Isolation of Antibiotic Resistant Plasmid Deoxyribonucleic Acid from MDR Diarrheal Pathogens

Cholera is an acute infectious disease in countries with poor sanitation. The main clinical symptom of cholera is gastroenteritis and a symptom of the disease includes mild to moderate dehydration, vomiting, fever and abdominal pain. Antibiotic drug resistance in V. cholera has become a serious problem mostly in developing countries and muti-drug resistance to different antibiotics as well as Tetracycline, Ampicillin, Kanamycin, Trimethoprim, Sulphonamides Streptomycin and Gentamicin. Multi-drug resistant V. cholera showed resistance against three or more three contrasting classes of antibiotics for recent decades. Fifty diarrheal samples were collected from the different hospitals in and around the Tirupur district. 25 positive V. cholerae were isolated. The isolates were characterized morphologically and biochemically. The confirmed strains were taken to decide susceptibility patterns by the disc diffusion method. Vibrio cholerae isolated in this study was subjected to an antibiogram against 10 commonly used antibiotics. All the tested isolates showed maximum resistance to Erythromycin (97%) and minimum resistance was noted in Trimethoprime (50%) respectively. Electrophoretic examination of plasmid DNA was carried out by Agarose gel Electrophoresis on 0.7%. More than 90% of resists showed were taken for plasmid isolation. The molecular weights of the fragments were evaluated by using a 10,000 bp DNA ladder and it was determined to be 1500 bp respectively. It is crucial to know the sensitivity and resistance pattern of any bacterial species for intercession an effective drug of choice in future

Insight into OroxylinA-7-O-β-d-Glucuronide-Enriched Oroxylum indicum Bark Extract in Oral Cancer HSC-3 Cell Apoptotic Mechanism: Role of Mitochondrial Microenvironment

Oroxylum indicum, of the Bignoniaceae family, has various ethnomedical uses such as an astringent, anti-inflammatory, anti-bronchitis, anti-helminthic and anti-microbial, including anticancer properties. The druggability of OI stem bark extract was determined by its molecular docking interactions with PARP and Caspase-3, two proteins involved in cell survival and death. Note that 50 µg/mL of Oroxylum indicum extract (OIE) showed a significant (p < 0.05%) toxicity to HSC-3 cells. MTT aided cell viability and proliferation assay demonstrated that 50 µg/mL of OIE displayed significant (p < 0.5%) reduction in cell number at 4 h of incubation time. Cell elongation and spindle formation was noticed when HSC-3 cells were treated with 50 µg/mL of OIE. OIE initiated DNA breakage and apoptosis in HSC-3 cells, as evident from DNA ladder assay and calcein/EB staining. Apoptosis potential of OIE is confirmed by flow cytometer and triple-staining (live cell/apoptosis/necrosis) assay. Caspase-3/7 fluorescence quenching (LANCE) assay demonstrated that 50 µg/mL of OIE significantly enhanced the RFU of caspases-3/7, indicating that the apoptosis potential of OIE is probably through the activation of caspases. Immuno-cytochemistry of HSC-3 cells treated with 50 µg/mL of OIE showed a significant reduction in mitochondrial bodies as well as a reduction in RFU in 60 min of incubation time. Immunoblotting studies clearly showed that treatment of HSC-3 cells with OI extract caused caspase-3 activation and PARP deactivation, resulting in apoptotic cell death. Overall, our data indicate that OIE is an effective apoptotic agent for human squamous carcinoma cells and it could be a future cancer chemotherapeutic target.

Construction of DNA ladder for determination of small size DNA fragments

DNA marker is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare recombinant plasmids pSY-60 which was partially digested by one restriction enzyme for generating DNA markers of 7 fragments from 60 to 420 bp. The procedure included a synthesis of 60 bp DNA fragment with EcoRI sites at both ends using PCR extension, self-ligation of the 60 bp fragments and subcloning the ligated product into plasmid, generating recombinant pSY-60. Once being cloned, 500 ng of 420 bp fragment purified from 100 µL PCR product was incompletely digested by EcoRI, sufficiently producing to 50 acrylamide gels. Our procedure for production of DNA markers could be simple, time saving and inexpensive in comparison with current ones widely used in most laboratories.

Names Targeting p65 to Inhibit Cas3 Transcription by Onjisaponin B for Radiation Damage Therapy in p65+/- Mice

Background: p65 is activated following radiation injury. The formation of p65 is regulated by Onjisaponin B (OB) in Alzheimer's disease models. In addition, there is a binding site for p65 in the promoter region of CAS3. In the present study, the use of OB as an intervention to modulate p65/Cas3 following radiation injury was studied.Methods: Cellular and animal experiments, immunofluorescence, HE staining, Western blotting, qRT-PCR, comet and DNA ladder assays, and flow cytometry were used to confirm the expression of p65 and Cas3.Results: The results demonstrated that if the expression of p65 was silenced in V79 and TC cells, OB did not significantly inhibit the activation of p65 or Cas3 following irradiation, or significantly inhibit the phosphorylation of p65 and its transfer into the nucleus. Overexpression of p65 in V79 and MTEC-1 cells resulted in OB significantly inhibiting the activation of p65 and Cas3, and the phosphorylation and translocation of p65 into the nucleus. In p65+/- mice, expression of the p65 gene was knocked down, leading to increased tissue apoptosis and inflammation, and serious tissue pathological changes. The inhibition of p65 activation by OB after exposure to radiation was not apparent in the thymus, but it was in the lung, indicating that OB has a regulatory effect on endogenous p65.Conclusions:In summary, OB interfered with radiation injury by targeting and regulating p65/Cas3. Therefore, it was confirmed that p65 is an important target molecule for the treatment of radiation injury.

Exercise Rehabilitation and/or Astragaloside Attenuate Amyloid-Beta Pathology by Reversing BDNF/TrkB Signaling Deficits and Mitochondrial Dysfunction

Background: Although there are numerous investigations regarding the beneficial effects of exercise rehabilitation (ER) or astragaloside (AST) after amyloid-beta (Ab) pathology, the mechanisms are still not well understood. We aim to assess whether ER and/or AST counteract Ab pathology via diminishing brain-derived neurotrophic factor (BDNF) signaling deficits and mitochondrial dysfunction. Methods: Ab1-42 were microinjected into the bilateral ventricles to induce Ab neuropathology in rats. The Alzet osmotic pump containing full of AST was implanted subcutaneously during surgery. The ER group of rats started at seven days post-surgery and lasted for four weeks. The ANA12 was administrated once per day to the endpoint of the experiments to antagonize the BDNF action. Neurobehavioral functions were evaluated by Y-maze, radial maze, and rotarod tests one day before surgery and 14 to 35 days post-surgery. Cortical and hippocampal expressions of both BDNF/TrkB and cathepsin D were determined by Western blotting method. The rat primary cultured cortical neurons were incubated with BDNF and/or AST and ANA12 followed by exposure to aggregated Ab for 24 hours. The cell viability (by MTT assay), mitochondrial membrane permeability and electrochemical potential (by JC-1 stain), DNA fragmentation (sub-G1 and DNA ladder assay), synaptic plasticity (by immunofluorescence stain), and pTrkB/pAkt/pGSK3b/b-catenin signaling (by Western blot) were determined.Results: ER and/or AST reversed neurobehavioral disorders, downregulation of cortical and hippocampal expression of both BDNF/TrkB and cathepsin D, neural pathology, Ab accumulation, and altered microglial polarization caused by Ab. In vitro studies also confirmed that topical application of BDNF and/or AST reversed the Ab-induced cytotoxicity, apoptosis, mitochondrial distress, synaptotoxicity, and decreased expression of p-TrkB, AKT, p-GSK3b, and b-catenin in altered rat cortical neurons. In particular, the beneficial effects of combined ER (or BDNF) and AST therapy in vivo and in vitro were superior to ER (or BDNF) or AST alone. Furthermore, we observed that any gains from ER (or BDNF) and/or AST could be significantly eliminated by ANA-12, a potent BDNF/TrkB antagonist. Conclusion: These results indicate that whereas ER (or BDNF) and/or AST attenuate Ab pathology by reversing BDNF/TrkB signaling deficits and mitochondrial dysfunction. In particular, AST can be an alternative therapy to replace ER.

Sclareol Inhibits Hypoxia-Inducible Factor-1α Accumulation and Induces Apoptosis in Hypoxic Cancer Cells

Purpose: The hypoxia in solid tumors is associated with the resistance to chemo/radiotherapy. Hypoxia-inducible factor-1 (HIF-1) plays a key role in cell remodeling to hypoxia. Therefore, the inhibition of HIF-1 accumulation is considered a hopeful strategy for the treatment of cancer. Here, we aimed to evaluate the geno- and cytotoxicity properties of sclareol, a natural bicyclic diterpene alcohol, on A549 cells in CoCl2-induced hypoxia. Methods: The cytotoxicity and apoptosis-inducing properties of sclareol on the A549 cell were evaluated using MTT assay and Annexin V/PI staining, respectively in hypoxia. DAPI staining, DNA ladder, and comet assay were used to evaluate the genotoxicity. Further, the qPCR technique was employed to assess the expression of HIF-1α, HIF-1β, and downstream target genes (GluT1, and Eno1). Finally, the level of HIF-1α protein was evaluated through Western blotting in sclareol-treated cells in hypoxia. Results: The inhibitory concentration (IC50) of sclareol against A549 cells was 8 μg/mL at 48 h in hypoxia. The genotoxicity of sclareol was confirmed in the cells treated with sclareol in hypoxia. Sclareol induced ~46% apoptosis and also necrosis in the hypoxic condition. The qPCR analyses showed an enhanced suppression of HIF-1α, HIF-1β, GluT1, and Eno1 due to the sclareol treatment in the hypoxia. Moreover, protein quantification analysis showed dose-dependently degradation of HIF-1α in hypoxia upon treatment with sclareol. Conclusion: The results obtained here indicate that sclareol possesses dose-dependent cytotoxicity effects against A549 cells in hypoxia through inhibition of HIF-1α protein accumulation, increasing cell sensitivity to intracellular oxygen levels, and disruption of cell adaptation to hypoxia.

Isolation and molecular detection of avian mycoplasmosis in selected areas of Mymensingh district in Bangladesh

Avian mycoplasmosis caused by several species of Mycoplasma including Mycoplasma gallisepticum, M synoviae, M. meleagridis and M. iowae. Among these Mycoplasma gallisepticum is the most important poultry pathogen in Bangladesh. For effective control of Mycoplasmosis, proper early diagnosis is the corner stone. The research work was designed, a total of 20 samples, lung exudates, swabs from trachea and air sacs were collected from dead birds of different poultry farms in Mymensingh district during October-December, 2007. Samples were collected in 10% buffered formalin for histopathological study. Swabs were collected in mycoplasma broth supplemented with supplement-G. Additionally Kanamycin solution was added to prevent the growth of gram–Ve bacteria and then the organisms were transferred into mycoplasma agar for isolation. Histopathological studies were conducted using routine procedure in Hematoxylin and Eosin stain. Isolated Mycoplasma were subjected to DNA extraction, Nested PCR was done using a commercial PCR kit. The histopathological study revealed the presence of mycoplasmal related tissue changes, such as severe congestion and infiltration of mononuclear cells in different organs. The extracted DNA accumulated at the upper position of DNA ladder as band without any smear formation. The DNA from avian mycoplasmas was amplified and gave amplified product of 975 bp by outer primer and 395 bp by inner primer which was much smaller than the expected size. In this study, preliminary results from field samples suggest that culture using mycoplasma agar and broth supplement with Supplement-G and Kanamycin solution could be useful for the isolation of pathogenic avian mycoplasmas. Asian J. Med. Biol. Res. 2021, 7 (2), 182-190

Quantitative spectrofluorometric assay detecting nuclear condensation and fragmentation in intact cells

At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6–48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.

Evaluation of pro-apoptotic potential of taxifolin against liver cancer

Liver cancer is the second most common cause of cancer-induced deaths worldwide. Liver cirrhosis and cancer are a consequence of the abnormal angio-architecture formation of liver and formation of new blood vessels. This angiogenesis is driven by overexpression of hypoxia-inducible factor 1-alpha (Hif1-α) and vascular endothelial growth factor (VEGF). Apart from this, protein kinase B (Akt) is also impaired in liver cancer. Despite the advancement in conventional treatments, liver cancer remains largely incurable. Nowadays, the use of naturally occurring anticancer agents particularly flavonoids is subject to more attention due to their enhanced physicochemical properties. Therefore, this study underlines the use of a natural anticancer agent taxifolin in the treatment of liver cancer using hepatocellular carcinoma cell line HepG2 and Huh7. The aim of our study is to devise a natural and efficient solution for the disease prevalent in Pakistan. The study involved the assessment of binding of ligand taxifolin using molecular docking. The binding of taxifolin with the proteins (Hif1-α, VEGF and Akt) was calculated by docking using Vina and Chimera. Further evaluation was performed by cell viability assay (MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay), colony formation assay, cell migration assay, DNA ladder assay and flow cytometry. To see whether taxifolin directly affected expression levels, analysis of gene expression of Hif1-α, VEGF and Akt was performed using real-time polymerase chain reaction (qPCR) and western blotting. In silico docking experiments revealed that these proteins showed favorable docking scores with taxifolin. Treatment with taxifolin resulted in the inhibition of the liver cancer growth and migration, and induced apoptosis in HepG2 and Huh7 cell lines at an inhibitory concentration (IC50) value of 0.15 µM and 0.22 µM, respectively. The expression of HIF1-α, VEGF and Akt was significantly reduced in a dose- dependent manner. The inhibitory effect of taxifolin on hepatic cells suggested its chemopreventive and therapeutic potential. The studied compound taxifolin exhibited pronounced pro-apoptotic and hepatoprotective potential. Our study has confirmed the pro-apoptotic potential of taxifolin in liver cancer cell lines and will pave a way to the use of taxifolin as a chemotherapeutic agent after its further validation on the animal models and humans based epidemiological studies.

The combination of berberine and methotrexate enhances anti-cancer effects in HeLa cancer cell line: A morphological study

Background: Co-administration of two or several either chemotherapeutic agents or conventional drugs as a combination treatment is the most effective method to increase therapeutic efficiency. Additive or synergistic influence are two mechanisms by which combination therapy causes a rise in optimal cancer therapy compared to a single treatment. Methods: Colorimetric assay was carried out to estimate the cytotoxicity of the combined system, followed by apoptosis assay to calculate the number of apoptotic cells. Both 4′,6-diamidino-2-phenylindole (DAPI) staining and DNA ladder were complemented tests to illuminate morphological changes and DNA fracturing on HeLa cancer cells. Statistical: Through Graph pad Prism 6.0 software. One-way ANOVA was used to determine the significance. A P-value of less than 0.05 was considered to be statistically significant. Results: In this study revealed that the combination of MTX and BER could inhibit the growth of HeLa cancer cells noticeably. Nevertheless, single BER and MTX were not as effective as a combined system to reduce cell viability at the same dose. Regarding the apoptosis induction and change in morphology of cancer cells’ nucleus, co-treatment of BER and MTX was more effective. The result was complemented with flow cytometry, DAPI staining and DNA ladder, which showed that BER+MTX depicted more anti-cancer effects. Conclusion: The combination therapy of HeLa cancer cells with BER and MTX showed high inhibition effect compared to other treated groups.