Search results for: in-vitro antidiabetic assays

Authors: T. Wresdiyati, S. Sa’diah, A. Winarto

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Diabetes mellitus (DM) is a metabolic disease that can be indicated by the high level of blood glucose. The objective of this study was to observe the antidiabetic properties of ethanolic extract of Indonesian Swietenia mahagoni Jacq. seed on the profile of pancreatic superoxide dismutase and β-cells in the alloxan- experimental diabetic rats. The Swietenia mahagoni seed was obtained from Leuwiliang-Bogor, Indonesia. Extraction of Swietenia mahagoni was done by using ethanol with maceration methods. A total of 25 male Sprague dawley rats were divided into five groups; (a) negative control group, (b) positive control group (DM), (c) DM group that was treated with Swietenia mahagoni seed extract, (d) DM group that was treated with acarbose, and (e) non-DM group that was treated with Swietenia mahagoni seed extract. The DM groups were induced by alloxan (110 mg/kgBW). The extract was orally administrated to diabetic rats 500 mg/kg/BW/day for 28 days. The extract showed hypoglycemic effect, increased body weight, increased the content of superoxide dismutase in the pancreatic tissue, and delayed the rate of β-cells damage of experimental diabetic rats. These results suggested that the ethanolic extract of Indonesian Swietenia mahagoni Jacq. seed could be proposed as a potential anti-diabetic agent.

Keywords: beta cells, diabetes, hypoglycemic, rat, Swietenia mahagoni

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Authors: Amir Ali, Laura Yael Mendoza

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The application of nanomaterials is becoming the most effective strategy of elicitation to produce a desirable level of plant biomass with complex medicinal compounds. This study was designed to check the influence of phytosynthesized iron nanoparticles (FeNPs) on physical growth characteristics, antioxidant status, and production of steviol glycosides of in vitro grown Stevia rebaudiana. Effect of different concentrations of iron nanoparticles replacement of iron sulfate in MS medium (stock solution) on invitro stevia plant growth following positive control (MS basal medium), negative control (iron sulfate devoid medium), iron sulfate devoid MS medium and supplemented with FeNPs at different concentrations (5.6 mg/L, 11.2 mg/L, 16.8 mg/L, 22.4 mg/L) was evaluated. The iron deficiency leads to a drastic reduction in plant growth. In contrast, applying FeNPs leads to improvement in plant height, leave diameter, improved leave morphology, etc., in a concentration-dependent manner. Furthermore, the stress caused by FeNPs at 16.8 mg/L in cultures produced higher levels of total phenolic content (3.7 ± 0.042 mg/g dry weight: DW) and total flavonoid content (1.9 ± 0.022 mg/g DW and antioxidant activity (78 ± 4.6%). In addition, plants grown in the presence of FeNPs at 22.4 mg/L resulted in higher enzymatic antioxidant activities (SOD = 3.5 ± 0.042 U/mg; POD = 2.6 ± 0.026 U/mg; CAT = 2.8 ± 0.034 U/mg and APx = 3.6 ± 0.043 U/ mg), respectively. Furthermore, exposure to a higher dose of FeNPs (22.4 mg/L) exhibited the maximum amount of stevioside (stevioside: 4.6 ± 0.058 mg/g (DW) and rebaudioside A: 4.9 ± 0.068 mg/g DW) as compared to other doses. The current investigation confirms the effectiveness of FeNPs in growth media. It offers a suitable prospect for commercially desirable production of S. rebaudiana biomass with higher sweet glycosides profiles in vitro.

Keywords: cell culture, stevia, iron nanoparticles, antioxidants

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Authors: Sadia Roshan, Kulsoom Sughra, Shazia Shamas, Shamaila Irum, Haleema Sadia

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To evaluate synergistic medicinal effects of Withania somnifera (Ashwaghandha) and Murraya koenigii (Curry pata) in vitro. Antimicrobial activity was determined using the disc diffusion method against five bacterial and two fungal strains. The antioxidant activity was evaluated by the DPPH assay. The antidiabetic activity was evaluated by alpha-glucosidase inhibition assay and alpha-amylase inhibition assay. Synergistic antibacterial activity was observed against all the strains of bacteria, either Gram-positive or Gram-negative and fungi under study conditions. The maximum antibacterial activity was displayed by combined extract against E. coli i.e. 26±0.4mm. Maximum antifungal activity was shown by combined extract against Aspergillus niger, i.e., 17.3±0.5mm. The antioxidant activity of the combined extract was also significant. Alpha-glucosidase inhibition and alpha-amylase inhibition assays also showed synergism. Results indicate that Withania somnifera and Murraya koengii have medicinal properties. The combined extract of both plants is more potent than their individual extracts, suggesting that these can work in synergism. The research suggests that different plant extracts could be used in combination to increase their medicinal activities by many folds, thus giving an insight into future use of herbal medication.

Keywords: withania somnifera, murraya koenigii, antimicrobial activity, gram-positive bacetria, gram-negative bacteria

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Authors: Teklay Gebrecherkos

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Background: Serological testing for SARS-CoV-2 plays an important role in epidemiological studies, in aiding the diagnosis of COVID-19 and assess vaccine responses. Little is known about the dynamics of SARS-CoV-2 serology in African settings. Here, we aimed to characterize the longitudinal antibody response profile to SARS-CoV-2 in Ethiopia. Methods: In this prospective study, a total of 102 PCR-confirmed COVID-19 patients were enrolled. We obtained 802 plasma samples collected serially. SARS-CoV-2 antibodies were determined using four lateral flow immune assays (LFIAs) and an electrochemiluminescent immunoassay. We determined longitudinal antibody response to SARS-CoV-2 as well as seroconversion dynamics. Results: Serological positivity rate ranged between 12%-91%, depending on timing after symptom onset. There was no difference in the positivity rate between severe and non-severe COVID-19 cases. The specificity ranged between 90%-97%. Agreement between different assays ranged between 84%-92%. The estimated positive predictive value (PPV) for IgM or IgG in a scenario with seroprevalence at 5% varies from 33% to 58%. Nonetheless, when the population seroprevalence increases to 25% and 50%, there is a corresponding increase in the estimated PPVs. The estimated negative-predictive value (NPV) in a low seroprevalence scenario (5%) is high (>99%). However, the estimated NPV in a high seroprevalence scenario (50%) for IgM or IgG is reduced significantly from 80% to 85%. Overall, 28/102 (27.5%) seroconverted by one or more assays tested within a median time of 11 (IQR: 9–15) days post symptom onset. The median seroconversion time among symptomatic cases tended to be shorter when compared to asymptomatic patients [9 (IQR: 6–11) vs. 15 (IQR: 13–21) days; p = 0.002]. Overall, seroconversion reached 100% 5.5 weeks after the onset of symptoms. Notably, of the remaining 74 COVID-19 patients included in the cohort, 64 (62.8%) were positive for antibodies at the time of enrollment, and 10 (9.8%) patients failed to mount a detectable antibody response by any of the assays tested during follow-up. Conclusions: Longitudinal assessment of antibody response in African COVID-19 patients revealed heterogeneous responses. This underscores the need for a comprehensive evaluation of serum assays before implementation. Factors associated with failure to seroconvert need further research.

Keywords: COVID-19, antibody, rapid diagnostic tests, ethiopia

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Authors: Priyanka Mokashi, Aparna Khanna, Nancy Pandita

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Diabetes mellitus is a chronic metabolic disorder which will be the 7th leading cause of death by 2030. The current line of treatment for the diabetes mellitus is oral antidiabetic drugs (biguanides, sulfonylureas, meglitinides, thiazolidinediones and alpha-glycosidase inhibitors) and insulin therapy depending upon the type 1 or type 2 diabetes mellitus. But, these treatments have their disadvantages, ranging from the developing of resistance to the drugs and adverse effects caused by them. Alternative to these synthetic agents, natural products provides a new insight for the development of more efficient and safe drugs due to their therapeutic values. Enicostema littorale blume (A. Raynal) is a traditional Indian plant belongs to the Gentianaceae family. It is widely distributed in Asia, Africa, and South America. There are few reports on Swrtiamarin, major component of this plant for its antidiabetic activity. However, the antidiabetic activity of flavonoids from E. littorale and their mechanism of action have not yet been elucidated. Flavonoids have a positive relationship with disease prevention and can act on various molecular targets and regulate different signaling pathways in pancreatic β-cells, adipocytes, hepatocytes and skeletal myofibers. They may exert beneficial effects in diabetes by (i) improving hyperglycemia through regulation of glucose metabolism in hepatocytes; (ii) enhancing insulin secretion and reducing apoptosis and promoting proliferation of pancreatic β-cells; (iii) increasing glucose uptake in hepatocytes, skeletal muscle and white adipose tissue (iv) reducing insulin resistance, inflammation and oxidative stress. Therefore, we have isolated four flavonoid rich fractions, Fraction A (FA), Fraction B (FB), Fraction C (FC), Fraction D (FD) from crude alcoholic hot (AH) extract from E. littorale, identified by LC/MS. Total eight flavonoids were identified on the basis of fragmentation pattern. Flavonoid FA showed the presence of swertisin, isovitexin, and saponarin; FB showed genkwanin, quercetin, isovitexin, FC showed apigenin, swertisin, quercetin, 5-O-glucosylswertisin and 5-O-glucosylisoswertisin whereas FD showed the presence of swertisin. Further, these fractions were assessed for their antidiabetic activity on stimulating glucose uptake in insulin-resistant HepG2 cell line model (IR/HepG2). The results showed that FD containing C-glycoside Swertisin has significantly increased the glucose uptake rate of IR/HepG2 cells at the concentration of 10 µg/ml as compared to positive control Metformin (0.5mM) which was determined by glucose oxidase- peroxidase method. It has been reported that enhancement of glucose uptake of cells occurs due the translocation of Glut4 vesicles to cell membrane through IR/IRS1/AKT pathway. Therefore, we have studied expressions of three genes IRS1, AKT and Glut4 by real-time PCR to evaluate whether they follow the same pathway or not. It was seen that the glucose uptake rate has increased in FD treated IR/HepG2 cells due to the activation of insulin receptor substrate-1 (IRS1) followed by protein kinase B (AKT) through phosphoinositide 3-kinase (PI3K) leading to translocation of Glut 4 vesicles to cell membrane, thereby enhancing glucose uptake and insulin sensitivity of insulin resistant HepG2 cells. Hence, the up-regulation indicated the mechanism of action through which FD (Swertisin) acts as antidiabetic candidate in the treatment of type 2 diabetes mellitus.

Keywords: E. littorale, glucose transporter, glucose uptake rate, insulin resistance

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Authors: Rajesh S. Mony, Vaidyaratnam Oushadhasala

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An attempt is made in this study to evolve a drug development modality based on classical Ayurvedic knowledge base as well as on modern scientific methodology. The present study involves (a) identification of a specific ailment condition, (b) the selection of a polyherbal formulation, (c) deciding suitable extraction procedure, (d) confirming the efficacy of the combination by in-vitro trials and (e) fixing up the recommended dose. The ailment segment selected is arthritic condition. The selected herbal combination is Kunturushka, Vibhitaki, Guggulu, Haridra, Maricha and Nirgundi. They were selected as per Classical Ayurvedic references, Authentified as per API (Ayurvedic Pharmacopeia of India), Extraction of each drug was done by different ratios of Hydroalcoholic menstrums, Invitro assessment of each extract after removing residual solvent for anti-Inflammatory, anti-arthritic activities (by UV-Vis. Spectrophotometer with positive control), Invitro assessment of each extract for COX enzyme inhibition (by UV-Vis. Spectrophotometer with positive control), Selection of the extracts was made having good in-vitro activity, Performed the QC testing of each selected extract including HPTLC, that is the in process QC specifications, h. Decision of the single dose with mixtures of selected extracts was made as per the level of in-vitro activity and available toxicology data, Quantification of major groups like Phenolics, Flavonoids, Alkaloids and Bitters was done with both standard Spectrophotometric and Gravimetric methods, Method for Marker assay was developed and validated by HPTLC and a good resolved HPTLC finger print was developed for the single dosage API (Active Pharmaceutical Ingredient mixture of extracts), Three batches was prepared to fix the in process and API (Active Pharmaceutical Ingredient) QC specifications.

Keywords: drug development, antiinflammatory, quality stardardisation, planar chromatography

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Authors: Meliani Samiha, Hassaine Sarah

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Figs are so much consumed in the Mediterranean region; they present a high nutritional value and also multiple therapeutic virtues. Our work contributes to the study of the antidiabetic activity of the common fig of the region of El Amra (AinDefla) Algeria. To do this, 20 Wistar rats female, divided into 4 lots, were used: Lot 1: 5 normal controls; Lot 2: 5 normal controls treated with dry fig juice at 20%; Lot 3: 5 diabetic controls; Lot 4: 5 diabetic controls treated with dry fig juice at 20%. The rats are rendered diabetic by intra-peritoneal injection of a streptozotocin solution. The blood glucose is measured after 1 hour, 2 hours, 3 hours and after 4 hours of the administration of the fig juice; it’s measured also on the 5th day, 8th day and 9th day of the beginning of the experiment. The determination of cholesterol and triglycerides blood is carried out at the beginning and the end of the study. On the 9th day, we recorded a very significant decrease of the blood sugar level of diabetic rats treated with dry fig juice. This blood glucose level normalized for 3 rats/5rats, we also recorded a decrease, but not significant, of cholesterol and triglycerides blood levels. In the short term (for 4 hours), an increase of blood sugar level, one hour after administration, for normal and diabetic rats. This increase is probably due to the high level of sugar content in the preparation. The blood glucose level is then corrected, four hours later. This may be the result of anti hyperglycemic effect of the active ingredients contained in the figs.

Keywords: antidiabetic, figs, hypoglycemia, streptozotocin

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Authors: H. Mayton, X. Yan, A. G. Taylor

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Infested tomato seeds were used to investigate the influence of Xanthomonas euvesicatoria on germination and seed to seedling transmission in a controlled environment and greenhouse assays in an effort to develop effective seed treatments and characterize seed borne transmission of bacterial leaf spot of tomato. Bacterial leaf spot of tomato, caused by four distinct Xanthomonas species, X. euvesicatoria, X. gardneri, X. perforans, and X. vesicatoria, is a serious disease worldwide. In the United States, disease prevention is expensive for commercial growers in warm, humid regions of the country, and crop losses can be devastating. In this study, four different infested tomato seed lots were extracted from tomato fruits infected with bacterial leaf spot from a field in New York State in 2017 that had been inoculated with X. euvesicatoria. In addition, vacuum infiltration at 61 kilopascals for 1, 5, 10, and 15 minutes and seed soaking for 5, 10, 15, and 30 minutes with different bacterial concentrations were used to artificially infest seed in the laboratory. For controlled environment assays, infested tomato seeds from the field and laboratory were placed othe n moistened blue blotter in square plastic boxes (10 cm x 10 cm) and incubated at 20/30 ˚C with an 8/16 hour light cycle, respectively. Infested tomato seeds from the field and laboratory were also planted in small plastic trays in soil (peat-lite medium) and placed in the greenhouse with 24/18 ˚C day and night temperatures, respectively, with a 14-hour photoperiod. Seed germination was assessed after eight days in the laboratory and 14 days in the greenhouse. Polymerase chain reaction (PCR) using the hrpB7 primers (RST65 [5’- GTCGTCGTTACGGCAAGGTGGTG-3’] and RST69 [5’-TCGCCCAGCGTCATCAGGCCATC-3’]) was performed to confirm presence or absence of the bacterial pathogen in seed lots collected from the field and in germinating seedlings in all experiments. For infested seed lots from the field, germination was lowest (84%) in the seed lot with the highest level of bacterial infestation (55%) and ranged from 84-98%. No adverse effect on germination was observed from artificially infested seeds for any bacterial concentration and method of infiltration when compared to a non-infested control. Germination in laboratory assays for artificially infested seeds ranged from 82-100%. In controlled environment assays, 2.5 % were PCR positive for the pathogen, and in the greenhouse assays, no infected seedlings were detected. From these experiments, X. euvesicatoria does not appear to adversely influence germination. The lowest rate of germination from field collected seed may be due to contamination with multiple pathogens and saprophytic organisms as no effect of artificial bacterial seed infestation in the laboratory on germination was observed. No evidence of systemic movement from seed to seedling was observed in the greenhouse assays; however, in the controlled environment assays, some seedlings were PCR positive. Additional experiments are underway with green fluorescent protein-expressing isolates to further characterize seed to seedling transmission of the bacterial leaf spot pathogen in tomato.

Keywords: bacterial leaf spot, seed germination, tomato, Xanthomonas euvesicatoria

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Authors: Ravid Inbar

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CaMKII (calcium-calmodulin dependent protein kinase II) makes up 2% of the protein in our brain and has a critical role in memory formation and long-term potentiation of neurons. Despite this, research has yet to uncover the role of one of the domains on the activation of this kinase. The following proposes to express the protein without the hub domain in E. coli, leaving only the kinase and regulatory segment of the protein. Next, a series of kinase assays will be conducted to elucidate the role the hub domain plays on CaMKII sensitivity to calcium/calmodulin activation. The hub domain may be important for activation; however, it may also be a variety of domains working together to influence protein activation and not the hub alone. Characterization of a protein is critical to the future understanding of the protein's function, as well as for producing pharmacological targets in cases of patients with diseases.

Keywords: CaMKII, hub domain, kinase assays, kinase + reg seg

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Authors: Jer Ping Ooi, Shah Rizal Bin Kasim, Nor Aini Saidin

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The objective of this study was to synthesize nano-sized β-tricalcium phosphate (β-TCP) powder and assess its cytotoxic effects on human osteoblast (hFOB1.19) by using four cytotoxicity assays, namely, lactose dehydrogenase (LDHe), tetrazolium hydroxide (XTT), neutral red (NR), and sulforhodamine B (SRB) assays. β-tricalcium phosphate (β-TCP) is a calcium phosphate compound commonly used as an implant material. To date, bulk-sized β-TCP is reported to be readily tolerated by the osteogenic cells and body based on in vitro, in vivo experiments and clinical studies. However, to what extent of nano-sized β-TCP will react in models as compared to bulk β-TCP is yet to be investigated. Thus, in this project, the cells were treated with nano β-TCP powder within a range of concentrations from 0 to 1000 μg/mL for 24, 48, and 72 h. The cytotoxicity tests showed that loss of cell viability ( > 50%) was high for hFOB1.19 cells in all assays. Cell cycle and apoptosis analysis of hFOB1.19 cells revealed that 50 μg/mL of the compound led to 30.5% of cells being apoptotic after 72 h of incubation, and the percentage was increased to 58.6% when the concentration was increased to 200 μg/mL. When the incubation time was increased from 24 to 72 h, the percentage of apoptotic cells increased from 17.3% to 58.6% when the hFOB1.19 were exposed with 200 μg/mL of nano β-TCP powder. Thus, both concentration and exposure duration affected the cytotoxicity effects of the nano β-TCP powder on hFOB1.19. We hypothesize that these cytotoxic effects on hFOB1.19 are related to the nano-scale size of the β-TCP.

Keywords: β-tricalcium phosphate, hFOB1.19, adipose-derived mesenchymal stem cells, cytotoxicity

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Authors: Xing Lu, Yi-ming Wu, Yan-hong Zhu, Zhen-feng Chen, Hong Liang, Yan Peng

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[Eu(BMQ)2(NO3)3(CH3OH)(H2O)] (1),and [Er(BMQ)2(NO3)3(CH3OH)(H2O)] (2),were synthesized. Compounds 1 and 2 exhibit a certain extent cytotoxicity against Hep G2, Hela 229, MGC80-3 and BEL-7404 cell lines invitro, with IC50 values in the14.51±1.41μM to 52.49±4.01μM range. Compound 1 exhibited significantly enhanced cytotoxicity against MGC80-3 cell line, comparing with free 3-(1H-benzimidazol-2-yl)-6-methyl-2(1H)- quinolinone. The binding abilities of 1 to DNA were stronger than that of 2. Intercalation is the most probable binding mode for both the complexes.

Keywords: quinolinone, Eu(II) complex, Er(III) complex, cytotoxicity.

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Authors: Kasipandi Muniyandi, Parimelazhagan Thangaraj

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Plants are important prospective wealth of a country, combination of local health care information about a specific plant together with data published by several groups of scientists, can help in deciding whether it should be considered acceptable for medicinal use. In the developed countries, too, plant-derived drugs may be of importance. The wide variety of ailments that are being treated with Plectranthus is an indication of the medicinal value of the genus. A number of species are not toxic and so may be taken orally, whilst others are used topically on the skin or as enemas. This study was designed to evaluate the different properties of Plectranthus stocksii and the aerial parts were collected and extracted with petroleum ether, chloroform, ethyl acetate, acetone and methanol by Soxhlet apparatus and finally macerated with hot water. The quantification assays revealed that, leaf methanol extract showed higher total phenolic (415.41 mg GAE/ g extract) and tannin (177.53 mg GAE/ g extract) contents whereas leaf ethyl acetate exhibited higher flavonoids (777.11 mg RE/ g extract) content. The antioxidant efficiency of the extracts was analyzed by various radical scavenging assays. Among the different antioxidant assays, leaf ethyl acetate extract showed higher free radical scavenging activities against DPPH (IC50 = 3.46 µg/mL), ABTS (27417.65 µM TE/ g extract), FRAP (152.17 mM Fe(II)E/ mg extract) NO• radical (21.46%) and Superoxide radical (IC50 = 24.16 µg/mL) assays. All the parts P. stocksii extracts showed significant protection against OH• induced DNA damage at 50 µg concentration. The HPLC analysis of leaf ethyl acetate extract revealed the presence of Quercetin (30.29 µg/mg of extract) was the major compound. Anticancer activity of leaf ethyl acetate extract showed better IC50 values were 48.87 and 36.08 µg/ mL against MCF-7 and Caco-2 respectively. From this study, P. stocksii can act as a potent antioxidant and cytotoxic antimicrobial agent. The scope for drug development from this plant is endless and there is undoubtedly a call for further research in pharmaceutical industries.

Keywords: antioxidant, cytotoxicity, phenolics, plectranthus stocksii

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Authors: Nondumiso Lushozi, Busisani Lembede, Eliton Chivandi

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Consumption of fructose increases the risk of obesity, nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome in children. Zingerone which is found in ginger has antidiabetic and antiobesogenic properties. Therefore, the aim of the study was to investigate the potential of orally administered zingerone to protect growing Sprague-Dawley rats (mimicking growing children) against high-fructose diet-induced metabolic derangements. Forty, 21-day old female Sprague-Dawley rats were randomly allocated and administered the following four treatments for 12 weeks: group I: standard rat chow (SR) + plain water (PW) + plain gelatine cube (PC). group II: SR + 20% (w/v) fructose solution (FS) + PC. group III: SR + FS + 100 mg/kg/day of fenofibrate in gelatine cube. group IV: SR+ FS + 20 mg/kg/day of zingerone in gelatine cube. The rats’ triglyceride, cholesterol, insulin & adiponectin concentration, visceral fat liver lipid content, homoeostasis model assessment of insulin resistance (HOMA-IR) and ability to handle glucose were determined. Oral administration of zingerone significantly increased (P<0.001) visceral fat and liver lipid content (P<0.001), respectively. Results from the study revealed that administration of 20% fructose solution did not induce metabolic dysfunction, however the zingerone treatment increased visceral fat and liver lipid content, all these lipid abnormalities are typical features of the metabolic syndrome, therefore the current study suggests that zingerone has no effect on metabolic dysfunction in adolescent females.

Keywords: antidiabetic, metabolic syndrome, zingerone, antiobesogenic

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Authors: Qi LI, Xu Lin, Nengming Lin

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Objective: The aim of this study was to investigate the role of desmocollin 2 (DSC2) protein in the proliferation, migration and drug resistance of lung cancer cells. Method: CCK-8 assays and colony formation assays were used to evaluate the effect of dsc2 regulation on cancer cell viability and colony formation. Transwell assays and wound healing assays were also performed. Cell flow double staining was used to detect the apoptosis rate of cells with DSC2, which was added cisplatin. Western blot assay was used to detect cell cycle, PI3k/Akt and apoptosis-related proteins. Results: Our data showed that dsc2 is upregulated in clinical lung cancer tissues compared with pericarcinomatous tissues, and it is differentially expressed in lung cancer cell lines. The down-regulation of dsc2 in A549 and H358 lung cancer cells significantly suppressed the cell proliferation, metastasis, and motility. In contrast, the opposite effects were observed in overexpression of dsc2 both in H23 and PC9 cell lines. In addition to lung adenocarcinoma cell lines, we also examined its expression in lung squamous cell lines, such as H226. Western blotting showed that dsc2 could reduce the level of phosphorylated Akt (Ser 473) and p-mTOR. Thus, it is speculated that dsc2 up-regulation promotes proliferation and invasiveness through activation of the PI3K/AKT pathway. Also, knockdown of dsc2 in A549 and H226 could significantly decreased in the levels of cyclinB and wee1 protein. Additionally, flow cytometry showed that dsc2 knockdown combined with cisplatin could significantly enhance cell apoptosis rate. Conclusion: These data suggest that dsc2 promotes the proliferation and migration of lung cancer cells in vitro. Also, the results suggested that dsc2 could affect the cell cycle and apoptosis of lung cells. Furthermore, knockdown of dsc2 could sensitize cisplatin in both lung adenocarcinoma and lung squamous cell lines. Thus we suggested that dsc2 can be used as a therapeutic target for lung cancer.

Keywords: desmocollin 2, cisplatin, lung cancer, PI3K/AKT, lung squamous cell

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Authors: Siobhan O’Shea, Sangeetha Vijaysri Nair, Hee Cheol Kim, Charles Thomas Nugent, Cheuk Yan William Tong, Sam Douthwaite, Andrew Worlock

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The Aptima® HIV-1 Quant Dx Assay is a fully automated assay on the Panther system. It is based on Transcription-Mediated Amplification and real time detection technologies. This assay is intended for monitoring HIV-1 viral load in plasma specimens and for the detection of HIV-1 in plasma and serum specimens. Nine-hundred and seventy nine specimens selected at random from routine testing at St Thomas’ Hospital, London were anonymised and used to compare the performance of the Aptima HIV-1 Quant Dx assay and Roche COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, v2.0. Two-hundred and thirty four specimens gave quantitative HIV-1 viral load results in both assays. The quantitative results reported by the Aptima Assay were comparable those reported by the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 with a linear regression slope of 1.04 and an intercept on -0.097. The Aptima assay detected HIV-1 in more samples than the Roche assay. This was not due to lack of specificity of the Aptima assay because this assay gave 99.83% specificity on testing plasma specimens from 600 HIV-1 negative individuals. To understand the reason for this higher detection rate a side-by-side comparison of low level panels made from the HIV-1 3rd international standard (NIBSC10/152) and clinical samples of various subtypes were tested in both assays. The Aptima assay was more sensitive than the Roche assay. The good sensitivity, specificity and agreement with other commercial assays make the HIV-1 Quant Dx Assay appropriate for both viral load monitoring and detection of HIV-1 infections.

Keywords: HIV viral load, Aptima, Roche, Panther system

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Authors: Vanessa Roulet, Marc Turini, Juliane Hey, Stéphanie Bord-Romeu, Emilie Bonzom, Mahmoud Badawi, Mohammed-Amine Chakir, Valérie Simon, Vanessa Viotti, Jérémie Gautier, Françoise Le Boulaire, Catherine Coignard, Claire Vincent, Sandrine Greaume, Isabelle Voisin

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Background: Beckman Coulter, Inc. has recently developed fully automated assays for the detection of HBsAg on a new immunoassay platform. The objective of this European multicenter study was to evaluate the performance of the ACCESS HBsAg and ACCESS HBsAg Confirmatory assays† on the recently CE-marked DxI 9000 ACCESS Immunoassay Analyzer. Methods: The clinical specificity of the ACCESS HBsAg and HBsAg Confirmatory assays was determined using HBsAg-negative samples from blood donors and hospitalized patients. The clinical sensitivity was determined using presumed HBsAg-positive samples. Sample HBsAg status was determined using a CE-marked HBsAg assay (Abbott ARCHITECT HBsAg Qualitative II, Roche Elecsys HBsAg II, or Abbott PRISM HBsAg assay) and a CE-marked HBsAg confirmatory assay (Abbott ARCHITECT HBsAg Qualitative II Confirmatory or Abbott PRISM HBsAg Confirmatory assay) according to manufacturer package inserts and pre-determined testing algorithms. False initial reactive rate was determined on fresh hospitalized patient samples. The sensitivity for the early detection of HBV infection was assessed internally on thirty (30) seroconversion panels. Results: Clinical specificity was 99.95% (95% CI, 99.86 – 99.99%) on 6047 blood donors and 99.71% (95%CI, 99.15 – 99.94%) on 1023 hospitalized patient samples. A total of six (6) samples were found false positive with the ACCESS HBsAg assay. None were confirmed for the presence of HBsAg with the ACCESS HBsAg Confirmatory assay. Clinical sensitivity on 455 HBsAg-positive samples was 100.00% (95% CI, 99.19 – 100.00%) for the ACCESS HBsAg assay alone and for the ACCESS HBsAg Confirmatory assay. The false initial reactive rate on 821 fresh hospitalized patient samples was 0.24% (95% CI, 0.03 – 0.87%). Results obtained on 30 seroconversion panels demonstrated that the ACCESS HBsAg assay had equivalent sensitivity performances compared to the Abbott ARCHITECT HBsAg Qualitative II assay with an average bleed difference since first reactive bleed of 0.13. All bleeds found reactive in ACCESS HBsAg assay were confirmed in ACCESS HBsAg Confirmatory assay. Conclusion: The newly developed ACCESS HBsAg and ACCESS HBsAg Confirmatory assays from Beckman Coulter have demonstrated high clinical sensitivity and specificity, equivalent to currently marketed HBsAg assays, as well as a low false initial reactive rate. †Pending achievement of CE compliance; not yet available for in vitro diagnostic use. 2023-11317 Beckman Coulter and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. All other trademarks are the property of their respective owners.

Keywords: dxi 9000 access immunoassay analyzer, hbsag, hbv, hepatitis b surface antigen, hepatitis b virus, immunoassay

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Authors: Bao-Fang Wen, Meng-Na Dai, Gao-Pei Zhu, Chen-Xi Zhang, Jing Sun, Xun-Bao Yin, Yu-Han Zhao, Hong-Wei Sun, Wei-Fen Zhang

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A drug-loaded nanoparticles containing berberine hydrochloride (BH/FA-CTS-NPs) was prepared. The physicochemical characterizations of BH/FA-CTS-NPs and the inhibitory effect on the HeLa cells were investigated. Folic acid-conjugated chitosan (FA-CTS) was prepared by amino reaction of folic acid active ester and chitosan molecules; BH/FA-CTS-NPs were prepared using ionic cross-linking technique with BH as a model drug. The morphology and particle size were determined by Transmission Electron Microscope (TEM). The average diameters and polydispersity index (PDI) were evaluated by Dynamic Light Scattering (DLS). The interaction between various components and the nanocomplex were characterized by Fourier Transform Infrared Spectroscopy (FT-IR). The entrapment efficiency (EE), drug-loading (DL) and in vitro release were studied by UV spectrophotometer. The effect of cell anti-migratory and anti-invasive actions of BH/FA-CTS-NPs were investigated using MTT assays, wound healing assays, Annexin-V-FITC single staining assays, and flow cytometry, respectively. HeLa nude mice subcutaneously transplanted tumor model was established and treated with different drugs to observe the effect of BH/FA-CTS-NPs in vivo on HeLa bearing tumor. The BH/FA-CTS-NPs prepared in this experiment have a regular shape, uniform particle size, and no aggregation phenomenon. The results of DLS showed that mean particle size, PDI and Zeta potential of BH/FA-CTS NPs were (249.2 ± 3.6) nm, 0.129 ± 0.09, 33.6 ± 2.09, respectively, and the average diameter and PDI were stable in 90 days. The results of FT-IR demonstrated that the characteristic peaks of FA-CTS and BH/FA-CTS-NPs confirmed that FA-CTS cross-linked successfully and BH was encapsulated in NPs. The EE and DL amount were (79.3 ± 3.12) % and (7.24 ± 1.41) %, respectively. The results of in vitro release study indicated that the cumulative release of BH/FA-CTS NPs was (89.48±2.81) % in phosphate-buffered saline (PBS, pH 7.4) within 48h; these results by MTT assays and wund healing assays indicated that BH/FA-CTS NPs not only inhibited the proliferation of HeLa cells in a concentration and time-dependent manner but can induce apoptosis as well. The subcutaneous xenograft tumor formation rate of human cervical cancer cell line HeLa in nude mice was 98% after inoculation for 2 weeks. Compared with BH group and BH/CTS-NPs group, the xenograft tumor growth of BH/FA-CTS-NPs group was obviously slower; the result indicated that BH/FA-CTS-NPs could significantly inhibit the growth of HeLa xenograft tumor. BH/FA-CTS NPs with the sustained release effect could be prepared successfully by the ionic crosslinking method. Considering these properties, block proliferation and impairing the migration of the HeLa cell line, BH/FA-CTS NPs could be an important compound for consideration in the treatment of cervical cancer.

Keywords: folic-acid, chitosan, berberine hydrochloride, nanoparticles, cervical cancer

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Authors: M. A. Munteanu, A. M. Lungu, A. I. Chivescu, V. Teodorescu, E. Tufanoiu, C. Nicolae, T. I. Nanea

Abstract:

Introduction: Sodium-glucose co-transporter 2 inhibitors (SGLT2i), which were first developed as antidiabetic medications, have demonstrated numerous positive benefits on the cardiovascular system, especially in the prevention of heart failure (HF). HF is a challenging, multifaceted disease that needs all-encompassing therapy. It should not be viewed as a limited form of heart illness but rather as a systemic disease that leads to multiple organ failure and death. SGLT2i is an extremely effective tool for treating HF by using its pleiotropic effects. In addition to its use in patients with diabetes mellitus who are at high cardiovascular risk or who have already experienced a cardiovascular event, SGLT2i administration has been shown to have positive effects on a variety of HF manifestations and stages, regardless of the patient's presence of diabetes mellitus. Material and Methods: According to the guide, 110 patients (83 males and 27 females) with heart failure with mildly reduced ejection fraction (HFmrEF), with T2D and neoplasia, were enrolled in the prospective study. The structural and functional state of the left ventricle myocardium and ejection fraction was assessed through echocardiography. Patients were randomized to receive once-daily dapagliflozin 10 mg. Results: Patients with HFmrEF were divided into 3 subgroups according to age. 7% (8) patients aged < 45 years, 35% (28) patients aged between 46-59 years, and 58% (74) patients aged> 60 years. The most prevalent comorbidities were hypertension (43.1%), coronary heart disease (40%), and obesity (33.2%). Study drug discontinuation and serious adverse events were not frequent in the subgroups, in either men or women, until now. Conclusions: SGLT-2 inhibitors are a novel class of antidiabetic agents that have demonstrated positive efficacy and safety outcomes in the setting of HFmrEF. Until now, in our study, dapagliflozin was safe and well-tolerated irrespective of sex.

Keywords: diabetes mellitus type 2, Sodium-glucose co-transporters-2 inhibitors, heart failure, neoplasia

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Authors: P. Gayathri, G. P. Jeyanthi

Abstract:

The bark of Polyalthia longifolia is used in ayurvedic system of medicine for the manangement of various ailments including diabetes mellitus. The bark of P. longifolia extracts was extracted using various polar and non-polar solvents and tested for inhibition of alpha-amylase and alpha-glucosidase among which the ethanolic extracts were found to be more potent. The ethanolic extracts of the bark were tested for the in vitro inhibition of alpha-amylase using starch as substrate and alpha-glucosidase using p-nitro phenyl alpha-D-gluco pyranoside as substrate to establish its in vitro antidiabetic effect. The mechanism of inhibition was determined by Dixon plot and Cornish-Bowden plot. The cytotoxic effect of the extract was tested on L6 and Vero cell-lines. The extract was partially purified by TLC. The individual effect of the ethanolic extract, TLC fractions and its combinatorial effect with insulin and glibenclamide on glucose uptake by rat hemi-diaphragm were studied.Results revealed that the ethanolic extracts of Polyalthia longifolia bark exhibited competitive inhibition of alpha-amylase and alpha-glucosidase. The extracts were also found not to be cytotoxic at the highest dose of 1 mg/mL. Glucose uptake study revealed that the extract alone and when combined with insulin, decreased the glucose uptake when compared to insulin control, however the purified TLC fractions exhibited significantly higher (p<0.05) glucose uptake by the rat hemi-diaphragm when compared to insulin. The study shows various possible mechanism of in vitro antidiabetic effect of the P. longifolia bark.

Keywords: alpha-amylase, alpha-glucosidase, dixon, cornish-bowden, L6 , Vero cell-lines, glucose uptake, polyalthia longifolia bark, ethanolic extract, TLC fractions

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Authors: Aditya Arya, Hairin Taha, Ataul Karim Khan, Nayiar Shahid, Hapipah Mohd Ali, Mustafa Ali Mohd

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This study has investigated the antidiabetic and antioxidant potential of Pseudovaria macrophylla bark extract on streptozotocin–nicotinamide induced type 2 diabetic rats. LCMS-QTOF and NMR experiments were done to determine the chemical composition in the methanolic bark extract. For in vivo experiments, the STZ (60 mg/kg/b.w, 15 min after 120 mg/kg/1 nicotinamide, i.p.) induced diabetic rats were treated with methanolic extract of Pseuduvaria macrophylla (200 and 400 mg/kg∙bw) and glibenclamide (2.5 mg/kg) as positive control respectively. Biochemical parameters were assayed in the blood samples of all groups of rats. The pro-inflammatory cytokines, antioxidant status and plasma transforming growth factor βeta-1 (TGF-β1) were evaluated. The histological study of the pancreas was examined and its expression level of insulin was observed by immunohistochemistry. In addition, the expression of glucose transporters (GLUT 1, 2 and 4) were assessed in pancreas tissue by western blot analysis. The outcomes of the study displayed that the bark methanol extract of Pseuduvaria macrophylla has potentially normalized the elevated blood glucose levels and improved serum insulin and C-peptide levels with significant increase in the antioxidant enzyme, reduced glutathione (GSH) and decrease in the level of lipid peroxidation (LPO). Additionally, the extract has markedly decreased the levels of serum pro-inflammatory cytokines and transforming growth factor beta-1 (TGF-β1). Histopathology analysis demonstrated that Pseuduvaria macrophylla has the potential to protect the pancreas of diabetic rats against peroxidation damage by downregulating oxidative stress and elevated hyperglycaemia. Furthermore, the expression of insulin protein, GLUT-1, GLUT-2 and GLUT-4 in pancreatic cells was enhanced. The findings of this study support the anti-diabetic claims of Pseudovaria macrophylla bark.

Keywords: diabetes mellitus, Pseuduvaria macrophylla, alkaloids, caffeic acid

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Authors: Tawqeer Ali Syed, Prakash Chandra

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This study aims to evaluate the antileishmanial activity of Nigella sativa black seed extract. This well-known plant extract was taken from the botanical garden of Kashmir. Materials and Methods: The methanolic extracts of these plants were screened for their antileishmanial activity against Leishmania major using 3‑(4.5‑dimethylthiazol‑2yl)‑2.5‑diphenyltetrazolium bromide assay or MTT assay. Results: The methanolic extract of Nigella sativa showed potential antileishmanial activity at an inhibition% value of 80.29% ± 0.65%. IC 50 was calculated after 48 hours to be 964.3 µg/ml. Conclusion: Considering these results, these medicinal plants from Kashmir could serve as potential drug sources for antileishmanial compounds.

Keywords: MTT assay, antileishmanial, cell viability, Nigella sativa

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Authors: Nichole Haag, Kimberly Velk, Tyler McCune, Chun Wu

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Methicillin/multiple-resistant Staphylococcus aureus (MRSA) are infectious bacteria that are resistant to common antibiotics. A previous in silico study in our group has identified a hypothetical protein SAV1226 as one of the potential drug targets. In this study, we reported the bioinformatics characterization, as well as cloning, expression, purification and kinetic assays of hypothetical protein SAV1226 from methicillin/vancomycin-resistant Staphylococcus aureus Mu50 strain. MALDI-TOF/MS analysis revealed a low degree of structural similarity with known proteins. Kinetic assays demonstrated that hypothetical protein SAV1226 is neither a domain of an ATP dependent dihydroxyacetone kinase nor of a phosphotransferase system (PTS) dihydroxyacetone kinase, suggesting that the function of hypothetical protein SAV1226 might be misannotated on public databases such as UniProt and InterProScan 5.

Keywords: Methicillin-resistant Staphylococcus aureus, dihydroxyacetone kinase, essential genes, drug target, phosphoryl group donor

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Authors: Md. Mizanur Rahman, Anquan Liu, Anna Frostegård, Johan Frostegård

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The role of the human immune system is essential in cardiovascular diseases and atherosclerosis. Activated cells in atherosclerosis produce abundant amounts of cytokines, but the exact mechanisms involved in the effects of these inflammatory cytokines are not clear in atherosclerosis. In a large clinical cohort, we have previously determined that antibodies against phosphorylcholine (anti-PC) are negatively and independently associated with both development of atherosclerosis and also a low risk of cardiovascular disease. Further, we reported that rheumatoid arthritis patients who were non-responders to TNF-inhibitors, where those with low anti-PC levels. Upon anti-TNF treatment, anti-PC levels increased. We, therefore, hypothesised that proinflammatory cytokines such as TNF could play a role in anti-PC regulation. Peripheral blood mononuclear cells (PBMC) were cultured with or without TNF and anti-TNF. The cell supernatants were collected after six days for ELISA measurements. In separate experiments, cells were cultured for 24 hours in both polystyrene plates and ELISPOT plates under a similar condition for ELISA and ELISPOT assays respectively. Total RNA was extracted after 6 hours of cell culture to perform RT-qPCR. Cell viability was confirmed by trypan blue staining and MTT assays. ELISA measurements detected less than 40% of anti-PC in TNF-treated cells, in comparison to control cells, whereas anti-PC production was recovered by anti-TNF treatment. ELISPOT assays showed that TNF suppresses anti-PC production by inhibiting anti-PC producing B-cells. In addition, RT-qPCR and ELISA showed that TNF also has effects also on B-cell activation as BAFF expression was inhibited by TNF treatment. Atherosclerosis is a major cause of cardiovascular diseases, but anti-PC is a protection marker for atherosclerosis development. Our findings show that TNF is a negative regulator of anti-PC production. Immune modulation and rising of anti-PC could be of major significance for the patients.

Keywords: anti-PC, Anti-TNF, atherosclerosis, cardiovascular diseases, phosphorylecholine

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Authors: Rutanachai Thaipratum

Abstract:

At present, it is widely-known that free radicals are the causes of illness such as cancers, coronary heart disease, Alzheimer’s disease and aging. One method of protection from free radical is the consumption of antioxidant-containing foods or herbs. Several analytical methods have been used for qualitative and quantitative determination of antioxidants. This project aimed to evaluate antioxidant activity of ethanolic and aqueous extracts from cabbage (Brassicca oleracea L. var. capitata L.) measured by DPPH and hydroxyl radical scavenging method. The results show that averaged antioxidant activity measured in ethanolic extract (µmol ascorbic acid equivalent/g fresh mass) were 7.316 ± 0.715 and 4.66 ± 1.029 as determined by DPPH and hydroxyl radical scavenging activity assays, respectively. Averaged antioxidant activity measured in aqueous extract (µmol ascorbic acid equivalent/g fresh mass) were 15.141 ± 2.092 and 4.955 ± 1.975 as determined by DPPH and hydroxyl radical scavenging activity assays respectively.

Keywords: free radical, antioxidant, cabbage, Brassica oleracea L. var. capitata L.

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Authors: Dubravka Štajner, Boris M. Popović, Saša Orlović, Ružica Ždero, Milan Popović, Aleksandra Popović

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Many plants possess antioxidant ingredients that provides efficacy by additive or synergistic activities. Present article highlights an antioxidant capacity of Serbian conifer plants. Antioxidant activities of the crude extracts were assessed using different assays. In this study, quantities of phenolic compounds (total phenols, flavonoids, tannins and proanthocyanidins), contents of photosynthetic pigments (chlorophyll a and b and carotenoids), soluble proteins and proline were examined. MDA quantities and ability of extracts to remove reactive nitrogen and oxygen species (RNOS) were also investigated. Furthermore, antioxidant activities of extracts against DPPH∙, ferric reducing antioxidant power, permanganate reducing antioxidant capacity were also determined. According to almost all used assays, antioxidant and scavenging capacities of silver fir (Abies alba Mill.), and Douglas fir (Pseudotsuga menziesii) were superior compared to spruce. Presented results implicated that leaves of Douglas fir and silver fir possessed outstanding antioxidant characteristics that could diminish damage caused by oxygen radicals which are responsible for many of the bodily changes and susceptibility to different diseases.

Keywords: conifers, antioxidant activity, reducing power, lipid peroxidation

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Authors: Luís R. Silva, Ana C. Gonçalves, Catarina Bento, Fábio Jesus, Branca M. Silva

Abstract:

Prunus avium Linnaeus, more known as sweet cherry, is one of the most appreciated fruit worldwide. Most of these quantities are produced in Fundão region, being Saco the cultivar most produced. Saco is very rich in bioactive compounds, especially phenolics, and presents great antioxidant capacity. The purpose of the present study was to investigate the chemical profile and biological potential, concerning antioxidant, anti-diabetic activity and protective effects towards erythrocytes by Saco sweet cherry collected from Fundão region (Portugal). The hydroethanolic extracts were prepared and passed through a C18 solid-phase extraction column. The phenolic profile analyzed by LC-DAD method allowed to the identification of 22 phenolic compounds, being 16 non-phenolics and 6 anthocyanins. In respect to non-coloured phenolics, 3-O-caffeoylquinic and ρ-coumaroylquinic acids were the main ones. Concerning to anthocyanins, cyanidin-3-O-rutinoside was found in higher amounts. Relatively to biological potential, Saco showed great antioxidant potential, through DPPH and NO radical assays, with IC50 =16.24 ± 0.46 µg/mL and IC50 = 176.69 ± 3.35 µg/mL for DPPH and NO, respectively. These results were similar to those obtained for ascorbic acid control (IC50 = 16.92 ± 0.69 and IC50 = 162.66 ± 1.31 μg/mL for DPPH and NO, respectively). In respect to antidiabetic potential, Saco revealed capacity to inhibit α-glucosidase in a dose-dependent manner (IC50 = 10.79 ± 0.40 µg/mL), being much active than positive control acarbose (IC50 = 306.66 ± 0.84 μg/mL). Additionally, Saco extracts revealed protective effects against ROO•-mediated toxicity generated by AAPH in human blood erythrocytes, inhibiting hemoglobin oxidation (IC50 = 38.57 ± 0.96 μg/mL) and hemolysis (IC50 = 73.03 ± 1.48 μg/mL), in a concentration-dependent manner. However, Saco extracts were less effective than quercetin control (IC50 = 3.10 μg/mL and IC50 = 0.7 μg/mL for inhibition of hemoglobin oxidation and hemolysis, respectively). The results obtained showed that Saco is an excellent source of phenolic compounds. These ones are natural antioxidant substances, which easily capture reactive species. This work presents new insights regarding sweet cherry antioxidant properties which may be useful for the future development of new therapeutic strategies for preventing or attenuating oxidative-related disorders.

Keywords: antioxidant capacity, health benefits, phenolic compounds, saco

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Authors: Neera Kapoor

Abstract:

Background: Knockdown Resistance (KDR) is one of the mechanisms of insecticide resistance in insects caused by the reduced target site sensitivity i.e. voltage gated sodium channel (VGSC) rendering it less sensitive to the toxic effects of DDT and pyrethroids. In this study, we evaluated insecticide susceptibility and its underlying KDR mechanism in eight Ae. aegypti and five Ae. albopictus field populations. Methodology: Field population was collected from four different geographical regions of India covering 18 districts of ten states. For genotyping of twelve KDR alleles in Ae. aegypti field populations, three PCR based assays were used; with DNA sequencing; ASPCR; PCR-RFLP. Genomic DNA was isolated, and three partial domains (II, III, and IV) of VGSC were amplified and sequenced. Results: Molecular screening for common KDR mutations, revealed the presence of five mutations viz. S989P, V1016G, T1520I, F1534C/L. Two novel mutations were observed, first at T1520 (ACC) residue where a C > T substitution at the second position of codon results in amino acid change to Isoleucine (ATC). Second mutation was an alternative point mutation at F1534 (TTC) residue where a substitution of T > C at the first position of codon results in an amino acid change to Leucine (CTC). ASPCRs were not accurate, so three PCR-RFLP assays were developed for genotyping of five KDR alleles in Ae. aegypti; viz. T1520I, F1534C/L. Representative samples of all genotypes (n=200) were sequenced to validate the newly developed PCR based assays for Ae. aegypti. Genotyping results showed that 989P is linked to 1016G and novel mutation 1520I was always found with 1534C allele. Conclusion: Present study confirmed the presence of DDT and pyrethroid resistance among Ae. aegypti populations in India and for the first time reported KDR mutations in this species from India including two novel mutations. Results of present study lead us to infer that, at least five KDR mutations (S989P, V1016G, T1530I, F1534C, and F1534L) can be seen as a potential marker for DDT/pyrethroid resistance.

Keywords: F1534C, F1534L, S989P, T1530I, V1016G

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Authors: Cristian Soitu, Alexander Feuerborn, Cyril Deroy, Alfonso Castrejon-Pita, Peter R. Cook, Edmond J. Walsh

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Microfluidics now stands as an academically mature technology after a quarter of a century research activities have delivered a vast array of proof of concepts for many biological workflows. However, translation to industry remains poor, with only a handful of notable exceptions – e.g. digital PCR, DNA sequencing – mainly because of biocompatibility issues, limited range of readouts supported or complex operation required. This technology exploits the domination of interfacial forces over gravitational ones at the microscale, replacing solid walls with fluid ones as building blocks for cell micro-environments. By employing only materials used by biologists for decades, the system is shown to be biocompatible, and easy to manufacture and operate. The method consists in displacing a continuous fluid layer into a pattern of isolated chambers overlaid with an immiscible liquid to prevent evaporation. The resulting fluid arrangements can be arrays of micro-chambers with rectangular footprint, which use the maximum surface area available, or structures with irregular patterns. Pliant, self-healing fluid walls confine volumes as small as 1 nl. Such fluidic structures can be reconfigured during the assays, giving the platform an unprecedented level of flexibility. Common workflows in cell biology are demonstrated – e.g. cell growth and retrieval, cloning, cryopreservation, fixation and immunolabeling, CRISPR-Cas9 gene editing, and proof-of-concept drug tests. This fluid-shaping technology is shown to have potential for high-throughput cell- and organism-based assays. The ability to make and reconfigure on-demand microfluidic circuits on standard Petri dishes should find many applications in biology, and yield more relevant phenotypic and genotypic responses when compared to standard microfluidic assays.

Keywords: fluid walls, micro-chambers, reconfigurable, freestyle

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Authors: Olaniyi Fawole, Umezuruike Opara

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Co-products obtained from pomegranate juice processing contain high levels of polyphenols with potential high added values. From value-addition viewpoint, the aim of this study was to evaluate the stability of polyphenolic concentrations in pomegranate fruit co-products in different solvent extracts and assess the effect on the total antioxidant capacity using the FRAP, DPPH˙ and ABTS˙+ assays during simulated in vitro digestion. Pomegranate juice, marc and peel were extracted in water, 50% ethanol (50%EtOH) and absolute ethanol (100%EtOH) and analysed for total phenolic concentration (TPC), total flavonoids concentration (TFC) and total antioxidant capacity in DPPH˙, ABST˙+ and FRAP assays before and after in vitro digestion. Total phenolic concentration (TPC) and total flavonoid concentration (TFC) were in the order of peel > marc > juice throughout the in vitro digestion irrespective of the extraction solvents used. However, 50% ethanol extracted 1.1 to 12-fold more polyphenols than water and ethanol solvents depending on co-products. TPC and TFC increased significantly in gastric digests. In contrast, after the duodenal, polyphenolic concentrations decreased significantly (p < 0.05) compared to those obtained in gastric digests. Undigested samples and gastric digests showed strong and positive relationships between polyphenols and the antioxidant activities measured in DPPH, ABTS and FRAP assays, with correlation coefficients (r2) ranging between 0.930 – 0.990 whereas, the correlation between polyphenols (TPC and TFC) and radical cation scavenging activity (in ABTS) were moderately positive in duodenal digests. Findings from this study also showed that the concentration of pomegranate polyphenols and antioxidant thereof during in vitro gastro-intestinal digestion may not reflect the pre-digested phenolic concentration. Thus, this study highlights the need to provide biologically relevant information on antioxidants by providing data reflecting their stability and activity after in vitro digestion.

Keywords: by-product, DPPH, polyphenols, value addition

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Authors: Prasanna Ramachandran, Kareem Graham, Helena Kiefel, Sunit Jain, Todd DeSantis

Abstract:

Microbes that reside inside the mammalian GI tract, commonly referred to as the gut microbiome, have been shown to have therapeutic effects in animal models of disease. We hypothesize that specific proteins produced by these microbes are responsible for this activity and may be used directly as therapeutics. To speed up the discovery of these key proteins from the big-data metagenomics, we have applied machine learning techniques. Using amino acid sequences of known epitopes and their corresponding binding partners, protein interaction descriptors (PID) were calculated, making a positive interaction set. A negative interaction dataset was calculated using sequences of proteins known not to interact with these same binding partners. Using Random Forest and positive and negative PID, a machine learning model was trained and used to predict interacting versus non-interacting proteins. Furthermore, the continuous variable, cosine similarity in the interaction descriptors was used to rank bacterial therapeutic candidates. Laboratory binding assays were conducted to test the candidates for their potential as therapeutics. Results from binding assays reveal the accuracy of the machine learning prediction and are subsequently used to further improve the model.

Keywords: protein-interactions, machine-learning, metagenomics, microbiome

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