Search

The 3 chymotrypsin-like protease (3CLpro) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is one of the primary targets for the development of antiviral drug therapies as it plays a critical role in viral replication. In this study, the gene encoding for SARS-CoV-2 3CLpro (918 bp) was amplified from the cDNA of the virus by polymerase chain reaction (PCR) and cloned into the pGEM-T vector. 3CLpro was then inserted into the expression vector pET28a at the end of the 6 histidine residue encoding sequence to form a fusion protein (6xHis-3CLpro). The 6xHis-3Clpro construct was successfully expressed in E. coli. The expression of 3CLpro was highest when E. coli BL21(DE3) RIL harboring pET28a-3CLpro vector was cultured in LB medium at 20 oC, induced by 1.0 mM Isopropyl thiogalactopyranosie (IPTG) when cell density measured by optical density at 600 nm (OD600) reached 0.7-0.8 and harvested after 24 hours of induction. The recombinant 3CLpro was purified by Ni-sepharose affinity chromatography under denaturation conditions. The purified 3CLpro showed to have a 41 kDa band on sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using polyclonal anti-3CLpro antibody and hydrolyzed a fluorescent specific substrate of 3CLpro after renaturation.

White spot syndrome virus (WSSV) is a double stranded DNA virus that causes WSS diseases for many crustaceans including the Penaeidae family shrimp. In this study, standard DNA for detection and quantification of WSSV by real-time polymerase chain reaction (qPCR) method was created. A specific gene fragment of 126 bp from WSSV genome was successfully amplified by PCR and cloned into pGEM-T vector. qPCR calibration curves using the created recombinant pGEM vector harboring 126 bp gene fragment as the standard DNA at concentrations of 3x102 to 3x108 copies/mL showed to have good linear regression with an efficiency of 99.1%, correlation coefficient R2 of 0.998 and the slope of -3.344. The recombinant vector was also used as a positive standard for detection and quantification of WSSV in a number of WSSV-infected shrimp samples and the values ranging from 3.69x103 copies/mL to 1.25x108 copies/mL WSSV were found in the collected samples.

Viral diseases are a serious issue for the shrimp aquaculture industry. White spot syndrome virus (WSSV) has been considered one of the most dangerous pathogens infecting cultured shrimp, causing a mortality rate as high as 100% within 7–10 days of viral infection. So far, several protocols have been applied to protect shrimp against virus attacks, but their protection efficiency is very limited. In this study, for the first time, three melanin-containing feedstuffs (F1, F2, and F3) were formulated and fed to cultured shrimp to investigate the ability of melanin to protect shrimp from WSSV. The obtained results showed that F2 had a protection rate of 64% at day 7 and 62% at day 10 after virus challenge. The protection ability of the feedstuff depended on the amount of melanin consumed by shrimp. Moreover, our results also demonstrated that the transcription level of the VP28 gene, which codes for the VP28 protein, a representative for the presence of WSSV, was significantly decreased in shrimp fed F2. Taken together, our study suggests that melanin-containing diets may be applied in aquaculture to protect shrimp against WSSV infection; further, combined protocols with the simultaneous use of melanin-containing diets and other protectants should be studied and applied to increase the protection efficiency.

Superroxide dismutase (SOD, EC.1.15.1.1) is the enzyme which dismutates superoxide radicals and plays an important role in protection of living cells against oxidative stress. SOD is also involved in immune response in shrimps. In this study, it was found that the total SOD activity of black tiger shrimp muscular tissues is 10 fold higher than that of the haemolymph, however, the specific activity of SOD in the shrimp haemolymph is 9.2 fold higher than that of muscular tissues. By using active gel electrophoresis, 2 different SOD forms were found in black tiger shrimps (one in muscular tissues and two in haemolymph).Using DE-52 cellulose and Q-Sepharose ion exchange column chromatography, one SOD (SOD1) from black tiger shrimp haemolymph was partially purified, and its purity was 31.2 times higher than that of the starting haemolymph. The SOD1 was shown to have mainly one protein band of approximately 24 kDa on SDS-PAGE. SOD1 was most active at 45oC and pH of 5.5. At a concentration of 5 mM, Mn2+ strongly activated SOD1 (up 200% activity), Ca2+ và Zn2+ could increase approximately 20% activity while Cu2+ inhibited more than 60% ativity of the enzyme.

Pigmented spore-forming bacterial strains were isolated from the gastrointestinal tracts of chickens for screening for heat-stable carotenoid-producing strains that could be applied as feed supplements. Of the seven heat-stable pigmented isolates screened, only two, yellow Sporosarcina saromensis CH1 and red-orange Bacillus aquimaris CH9, produced pigments with typical carotenoid absorbance peaks (400–500 nm). The CH9 carotenoids exhibited higher scavenging activity (73.2%) of DPPH free radicals than the CH1 carotenoids (35.9%) and carotenoids of the reference B. indicus HU36 strain (78.4%), in comparison to 100% activity of acid ascorbic at 18.75 M as the standard. The CH9 strain produced high levels of carotenoids (439 g [g DW]-1) and formed nearly 100% spores, whereas the CH1 strain produced low levels of carotenoids (92 g [g DW]-1) and only achieved 30% sporulation. Chromatographic and spectral profiles of the carotenoids found in CH9 indicated the presence of as many as 11 different carotenoid types closely related to 1-HO-demethylspheroidene and keto/hydroxyl derivatives of  carotene. We successfully produced concentrated orange CH9 spore powder at a high concentration of 6.1 × 1011 CFU g-1; these spores were much more heat-stable (66% survival at 80°C for 20 min) than the reference B. indicus HU36 spores (9% survival at 50°C for 20 min). In conclusion, B. aquimaris CH9 is a promising probiotic carotenoid-producing strain, with heat-stable spores that should withstand the heat-treatment processing required for feed and food supplement production.

SiO2-coated Fe3O4 nanoparticles (Fe3O4@SiO2 NPs) were successfully synthesized using ultrasound in order to extract DNA from cancer tissues for application in diagnostics. The core 10.7-nm-diameter Fe3O4 nanoparticles were synthesized by co-precipitation of Fe3+ and Fe2+ as reaction substrates and NH4OH as precipitant, then coated with a thin layer of amorphous silica by a modified Stober method. Further SiO2 coating using alkaline hydrolysis of tetraethyl orthosilicate in ethanol and water mixture was accelerated in the presence of a 37-kHz ultrasound, resulting in the NPs having different sizes of 14.5 nm (version M1), 24.4 nm (version M2), and 34.9 nm (version M3) with saturation magnetization values of 50.2 emu/g, 18.6 emu/g, 10.3 emu/g, respectively. Among the three Fe3O4@SiO2 NPs versions, the M1 NPs allowed extraction of DNAs from 10 mg formalin-fixed and paraffin-embedded (FFPE) tissues of nasopharyngeal carcinoma patients with the highest recovery of about 100–500 ng/μl and good purity (A260/A280: 1.8–1.9). The extracted DNAs could be used as templates for downstream amplification of 252-bp sequencing specifically for the Braf cancer biomarker gene using polymerase chain reaction (PCR), as well as detection of the pathogenic Epstein–Barr virus (EBV) and the human papilloma-virus (HPV) using real-time PCR. DNA extraction recoveries of both EBV and HPV using Fe3O4@SiO2 NPs M1 were significantly better that those using commercialized Fe3O4@SiO2 microbeads, as indicated by lower threshold cycles of all fluorescent signals including fluorescein amidite (FAM) dye representative for EBV infection, hexachlorofluorescein (HEX) dye representative for β-globin (internal control), and SYBR Green dye representative for HPV infection in tested clinical samples from patients with nasopharyngeal carcinoma (NPC).

Mitochondrial genome A3243G mutation in the tRNALeu(UUR) encodinggene (MTTL)is the main cause of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS). This mutation exists in heteroplasmic form and severity of the disease is affected by many factors including heteroplasmy level. In this study, a pediatric proband (female, 8 years old) was found to carry A3243G mutation at 77.6% of heteroplasmy by using PCR-RFLP in combination with real-time PCR. The results of the A3243G mutation analysis of the proband’s family showed that her mother without any symptoms of encephalopathyalso carried the mutation at 7.9% of heteroplasmy whereas the mutation was not found in the proband’s healthy father and healthy sister, indicating that the proband received the A3243G mutation from her mother and the expression of MELAS syndromes depended on the level of heteroplasmy.

Gold nanoparticles (GNPs) have been synthesized by a chemical reduction method using sodium borohydride and functionalized with amino groups on their surface. The GNPs were then conjugated with the anti-HER2 human antibody (trastuzumab) for detecting breast cancer cells. The specific binding of trastuzumab-conjugated GNPs onto the breast cancer cells (KPL4 line) were observed by bright-field and dark-field microscopy and were for the first time observed by scanning electron microscopy and energy dispersive X-ray scanning. [DOI: 10.1380/ejssnt.2011.544]

Magnetic nanoparticles with a diameter of 15 nm prepared by using the coprecipitation method have been applied to enforce the sedimentation of the solid waste, to adsorb the arsenic ions in water, to increase the DNA concentration by using a magnetic field for the electrochemical DNA sensor and to separate the helper CD4+ T cells to determine the number of the cells in blood. A combination of magnetic nanoparticles and alum makes the solid waste in water under a magnetic field aggregate a dozen times faster than under the gravity alone. A concentration of 0.1 mg/l of arsenic in water was reduced to a value lower than the permissible concentration of 0.01 mg/l after few minutes of stirring. The particles functionalized with 3-aminopropyl triethoxysilane were used to enrich the DNA of the Herpes virus, which extended the sensitivity of an electrochemical sensor down to a concentration lower than nM/l. The particles coated with fluorescent-labeled antiCD4 antibody were used to count the helper CD4+ T cells. The fluorescence signals of the particle/cell system were two times stronger than those of the fluorescence antiCD4 cell system. This can be used for the treatment of an HIV-infected patient with a simple fluorescent microscope. Author

Vemurafenib has recently been used as drug for treatment of melanomas with BRAFV600E mutation. Unfortunately, treatment with only vemurafenib has not been sufficiently effective, with recurrence after a short period. In this study, three vemurafenib-resistant BRAFV600E melanoma cell lines, A375PR, A375MR and SKMEL-28R, were established from the original A375P, A375M and SKMEL-28 cell lines. Examination of the molecular mechanisms showed that the phosphorylation levels of MEK and ERK, which play key roles in the RAS/RAF/MEK/ERK signaling pathway, were reduced in these three cell lines, with increased phosphorylation levels of pAKTs limited to SKMEL-28R cells. Treatment of SKMEL-28R cells with 100 nM paclitaxel resulted in increased apoptosis and decreased cellular proliferation, invasion and colony formation via reduction of expression levels of EGFR and pAKTs. Moreover, vemurafenib-induced pAKTs in SKMEL-28R were decreased by treatment with an AKT inhibitor, MK-2206. Taken together, our results revealed that resistance mechanisms of BRAFV600E-mutation melanoma cells to vemurafenib depended on the cell type. Our results suggested that paclitaxel should be considered as a drug in combination with vemurafenib to treat melanoma cells.

Proline (Pro) and Trehalose (Tre) function as compatible solutes and are upregulated in plants under abiotic stress. They play an osmoprotective role in physiological responses, enabling the plants to better tolerate the adverse effects of abiotic stress. We investigated the effect of exogenous Pro and Tre (10 mM) in seedlings of Thai aromatic rice (cv. KDML105; salt-sensitive) during salt stress and subsequent recovery. Salt stress (S, NaCl) resulted in growth reduction, increase in the Na(+)/K(+) ratio, increase in Pro level and up-regulation of Pro synthesis genes (pyrroline-5-carboxylatesynthetase, P5CS; pyrroline-5-carboxylate reductase, P5CR) as well as accumulation of hydrogen peroxide (H(2)O(2)), increased activity of antioxidative enzymes (superoxide dismutase, SOD; peroxidase, POX; ascorbate peroxidase, APX; catalase, CAT) and transcript up-regulation of genes encoding antioxidant enzymes (Cu/ZnSOD, MnSOD, CytAPX, CatC). Under salt stress, exogenous Pro (PS; Pro+NaCl) reduced the Na(+)/K(+) ratio, further increased endogenous Pro and transcript levels of P5CS and P5CR, but decreased the activity of the four antioxidant enzymes. The transcription of genes encoding several antioxidant enzymes was upregulated. Exogenous Tre (TS; Tre+NaCl) also reduced the Na(+)/K(+) ratio and strongly decreased endogenous Pro. Transcription of P5CS and P5CR was upregulated, the activities of SOD and POX decreased, the activity of APX increased and the transcription of all antioxidant enzyme genes upregulated. Although exogenous osmoprotectants did not alleviate growth inhibition during salt stress, they exhibited a pronounced beneficial effect during recovery period showing higher percentage of growth recovery in PS (162.38%) and TS (98.43%) compared with S (3.68%). During recovery, plants treated with PS showed a much greater reduction in endogenous Pro than NaCl-treated (S) or Tre-treated plants (TS). Increase in CAT activity was most related to significant reduction in H(2)O(2), particularly in the case of PS-treated plants. Advantageous effects of Pro were also associated with increase in APX activity during recovery.

Colloidal sphere-like gold nanoparticles were prepared from an HAuCl4 aqueous solution by the chemical reduction method and by using X-ray irradiation, while rod-like gold nanoparticles were synthesised according to the seed-mediated growth method and by sonoelectrochemistry. Gold nanoparticles exhibit fcc structure. Sphere-like gold nanoparticles have a diameter of 20-60 nm, while rod-like gold nanoparticles have an aspect ratio of 2–4. In UV-vis spectra, the absorption bands related to surface plasmon resonance were observed. Co-Pt nanoparticles of 5-20 nm were prepared by electrochemistry, while Fe-Pt nanoparticles with a size of 5-10 nm were prepared by sonoelectrochemistry. After annealing, these magnetic nanoparticles showed a high coercivity. The gold nanoparticles were functionalised for detecting breast cancer cells.