In vitro profiling and molecular dynamics simulation studies of berberine loaded MCM-41 mesoporous silica nanoparticles to prevent neuronal apoptosis.

Neuronal loss in Alzheimer's disease has been reported to display features of apoptosis, pyroptosis (programmed necrosis), or necroptosis. This study thoroughly examines the production and characterization of MCM-41 based berberine (BBR)-loaded porous silica nanoparticles (MSNs) by a modified Stöber method, focusing on their possible role in inhibiting the apoptotic process. Particle size, polydispersity index, morphology, drug loading, zeta potential, entrapment efficiency, and drug release were examined. The formulation was analyzed using various spectroscopic techniques. The surface area was computed by the Brunauer-Emmett-Teller plot. Computational models were developed for molecular dynamics simulation studies. A small PDI value indicated an even distribution of particles at nanoscale sizes (80-100 nm). Results from XRD and SEAD experiments confirmed the amorphous nature of BBR in nanoparticles. Nanoparticles had high entrapment (75.21 ± 1.55%) and drug loading (28.16 ± 2.5%) efficiencies. A negative zeta potential value (-36.861.1 mV) indicates the presence of silanol groups on the surface of silica. AFM findings reveal bumps due to the surface drug that contributed to the improved roughness of the MSNs-BBR surface. Thermal gravimetric analysis confirmed the presence of BBR in MSNs. Drug release was controlled by simple diffusion or quasi-diffusion. Molecular dynamics simulations confirmed the existence of diffused drug molecules. Cellular studies using SH-SY-5Y cells revealed dose-dependent growth inhibition. Fragmented cell nuclei and nuclear apoptotic bodies in DAPI-stained cells exposed to nanoparticles showed an increase in apoptotic cells. Flow cytometry analysis demonstrated a lower red-to-green ratio in SH-SY-5Y cells treated with nanoparticles. This suggests improved mitochondrial health, cellular viability restoration, and prevention of the apoptotic process. This study provides essential data on the synthesis and potential of MSNs loaded with BBR, which may serve as a viable therapeutic intervention for conditions associated with apoptosis.

MicroRNA-29b-3p degenerates terminally differentiated dopaminergic SH-SY5Y cells by perturbation of mitochondrial functions.

Mitochondrial dysfunction is the main cause of gradual deterioration of structure and function of neuronal cells, eventually resulting in neurodegeneration. Studies have revealed a complex interrelationship between neurotoxicant exposure, mitochondrial dysfunction, and neurodegenerative diseases. Alteration in the expression of microRNAs (miRNAs) has also been linked with disruption in mitochondrial homeostasis and bioenergetics. In our recent research (Cellular and Molecular Neurobiology (2023) https://doi.org/10.1007/s10571-023-01362-4), we have identified miR-29b-3p as one of the most significantly up-regulated miRNAs in the blood of Parkinson's patients. The findings of the present study revealed that neurotoxicants of two different natures, that is, arsenic or rotenone, dramatically increased miR-29b-3p expression (18.63-fold and 12.85-fold, respectively) in differentiated dopaminergic SH-SY5Y cells. This dysregulation of miR-29b-3p intricately modulated mitochondrial morphology, induced oxidative stress, and perturbed mitochondrial membrane potential, collectively contributing to the degeneration of dopaminergic cells. Additionally, using assays for mitochondrial bioenergetics in live and differentiated SH-SY5Y cells, a reduction in oxygen consumption rate (OCR), maximal respiration, basal respiration, and non-mitochondrial respiration was observed in cells transfected with mimics of miR-29b-3p. Inhibition of miR-29b-3p by transfecting inhibitor of miR-29b-3p prior to exposure to neurotoxicants significantly restored OCR and other respiration parameters. Furthermore, we observed that induction of miR-29b-3p activates neuronal apoptosis via sirtuin-1(SIRT-1)/YinYang-1(YY-1)/peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α)-regulated Bcl-2 interacting protein 3-like-dependent mechanism. Collectively, our studies have shown the role of miR-29b-3p in dysregulation of mitochondrial bioenergetics during degeneration of dopaminergic neurons via regulating SIRT-1/YY-1/PGC-1α axis.

Expression of miR-145 and miR-18b in Peripheral Blood Samples of Head and Neck Cancer Patients.

Head and neck squamous cell carcinomas (HNSCC) is one of the most prevalent type of cancer known in Indian population. Studies are needed to identify the early biomarkers for HNSCC. MicroRNAs (miRNAs) are non-coding RNA molecules, expression of which can be used as biomarker for early diagnosis of HNSCC. For miRNA profiling total RNA, which also contained small RNAs were isolated from ten HNSCC tissue samples and adjacent control. Purity and concentration of eluted RNA was assessed using the NanoDrop1000® spectrophotometer, Reverse Transcription reaction was carried out with megaplex RT primers of pool A and pool B and the expression of selected miRNAs (miR-143/145 and miR-18a/b) was measured using TaqMan primers specific for mature miRNAs. Our study showed dramatic downregulation in expression of two miRNAs, miR-18b and miR-145 in blood samples of HNSCC patients, which are inhibitor of tumorigenesis and can be targeted as biomarker of HNSCC pathogenesis therefore developing avenues for miRNA role in prognosis and therapeutics. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-023-01119-2.

Implications of In Vitro Multi-Serine Phosphorylation of Alpha-Synuclein in Aggregation and Cytotoxicity.

Post-translational modifications guide the functional diversity and identity of proteins. Phosphorylation is one such post-translational modification that has been reported in pathological proteins related to various neurodegenerative disorders such as α-synuclein (α-syn) phosphorylation in Parkinson's disease and other synucleinopathies. In α-syn, the phosphorylation has mostly been observed at S129; however, the occurrence of other serine modifications at S9, S42, and S87 is partially explored. In pathogenic conditions, where α-syn is phosphorylated by complex kinase pathways, multi-site modifications may happen and alter the mechanism of α-syn aggregation. Here, using Polo-like kinase 2 and G-protein coupled receptor kinase 4, the in vitro phosphorylation of α-syn was performed, which revealed multi-serine phosphorylation. Mass spectrometry with customized proteolytic digestion showed prominent phosphorylation at S129 and modifications at S87 and S42 with PLK2 and S87 with GRK4. The phosphorylation at the identified serine residues was further validated with NMR and western blotting. Multi-serine phosphorylation aggravates the aggregation potential of monomeric α-syn, seeding capacity, and cytotoxicity in the SH-SY5Y cell line. This study proposes evidence for in vitro multi-site phosphorylation and its significance in α-syn aggregation, toxicity, and related pathogenesis.

Shift and longtime light induces endometrioid adenocarcinoma via activation of PKC-α/Akt pathway in female golden hamster: Involvement of altered Aanat and Bmal1 rhythm.

Female night-workers get exposed to frequent light shifts, hence have altered circadian rhythm and are at high risk of endometrial cancer; the underlying mechanism however is still not clear. We, therefore examined the effect of long light exposure (16L:8D, LD1) and regular shift (8 h) in long nighttime (LD2) on endometrial changes of female golden hamsters. Morphometric analysis, scanning electron microscopy imaging, alcian blue staining, and cytological nuclear atypia of endometrial stromal cells confirmed the incidence of endometrial adenocarcinoma in LD2 exposed hamsters. But, less severe pathomorphological alterations were noted in uterus of LD1 exposed hamsters. Altered Aanat and Bmal1 mRNA, melatonin rhythm, downregulation of important marker gene of adenocarcinoma like Akt, 14-3-3, and PR protein expression and upregulation PKCα, pAkt-S473 and vascular epithelial growth factor (VEGF) were observed in LD2 exposed hamsters suggesting the endometrial adenocarcinoma. Further, our western blot analysis supported the immunohistochemical localization of PR, PKCα, and VEGF in uterine tissues along low progesterone. Overall, our data indicates that light shift and long light exposure potentially induced endometrioid adenocarcinoma via activation of PKC-α/Akt pathway in female hamsters. Therefore, duration of light is essential for female normal uterine function.

Transcriptomics and Proteomics Approach for the Identification of Altered Blood microRNAs and Plasma Proteins in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder caused by the selective destruction of dopaminergic neurons (DA-nergic). Clinically, PD is diagnosed based on developing signs and symptoms. A neurological and physical examination and sometimes medical and family history also help in the diagnosis of PD. However, most of these features are visible when more than 80% of the dopaminergic neurons have degenerated. An understanding of the selective degeneration process at the cellular and molecular level and the development of new biomarkers are required for effective PD management. Several studies have been carried out using a selected set of miRNAs/ mRNAs and proteins to develop biomarkers of PD; however, an unbiased and combined miRNA-protein profiling study was required to identify the markers of progressive and selected degeneration of dopaminergic neurons in PD patients. In the present study, we have carried out global protein profiling through LC-MS/MS and miRNA profiling by using a "brain-specific" miRNA array panel of 112 miRNAs in PD patients and healthy controls to find the unprejudiced group of proteins and miRNAs that are deregulating in PD. In the whole blood samples of PD patients compared to healthy controls, the expression of 23 miRNAs and 289 proteins was significantly increased, whereas the expression of 4 miRNAs and 132 proteins was considerably downregulated. Network analysis, functional enrichment, annotation, and analysis of miRNA-protein interactions were also performed as part of the bioinformatics investigation of the discovered miRNAs and proteins revealing several pathways that lead to PD development and pathogenesis. Based on the analysis of miRNA and protein profiling, we have identified four miRNAs (hsa-miR-186-5p, miR-29b, miR-139 & has-miR-150-5p) and four proteins (YWHAZ, PSMA4, HYOU1, & SERPINA1), which can be targeted for the development of new biomarkers of PD. In vitro studies have identified the role of miR-186-5p in regulating the levels of the YWHAZ/YWHAB & CALM2 gene, which has shown maximum downregulation in PD patients and is known for its role in neuroprotection from apoptotic cell death & calcium regulation. In conclusion, our research has identified a group of miRNA-proteins that can be developed as PD biomarkers; however, future studies on the release of these miRNAs and proteins in extracellular vesicles circulating in the blood of PD patients can further validate these as specific biomarkers of PD.

A novel single sensor hemoglobin domain from the thermophilic cyanobacteria Thermosynechococcus elongatus BP-1 exhibits higher pH but lower thermal stability compared to globins from mesophilic organisms.

Thermosynechococcus elongatus-BP1 belongs to the class of photoautotrophic cyanobacterial organisms. The presence of chlorophyll a, carotenoids, and phycocyanobilin are the characteristics that categorize T. elongatus as a photosynthetic organism. Here, we report the structural and spectroscopic characteristics of a novel hemoglobin (Hb) Synel Hb from T.elongatus, synonymous with Thermosynechococcus vestitus BP-1. The X-ray crystal structure (2.15 Å) of Synel Hb suggests the presence of a globin domain with a pre-A helix similar to the sensor domain (S) family of Hbs. The rich hydrophobic core accommodates heme in a penta-coordinated state and readily binds an extraneous ligand (imidazole). The absorption and circular dichroic spectral analysis of Synel Hb reiterated that the heme is in FeIII+ state with a predominantly α-helical structure similar to myoglobin. Synel Hb displays higher resistance to structural perturbations induced via external stresses like pH and guanidium hydrochloride, which is comparable to Synechocystis Hb. However, Synel Hb exhibited lower thermal stability compared to mesophilic hemoglobins. Overall, the data is suggestive of the structural sturdiness of Synel Hb, which probably corroborates its origin in extreme thermophilic conditions. The stable globin provides scope for further investigation and may lead to new insights with possibilities for engineering stability in hemoglobin-based oxygen carriers.

Crosstalk Between miRNA and Protein Expression Profiles in Nitrate-Exposed Brain Cells.

Growing evidence reported a strong association between the nitrate ingestion and adverse health consequences in humans, including its detrimental impact on the developing brain. The present study identified miRNAs and proteins in SH-SY5Y human neuroblastoma cells and HMC3 human microglial cells using high-throughput techniques in response to nitrate level most prevalent in the environment (India) as X dose and an exceptionally high nitrate level as 5X dose that can be reached in the near future. Cells were exposed to mixtures of nitrates for 72 h at doses of X and 5X, 320 mg/L and 1600 mg/L, respectively. OpenArray and LCMS analysis revealed maximum deregulation in miRNAs and proteins in cells exposed to 5X dose. Top deregulated miRNAs include miR-34b, miR-34c, miR-155, miR-143, and miR-145. The proteomic profiles of both cell types include proteins that are potential targets of deregulated miRNAs. These miRNAs and their targeted proteins involve in multiple functions, including metabolic processes, mitochondrial functions, autophagy, necroptosis, apoptosis, neuronal disorders, brain development, and homeostasis. Furthermore, measuring mitochondrial bioenergetics in cells exposed to nitrate revealed that a 5X dose causes a significant reduction in oxygen consumption rate (OCR) and other bioenergetic parameters in both cell types. In summary, our studies have demonstrated that a 5X dose of nitrate significantly alters cellular physiology and functions by deregulating several miRNAs and proteins. However, X dose of nitrate has not caused any adverse effects on any cell type.

Effect of carbon quantum dots derived from extracts of UV-B-exposed Eclipta alba on alcohol-induced liver cirrhosis in Golden Hamster.

The Eclipta alba plant is considered hepatoprotective, owing to its phytoconstituents wedelolactone. In the current study, effect of elevated ultraviolet-B (eUV-B) radiation was investigated on biochemical, phytochemical, and antioxidative enzymatic activities of E. alba (Bhringraj) plant. The UV-B exposure resulted in an increase in oxidative stress, which has caused an imbalance in phytochemical, biochemical constituents, and induced antioxidative enzymatic activities. It was observed that the UV-B exposure promoted wedelolactone yield by 23.64%. Further, the leaf extract of UV-B-exposed plants was used for the synthesis of carbon quantum dots (CQDs) using low cost, one-step hydrothermal technique and its biocompatibility was studied using in vitro MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay on HepG2 liver cell line. It revealed no toxicity in any treatment groups in comparison to the control. Both CQDs and leaf extract were orally administered to the golden hamster suffering from alcohol-induced liver cirrhosis. In the morphometric study, it was clearly observed that a combination of UV-B-exposed leaf extract and synthesized CQDs delivered the best result with maximum recovery of liver tissues. The present study reveals the positive impact of UV-B exposure on the medicinally important plant, increased yield of wedelolactone, and its enhanced hepatoprotective efficacy for the treatment of damaged liver tissues.

Flavonoids as Promising Neuroprotectants and Their Therapeutic Potential against Alzheimer's Disease.

Alzheimer's disease (AD) is one of the serious and progressive neurodegenerative disorders in the elderly worldwide. Various genetic, environmental, and lifestyle factors are associated with its pathogenesis that affect neuronal cells to degenerate over the period of time. AD is characterized by cognitive dysfunctions, behavioural disability, and psychological impairments due to the accumulation of amyloid beta (Aß) peptides and neurofibrillary tangles (NFT). Several research reports have shown that flavonoids are the polyphenolic compounds that significantly improve cognitive functions and inhibit or delay the amyloid beta aggregation or NFT formation in AD. Current research has uncovered that dietary use of flavonoid-rich food sources essentially increases intellectual abilities and postpones or hinders the senescence cycle and related neurodegenerative problems including AD. During AD pathogenesis, multiple signalling pathways are involved and to target a single pathway may relieve the symptoms but not provides the permanent cure. Flavonoids communicate with different signalling pathways and adjust their activities, accordingly prompting valuable neuroprotective impacts. Flavonoids likewise hamper the movement of obsessive indications of neurodegenerative disorders by hindering neuronal apoptosis incited by neurotoxic substances. In this short review, we briefly discussed about the classification of flavonoids and their neuroprotective properties that could be used as a potential source for the treatment of AD. In this review, we also highlight the structural features of flavonoids, their beneficial roles in human health, and significance in plants as well as their microbial production.

Circadian desynchronization in pregnancy of Golden hamster following long time light exposure: Involvement of Akt/FoxO1 pathway.

Coordination between central and peripheral reproductive clocks in females is poorly understood. Long light is having a hazardous effect on reproductive health. Hence, explored the effect of long-time light exposure (LLD; 16L:8D) on the central and peripheral reproductive (ovary and uterus) clock genes (Bmal1, Clock, Per1, Per2, Cry1 and Cry2) and its downstream regulators (Aanat, Egf, Cx26, Cx43, ERα, pAktS-473, pAktT-308, pFoxO1T-24, 14-3-3, HoxA10, HoxA11 and Pibf) expression in non-pregnant and pregnant Golden hamster. Young adult Golden hamsters were exposed to LLD for 30 days and then were mated. We observed that LLD exposure increased the thickness of the endometrium and reduced myometrium thickness, resembling uterine adenomyosis. In non-pregnant females LLD altered the expressions of clock genes in suprachiasmatic nuclei (SCN), ovary and the uterus along with serum estradiol rhythm. LLD upregulated Egf and downregulated Aanat, Cx26, and Cx43 mRNA levels in uterus. LLD upregulated Akt/FoxO1 phosphorylation and 14-3-3 expressions in the uterus of nonpregnant females. LLD exposure to pregnant females lowered serum progesterone, Aanat, Pibf, Hoxa10, and Hoxa11 mRNA expressions on D4 (peri-implantation) and D8 (post-implantation) resulting in a low implantation rate on D8 (post-implantation). Hence it is evident that the frequent pregnancy anomalies noted under a long light schedule might be due to desynchronization in Aanat, Pibf, Hoxa10, and Hoxa11 as well as the central and peripheral clock genes (Bmal1, Clock, Per1, Per2, Cry1 and Cry2). LLD exposure desynchronized the central and peripheral reproductive clock affecting uterine physiology via Akt/FoxO1 pathway in Golden hamsters. Thus, LLD is a risk factor for female reproductive health and fertility.

Melatonin induces apoptosis and cell cycle arrest in cervical cancer cells via inhibition of NF-κB pathway.

Cervical cancer is the most prevalent cancer in females. Melatonin, a neurohormone has been documented as a promising therapeutic molecule for cervical cancer. However, the underlying molecular mechanism is not known. We explored the dose-dependent anti-tumor response of melatonin against cervical cancer cell lines, HeLa (HPV-18 positive) and SiHa (HPV-16 positive). The anti-cancer effect of melatonin was evaluated by MTT assay, cell imaging, colony formation, DAPI, AO/PI, LDH, Flow cytometry, scratch assay, western blot analysis and real-time PCR. Results of DAPI, AO/PI, LDH, and Annexin/PI staining revealed that melatonin induces apoptosis. The results of cell cycle analysis revealed that melatonin arrests the HeLa and SiHa cells in sub-G1 and G1 phases, respectively. Western blot analysis revealed that melatonin downregulated the expression of pro-inflammatory transcription factor, NF-κB and the expression of COX-2 protein, a key mediator in cell proliferation. In addition, melatonin downregulated the expression of an invasive marker, MMP-9, an antiapoptotic protein, Bcl-2, and upregulated the expression of pro-apoptotic protein, Bax at both transcriptional and translational levels. Overall, the results suggest that melatonin exhibited strong anti-cancer therapeutic potential against human cervical cancer cell line progression possibly through inhibition of NF-κB signalling pathway.

Continuous artificial light potentially disrupts central and peripheral reproductive clocks leading to altered uterine physiology and reduced pregnancy success in albino mice.

AIMS: The mechanism behind clock coordination in female reproductive disorders is poorly understood despite the known importance of coordinated and synchronized timing of central and clocks in reproductive organs. We investigated the effect of continuous artificial light (LL) on the central and peripheral reproductive clock gene (Bmal1, Clock, Per1, Per2 and Cry1) and its downstream regulators (Hgf, PR-A and HOXA10) during non-pregnancy and pregnancy phases of female mice. MAIN METHODS: Mice (n = 60) in two sets, were maintained under continuous light (LL) and natural day cycle (LD;12L: 12D) for both non-pregnant and pregnant study. Tissues from hypothalamus-containing SCN, ovary, uterus and serum were collected at different zeitgeber time points (ZT; at 4-h intervals across 24-h periods). KEY FINDINGS: LL exposure desynchronized the expressions of the clock mRNAs (Bmal1, Clock, Per1, Per2 and Cry1) in SCN, ovary, and uterus along with Hgf mRNA rhythm. LL significantly increased the thickness of endometrial tissues. Furthermore, the pregnant study revealed lower serum progesterone level during peri- and post-implantation under LL along with downregulated expression of progesterone receptor (PR) as well as progesterone dependent uterine Homeobox A-10 (Hoxa10) proteins with lowered pregnancy outcomes. SIGNIFICANCE: Our result suggests that LL disrupted the circadian coordination between central and clock genes in reproductive tissue leading to interrupted uterine physiology and altered pregnancy in mice. This led us to propose that duration of light exposure at work-places or home for females is very important in prevention of pregnancy anomalies.

B-doped SnO2 nanoparticles: a new insight into the photocatalytic hydrogen generation by water splitting and degradation of dyes.

Boron-doped SnO2 (B:SnO2) has been synthesized via a facile wet chemical method to deal with increasing energy demand and environment-related issues. Powder XRD confirmed the rutile phase of the synthesized B:SnO2 nanoparticles. Energy dispersive X-ray analysis and elemental mapping confirmed 1% B doping into SnO2 lattice. A red shift was observed during the analysis of Raman and FTIR spectral data. The bands in FTIR and Raman spectra confirmed the in-plane and bridging oxygen vacancies in SnO2 lattice introduced due to B doping. These nanoparticles showed proficiency in photocatalytic hydrogen generation and degradation of crystal violet (CV) and rhodamine B (RhB) dyes. The degradation of CV and RhB dyes in the presence of B:SnO2 NPs and ethane-1,2-diaminetetracetic acid (EDTA) was found to be 83 and ~ 100%, respectively. To escalate the efficiency of dye degradation, the experiment was performed with different sacrificial agents (EDTA, methanol, and triethanolamine). The maximum hydrogen production rate (63.6184 µmol g-1 h-1) was observed for B:SnO2 along with Pd as co-catalyst, and methanol and EDTA solution as sacrificial agents.

Transcriptomic and proteomic insights into patulin mycotoxin-induced cancer-like phenotypes in normal intestinal epithelial cells.

Patulin (PAT) is a natural contaminant of fruits (primarily apples) and their products. Significantly, high levels of contamination have been found in fruit juices all over the world. Several in vitro studies have demonstrated PAT's ability to alter intestinal structure and function. However, in real life, the probability of low dose long-term exposure to PAT to humans is significantly higher through contaminated food items. Thus, in the present study, we have exposed normal intestinal cells to non-toxic levels of PAT for 16 weeks and observed that PAT had the ability to cause cancer-like properties in normal intestinal epithelial cells after chronic exposure. Here, our results showed that chronic exposure to low doses of PAT caused enhanced proliferation, migration and invasion ability, and the capability to grow in soft agar (anchorage independence). Moreover, an in vivo study showed the appearance of colonic aberrant crypt foci (ACFs) in PAT-exposed Wistar rats, which are well, establish markers for early colon cancer. Furthermore, as these neoplastic changes are consequences of alterations at the molecular level, here, we combined next-generation RNA sequencing with liquid chromatography mass spectrometry-based proteomic analysis to investigate the possible underlying mechanisms involved in PAT-induced neoplastic changes.

Studies on Regulation of Global Protein Profile and Cellular Bioenergetics of Differentiating SH-SY5Y Cells.

The SH-SY5Y cells differentiated by sequential exposure of retinoic acid (RA) and brain-derived neurotrophic growth factor (BDNF) are a well-employed cellular model for studying the mechanistic aspects of neural development and neurodegeneration. Earlier studies from our lab have identified dramatic upregulation (77 miRNAs) and downregulation (17 miRNAs) of miRNAs in SH-SY5Y cells differentiated with successive exposure of RA + BDNF and demonstrated the essential role of increased levels of P53 proteins in coping with the differentiation-induced changes in protein levels. In continuation to our earlier studies, we have performed unbiased LC-MS/MS global protein profiling of naïve and differentiated SH-SY5Y cells and analyzed the identified proteins in reference to miRNAs identified in our earlier studies to identify the cellular events regulated by both identified miRNAs and proteins. Analysis of LC-MS/MS data has shown a significant increase and decrease in levels of 215 and 163 proteins, respectively, in differentiated SH-SY5Y cells. Integrative analysis of miRNA identified in our previous studies and protein identified in the present study is carried out to discover novel miRNA-protein regulatory modules to elucidate miRNA-protein regulatory relationships of differentiating neurons. In silico network analysis of miRNAs and proteins deregulated upon SH-SY5Y differentiation identified cell cycle, synapse formation, axonogenesis, differentiation, neuron projection, and neurotransmission, as the topmost involved pathways. Further, measuring mitochondrial dynamics and cellular bioenergetics using qPCR and Seahorse XFp Flux Analyzer, respectively, showed that differentiated cells possess increased mitochondrial dynamics and OCR relative to undifferentiated cells. In summary, our studies have identified a novel set of proteins deregulated during neuronal differentiation and establish the role of miRNAs identified in earlier studies in the regulation of proteins identified by LC-MS/MS-based global profiling of differentiating neurons, which will help in future studies related to neural development and neurodegeneration.

Identification of Altered Blood MicroRNAs and Plasma Proteins in a Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is the age-related neurological disorder characterized by the degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). PD is based on motor deficits which start to appear when up to 80% of the DA neurons of SNpc have been lost. Effective management of PD requires the development of novel biomarkers. Therefore, the present study aimed to characterize biomarkers of PD using miRNomics, proteomics, and bioinformatics approaches. Rats exposed to rotenone (2.5 mg/kg b.wt) for 2 months were used as an animal model to identify the unbiased set of miRNAs and proteins deregulated in blood samples. OpenArray, a real-time PCR-based array, is used for high-throughput profiling of miRNAs, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to carry out the global protein profiling. Systematic bioinformatics analysis of miRNAs and proteins was also performed, including annotation, functional classification and functional enrichment, network analysis, and miRNA-protein interaction analysis. Expression of 19 miRNAs and 96 proteins was significantly upregulated in the blood, while 22 proteins were significantly downregulated in blood samples of rotenone-exposed rats. In silico pathway analysis of deregulated proteins and miRNAs in rotenone-exposed rats has identified multiple pathways leading to PD. In summary, we have identified a set of miRNAs (miR-144, miR-96, and miR-29a) and proteins (PLP1, TUBB4A, and TUBA1C), which can be used as a potential biomarker of PD, while further validation required large human population studies.

4-chloro eugenol interacts synergistically with artesunate against drug resistant P. falciparum inducing oxidative stress.

4-chloro eugenol (4CE), a semisynthetic analog of phytomolecule eugenol exhibited potent antiplasmodial activity with IC50 in the range of 1.5-5 µM against sensitive as well as drug resistant strain of P. falciparum. This analog also showed synergy with a clinically used antimalarial drug artesunate and was able to curtail the IC50 of artesunate up to 4-5 folds. Although, 4CE did not show any effect on heme polymerization, the most common drug target in the malaria parasite, it could increase the level of reactive oxygen species (ROS) and reactive nitrogen species (RNS) alone as well as in combination with artesunate. Further, 4CE induced oxidative stress was observed to affect the macromolecules in terms of DNA damage, protein carbonylation and lipid peroxidation. At the physiological level, cellular organelles like mitochondria and endoplasmic reticulum were observed to be get affected by 4CE in terms of membrane depolarization and calcium ion leakage respectively. These observations could be validated by expression analysis of oxidative stress responsive genes and proteins. Further, in in vivo assay, 4CE showed significant chemo-suppression of parasitemia as well as an increase in mean survival time in the murine malaria model. Interestingly, in combination with artesunate, 4CE showed higher chemo-suppression as well as enhanced mean survival time at a much lower concentrations of both the partners as compared to an individual dose of artesunate and 4CE. A combination of 4CE and artesunate was also observed to attenuate cerebral malaria pathogenesis.

Synthesis and characterization of novel protein nanodots as drug delivery carriers with an enhanced biological efficacy of melatonin in breast cancer cells.

Melatonin is a potent antioxidant, chemotherapeutic and chemo preventive agent against breast cancer. However, its short half-life is one of the major limitations in its application as a therapeutic drug. To overcome this issue, the green-emitting protein nanodot (PND) was synthesized by a one-step hydrothermal method for loading melatonin. The synthesized pH-7 and pH-2 PND showed a quantum yield of 22.1% and 14.0%, respectively. The physicochemical characterization of both PNDs showed similar morphological and functional activities. Furthermore, the biological efficacy of melatonin-loaded PND (MPND) was evaluated in a breast cancer cell line (MDA-MB-231) for live-cell imaging and enhanced nano-drug delivery efficacy. Interestingly, the permeability of neutral pH PND in both cell cytoplasm and nucleus nullifies the limitations of real-time live-cell imaging, and ensures nuclear drug delivery efficacy. Neutral pH PND showed better cell viability and cytotoxicity as a fluorescence bioimaging probe compared to acidic PND. The bioavailability and cell cytotoxicity effect of MPND on MDA-MB-231 breast cancer cells were studied through confocal and migration assay. Results showed that MPND causes enhanced bioavailability, better cellular uptake, and inhibition of the migration of breast cancer cells as compared to the drug alone. Besides, the synthesized MPND showed no sign of fluorescence quenching even at a high concentration of melatonin, making it an ideal nanocarrier for bioimaging and drug delivery.

A combined microRNA and proteome profiling to investigate the effect of ZnO nanoparticles on neuronal cells.

Zinc oxide nanoparticles (ZnO NPs) are one of the most broadly used engineered nanomaterials. The toxicity potential of ZnO NPs has been explored in several studies; however, its neurotoxicity, especially its molecular mechanism, has not been studied in depth. In this study, we have used a cellular model of neuronal differentiation (nerve growth factor differentiated PC12 cells) to compare the effect of ZnO NPs exposure on neuronal (differentiated or mature neurons) and non-neuronal (undifferentiated) cells. Our studies have shown that the noncytotoxic concentration of ZnO NPs causes neurite shortening and degeneration in differentiated PC12 cells. Brain-specific microRNA (miRNA) array and liquid chromatography with tandem mass spectrometry (LC-MS/MS) are used to carry out profiling of miRNAs and proteins in PC12 cells exposed with ZnO NPs. Exposure of ZnO NPs produced significant deregulation of a higher number of miRNAs (15) and proteins (267) in neuronal cells in comparison to miRNAs (8) and proteins (207) of non-neuronal cells (8). In silico pathway analysis of miRNAs and proteins deregulated in ZnO NPs exposed differentiated PC12 cells have shown pathways leading to neurodegenerative diseases and mitochondrial dysfunctions are primarily targeted pathways. Further, a bioenergetics study carried out using Seahorse XFp metabolic flux analyzer has confirmed the involvement of mitochondrial dysfunctions in ZnO NPs exposed differentiated PC12 cells. In conclusion, differentiated PC12 cells (neuronal) were found more vulnerable than undifferentiated (non-neuronal PC12 cells) toward the exposure of ZnO NPs and deregulation of miRNAs and mitochondrial dysfunctions play a significant role in its toxicity.