Validation of Protein Biomarker Candidates for Diagnosis of HBV induced HCC

Hepatocellular carcinoma is a major contributor to the global cancer burden. It affects millions of people in Pakistan on a yearly basis. Furthermore, HCC is linked to viral infections Hepatitis B and C, which account for roughly 87 percent of HCC cases in Pakistan. HCC is identified using imaging techniques such as MRI, Ultrasound, and histology, which have radiation hazards and frequently need expensive healthcare systems that are less available in most of the developing countries. Novel HCC biomarkers are being developed as part of a large research project aimed at detecting the disease early. These include the creation of biomarkers based on HCC patients' transcriptome and proteomic profiles. Circulating proteins, which are easily detected in body fluids, including blood serum, may thus provide an opportunity for the development of HCC biomarkers. Blood-based serum biomarkers must be developed for easy, non-invasive, and early detection of HCC. In conjunction with imaging techniques, alpha-fetoprotein (AFP) has been used to detect HCC, although it has little clinical usefulness. Also, the reported AFP negative results make its utility meager. Multiple circulating proteins have been studied as biomarker possibilities for HCC diagnosis in recent years. In this study, Blood serum was used to validate three novel protein biomarker candidates to detect HBV induced HCC that had previously been predicted using a bioinformatics methodology. Proteins named C6, C8A and C8B were measured in the serum of 22 HCC patients infected with HBV in Pakistani population and compared to AFP levels using quantitative ELISA. C8A possesses considerable biomarker potential, with 95.45 percent specificity and 77.27% sensitivity with 0.933 Area Under the Curve (AUC), whereas C6 and C8B showed poor biomarker potential. Hence, C8A demonstrated great promise as a circulating blood-based protein biomarker for HBV induced HCC diagnosis. View Article DOI: 10.47856/ijaast.2022.v09i03.00

Strategies and challenges with the microbial conversion of methanol to high-value chemicals

As alternatives to traditional fermentation substrates, methanol (CH3OH), carbon dioxide (CO2) and methane (CH4) represent promising one-carbon (C1) sources that are readily available at low-cost and share similar metabolic pathway. Of these C1 compounds, methanol is used as a carbon and energy source by native methylotrophs, and can be obtained from CO2 and CH4 by chemical catalysis. Therefore, constructing and rewiring methanol utilization pathways may enable the use of one-carbon sources for microbial fermentations. Recent bioengineering efforts have shown that both native and nonnative methylotrophic organisms can be engineered to convert methanol, together with other carbon sources, into biofuels and other commodity chemicals. However, many challenges remain and must be overcome before industrial-scale bioprocessing can be established using these engineered cell refineries. Here, we provide a comprehensive summary and comparison of methanol metabolic pathways from different methylotrophs, followed by a review of recent progress in engineering methanol metabolic pathways in vitro and in vivo to produce chemicals. We discuss the major challenges associated with establishing efficient methanol metabolic pathways in microbial cells, and propose improved designs for future engineering

The Pichia pastoris transmembrane protein GT1 is a glycerol transporter and relieves the repression of glycerol on AOX1 expression

Promoter of alcohol oxidase I (PAOX1) is the most efficient promoter involved in the regulation of recombinant protein expression in Pichia pastoris (P. pastoris). PAOX1 is tightly repressed by the presence of glycerol in the culture medium; thus, glycerol must be exhausted before methanol can be taken up by P. pastoris and the expression of the heterologous protein can be induced. In this study, a candidate glycerol transporter (GT1, GeneID: 8197545) was identified, and its role was confirmed by further studies (e.g. bioinformatics analysis, heterologous complementation in Schizosaccharomyces pombe (S. pombe)). When GT1 is co-expressed with enhanced green fluorescent protein (EGFP), it localizes to the membrane and S. pombe carrying gt1 but not the wild-type strain can grow on medium containing glycerol as the sole carbon source. The present study is the first to report that AOX1 in the X-33gt1 mutant can achieve constitutive expression in medium containing glycerol; thus, knocking down gt1 can eliminate the glycerol repression of PAOX1 in P. pastoris. These results suggest that the glycerol transporter may participate in the process of PAOX1 inhibition in glycerol medium

Cooperation of DLC1 and CDK6 Affects Breast Cancer Clinical Outcome

Peer reviewe

Correlation Between Protein Primary Structure and Soluble Expression Level of HSA dAb in Escherichia coli

Izoelektrična točka, duljina molekule, molekularna masa i slijed aminokiselina bitno utječu na topljivost proteina. U ovom smo se radu fokusirali na sastav aminokiselina i ispitali one koje najviše utječu na razinu ekspresije topljivog protutijela albumina iz ljudskog seruma (HSA dAb). Grupiranjem i primjenom linearnog modela analizirana je topljivost 65 varijanti proteina. Bitan utjecaj na ekspresiju topljivog protutijela dAb imale su specifične kombinacije aminokiselina, i to (S, R, N, D, Q) u supernatantu, (G, R, C, N, S) u lizatu peleta i (R, S, G) u ukupnom topljivom protutijelu dAb. Od 20 aminokiselina, arginin je imao negativan, a glicin i serin su imale pozitivan učinak na razinu ekspresije topljivog proteina. Preciznost linearnog modela predviđanja topljivosti proteina bila je 80 %. Zaključeno je da se povećanjem udjela polarnih aminokiselina, osobito glicina i serina, te smanjenjem udjela arginina bitno povećala ekspresija topljivog proteina HSA dAb.It is widely accepted that features such as pI, length, molecular mass and amino acid (AA) sequence have a significant influence on protein solubility. Here, we mainly focused on AA composition and explored those that most affected the soluble expression level of human serum albumin (HSA) domain antibody (dAb). The soluble expression and sequence of 65 dAb variants were analysed using clustering and linear modelling. Certain AAs significantly affected the soluble expression level of dAb, with the specific AA combinations being (S, R, N, D, Q), (G, R, C, N, S) and (R, S, G); these combinations respectively affected the dAb expression level in the broth supernatant, the level in the pellet lysate and total soluble dAb. Among the 20 AAs, R displayed a negative influence on the soluble expression level, whereas G and S showed positive effects. A linear model was built to predict the soluble expression level from the sequence; this model had a prediction accuracy of 80 %. In summary, increasing the content of polar AAs, especially G and S, and decreasing the content of R, was helpful to improve the soluble expression level of HSA dAb

Oxidative stress in submerged cultures of a recombinant Aspergillus niger (BI-D)

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Introduction to bioreactors of shake-flask inocula leads to development of oxidative stress in Aspergillus niger

Inoculation of bioreactors with shake-flask cultures present the organism with an immediate shift from an environment with little O2 to one in which O2 is typically at 100% saturation. The inoculation of such shake-flasks cultures into bioreactors sparged with 1 vvm air or 1 vvm air/O2 mix i.e. 50% O2 enrichment is an oxidatively stressful event, as judged by immediate increases in the intracellular concentrations of superoxide anion radical (O2·−) (from 4,600 to 11,600 RLU mg DCW−1 and 5,500 to 23,000 RLU mg DCW−1 respectively) and changes in the activities of the major antioxidant enzymes superoxide dismutase and catalase in all cultures. There are further effects on metabolic indices, particularly decreased nutrient consumption in oxygenated cultures (from 0.16 to 0.12 g starch g DCW h−1) and decreased protein production, indicating that inoculation of the bioreactor exerts a global burden on the cellular metabolic networks

Development of a secretory expression system with high compatibility between expression elements and an optimized host for endoxylanase production in Corynebacterium glutamicum

Abstract Background In terms of protein production, the internal environment of the host influences the activity of expression elements, thus affecting the expression level of the target protein. Native expression elements from a specific strain always function well in the original host. In the present study, to enhance the endoxylanase (XynA) production level in Corynebacterium glutamicum CGMCC1.15647 with its native expression elements, approaches to reduce host expression obstacles and to promote expression were evaluated. Results We identified the signal peptide of CspB2 in C. glutamicum CGMCC1.15647 by MALDI-TOF and applied it along with its promoter for the production of endoxylanase (XynA) in this strain. The native cspB2 promoter and cspB2 signal peptide are superior to the well-used cspB1 promoter and cspA signal peptide for XynA expression in C. glutamicum CGMCC1.15647, and expression in this strain is superior to the expression in C. glutamicum ATCC13032. The highest XynA secretion efficiency level in deep 24-well plates level (2492.88 U/mL) was achieved by disruption of the cell wall protein CspB2 and the protease ClpS, chromosomal integration of xynA and coexisting plasmid expression, which increased expression 11.43- and 1.35-fold compared to that of chromosomal expression and pXMJ19-xynA-mediated expression in the original strain, respectively. In fed-batch cultivation, the highest XynA accumulation (1.77 g/L) was achieved in the culture supernatant after 44 h of cultivation. Conclusion Adaptation between the expression elements and the host is crucial for XynA production in C. glutamicum CGMCC1.15647. Strategies including host optimization, chromosomal integration, and coexistence of plasmids were useful for efficient protein production in C. glutamicum

Development of a Chemiluminescence Immunoassay for Quantification of 25-Hydroxyvitamin D in Human Serum

In this study, a chemiluminescence immunoassay (CLIA) for human serum 25-hydroxyvitamin D (25(OH)D) was established by a competition model. In serum, more than 99% of total circulating 25(OH)D binds to protein and less than 1% of 25(OH)D is in free form (Jassil et al., 2017). Before measuring concentration of 25(OH)D in serum, a releasing procedure should be conducted. A new reagent is used to release binding 25(OH)D to free form. Streptavidin (SA) was labeled to magnetic beads by a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) method. Biotinylated VD was used as a competitor of 25(OH)D in samples. Anti-VD antibody (aby) was labeled to horseradish peroxidase (HRP) by EDC to react with 25(OH)D and biotinylated-VD molecules. The pretreated samples or standards were added into the reaction tube with biotin-VD and anti-VD aby-HRP, free 25(OH)D in the sample competes with biotinylated VD for binding to anti-VD aby-HRP, an SA-labeled magnetic particle is added to isolate the signal-generating complex, and the signal is inversely proportional to the 25(OH)D concentration in the sample. The method established shows good thermostability and performance. The limitation of detection (LoD) is 1.43 ng/mL. The intra-assay coefficient of variation (CV) is 3.66%–6.56%, the interassay CV is 4.19%–7.01%, and the recovery rate is 93.22%–107.99%. Cross-reactivity (CR) was remarkably low with vitamin D2, vitamin D3, 1, 25-dihydroxyvitamin D3, and 1, 25-dihydroxyvitamin D2. At the same time, the cross-reaction values with 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were 97% and 100%, respectively. The developed method shows good correlation with the total VD product from Roche and DiaSorin. 1096 clinical patient samples were measured with developed reagent kit in this study. 7 types of disease were involved, and the concentration of 25(OH)D is less than 30 ng/mL in 94.98% of patients