Intranasally Administered EVs from hiPSC-derived NSCs Alter the Transcriptomic Profile of Activated Microglia and Conserve Brain Function in an Alzheimer's Model.

Antiinflammatory extracellular vesicles (EVs) derived from human induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs) hold promise as a disease-modifying biologic for Alzheimer's disease (AD). This study directly addressed this issue by examining the effects of intranasal administrations of hiPSC-NSC-EVs to 3-month-old 5xFAD mice. The EVs were internalized by all microglia, which led to reduced expression of multiple genes associated with disease-associated microglia, inflammasome, and interferon-1 signaling. Furthermore, the effects of hiPSC-NSC-EVs persisted for two months post-treatment in the hippocampus, evident from reduced microglial clusters, inflammasome complexes, and expression of proteins and/or genes linked to the activation of inflammasomes, p38/mitogen-activated protein kinase, and interferon-1 signaling. The amyloid-beta (Aß) plaques, Aß-42, and phosphorylated-tau concentrations were also diminished, leading to better cognitive and mood function in 5xFAD mice. Thus, early intervention with hiPSC-NSC-EVs in AD may help maintain better brain function by restraining the progression of adverse neuroinflammatory signaling cascades.

Extracellular vesicles from hiPSC-NSCs can prevent peripheral inflammation-induced cognitive dysfunction with inflammasome inhibition and improved neurogenesis in the hippocampus.

Extracellular vesicles (EVs) released by human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSCs) are enriched with miRNAs and proteins capable of mediating robust antiinflammatory activity. The lack of tumorigenic and immunogenic properties and ability to permeate the entire brain to incorporate into microglia following intranasal (IN) administrations makes them an attractive biologic for curtailing chronic neuroinflammation in neurodegenerative disorders. We tested the hypothesis that IN administrations of hiPSC-NSC-EVs can alleviate chronic neuroinflammation and cognitive impairments induced by the peripheral lipopolysaccharide (LPS) challenge. Adult male, C57BL/6J mice received intraperitoneal injections of LPS (0.75 mg/kg) for seven consecutive days. Then, the mice received either vehicle (VEH) or hiPSC-NSC-EVs (~ 10 × 109 EVs/administration, thrice over 6 days). A month later, mice in all groups were investigated for cognitive function with behavioral tests and euthanized for histological and biochemical studies. Mice receiving VEH after LPS displayed deficits in associative recognition memory, temporal pattern processing, and pattern separation. Such impairments were associated with an increased incidence of activated microglia presenting NOD-, LRR-, and pyrin domain containing 3 (NLRP3) inflammasomes, elevated levels of NLRP3 inflammasome mediators and end products, and decreased neurogenesis in the hippocampus. In contrast, the various cognitive measures in mice receiving hiPSC-NSC-EVs after LPS were closer to naive mice. Significantly, these mice displayed diminished microglial activation, NLRP3 inflammasomes, proinflammatory cytokines, and a level of neurogenesis matching age-matched naïve controls. Thus, IN administrations of hiPSC-NSC-EVs are an efficacious approach to reducing chronic neuroinflammation-induced cognitive impairments.

Intranasally administered extracellular vesicles from human induced pluripotent stem cell-derived neural stem cells quickly incorporate into neurons and microglia in 5xFAD mice.

Introduction: Extracellular vesicles (EVs) released by human-induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs) have robust antiinflammatory and neurogenic properties due to therapeutic miRNAs and proteins in their cargo. Hence, hiPSC-NSC-EVs are potentially an excellent biologic for treating neurodegenerative disorders, including Alzheimer's disease (AD). Methods: This study investigated whether intranasally (IN) administered hiPSC-NSC-EVs would quickly target various neural cell types in the forebrain, midbrain, and hindbrain regions of 3-month-old 5xFAD mice, a model of ß-amyloidosis and familial AD. We administered a single dose of 25 × 109 hiPSC-NSC-EVs labeled with PKH26, and different cohorts of naïve and 5xFAD mice receiving EVs were euthanized at 45 min or 6 h post-administration. Results: At 45 min post-administration, EVs were found in virtually all subregions of the forebrain, midbrain, and hindbrain of naïve and 5xFAD mice, with predominant targeting and internalization into neurons, interneurons, and microglia, including plaque-associated microglia in 5xFAD mice. EVs also came in contact with the plasma membranes of astrocytic processes and the soma of oligodendrocytes in white matter regions. Evaluation of CD63/CD81 expression with the neuronal marker confirmed that PKH26 + particles found within neurons were IN administered hiPSC-NSC-EVs. At 6 h post-administration, EVs persisted in all cell types in both groups, with the distribution mostly matching what was observed at 45 min post-administration. Area fraction (AF) analysis revealed that, in both naïve and 5xFAD mice, higher fractions of EVs incorporate into forebrain regions at both time points. However, at 45 min post-IN administration, AFs of EVs within cell layers in forebrain regions and within microglia in midbrain and hindbrain regions were lower in 5xFAD mice than naïve mice, implying that amyloidosis reduces EV penetrance. Discussion: Collectively, the results provide novel evidence that IN administration of therapeutic hiPSC-NSC-EVs is an efficient avenue for directing such EVs into neurons and glia in all brain regions in the early stage of amyloidosis. As pathological changes in AD are observed in multiple brain areas, the ability to deliver therapeutic EVs into various neural cells in virtually every brain region in the early stage of amyloidosis is attractive for promoting neuroprotective and antiinflammatory effects.

Investigation of the hydration process and biological activity of a novel nanosilver incorporated dicalcium silicate based retrograde filling material.

Background: Although several materials have been used for retrograde filling following apical surgeries, there is no consensus on a single best material. Tricalcium silicate-based types of cement have been developed as root-end filling materials mainly due to tricalcium silicate's hydraulic properties. However, its unfavorable setting characteristics and minimal antimicrobial properties have necessitated the introduction of new additives into the existing commercially available materials. To design an affordable product based on a dicalcium silicate with a shorter set time, minimal cytotoxic complications, and enhanced antibacterial activity, we developed a new endodontic cement from pure raw materials, intending to satisfy the prerequisites of ideal retrograde material. Methods: The composition of the experimental calcium silicate-based cement included the addition of calcium chloride and silver nanoparticles in varying concentrations. Structural characterization was carried out using energy dispersive analysis by X-rays using scanning electron microscope (EDAX SEM) and hydration characteristics were performed using an X-ray diffractometer (XRD). The experimental material was further evaluated for biocompatibility using MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)assay and antibacterial activity was evaluated using an agar diffusion test against Enterococcus faecalis. Results: The structural characterization and hydration characteristics revealed that the experimental cement was dicalcium silicate based with favorable biocompatibility and enhanced antibacterial activity. Tricalcium silicate based mineral trioxide aggregate (MTA) also had favourable biocompatibility, however, its antibacterial activity was significantly decreased when compared to the novel cement. Conclusion: All hydraulic cements that are available in the dental market are predominantly tricalcium silicate-based materials. There has been no evidence in the literature to date wherein it has been explored whether a dicalcium silicate-based hydraulic cement can solely be used in root-end cavities. The findings of the study revealed a dicalcium silicate based retrograde filling material with favourable biocompatibility exhibited immediately as well as in the set samples. Incorporation of silver nanoparticles boosted the antibacterial activity when compared to that of ProRoot MTA. This material could potentially reinstate the usual hype created with tricalcium silicate types of cement since dicalcium silicate cements also exhibit similar properties.

Intranasally administered human MSC-derived extracellular vesicles inhibit NLRP3-p38/MAPK signaling after TBI and prevent chronic brain dysfunction.

Traumatic brain injury (TBI) leads to lasting brain dysfunction with chronic neuroinflammation typified by nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) inflammasome activation in microglia. This study probed whether a single intranasal (IN) administration of human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs) naturally enriched with activated microglia-modulating miRNAs can avert chronic adverse outcomes of TBI. Small RNA sequencing confirmed the enrichment of miRNAs capable of modulating activated microglia in hMSC-EV cargo. IN administration of hMSC-EVs into adult mice ninety minutes after the induction of a unilateral controlled cortical impact injury resulted in their incorporation into neurons and microglia in both injured and contralateral hemispheres. A single higher dose hMSC-EV treatment also inhibited NLRP3 inflammasome activation after TBI, evidenced by reduced NLRP3, apoptosis-associated speck-like protein containing a CARD, activated caspase-1, interleukin-1 beta, and IL-18 levels in the injured brain. Such inhibition in the acute phase of TBI endured in the chronic phase, which could also be gleaned from diminished NLRP3 inflammasome activation in microglia of TBI mice receiving hMSC-EVs. Proteomic analysis and validation revealed that higher dose hMSC-EV treatment thwarted the chronic activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway by IL-18, which decreased the release of proinflammatory cytokines. Inhibition of the chronic activation of NLRP3-p38/MAPK signaling after TBI also prevented long-term cognitive and mood impairments. Notably, the animals receiving higher doses of hMSC-EVs after TBI displayed better cognitive and mood function in all behavioral tests than animals receiving the vehicle after TBI. A lower dose of hMSC-EV treatment also partially improved cognitive and mood function. Thus, an optimal IN dose of hMSC-EVs naturally enriched with activated microglia-modulating miRNAs can inhibit the chronic activation of NLRP3-p38/MAPK signaling after TBI and prevent lasting brain dysfunction.

Microbial Sterility Testing for Mesenchymal Progenitor Cells Derived from Human Osteoarthritis and Rheumatoid Arthritis Cartilage.

BACKGROUND: The goal is to evaluate the microbial sterility of mesenchymal progenitor cells (MPCs)-derived from osteoarthritis (OA) and rheumatoid arthritis (RA) articular cartilages. METHODS: Contaminants, including bacteria and fungi in MPC cultures were initially evaluated by inoculation culture methods and then affirmed through the amplification of 16S ribosomal DNA (rDNA) and internally transcribed spacer (ITS) regions, respectively, using polymerase chain reaction (PCR). Further, the mollicutes, if any, were identified by genus-specific 16S rDNA, and the positive samples were reamplified using species-specific primers. RESULTS: No bacteria or fungi were found to be compromising the sterility of MPCs (n = 20) assessed by both traditional culture methods and PCR. However, two in early passages and three in later passages of MPCs had the presence of mollicutes. Further rescreening for species of mollicutes indicated the presence of Mycoplasma hyorhinis, M. salivarium, and M. arginine. CONCLUSIONS: The PCR methods employed in this study could be beneficial as a rapid sterility testing of cell lines.

Drug Release and Cytotoxicity of Hyaluronic Acid and Zinc Oxide Gels, An In-Vitro Study.

Hyaluronic acid (HA) is a naturally occurring biopolymer, with a remarkable wound healing property. Zinc-oxide non-eugenol is a material widely used for periodontal dressing in dentistry. However, it has been reported that zinc oxide non-eugenol is toxic to osteoblasts and fibroblasts. Hence, the present study aimed to evaluate the drug release and cytotoxicity of HA and zinc-oxide gels. Hydrogels of HA and zinc oxide were formulated with carbopol as a carrier. In vitro drug release was performed by UV spectrophotometry, dialysis, and vial bag methods. Cytotoxicity assessment of HA and zinc-oxide gels was performed in human periodontal ligament fibroblasts (HPdLF) and human gingival fibroblasts (hGFs). An inverted phase-contrast microscope was used to assess the morphological changes. At 24 and 48 hr, HPdLF cells showed the highest viability in 0.1% low molecular weight-HA (LMW-HA) with a median value of 131.9, and hGFs showed the highest viability in 5% LMW-HA with a median of 129.56. The highest viability of HPdLF cells was observed in 5% high molecular weight-HA (HMW-HA), with a median value of 127.11. hGFs showed the highest viability in 1% HMW-HA with a median value of 97.99. Within the limitations of the present study, we concluded that LMW-HA is more efficient than HMW-HA. Both HPdLF and hGF cells showed complete cell morbidity with zinc-oxide hydrogels. Therefore, zinc oxide-based gels in concentrations as low as 9% could be toxic intraorally to soft tissues that harbor gingival and periodontal ligament fibroblasts.

Proficiency of Extracellular Vesicles From hiPSC-Derived Neural Stem Cells in Modulating Proinflammatory Human Microglia: Role of Pentraxin-3 and miRNA-21-5p.

Extracellular vesicles (EVs) shed by human-induced pluripotent stem cell (hiPSC)-derived neural stem cells (hNSC-EVs) have shown potent antiinflammatory properties in a mouse macrophage assay and a mouse model of acute neuroinflammation. They can also quickly permeate the entire brain after intranasal administration, making them attractive as an autologous or allogeneic off-the-shelf product for treating neurodegenerative diseases. However, their ability to modulate activated human microglia and specific proteins and miRNAs mediating antiinflammatory effects of hNSC-EVs are unknown. We investigated the proficiency of hNSC-EVs to modulate activated human microglia and probed the role of the protein pentraxin 3 (PTX3) and the miRNA miR-21-5p within hNSC-EVs in mediating the antiinflammatory effects. Mature microglia generated from hiPSCs (iMicroglia) expressed multiple microglia-specific markers. They responded to lipopolysaccharide (LPS) or interferon-gamma challenge by upregulating tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) mRNA expression and protein release. iMicroglia also exhibited proficiency to phagocytose amyloid-beta (Aß). The addition of hNSC-EVs decreased TNF-α and IL-1ß mRNA expression and the release of TNF-α and IL-1ß by LPS-stimulated iMicroglia (proinflammatory human Microglia). However, the antiinflammatory activity of hNSC-EVs on LPS-stimulated microglia was considerably diminished when the PTX3 or miR-21-5p concentration was reduced in EVs. The results demonstrate that hNSC-EVs are proficient for modulating the proinflammatory human microglia into non-inflammatory phenotypes, implying their utility to treat neuroinflammation in neurodegenerative diseases. Furthermore, the role of PTX3 and miR-21-5p in the antiinflammatory activity of hNSC-EVs provides a new avenue for improving the antiinflammatory effects of hNSC-EVs through PTX3 and/or miR-21-5p overexpression.

Comparison of Cellular and Differentiation Characteristics of Mesenchymal Stem Cells Derived from Human Gingiva and Periodontal Ligament.

Objectives: Dental tissues possess multipotent stem cells with varying biological properties. The present study was aimed to establish a primary culture of human gingiva-derived mesenchymal stem cells (GMSCs) and periodontal ligament-derived stem cells (PDLSCs) from periodontally healthy subjects and compare their biological characteristics. Materials and Methods: Gingival and periodontal ligament (PDL) tissues were collected from extracted premolar teeth of five healthy subjects and primary cultures were established. Basic biological characteristics, such as cell morphology, viability, proliferation capacity, and colony-forming units, and in vitro osteogenic and adipogenic differentiation potential were performed at passage 3 of GMSCs and PDLSCs. This was followed by immuno-phenotyping and flow cytometric analysis for identification of positive mesenchymal stem cell (MSC) markers, such as CD73, CD90, and CD105, and negative markers CD45 and CD34. Statistical Analysis Used: One-way analysis of variance (ANOVA). Results: Primary cultures of GMSCs and PDLSCs were successfully established. Cells exhibited a fibroblast-like morphology with a homogeneous population at passage 3. Cells derived from both tissues were highly viable (>95%), proliferative, and capable of forming colonies. Both cells did not exhibit any noticeable differences in cellular properties. Immunofluorescence and flow cytometric analyses showed positivity for MSC markers, CD73, CD90, and CD105, and negativity for CD34 and CD45. Furthermore, GMSCs and PDLSCs were capable of differentiating in vitro into osteocytes as evidenced by Alizarin red-S staining, and adipocytes as demonstrated by oil red O staining. Conclusions: The results of the present study indicate that both GMSCs and PDLSCs have similar cellular characteristics and mesenchymal differentiation potential. Therefore, they may serve as an equally potent source of stem cells for use in cell-based periodontal therapies.

Assessment of proliferation, clonogenic assay, and osteogenic differentiation of human periodontal ligament stem cells following application of orthodontic forces.

Context: The proliferation and differentiation of human periodontal ligament stem cells (hPDLSC) into other cell types are also mediated by mechanical stresses; they might offer therapeutic benefits in tissue regeneration and angiogenesis. Objectives: The study was planned to assess the proliferation, clonogenic potential, and osteogenic differentiation of human periodontal ligament stem cells (PDLSC) following the application of light and heavy orthodontic forces. Materials and Methods: A couple forces of 50 gm (light force) were applied on the 1st premolar on the one side and 250 gm (heavy force) on the contralateral side in the upper arch of patients requiring orthodontic treatment with extraction of all 1st premolars. After 30 days, periodontal tissues were scrapped from extracted teeth for the establishment of PDLSC in vitro. PDLC from the lower premolar teeth where no orthodontic force was applied acted as the control group. Morphology, viability, proliferating rate and population doubling time, clonogenicity, and alkaline phosphatase activity were analysed. Result: The osteogenic potential was confirmed by Alizarin red staining and the expression of the osteogenic markers by qRT-PCR. The morphology, growth kinetics, potency, and osteogenic lineage characteristics inferred the application of high force reduced the proliferative ability and osteogenesis of PDLSC, though the difference was not significant. Conclusion: The established PDLSCs demonstrated their MSC-like properties based on morphology, growth kinetics, colony forming ability, and AP activity. The culture-expanded PDLSCs showed their differentiation potential into osteocytes. The application of high force reduced the proliferative ability and osteogenesis of PDLSCs, variations were not significant.

Characterization and evaluation of ascorbic acid-induced cell sheet formation in human periodontal ligament stem cells: An in vitro study.

OBJECTIVES: Periodontal ligament-derived stem cells (PDLSCs) are regarded as a viable option for periodontal regeneration using cell sheet technology. The objective of the present in vitro study was to characterize human PDLSCs based on their phenotypic and biological properties and to evaluate the ascorbic acid (AA or vitamin C)-induced cell sheet by analyzing the molecular markers. METHODS: PDLSCs were established from premolars, and their morphology, viability, proliferation, phenotypic marker expression, and ability to differentiate into osteocytes and adipocytes were analyzed. PDLSCs were then induced to form cell sheets using 100 µM AA, and gene expression was examined by real-time polymerase chain reaction. RESULTS: PDLSCs showed fibroblastic morphology with >95% viability. The cells were highly proliferative and positive for surface antigens CD29, CD73, and CD90 but negative for CD34 and CD45. They were capable of differentiating into osteocytes and adipocytes. Induction with 100 µM AA transformed PDLSCs into two-to three-layered cell sheets. There was no significant upregulation in ALP and RUNX2 expression in the AA-induced cell sheet. However, the expression levels of late osteoblast differentiation marker (bone gamma-carboxy glutamate protein); cementogenic markers (cementum attachment protein and CP23), and genes encoding extracellular matrix (ECM) proteins [collagen type 1 alpha 1 and integrin beta 1) were higher in AA-induced cell sheets by PDLSCs. CONCLUSIONS: The stimulating effect of AA on cell sheet formation by PDLSCs was confirmed by the expression of typical markers involved in osteogenesis/cementogenesis and ECM secretion, which makes this procedure a prospective option for periodontal tissue regeneration applications.

Minimal influence of chronic inflammation on the potency and differentiation characteristics of gingiva-derived mesenchymal stem cells-An in vitro study.

OBJECTIVE: Gingiva-derived mesenchymal stem cells (GMSCs) have been identified and characterized from healthy tissues. However, reports on the influence of chronic inflammation on their stemness characteristics are sparse. The present study evaluated the potency and differentiation ability of GMSCs from periodontally healthy GMSC (H-GMSC) and inflamed GMSC (I-GMSC) tissues. MATERIALS AND METHODS: Established H-GMSCs and I-GMSCs were evaluated on their potency characteristics, such as morphology, viability, proliferation rate, population doubling time, colony-forming ability, expression of stemness markers, and mesenchymal differentiation potential. RESULTS: H-GMSCs and I-GMSCs exhibited fibroblast-like morphology and showed >95% viability with high proliferation potential and shorter doubling time. H-GMSCs showed fewer and smaller colonies, whereas I-GMSCs exhibited multiple and larger colonies. The evaluation of stemness markers revealed that both H-GMSCs and I-GMSCs were weakly positive for stage-specific embryonic antigen-4, Stro1, and CD105 (Endoglin), strongly positive for CD73 and CD90, and negative for the hematopoietic cell markers, CD34 and CD45. H-GMSCs showed a slightly higher osteogenic potential when compared to I-GMSCs, while I-GMSCs had a higher adipogenic potential than H-GMSCs. CONCLUSION: The findings showed that the inflammatory environment might have a stimulatory effect on the growth kinetics and ability of colony formation in GMSCs. However, varied osteogenic and adipogenic differentiation was observed between H-GMSCs and I-GMSCs.

Comparison of stem cells from human exfoliated deciduous posterior teeth with varying levels of root resorption.

BACKGROUND: Stem cells from human exfoliated deciduous teeth (SHED) are regarded as an attractive cell source for tissue regeneration. However, the effect of different levels of root resorption on the characteristics of SHED remains less understood. Thus, the tooth source that is most suitable for the isolation of SHEDs needs to be determined. To compare cellular and biological characteristics of stem cells from human exfoliated deciduous posterior teeth with varying levels of root resorption. METHODS: The pulp was obtained from the deciduous posterior teeth depending on the level of root resorption, and isolated SHEDs were grouped as follows: Teeth with 0 to 1/3rd root resorption as SHEDs (G1) and 1/3rd to 2/3rd root resorption as SHEDs (G2). Teeth were also collected from >2/3rd root resorption status, but failed to establish primary culture of SHED as the availability of pulp tissue was too less. Later, isolated SHEDs were compared on their morphology, viability, growth kinetics, colony-forming ability, expression of cell surface markers and in vitro differentiation into osteocytes and adipocytes. RESULTS: No major differences were observed in terms of cellular morphology, viability, proliferation rate, colony-forming ability, cell surface markers expression, and mesenchymal lineage differentiation of SHEDs isolated from posterior teeth with 0 to 1/3rd and 1/3rd to 2/3rd root resorption. However, SHED from teeth with 0 to 1/3rd root resorption (G1) displayed relatively higher proliferation capacity and expression of selected markers. CONCLUSIONS: Collectively, SHEDs (G1) and SHEDs (G2) showed comparable cellular and biological characteristics that enable their possible applications in regenerative therapies.

Influence of human teeth matrix on the cellular and biological properties of dental pulp stem cells - An in vitro study.

BACKGROUND: A major challenge in bone tissue regeneration is the use of right combination of stem cells with osteoinductive biomaterials. Hence, the present in vitro study was aimed at evaluating the effect of mineralized teeth matrix (MTM) and demineralized teeth matrix (DTM) on the selected cellular and biological characteristics of human dental pulp stem cells (DPSCs). METHODS: Established DPSCs were cultured in conditioned media (CM) of MTM and DTM and analyzed on their morphology, proliferation rate, population doubling time (PDT), viability, migration ability, ploidy and expression of cell surface markers, Further, the effect of MTM and DTM on the biocompatibility and osteogenic differentiation ability of DPSCs was evaluated. RESULTS: The DPSCs exhibited a fibroblast-like morphology with >80% viability. Cells were highly proliferative with an average PDT of 61 â€‹± â€‹12 â€‹h. A greater proliferation of DPSCs in the scratched area was observed when cultured in CM of teeth matrix compared to the cells in basal media. Moreover, no chromosomal abnormalities were induced during the culture of DPSCs. Flow cytometry analysis showed that DPSCs in basal media and CM of MTM and DTM were positive for CD29, CD44, CD73, CD90 (>70%), and negative for CD34 and CD45 (<0.1%). Alizarin red staining showed the higher deposition of mineralized nodules in DPSCs cultured with DTM compared to MTM. CONCLUSION: MTM and DTM-derived CM enhanced the proliferation and selected phenotypic markers expression with no chromosomal abnormalities in DPSCs. In addition, both matrices were biocompatible with DPSCs and increased the osteogenic differentiation through higher nodule formation.

Characterization of Stem Cells from Human Exfoliated Deciduous Anterior Teeth with Varying Levels of Root Resorption.

BACKGROUND: Deciduous teeth undergo the physiologic process of resorption, during which the remnant pulp undergoes activation. However, the quality of stem cells obtained at various stages of root resorption has not been documented. OBJECTIVE: To isolate and characterize stem cells from deciduous teeth with varying levels of root resorption. STUDY DESIGN: Healthy primary anterior teeth were extracted according to the treatment needs of the patient. The teeth were categorized into SHED(1/3)- teeth with 0 to 1/3rd root resorption, SHED(2/3)- teeth with 1/3rd to 2/3rd root resorption, and SHED(COMP)- teeth with more than 2/3rd root resorption. SHED were characterized based on their morphology, viability, proliferation rate, population doubling time, expression of cell surface markers, and in vitro differentiation potential into osteocytes and adipocytes. RESULTS: SHED from all three groups demonstrated largely similar morphological and cellular characteristics. However, SHED(2/3) showed relatively better characteristics in terms of growth kinetics and phenotypic marker expression. Also, the differentiation ability for osteogenic and adipogenic cell lineages was slightly higher in SHED(1/3) and SHED(2/3) compared with SHED(COMP). CONCLUSION: Based on the cellular, phenotypic and biological characteristics, it is suggested that SHED (2/3) could be a useful source for tissue regeneration, and warrants further investigations.

Inhibition of Ehrlich ascites cancer, hypoxia-inducible factor-1 alpha, and the kinase insert domain-containing receptor/fms-like tyrosine kinase-binding domains of vascular endothelial growth factor by Thiazole Acetamide Derivatives.

BACKGROUND: Tumor cells that have the ability to express vascular endothelial growth factor (VEGF) are more competent to growth and metastasize by the adequate amount of blood and oxygen supply by the blood vessels to the growing mass of cells. Hypoxic tumors are known for its aggressiveness and resistance to the treatment. Targeting VEGF and hypoxia-inducible factor-1 alpha (HIF-1α) is an attractive strategy to interrupt the multiple pathways crucial for tumor growth. In the present study, two thiazole acetamide derivative's anticancer property, anti VEGF and HIF-1α inhibitory property were investigated. METHODOLOGY: Two thiazole acetamide compounds were synthesized, TA1 and TA2 and its anticancer property was studied in Erlich's ascites cancer cells. To evaluate the anticancer property the assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA diffusion assay for apoptosis, and lactate dehydrogenase leakage assay were carried out. The cell culture media was used to assess the secreted VEGF level. Molecular docking studies were performed to analyze the binding efficiency of the study compounds to the kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase (FLT)-binding domains of VEGF protein. HIF-1α inhibitory study was performed by flow cytometry analysis using HUVEC cell line. RESULTS: The study compounds inhibited HIF-1α and VEGF secretion, these data shown positive prop up for the anticancer property of the derivatives. The docking studies showed moderate binding of study compounds to KDR and FLT-binding domains of VEGF protein. CONCLUSION: These results conclude the anticancer and anti-angiogenic property of the synthesized thiazole-acetamide derivatives.

Sex determination from the pulp tissue of deciduous teeth exposed to natural soil and wet clay - A PCR study.

CONTEXT: Dental tissue remains are the toughest, and chemically, the most stable tissue in the body. Its high resilience in the events of fire and bacterial decomposition makes them vital for DNA analysis by PCR method. AIMS: Determination of sex of children through molecular analysis of pulp tissue of exfoliated deciduous teeth stored in different media and analyzed after a different time period. SETTINGS AND DESIGN: Sixty samples of deciduous teeth were divided into three groups. Group IA and Group IIA were stored in natural soil and wet clay for 1 month, respectively. Group IB and Group IIB were stored in natural soil and wet clay for 6 months, respectively. Group III was analyzed immediately after extraction. METHODS AND MATERIAL: Sex determination was carried out in five steps: Pulp tissue removal, DNA isolation, DNA quantification, PCR amplification, Sex determination. X and Y specific chromosomes from each sample were amplified and compared. STATISTICAL ANALYSIS USED: Kruskal-Wallis test, Dunn's test, and Wilcoxon signed rank test. RESULTS: Group III revealed the highest amount of DNA quantified. Amount of DNA quantified after 6 months of storage in natural soil and wet clay decreased in both the groups with the samples stored in wet clay showing a maximum decrease. Results of the PCR analysis also showed 100% accuracy rate in the samples of Group III. CONCLUSIONS: Sex determination from pulp tissue depends a lot on the quality and quantity of DNA extracted. Sex could be effectively determined among the samples evaluated immediately after extraction. This ability decreases as the storage condition changes and the time period increases. Samples stored in wet clay were found to show the least sex identification ability than dry soil.

Cytogenetic and cytokine profile in elderly patients with cytopenia.

In the elderly with cytopenia, the diagnosis of myelodysplastic syndrome (MDS) may be missed. Cytokine levels contribute to the pathology of MDS. Hence, the objectives were to evaluate cytogenetic profile as a prognostic indicator in risk stratification and cytokine levels as a screening tool in patients with cytopenia for diagnosis. Over 2 years (2016-2018), 150 elderly patients were screened. MDS diagnosis was confirmed by morphology. Interleukin-2 (IL-2) and IL-6 levels were assessed in 50 patients, and karyotyping was performed in 20 confirmed cases of MDS. Age-matched healthy controls were used for comparison of cytokine levels. Among 150 patients, 88.6% had anemia, including nutritional anemia (51.2%). MDS diagnosis was confirmed in 35 patients. In 15 patients, unexplained cytopenia (UC) was present. Karyotyping in 20 MDS patients was normal in 15 (75%) patients and revealed a complex karyotype in four (20%) patients and double chromosomal abnormality in one (5%) patient. The Revised International Prognostic Scoring System (IPSS-R) scored 91% in the low-risk group and 9% (n = 3) in the high-risk group; the latter three developed acute myeloid leukemia (AML) and two of them had a 7q deletion. Among the 15 cases of UC, one patient died from refractory anemia. No significant difference in levels of IL-2 and IL-6 were found between MDS and UC patients when compared with healthy controls, as well as between different risk groups and karyotypes. A significant difference in IL-2 levels was found in MDS patients with disease progression and with stable disease. On the basis of the findings, it is suggested that IL-2 levels will help in predicting disease progression.

Comparison of response surface methodology and artificial neural network to enhance the release of reducing sugars from non-edible seed cake by autoclave assisted HCl hydrolysis.

In the current investigation, statistical approaches were adopted to hydrolyse non-edible seed cake (NESC) of Pongamia and optimize the hydrolysis process by response surface methodology (RSM). Through the RSM approach, the optimized conditions were found to be 1.17%v/v of HCl concentration at 54.12 min for hydrolysis. Under optimized conditions, the release of reducing sugars was found to be 53.03 g/L. The RSM data were used to train the artificial neural network (ANN) and the predictive ability of both models was compared by calculating various statistical parameters. A three-layered ANN model consisting of 2:12:1 topology was developed; the response of the ANN model indicates that it is precise when compared with the RSM model. The fit of the models was expressed with the regression coefficient R2, which was found to be 0.975 and 0.888, respectively, for the ANN and RSM models. This further demonstrated that the performance of ANN was better than that of RSM.

Haematopoietic, Antioxidant and Membrane Stabilizing Property of Diallyl Disulphide in Irradiated Mice.

INTRODUCTION: Diallyl disulphide is an organo-sulphur compound which is present in garlic and responsible for the characteristic odor of garlic. It is known for its anticancer and invitro membrane stabilizing properties. AIM: The main aim was to evaluate the haematopoietic, antioxidant and membrane stabilizing property of diallyl disulfide in irradiated mice. MATERIALS AND METHODS: Mice were grouped into 6 groups as control, drug control, radiation control and drug pre-treatment groups (i.e. drug administration + radiation group) The mice were fed orally for 15 consecutive days and on the 15(th) day, one hour after drug administration, the mice were irradiated with 6Gy electron beam radiation. The changes in blood cell count, total antioxidant levels, malondialdehyde and reduced glutathione levels were determined. The immunomodulatory response of DADS to the radiological effects was determined by the estimation of IL-6 levels. RESULTS: A significant improvement in pre-drug treatment group when compared to control groups in the haemoglobin, red blood cell count, white blood cell count, haematocrit and platelet counts was observed. There is an increased level of interleukin-6 in the drug treated groups compared to the radiation control. An increase in the malondialdehyde levels and decrease in the glutathione levels in the irradiated group indicate increased lipid peroxidation and oxidative stress, whereas, there is a significant reduction in the malondialdehyde levels and increased glutathione levels in the drug pre-treatment groups showing membrane stabilization. CONCLUSION: Thus DADS proves to be an effective haematopoietic and antioxidative agent to counter radiation induced haematopoietic suppression and oxidative stress.