Cell Lysate Supernatant Research Articles

A novel Affi-Cova magnetic nanoparticles for one-step covalent immobilization of His-tagged enzyme directly from crude cell lysate

Owing to the rapid advancement of in vitro synthetic biology, functional carriers capable of covalently binding target proteins from crude lysates under mild conditions have garnered escalating attention. Herein, a magnetic nanoparticle with affinity/covalent bifunction (MNP@Affi-Cova) was developed for the direct covalent immobilization of the recombinant enzyme of His-tagged birA (r-birA) from crude cell lysates in a single step. This innovative approach is attributed to the presence of chelated Ni2+ ions and epoxy groups on the surface of the beads. The fabricated magnetic nanoparticles were characterized by SEM, FT-IR spectrum, and zeta potential. The application conditions and stability of the MNP@Affi-Cova beads were systematically evaluated. Notably, the MNP@Affi-Cova beads exhibited a covalent capture efficiency of 91.25 μg r-birA/mg beads from a cell lysate supernatant containing 2.62 mg/mL crude protein. The immobilized r-birA exhibited significantly enhanced pH and thermal stability compared to the free counterpart. Additionally, the reusability of the immobilized r-birA on MNP@Affi-Cova demonstrated the retention of 76.1 % of its initial activity over ten cycles. These results suggest that the MNP@Affi-Cova presents considerable potential as a support for the covalent immobilization of recombinant His-tagged enzymes directly from crude lysates, thereby circumventing the labor-intensive purification process typically required before enzyme immobilization.

Biogenesis of bacterial cellulose/xanthan/CeO2NPs composite films for active food packaging

The increasing significance of biopolymer-based food packaging can be attributed to its biodegradability and independence from petroleum-derived materials. Concurrently, metal oxide nanoparticles (NPs) have gained prominence as effective antimicrobial agents against both wild-type and antibiotic-resistant microbes. In this study, cerium oxide or ceria, CeO2, nanoparticles with an average diameter of 50 nm were synthesized via a green method utilizing Vibrio sp. VLC cell lysate supernatant. The synthesized CeO2 NPs displayed remarkable antimicrobial properties, inhibiting the growth of Escherichia coli and Staphylococcus aureus by 93.7 % and 98 %, respectively. To enhance the potential of bacterial cellulose (BC) for advanced applications, we developed a BC/xanthan/CeO2 nanocomposite using both ex situ and in situ techniques. The integration of CeO2 NPs within the nanocomposite structure not only improved the inherent properties of BC, but also rendered it suitable for use in active food packaging systems. The nanocomposite exhibited no significant cytotoxicity on the human dermal fibroblast (HDF) cells, confirming its safety. Nanocomposites containing biogenically synthesized CeO2 NPs demonstrated exceptional efficacy for reducing microbial contamination. Bread samples coated with nanocomposite films displayed no signs of microbial growth. These results support the application of BC/xanthan/CeO2 nanocomposites as suitable and effective coating materials for antimicrobial food packaging applications.

Expression of a recombinant endolysin from bacteriophage CAP 10-3 with lytic activity against Cutibacterium acnes

The bacteriophage CAP 10-3 forming plaques against Cutibacterium acnes which causes skin acne was previously isolated from human skin acne lesion. Incomplete whole genome sequence (WGS) of the bacteriophage CAP 10-3 was obtained and it had 29,643 bp long nucleotide with 53.86% GC content. The sequence was similar to C. acnes phage PAP 1-1 with a nucleotide sequence identity of 89.63% and the bacteriophage belonged to Pahexavirus. Bioinformatic analysis of the WGS predicted 147 ORFs and functions of 40 CDSs were identified. The predicted endolysin gene of bacteriophage CAP 10-3 was 858 bp long which was deduced as 285 amino acids (~ 31 kDa). The protein had the highest similarity with amino acid sequence of the endolysin from Propionibacterium phage PHL071N05 with 97.20% identity. The CAP 10-3 endolysin gene was amplified by PCR with primer pairs based on the gene sequence, cloned into an expression vector pET-15b and transformed into Escherichia coli BL21(DE3) strain. The predicted protein band (~ 33 kDa) for the recombinant endolysin was detected in an SDS-PAGE gel and western blot assay. The concentrated supernatant of cell lysate from E. coli BL21(DE3) (pET-15b_CAP10-3 end) and a partially purified recombinant CAP 10-3 endolysin showed antibacterial activity against C. acnes KCTC 3314 in a dose-dependent manner. In conclusion, the recombinant CAP 10-3 endolysin was successfully produced in E. coli strain and it can be considered as a therapeutic agent candidate for treatment of human skin acne.

Analysis of the molecular pathogenesis of hereditary protein C deficiency due to a p.Gly86Asp variant of the PROC gene

To explore the molecular pathogenesis of hereditary protein C (PC) deficiency due to a p.Gly86Asp variant of the PROC gene through in vitro expression experiment. Wild type and Gly86Asp mutant expression plasmids of PC were constructed and respectively transfected into HEK 293FT cells. Total RNA was extracted from the transfected cells, and the expression of PROC gene was determined by quantitative real-time PCR (qRT-PCR). PC antigen (PC:Ag) in the supernatant of cell culture and cell lysate was determined by enzyme-linked immunosorbent assay (ELISA), and the level of PC protein was detected by Western blotting. qRT-PCR has detected no significant difference in the transcription level of wild-type and mutant-type PC. Compared with the wild type, the level of mutant PC:Ag in the supernatant and cell lysate were 81.3%±2.6% and 110.0%±2.8%, respectively. No difference was detected in the molecular weight between the wild-type and mutant-type PC by Western blotting. The PC content of mutant type was higher than wild-type in cell lysate, while the opposite was found with the cell culture supernatant. The impaired secretion by mutant PC may be the molecular mechanism of PC deficiency caused by the p.Gly86Asp variant.

Inhibition of Acanthamoeba polyphaga by chlorhexidine-mediated oxidative stress response

Acanthamoeba keratitis is a severe corneal infection caused by a ubiquitous opportunistic protozoan pathogen known as acanthamoeba. For the last decade, the approach to treating this infection typically includes the use of polyhexamethylene biguanide (0.02%) and/or chlorhexidine (Chx) (0.02%). Although chlorhexidine is reportedly effective, its mode of action towards this type of cell is not clear. The aim of this work was to study the effect of chlorhexidine on the oxidative status of Acanthamoeba polyphaga. The effect of chlorhexidine (Chx) on the oxidative state of Acanthamoeba polyphaga was studied using different antiradical methods including ABTS, DPPH and FRAP and measuring the activity of a couple of antioxidant enzyme namely SOD, NADH-FRD and SDH. The chlorhexidine was able to induce oxidative imbalance in cells by over expression of reactive oxygen species and/or inhibiting the antioxidant enzymes. In addition to enhancing the antiradical activity in response to oxidative stress, the present drug was able to reduce the activity of two antioxidant enzymes, superoxide dismutase (SOD) and reduced flavin adenine dinucleotide-fumarate reductase (NADH-FRD), to 30% and 40%, respectively. We could observe an increase of the antiradical capacity of cell's lysate supernatant, to cope with the overproduction of ROS. The imbalance state The inhibition of both SOD and NADH-FRD activities could have a major role in cell oxidative imbalance.

Investigation of the Uptake and Transport of Aspirin Eugenol Ester in the Caco-2 Cell Model.

Background: Aspirin eugenol ester (AEE) is a novel medicinal compound synthesized by esterification of aspirin with eugenol using the prodrug principle. AEE has the pharmacological activities of being anti-inflammatory, antipyretic, analgesic, anti-cardiovascular diseases, and anti-oxidative stress However, its oral bioavailability is poor, and its intestinal absorption and transport characteristics are still unknown. Objective: The purpose of this study was to investigate the uptake and transport mechanisms of AEE in Caco-2 cells. Methods: The effects of time, concentration, and temperature on the transport and uptake of AEE were studied. Results: The results showed that a higher concentration of salicylic acid (SA) was detected in the supernatant of cell lysates and cell culture medium, while AEE was not detected. Therefore, the content change of AEE was expressed as the content change of its metabolite SA. In the uptake experiment, when the factors of time, concentration, and temperature were examined, the uptake of SA reached the maximum level within 30 min, and there was concentration dependence. In addition, low temperature (4°C) could significantly reduce the uptake of SA in Caco-2 cells. In the transport experiment, under the consideration of time, concentration, and temperature, the transepithelial transport of SA from AP-BL and BL-AP sides was time-dependent. The amount of SA transported in Caco-2 cells increased with the increase of concentration, but the transmembrane transport rate had no correlation with the concentration. This phenomenon may be due to the saturation phenomenon of high concentration. The efflux ratio (ER) was less than 1, which indicated that their intestinal transport mechanism was passive transport. Moreover, the temperature had a significant effect on the transport of AEE. Conclusion: In summary, intestinal absorption of AEE through Caco-2 cell monolayers was related to passive transport. The uptake and transport of AEE were concentration-dependent, and temperature significantly affected their uptake and transport. The absorption and transport characteristics of AEE may contribute to the exploration of mechanisms of absorption and transport of chemosynthetic drugs in vitro.

Carrier-Free Deferoxamine Nanoparticles against Iron Overload in Brain

Carrier-Free Deferoxamine Nanoparticles against Iron Overload in Brain

Liraglutide, a glucagon-like peptide-1 receptor agonist, induces ADAM10-dependent ectodomain shedding of RAGE via AMPK activation in human aortic endothelial cells

AimsGlucagon-like peptide-1 alleviates the deleterious effects of advanced glycation end products (AGEs), but the underlying mechanisms are not fully understood. In this study, we investigated the protective mechanism using liraglutide, a glucagon-like peptide-1 receptor agonist, in cultured human aortic endothelial cells (HAECs). Main methodsFollowing liraglutide treatment in HAECs, the receptor for AGEs (RAGE) was measured in both cell lysate and culture supernatant, the cytosolic free Ca2+ level was monitored using Fluo-4 AM, the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was analyzed, and immunofluorescence staining was used to visualize a disintegrin and metalloprotease 10 (ADAM10) on the cell surface. Key findingsLiraglutide (100 nM) induced ectodomain shedding of RAGE within 30 min and inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) induced by AGEs of bovine serum albumin (AGE-BSA). Further experiments revealed that liraglutide rapidly increases extracellular Ca2+ influx through L-type calcium channels and activates AMPK, resulting in the translocation of ADAM10 to the cell surface, whereas siRNA-mediated ADAM10 depletion prevents liraglutide-induced ectodomain shedding of RAGE and eliminates liraglutide's inhibitory effect on AGE-BSA-induced ICAM-1 expression. Moreover, compound C-mediated AMPK inhibition and siRNA-mediated AMPK depletion both prevented ADAM10 translocation to the cell surface and ADAM10-mediated ectodomain shedding of RAGE. SignificanceLiraglutide reduces the number of intact RAGE on the cell surface by inducing ADAM10-mediated ectodomain shedding, which decreases the inflammatory effects of AGEs. AMPK activated by extracellular Ca2+ influx is critically involved in the translocation of ADAM10 to the cell surface, where it cleaves RAGE.

Recombinant Escherichia coli Cell Lysates as a Low-Cost Alternative for Vaccines Against Veterinary Clostridial Diseases.

This chapter describes a practical, industry-friendly, and efficient vaccine protocol based on the use of Escherichia coli cell fractions (inclusion bodies or cell lysate supernatant) containing the recombinant antigen. This approach was characterized and evaluated in laboratory and farm animals by the seroneutralization assay in mice, thereby showing to be an excellent alternative to induce a protective immune response against clostridial diseases.

Hypoxia-inducible transcription factor-1\u03b1 inhibition by topotecan protects against lipopolysaccharide-induced inflammation and apoptosis of cardiomyocytes

BackgroundMyocarditis, an inflammatory disease of the myocardium, is a serious hazard to human life due to the expansion of inflammatory lesions in the myocardium. The aim of this study was to investigate the role of hypoxia-inducible transcription factor (HIF)-1α and its inhibitor topotecan in the pathogenesis of myocarditis.MethodsH9c2 cardiomyoblasts was stimulated with lipopolysaccharide (LPS) to simulate myocarditis model in vitro. The levels of myocardial damage markers were determined using commercially available kits. Western blotting was used to evaluate HIF-1α expression after LPS challenge. Then, after HIF-1α silencing, the contents of inflammatory factors were determined with enzyme-linked immunosorbent assay (ELISA). Cell viability was tested by means of a cell counting kit-8 (CCK-8) assay. Cell apoptosis was assessed by flow cytometry, and the expression of apoptotic proteins was examined using western blot analysis. Subsequently, HIF-1α was overexpressed and topotecan was employed to treat H9c2 cells under LPS exposure condition. The biological functions were detected again.ResultsLPS significantly elevated the levels of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB) and cardiac troponin-I (cTn-I) in supernatant of H9c2 cell lysates. Additionally, LPS led to the notably upregulated expression of HIF-1α. HIF-1α-knockdown markedly decreased the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 compared with the LPS-induced group. Moreover, the cell viability was conspicuously enhanced and cell apoptotic ratio was remarkably reduced, accompanied by downregulated expression of Bax, Bim, caspase 3 and caspase 9 after HIF-1α silencing. Consistently, HIF-1α gain-of-function significantly promoted the production of inflammatory cytokines and cell apoptosis, which was partially counteracted by topotecan administration.ConclusionTo conclude, these findings demonstrated that HIF-1α inhibition by topotecan ameliorates LPS-induced myocarditis in vitro, providing a new approach in the treatment of myocarditis.

Streptococcus infantarius 25124 isolated from pozol produces a high molecular weight amylopullulanase, a key enzyme for niche colonization

Abstract Pozol is a beverage made with maize dough that is prepared after boiling the kernels in limewater, causing a decrease in soluble sugars, with starch being the main fermentable carbohydrate in the dough. Previously, Streptococcus infantarius ssp. infantarius 25124 (Sii-25124) was identified as the most amylolytic bacteria isolated in this product. Analysis of Sii-25124 amylolytic enzymes revealed two amylases, a cytoplasmic α-amylase of 55.7 kDa and an extracellular amylopullulanase of 246.3 kDa, with two catalytic domains, one typical of an α-amylase and another typical of a pullulanase/glycogen debranching enzyme. Characterization of the joint activity of both enzymes using Sii-25124 cell lysate supernatant demonstrated stability between 30 °C and 45°C, and pH stability in a range between 6.8 and 8.0. The joint activity of Sii-25124 amylases showed a fast production of reducing sugars when starch was used as the substrate. In contrast, reducing sugar production from amylopectin was lower, but it steadily increased throughout the reaction time. The amylopullulanase produced by Sii-25124 hydrolyzes the starch in the dough to produce low molecular weight oligosaccharides, which may be transported into Sii-25124 cells, so that intracellular α-amylase hydrolyzes them to mono- and disaccharides. Amylopullulanase production by Sii-25124 could be an example of a specialized enzyme that successfully dominates starchy food fermentation.

Molecular cloning, expression and characterization of aspartyl protease inhibitor from Ancylostoma ceylanicum.

Aspartyl protease inhibitors (APIs) from parasitic intestinal nematodes are highly immunogenic and have been suggested as potential vaccine antigens. Ac-API-1 from Ancylostoma caninum showed strong immunogenicity and its polyclonal antibodies could specifically recognize the excretory/secretory products of adult worms. However, little is known about molecular characteristics and biological function of API from Ancylostoma ceylanicum (Ace-API). In this study, the Ace-API mature peptide coding sequence was cloned and expressed, and molecular characteristics of its full length sequence were analyzed. Ace-API cDNA was 684bp in length, which encoded 228 amino acids. The similarity of the Ace-API amino acid sequence to Ac-API-1 and Adu-API-1 was 96.93% and 96.49%, respectively, and they clustered together in the phylogenetic tree. Escheria coli-expressed recombinant protein was mainly soluble in the supernatant of bacterial cell lysate. Western blot showed that Ace-API protein had good reactivity to the serum of infected dogs. Pepsin inhibition assay revealed that the recombinant protein had inhibitory activity on pepsin. Immunofluorescence results demonstrated that Ace-API was mainly localized to the epidermis, excretory glands, and pseudocoelomic fluid of the adult. Using the quantitative real-time PCR, the expression of Ace-api mRNA in adults was significantly higher than that in the third stage (L3) larvae. Together, these data indicate that Ace-API is secreted extracellularly by the parasite, and might play a role in protecting the parasite against the proteolytic digestion by the host proteases, which stimulate further studies to explore this protein as a potential hookworm vaccine candidate.

Expression of Aspergillus niger glucose oxidase in Pichia pastoris and its antimicrobial activity against Agrobacterium and Escherichia coli.

The gene encoding glucose oxidase from Aspergillus niger ZM-8 was cloned and transferred to Pichia pastoris GS115, a transgenic strain P. pastoris GS115-His-GOD constructed. The growth curve of P. pastoris GS115-His-GOD was consistent with that of Pichia pastoris GS115-pPIC9K under non-induced culture conditions. Under methanol induction conditions, the growth of the GOD-transgenic strain was significantly lowered than P. pastoris GS115-pPIC9K with the induced-culture time increase, and the optical densities of GOD-transgenic strain reached one-third of that of the P. pastoris GS115-pPIC9K at 51 h. The activity of glucose oxidase in the cell-free supernatant, the supernatant of cell lysate, and the precipitation of cell lysate was 14.3 U/mL, 18.2 U/mL and 0.48 U/mL, respectively. The specific activity of glucose oxidase was 8.3 U/mg, 6.52 U/mg and 0.73 U/mg, respectively. The concentration of hydrogen peroxide formed by glucose oxidase from supernatant of the fermentation medium, the supernatant of the cell lysate, and the precipitation of cell lysate catalyzing 0.2 M glucose was 14.3 μg/mL, 18.2 μg/mL, 0.48 μg/mL, respectively. The combination of different concentrations of glucose oxidase and glucose could significantly inhibit the growth of Agrobacterium and Escherichia coli in logarithmic phase. The filter article containing supernatant of the fermentation medium, supernatant of the cell lysate, and precipitation of cell lysate had no inhibitory effect on Agrobacterium and E. coli. The minimum inhibitory concentration of hydrogen peroxide on the plate culture of Agrobacterium and E. coli was 5.6 × 103 μg/mL and 6.0 × 103 μg/mL, respectively.

Colonization and Probiotic Effect of Metschnikowia sp. C14 in the Intestine of Juvenile Sea Cucumber, Apostichopus japonicus

Viable cell count was used to determine whether Metschnikowia sp. C14 can colonize the intestine of juvenile sea cucumber Apostichopus japonicus . Sea cucumber individuals were divided into two groups, which were fed the control diet for 38 days or the C14-supplemented diet at 10 5 cells g −1 diet for 28 days, then the control diet from day 29 to day 38. The number of C14 cells in the intestine of sea cucumber fed the C14-supplemented diet significantly increased from day 7 to day 28, and decreased from day 29 to day 38. Sea cucumber fed with the diet containing C14 showed a significant increase in trypsin activity and lipase activity from day 21 to day 33 compared with the control. Feeding C14 significantly improved the phagocytic activity and respiratory burst in coelomocytes from day 21 to day 35 and from day 14 to day 38, respectively. In addition, there was an obvious enhancement in lysozyme activity (from day 21 to day 38 or day 33), phenoloxidase activity (from day 21 to day 28) and total nitric oxide synthase activity (from day 14 to day 38) in coelomic fluid supernatant and/or coelomocyte cell lysate supernatant compared with the control. There were significant positive correlations between the number of C14 cells colonizing the intestine and trypsin activity of the intestine, lysozyme activity of the coelomic fluid supernatant and coelomocyte lysate supernatant from sea cucumber. These data suggested that the number of C14 cells should be maintained at 10 5 cfu (colony-forming units) g −1 intestine material for the maximum benefit.

Nitrate Reductase mediated synthesis of surface passivated nanogold as broad-spectrum antibacterial agent

The green synthesis of gold nanoparticles has attracted tremendous interest owing to their unique physicochemical properties and widespread applications which are primarily size-dependent. The stability, less reaction time, and use of biological resources as novel nanofactories as an alternative to conventional synthesis strategies are the main objectives of green synthesis approaches. However, to attain size-controlled synthesis from the biogenic route is still a challenge. Hence, use of nontoxic stabilizers becomes increasingly essential. Herein, we describe an emerging, simple, nonconventional approach to synthesize stable and size-controlled biogenic nanogold using cell lysate supernatant containing nitrate reductase of Bacillus licheniformis in the presence of Tween 20 and dodecanethiol respectively. The face-centered central composite design used for the optimization of gold nanoparticles (AuNPs) biosynthesis. The maximum AuNPs biosynthesis obtained using the optimized media variables, glucose (2.1 g/L), peptone (14.05 g/L), yeast extract (4.14 g/L), and potassium nitrate (3.91 g/L) was 0.769 a.u. Highly stable monodispersed nanogold of 10.4 ± 0.6 nm and 12.5 ± 0.9 nm sizes arranged in ordered self-assembly was obtained. The stability profile and kinetics of bioreduction was evaluated with respect to time, and the involvement of the nitrate reductase enzyme in bioreduction was validated by inhibitor study. The physicochemical properties of biogenic nanoparticles were characterized using multiple spectroscopy and microscopy techniques. The obtained nanogold demonstrated excellent bactericidal property against both gram-negative and gram-positive bacteria in size-dependent manner and thus could find tremendous utility in clinical, biological, and environmental applications as a broad-spectrum antibacterial agent.

Interleukin 10 Induces the Expression of Membrane-Bound HLA- G and the Production of Soluble HLA-G on HeLa CCL-2 Cells.

BACKGROUND:Interleukin-10 is a cytokine that has a pleiotropic effect on the immune system and inflammation. IL-10 can contribute to the anti-tumour immune response by increasing HLA-G expression.AIM:This study aimed to determine the effect of IL-10 induction on membrane HLA-G expression and soluble HLA-G production of HeLa CCL-2 cells.METHODS:HeLa CCL-2 cells were cultured in the well plate and divided into 4 groups consist of 1 control group that was not induced by IL-10 and 3 treatment groups that were induced by IL-10 500 ng/ml, 1000 ng/ml and 2000 ng/ml respectively. All groups were incubated for 48 hours in a 37°C incubator at 5% CO2 atmospheric pressure. HLA-G measurements were carried out both in cell lysate and cell culture supernatant using ELISA and in membrane-bound using immunofluorescence method. The expression of HLA-G in membrane-bound calculated using the ImageJ application. Data obtained were analysed by ANOVA and LSD test.RESULTS:In the control group, the HLA-G level in the culture supernatant was higher than in cell lysate (p = 0.000), as well as in all treatment groups (p = 0.000). There were significant differences between the treatment group (p = 0.000) and within the treatment group (p = 0.000) at HLA levels. The highest expression of HLA-G in HeLa cell membranes found in cell culture induced by IL-10 concentrations of 500 ng/ml, i.e., 59.28 AU in view. HLA-G membrane expression in the IL-10 1000 ng/ml induced group was significantly different from all treatment groups (p = 0.000).CONCLUSION:HeLa CCL-2 cells express HLA-G on the membrane and release dissolved HLA-G without induction of IL-10 although IL-10 induction augments the presence and the production of HLA-G in HeLa CCl-2 cells.

Genistein Decreases APP/tau Phosphorylation and Ameliorates Aβ Overproduction Through Inhibiting CIP2A.

Upregulation of Cancerous Inhibitor of PP2A (CIP2A) plays an important role in disease-related phosphorylation of tau/APP and tau pathology/Aβ overproduction through inhibiting PP2A in AD brain. Genistein has been shown to potently reduce CIP2A in experimental cancer treatment research. Whether Genistein can ameliorate AD pathology through targeting CIP2A needs further investigation. The inhibitory effects of Genistein on tau/APP phosphorylation and Aβ overproduction in AD cell models have been explored. HEK293-T cells were co-transfected with CIP2A and APP plasmids, or CIP2A and tau plasmids, with Genistein incubation at 0, 30, 60 or 120 µM for 48 h, cell viability and PP2A activities were measured. HEK293-T cells with CIP2A/APP overexpression treated with Genistein at 30 µM for 48 h were collected and lyzed for Western blotting detection of CIP2A, PP2Ac, APP-T668, total APP, PS1, BACE1, sAPPα and sAPPβ. Aβ40 and Aβ42 levels in cell supernatant, soluble fraction (RIPA) and insoluble fraction (formic acid soluble) of cell lysates were measured by ELISA. HEK293-T cells with CIP2A/tau overexpression treated with Genistein at 30 µM for 48 h were collected for Western blotting detection of CIP2A, PP2Ac, tau-S396, tau-S404 and total tau. Genistein effectively reduced CIP2A expression, and restored PP2A activities both in CIP2A/APP, CIP2A/tau co-expressed cells. Genistein reduced APP phosphorylation at T668 site and inhibited Aβ production. Meantime, Genistein ameliorated tau hyperphosphorylation through repressing the inhibitory effect of CIP2A on PP2A. CIP2A is a target of Genistein in AD therapy. Genistein reduces APP/tau hyperphosphorylation and Aβ production through inhibiting the effect of CIP2A on PP2A.

Multifunctional CuO nanoparticles with cytotoxic effects on KYSE30 esophageal cancer cells, antimicrobial and heavy metal sensing activities

In this work, fluorescent copper oxide nanoparticles (CuO NPs) were green synthesized using viable cells, cell lysate supernatant (CLS) and protein extracts of luminescent Vibrio sp. VLC. Biogenic CuO NPs were then characterized by XRD, FTIR, UV/Vis spectroscopy, TEM, DLS, and PL spectroscopy. Results showed that CLS method was more efficient for CuO NPs production, therefore CuO NPs synthesized by this method from copper sulfate (CuO NPs-1) and/or copper nitrate (CuO NPs-2) were used for further studies. The crystallite size of polydispersed CuO NPs-1 and CuO NPs-2 were about 8.83 and 8.77 nm, respectively indicating their suitability for biological applications. Antibacterial activity of CuO NPs was determined using broth microdilution, well diffusion agar, and time-kill curves methods. Both CuO NP-1 and CuO NP-2 inhibited bacterial growth at the minimum inhibitory concentration (MIC) of 625 mg/L except St. mutants (MIC = 1250 mg/L). Emission of fluorescent light from the surface of NPs was increased when exposed to Cd2+, As2+ and Hg2+ ions but decreased by Pb2+ ions. Results showed that CuO NP-1 had anticancer properties against KYSE30 esophageal cancer cell line (IC50 = 13.96 mg/L) while no higher cytotoxic effects were observed on Human Dermal Fibroblasts (HDF) (IC50 = 48.88 mg/L).

BIOGENIC SYNTHESIS AND CHARACTERIZATION OF GOLD NANOPARTICLES BY A NOVEL MARINE BACTERIA MARINOBACTER ALGICOLA: PROGRESSION FROM NANOSPHERES TO VARIOUS GEOMETRICAL SHAPES

Metal nanoparticle biosynthesis is gaining impetus as the process is cost-effective and ecofriendly with a variety of application of them in biomedical field. In this study, biosynthesis approach for the synthesis of gold nano particles (GNPs) has been investigated using marine bacteria, Marinobacter algicola isolated from Indian sector of Southern Ocean. The biosynthesis of GNP was the obtained by adding HAuCl4–1mM, to the bacterial cell lysate supernatant (CLS) maintained at pH 7.0, incubated at 30 °C for 24 h. The bio-reduction of auric chloride was supposedly accomplished by enzyme nitrate reductase from the bacterial cellular bio mass, and was established using the FTIR data. GNP characterization from SEM, AFM and TEM exhibited formation of various shaped GNPs like spherical, triangular, pentagonal and hexagonal, possibly from enzyme nitrate reductase which is relatively different from GNPs synthesized so far reporting only single shape from single enzyme. XRD analysis determined the FCC structure of GNPs. DLS result display the GNPs formed is of mean size of 4-168 nm with an average of 74 nm. Being able to control the shapes of nanoparticles as a time dependent factor can be considered path breaking leading to its multipurpose biomedical application. The present research also studied the antimicrobial effect of GNPs on various pathogens.

Enriched environment promotes post-stroke neurogenesis through NF-κB-mediated secretion of IL-17A from astrocytes

Enriched environment (EE) has been shown to promote post-stroke neurogenesis and functional recovery. However, the underlying molecular mechanisms remains poorly understood. Male C57BL/6 mice underwent 60-min middle cerebral artery occlusion (MCAO) followed by reperfusion, after which mice were housed in either standard environment (SE) or EE. We found that post-ischemic EE exhibited reduced protein level of nuclear factor κB (NF-κB)/p65 in cytoplasm and increased its expression correspondingly in nucleus at 28 days post-ischemia (dpi). However, post-ischemic EE had no effects on terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)-positive cells in ischemic hemisphere at 28dpi. EE mice treated with NF-kB inhibitor Bay11-7082 had decreased subventricular zone (SVZ) neural precursor cells (NPCs) proliferation, neuronal differentiation and subsequent functional recovery after stroke at 28dpi. Bay11-7082 treatment attenuated the promoting effects of post-ischemic EE on interleukin 17A (IL-17A) messenger RNA (mRNA) and protein expression at 28dpi. Furthermore, our in vitro data revealed that in primary astrocyte cultures addition of Bay11-7082 markedly decreased the expression of IL-17A in both the cell lysate and culture supernatant of activated astrocytes. Blockade of IL-17A with neutralizing antibody abrogated the promoting role of EE in NPCs proliferation derived from SVZ, neuronal differentiation and subsequent functional recovery after stroke. Thus, our results reveal a previously uncharacterized property of NF-κB/IL-17A signaling pathway in EE-mediated neurogenesis and functional recovery after ischemic stroke.