Abstract: 5-FU is a chemotherapeutic agent that has been used in colorectal cancer (CRC) treatment. However, it is usually associated with the acquired resistance, which decreases the therapeutic effects of 5-FU. miR-200c is involved in chemotherapeutic drug resistance, but its mechanism is not fully understood. In this study, the effect of inhibition of miR-200c in sensitivity of HCT-116 CRC cells to 5-FU was evaluated. HCT-116 cells were transfected with LNA-anti- miR-200c for 48 h. mRNA expression of miR-200c was evaluated using quantitative real- time PCR. The protein expression of phosphatase and tensin homolog (PTEN) and E-cadherin were analyzed by western blotting. Annexin V and propidium iodide staining assay were applied for apoptosis detection. The caspase-3 activation was evaluated by an enzymatic assay. The results showed LNA-anti-miR-200c inhibited the expression of PTEN and E-cadherin protein, apoptosis and activation of caspase 3 compared with control cells. In conclusion, these results suggest that miR-200c as a prognostic marker can overcome to 5-FU chemoresistance in CRC.
Abstract: Production of specific antibody responses against hTSH is a cumbersome process due to the high identity between the hTSH and the other members of the glycoprotein hormone family (FSH, LH and HCG) and the high identity between the human hTSH and host animals for antibody production. Therefore, two polyclonal antibodies were purified against two recombinant proteins. Four possible ELISA tests were designed based on these antibodies. These ELISA tests were checked against hTSH and other glycoprotein hormones, and their sensitivity and specificity were assessed. Bioinformatics tools were used to analyze the immunological properties. After the immunogen region selection from hTSH protein, c terminal of B hTSH was selected and applied. Two recombinant genes, with these cut pieces (first: two repeats of C terminal of B hTSH, second: tetanous toxin+B hTSH C terminal), were designed and sub-cloned into the pET32a expression vector. Standard methods were used for protein expression, purification, and verification. Thereafter, immunizations of the white New Zealand rabbits were performed and the serums of them were used for antibody titration, purification and characterization. Then, four ELISA tests based on two antibodies were employed to assess the hTSH and other glycoprotein hormones. The results of these assessments were compared with standard amounts. The obtained results indicated that the desired antigens were successfully designed, sub-cloned, expressed, confirmed and used for in vivo immunization. The raised antibodies were capable of specific and sensitive hTSH detection, while the cross reactivity with the other members of the glycoprotein hormone family was minimum. Among the four designed tests, the test in which the antibody against first protein was used as capture antibody, and the antibody against second protein was used as detector antibody did not show any hook effect up to 50 miu/l. Both proteins have the ability to induce highly sensitive and specific antibody responses against the hTSH. One of the antibody combinations of these antibodies has the highest sensitivity and specificity in hTSH detection.
Abstract: Radiation therapy is an effective vital strategy used globally in the treatment of cervical cancer. However, radiation efficacy principally depends on the radiosensitivity of the tumor, and not all patient exhibit significant response to irradiation. A radiosensitive tumor is easier to cure than a radioresistant tumor which later advances to local recurrence and metastasis. Herbal polyphenols are gaining attention for exhibiting radiosensitization through various signaling. Current work focuses to study the radiosensitization effect of ellagic acid (EA), on HeLa cells. EA intermediated radiosensitization of HeLa cells was due to the induction γ-H2AX foci formation, G1 phase cell cycle arrest, and loss of reproductive potential, growth inhibition, drop in the mitochondrial membrane potential and protein expression studies that eventually induced apoptosis. Irradiation of HeLa in presence of EA (10 μM) to doses of 2 and 4 Gy γ-radiation produced marked tumor cytotoxicity. EA also demonstrated radio-protective effect on normal cell, NIH3T3 and aided recovery from the radiation damage. Our results advocate EA to be an effective adjuvant for improving cancer radiotherapy as it displays striking tumor cytotoxicity and reduced normal cell damage instigated by irradiation.
Abstract: Agriculture is the backbone of economy of Pakistan
and cotton is the major agricultural export and supreme source of raw
fiber for our textile industry. To combat severe problems of insect
and weed, combination of three genes namely Cry1Ac, Cry2A and
EPSPS genes was transferred in locally cultivated cotton variety
MNH-786 with the use of Agrobacterium mediated genetic
transformation. The present study focused on the molecular screening
of transgenic cotton plants at T3 generation in order to confirm
integration and expression of all three genes (Cry1Ac, Cry2A and
EPSP synthase) into the cotton genome. Initially, glyphosate spray
assay was used for screening of transgenic cotton plants containing
EPSP synthase gene at T3 generation. Transgenic cotton plants which
were healthy and showed no damage on leaves were selected after 07
days of spray. For molecular analysis of transgenic cotton plants in
the laboratory, the genomic DNA of these transgenic cotton plants
were isolated and subjected to amplification of the three genes. Thus,
seventeen out of twenty (Cry1Ac gene), ten out of twenty (Cry2A
gene) and all twenty (EPSP synthase gene) were produced positive
amplification. On the base of PCR amplification, ten transgenic plant
samples were subjected to protein expression analysis through
ELISA. The results showed that eight out of ten plants were actively
expressing the three transgenes. Real-time PCR was also done to
quantify the mRNA expression levels of Cry1Ac and EPSP synthase
gene. Finally, eight plants were confirmed for the presence and active
expression of all three genes at T3 generation.
Abstract: Biopharmaceuticals manufacturing is one of the major economic activities worldwide. Ninety-three percent of the workforce in a biomanufacturing environment concentrates in production-related areas. As a result, strategic collaborations between industry and academia are crucial to ensure the availability of knowledgeable workforce needed in an economic region to become competitive in biomanufacturing. In the past decade, our institution has been a key strategic partner with multinational biotechnology companies in supplying science and engineering graduates in the field of industrial biotechnology. Initiatives addressing all levels of the educational pipeline, from K-12 to college to continued education for company employees have been established along a ten-year span. The Amgen BioTalents Program was designed to provide undergraduate science and engineering students with training in biomanufacturing. The areas targeted by this educational program enhance their academic development, since these topics are not part of their traditional science and engineering curricula. The educational curriculum involved the process of producing a biomolecule from the genetic engineering of cells to the production of an especially targeted polypeptide, protein expression and purification, to quality control, and validation. This paper will report and describe the implementation details and outcomes of the first sessions of the program.
Abstract: P16/INK4A is tumor suppressor protein that plays a critical role in cell cycle regulation. Loss of P16 protein expression has been implicated in pathogenesis of many cancers, including lymphoma. Therefore, we sought to investigate if loss of P16 protein expression is associated with lymphoma and/or any specific lymphoma subtypes (Hodgkin-s lymphoma (HL) and nonHodgkin-s lymphoma (NHL)). Fifty-five lymphoma cases consisted of 30 cases of HL and 25 cases of NHL, with an age range of 3 to 78 years, were examined for loss of P16 by immunohistochemical technique using a specific antibody reacting against P16. In total, P16 loss was seen in 33% of all lymphoma cases. P16 loss was identified in 47.7% of HL cases. In contrast, only 16% of NHL showed loss of P16. Loss of P16 was seen in 67% of HL patients with 50 years of age or older, whereas P16 loss was found in only 42% of HL patients with less than 50 years of age. P16 loss in HL is somewhat higher in male (55%) than in female (30%). In subtypes of HL, P16 loss was found exclusively in all cases of lymphocyte depletion, lymphocyte predominance and unclassified cases, whereas P16 loss was seen in 39% of mixed cellularity and 29% of nodular sclerosis cases. In low grade NHL patients, P16 loss was seen in approximately one-third of cases, whereas no or very rare of P16 loss was found in intermediate and high grade cases. P16 loss did not show any correlation with age or gender of NHL patients. In conclusion, the high rate of P16 loss seen in our study suggests that loss of P16 expression plays a critical role in the pathogenesis of lymphoma, particularly with HL.
Abstract: In this study, three strains of Vibrio parahaemolyticus
(690, BCRC 13023 and BCRC 13025) were subjected to acid
adaptation at pH 5.5 for 90 min. The survival of acid-adapted and
non-adapted V. parahaemolyticus strains under simulated gastric
condition and their protein expression profiles were investigated.
Results showed that acid adaptation increased the survival of the test
V. parahaemolyticus strains after exposure to simulated gastric juice
(pH 3). Additionally, acid adaptation also affected the protein
expression in these V. parahaemolyticus strains. Nine proteins,
identified as atpA, atpB, DnaK, GroEL, OmpU, enolase,
fructose-bisphosphate aldolase, phosphoglycerate kinase and
triosephosphate isomerase, were induced by acid adaptation in two or
three of the test strains. These acid-adaptive proteins may play
important regulatory roles in the acid tolerance response (ATR) of V.
parahaemolyticus.
Abstract: Supplementation of palm vitamin E has been reported
to prevent loss of bone density in ovariectomised female rats. The
mechanism by which palm vitamin E exerts these effects is still
unknown. We hypothesized that palm vitamin E may act by
preventing the protein expression changes. Two dimensional poly
acyrilamide gel electrophoresis (2-D PAGE) and PD Quest software
genomic solutions Investigator (proteomics) was used to analyze the
differential protein expression profile in femoral and humeri bones
harvested from three groups of rats; sham-operated rats (SO),
ovariectomised rats (Ovx) and ovariectomised rats supplemented for
2 months with palm vitamin E. The results showed that there were
over 300 valued spot on each of the groups PVE and OVX as
compared to about 200 in SO. Comparison between the differential
protein expression between OVX and PVE groups showed that ten
spots were down –regulated in OVX but up-regulated in PVE. The
ten differential spots were separately named P1-P10. The
identification and understanding of the pathway of the differential
protein expression among the groups is ongoing and may account for
the molecular mechanism through which palm vitamin E exert its
anti-osteoporotic effect.
Abstract: The two-dimensional gel electrophoresis method
(2-DE) is widely used in Proteomics to separate thousands of proteins
in a sample. By comparing the protein expression levels of proteins in
a normal sample with those in a diseased one, it is possible to identify
a meaningful set of marker proteins for the targeted disease. The major
shortcomings of this approach involve inherent noises and irregular
geometric distortions of spots observed in 2-DE images. Various
experimental conditions can be the major causes of these problems. In
the protein analysis of samples, these problems eventually lead to
incorrect conclusions. In order to minimize the influence of these
problems, this paper proposes a partition based pair extension method
that performs spot-matching on a set of gel images multiple times and
segregates more reliable mapping results which can improve the
accuracy of gel image analysis. The improved accuracy of the
proposed method is analyzed through various experiments on real
2-DE images of human liver tissues.
Abstract: Hyperglycaemia is a key factor that contributes to the
development of diabetes-related microvascular disease and a major
risk factor for endothelial dysfunction. In the current study, we have
explored glucose-induced abnormal intracellular calcium (Ca2+
i)
homeostasis in mouse microvessel endothelial cells (MMECs) in
high glucose (HG) (40mmol/L) versus control (low glucose, LG) (11
mmol/L). We demonstrated that the exposure of MMECs to HG for 3
days did not change basal Ca2+
i, however, there was a significant
increase of acetylcholine-induced Ca2+ entry. Western blots
illustrated that exposure to HG also increased STIM1 (Stromal
Interaction Molecule 1), but not Orai1 (the pore forming subunit),
protein expression levels. Although the link between HG-induced
changes in STIM1 expression, enhanced Ca2+ entry and endothelial
dysfunction requires further study, the current data are suggestive
that targeting these pathways may reduce the impact of HG on
endothelial function.
Abstract: Highly pathogenic avian influenza (HPAI) H5N1 viruses have created demand for a cost-effective vaccine to prevent a pandemic of the disease. Here, we report that Trichoplusia ni (T. ni) larvae can act as a cost-effective bioreactor to produce recombinant HA5 (rH5HA) proteins as an potential effective vaccine for chickens. To facilitate the recombinant virus identification, virus titer determination and access the infected larvae, we employed the internal ribosome entry site (IRES) derived from Perina nuda virus (PnV, belongs to insect picorna like Iflavirus genus) to construct a bi-cistronic baculovirus expression vector that can express the rH5HA protein and enhanced green fluorescent protein (EGFP) simultaneously. Western blot analysis revealed that the 70 kDa rH5HA protein and partially cleaved products (40 kDa H5HA1) were generated in T. ni larvae infected with recombinant baculovirus carrying the H5HA gene. These data suggest that the baculovirus-larvae recombinant protein expression system could be a cost-effective platform for H5N1 vaccine production.