 130 articles obtained, 122 duplicates or irrelevant articles were eliminated. Following the full-text reading of eight articles, five articles were selected based on eligibility criteria. The five studies reported that a toothbrush is a good source of DNA irrespective of the time interval. In a few studies some samples were not sufficient for complete DNA profiling due to factors such as the method of DNA extraction. CONCLUSION: Although a toothbrush is an excellent source of DNA for human and gender identification, future studies with a larger sample size, appropriate control group, and standardized technique of DNA extraction need to be conducted. Additionally, factors influencing the quantity and quality of DNA in toothbrushes need to be determined with standardized techniques.

D-Pinitol treatment induced the apoptosis in human leukemia MOLT-4 cells by improved apoptotic signaling pathway.

Cancer is still remain as a global burden with the 18.1 million and 9.6 million new cases and mortlities, respectively estimated globally. Leukemia may arise at all ages varied from the infants to elders. In this exploration, we planned to evaluate the antiproliferative effect of D-pinitol on human leukemia MOLT-4 cells. Anticancer potential of D-pinitol was examined using MTT assay. Reactive oxygen species (ROS) generation was studied by fluorescence microscopic method using DCFH-DA staining. Apoptotic morphological alterations were determined by dual staining (acridine orange and ethidium bromide). Western blot and ELISA methods were employed to study apoptotic protein expression. D-pinitol treatment significantly induced cytotoxicity in human leukemia MOLT-4 cells. We observed that D-pinitol induces the generation of ROS in MOLT-4 cells. Further, we noticed that D-pinitol significantly induced apoptosis in a dosage dependent manner. Moreover, western blot and ELISA based analysis revealed that D-pinitol elevated the Bax, Caspase-3, Caspase-9 and attenuated the Bcl-2 expression in leukemic cancer cell. Our findings suggest that D-pinitol treatment induces the apoptosis in human leukemic cells by generating intracellular ROS and modulating apoptotic protein expression.