Journal, genetic engineering & biotechnology最新文献

Background: The existence of mutated Delta (B.1.617.2) variants of SARS-CoV-2 causes rapid transmissibility, increase in virulence, and decrease in the effectiveness of public health. Majority of mutations are seen in the surface spike, and they are considered as antigenicity and immunogenicity of the virus. Hence, finding suitable cross antibody or natural antibody and understanding its biomolecular recognition for neutralizing surface spike are crucial for developing many clinically approved COVID-19 vaccines. Here, we aim to design SARS-CoV-2 variant and hence, to understand its mechanism, binding affinity and neutralization potential with several antibodies.

Results: In this study, we modelled six feasible spike protein (S1) configurations for Delta SARS-CoV-2 (B.1.617.2) and identified the best structure to interact with human antibodies. Initially, the impact of mutations at the receptor-binding domain (RBD) of B.1.617.2 was tested, and it is found that all mutations increase the stability of proteins (ΔΔG) and decrease the entropies. An exceptional case is noted for the mutation of G614D variant for which the vibration entropy change is found to be within the range of 0.133-0.004 kcal/mol/K. Temperature-dependent free energy change values (ΔG) for wild type is found to be - 0.1 kcal/mol, whereas all other cases exhibit values within the range of - 5.1 to - 5.5 kcal/mol. Mutation on spike increases the interaction with the glycoprotein antibody CR3022 and the binding affinity (CLUSpro energy =  - 99.7 kcal/mol). The docked Delta variant with the following antibodies, etesevimab, bebtelovimab, BD-368-2, imdevimab, bamlanivimab, and casirivimab, exhibit a substantially decreased docking score (- 61.7 to - 112.0 kcal/mol) and the disappearance of several hydrogen bond interactions.

Conclusion: Characterization of antibody resistance for Delta variant with respect to the wild type gives understanding regarding why Delta variant endures the resistance boosted through several trademark vaccines. Several interactions with CR3022 have appeared compared to Wild for Delta variant, and hence, it is suggested that modification on the CR3022 antibody could further improve for the prevention of viral spread. Antibody resistance decreased significantly due to numerous hydrogen bond interactions which clearly indicate that these marketed/launched vaccines (etesevimab) will be effective for Delta variants.

Background: The cytochrome b (Cyt b) gene is one of the most studied mitochondrial DNA (mtDNA) genes to determine sheep's genetic profile. This study aimed to determine the genetic diversity and relationships of several Indonesian local sheep populations on Java Island, Indonesia, based on the mtDNA Cyt b gene sequences. Blood samples were collected from forty-one individual sheep in seven populations of Indonesia local sheep breeds on Java Island (Priangan = 6, Garut = 6, Batur = 7, Wonosobo = 5, Javanese Thin-Tailed/JTT = 7, Javanese Fat-Tailed/JFT = 5, and Sapudi = 5). DNA extraction was performed on blood samples, and the mtDNA Cyt b gene was amplified using specific primers (Alek-CBF: 5'-CAACCCCACCACTTACAA-3' and Alek-CBR: 5'-CCTTGAGTCTTAGGGAGGTT-3'). The polymerase chain reaction (PCR) products were then sequenced, and data were analyzed using the MEGA version 7.0, DNA SP version 6.0, and NTSYS-pc version 2.11 software.

Results: A total of 1140 bp complete mtDNA Cyt b gene sequences in this study obtained 1134 monomorphic sites (I), six polymorphic sites (V), one segregation site (S), and five parsimony informative sites (P) with a nucleotide diversity (Pi), the average number of nucleotide differences (K), and sequence conservation (SC) were 0.00119, 1.35610, and 0.9947, respectively. There were six haplotypes consisting of two unique haplotypes and four shared haplotypes with a haplotype diversity (Hd) of 0.5415. The genetic distance within and between populations ranged from 0.0000 to 0.0016 and 0.0000 to 0.0020, respectively. Wonosobo, JFT, and Sapudi sheep have the closest relationship, and then these three breeds were close to JTT sheep, followed by Batur, Priangan, and Garut sheep. Two haplogroups have been found based on the Ovine haplogroup clustering. All Wonosobo, JTT, JFT, Sapudi sheep, and most Batur sheep were clustered into haplogroup B. In contrast, Garut sheep were mostly clustered into haplogroup A, while Priangan sheep were clustered into both haplogroups with the same percentage.

Conclusion: Seven Indonesian local sheep breeds on Java Island have a close relationship clustered into two haplogroups, namely haplogroups A and B. Most Indonesian local sheep breeds on Java Island in this study were clustered into haplogroup B, except for Garut and Priangan sheep.

Background: Seaweeds are a viable bioresource for suffering plants against salt stress, as they abundant in nutrients, hormones, vitamins, secondary metabolites, and many other phytochemicals that sustain plants' growth under both typical and stressful situations. The alleviating capacity of extracts from three brown algae (Sargassum vulgare, Colpomenia sinuosa, and Pandia pavonica) in pea (Pisum sativum L.) was investigated in this study.

Methods: Pea seeds were primed for 2 h either with seaweed extracts (SWEs) or distilled water. Seeds were then subjected to salinity levels of 0.0, 50, 100, and 150 mM NaCl. On the 21st day, seedlings were harvested for growth, physiological and molecular investigations.

Results: SWEs helped reduce the adverse effects of salinity on pea, with S. vulgare extract being the most effective. Furthermore, SWEs diminished the effect of NaCl-salinity on germination, growth rate, and pigment content and raised the osmolytes proline and glycine betaine levels. On the molecular level, two low-molecular-weight proteins were newly synthesized by the NaCl treatments and three by priming pea seeds with SWEs. The number of inter-simple sequence repeats (ISSR) markers increased from 20 in the control to 36 in 150 mM NaCl-treated seedlings, including four unique markers. Priming with SWEs triggered more markers than the control, however about ten of the salinity-induced markers were not detected following seed priming before NaCl treatments. By priming with SWEs, seven unique markers were elicited.

Conclusion: All in all, priming with SWEs alleviated salinity stress on pea seedlings. Salinity-responsive proteins and ISSR markers are produced in response to salt stress and priming with SWEs.

Background: Hepatitis E virus (HEV) is the cause of a liver disease hepatitis E. The translation product of HEV ORF2 has recently been demonstrated as a protein involved in multiple functions besides performing its major role of a viral capsid. As intrinsically disordered regions (IDRs) are linked to various essential roles in the virus's life cycle, we analyzed the disorder pattern distribution of the retrieved ORF2 protein sequences by employing different online predictors. Our findings might provide some clues on the disorder-based functions of ORF2 protein that possibly help us in understanding its behavior other than as a HEV capsid protein.

Results: The modeled three dimensional (3D) structures of ORF2 showed the predominance of random coils or unstructured regions in addition to major secondary structure components (alpha helix and beta strand). After initial scrutinization, the predictors VLXT and VSL2 predicted ORF2 as a highly disordered protein while the predictors VL3 and DISOPRED3 predicted ORF2 as a moderately disordered protein, thus categorizing HEV-ORF2 into IDP (intrinsically disordered protein) or IDPR (intrinsically disordered protein region) respectively. Thus, our initial predicted disorderness in ORF2 protein 3D structures was in excellent agreement with their predicted disorder distribution patterns (evaluated through different predictors). The abundance of MoRFs (disorder-based protein binding sites) in ORF2 was observed that signified their interaction with binding partners which might further assist in viral infection. As IDPs/IDPRs are targets of regulation, we carried out the phosphorylation analysis to reveal the presence of post-translationally modified sites. Prevalence of several disordered-based phosphorylation sites further signified the involvement of ORF2 in diverse and significant biological processes. Furthermore, ORF2 structure-associated functions revealed its involvement in several crucial functions and biological processes like binding and catalytic activities.

Conclusions: The results predicted ORF2 as a protein with multiple functions besides its role as a capsid protein. Moreover, the occurrence of IDPR/IDP in ORF2 protein suggests that its disordered region might serve as novel drug targets via functioning as potential interacting domains. Our data collectively might provide significant implication in HEV vaccine search as disorderness in viral proteins is related to mechanisms involved in immune evasion.

Background: Actinomycetes isolated from marine habitats are known to have the potential for novel enzymes that are beneficial in the industry. In-depth knowledge is necessary given the variety of this bacterial group in Indonesia and the lack of published research. Actinomycetes isolates (BLH 5-14) obtained from marine sediments of Sarena Kecil, Bitung, North Sulawesi, Indonesia, showed an ability to produce pectinase and xylanase that have equal or even higher potential for pectic-oligosaccharides (POS) and xylooligosaccharides (XOS) production from raw biomass than from commercial substrates. This study's objective was to characterize both enzymes to learn more for future research and development.

Results: Pectinase had the highest activity on the 6^th day (1.44±0.08 U/mL) at the optimum pH of 8.0 and optimum temperature of 50 °C. Xylanase had the maximum activity on the 6^th day (4.33±0.03 U/mL) at optimum pH 6.0 and optimum temperature 60 °C. Hydrolysis and thin layer chromatography also showed that pectinase was able to produce monosaccharides such as galacturonic acid (P1), and xylanase was able to yield oligosaccharides such as xylotriose (X3), xylotetraose (X4), and xylopentaose (X5). BLH 5-14 identified as the genus Streptomyces based on the 16S rDNA sequences and the closely related species Streptomyces tendae (99,78%).

Conclusions: In the eco-friendly paper bleaching industry, Streptomyces tendae has demonstrated the potential to create enzymes with properties that can be active in a wide range of pH levels. The oligosaccharides have the potential as prebiotics or dietary supplements with anti-cancer properties. Further research is needed to optimize the production, purification, and development of the application of pectinase and xylanase enzymes produced by Actinomycetes isolates.

Background: Phoebe goalparensis is an endemic forest species of North East India that belongs to Lauraceae family. P. goalparensis is used as timbers yielding plants for commercial importance in the local furniture markets of North East India. A rapid in vitro micropropagation protocol was established by using apical and axillary shoot tips on Murashige and Skoog medium with varied concentrations of plant growth regulators.

Results: In this study, 5.0 mg/l BAP augmented medium was chosen as the best for shoot multiplication of the plant. However, IBA (2.0 mg/l) was the most responsive for root induction. Moreover, 70% of root induction was recorded during rooting experiment and 80-85% survivability was observed during the acclimatization of this species. Clonal fidelity of P. goalparensis was determined with ISSR marker and it was observed that in vitro raised plantlets were polymonomorphic.

Conclusion: Hence, an efficient protocol with high proliferation and rooting was established for P. Goalparensis that could aid in massive propagation in future.

Background: The Ag-NPs by green synthesis has a notable interest because of their eco-friendliness, economic views, feasibility, and applications in a wide range. Herein, native plants of Jharkhand (Polygonum plebeium, Litsea glutinosa, and Vangueria spinosus) were selected for the current work of Ag-NP synthesis and further antibacterial activity. Green synthesis was performed for Ag-NPs using Silver nitrate solution as precursor and the dried leaf extract performs as a reductant and stabilizer here.

Result: Visually Ag-NP formation was observed along with a colour change and confirmed by UV-visible spectrophotometry on which an absorbance peak occurs at around 400-450nm. Further characterization was done on DLS, FTIR, FESEM, and XRD. Size around 45-86 nm of synthesized Ag-NPs was predicted through DLS. The synthesized Ag-NPs exhibited significant antibacterial activity against Bacillus subtilis (Gram-positive bacteria) and Salmonella typhi (Gram-negative bacteria). The finest antibacterial activity was disclosed by the Ag-NPs synthesized by Polygonum plebeium extract. The diameter of the zone of inhibition in the bacterial plate measured was 0-1.8 mm in Bacillus and 0-2.2 mm in Salmonella typhi. Protein-Protein interaction study was performed to study the effect of Ag-NPs towards different antioxidant enzyme system of bacterial cell.

Conclusion: Present work suggest the Ag-NPs synthesized from P. plebeium were more stable for long term and might have prolonged antibacterial activity. In the future, these Ag-NPs can be applied in various fields like antimicrobial research, wound healing, drug delivery, bio-sensing, tumour/cancer cell treatment, and detector (detect solar energy). Schematic representation of Ag-NPs green synthesis, characterization, antibacterial activity and at the end, in silico study to analyse the mechanism of antibacterial activity.

Background: Glutathione s-transferases (GSTs) perform an essential role in detoxification of xenobiotics and endogenous compounds via their conjugation to reduce glutathione.

Results: A GST enzyme, designated tick larvae glutathione S transferase (TLGST), was purified from larvae of the camel tick Hyalomma dromedarii via ammonium sulfate precipitation, glutathione-Sepharose affinity column and Sephacryl S-300 chromatography. TLGST-specific activity was found to be 1.56 Umg^-1 which represents 39 folds and 32.2% recovery. The molecular weight of TLGST purified from camel tick larvae was found as 42 kDa by gel filtration. TLGST has a pI value of 6.9 and was found a heterodimeric protein of 28 and 14 kDa subunits as detected on SDS-PAGE. The Lineweaver-Burk plot calculated the km for CDNB to be 0.43 mM with Vmax value of 9.2 Umg^-1. TLGST exhibited its optimal activity at pH 7.9. Co²⁺, Ni²⁺ and Mn²⁺ increased the activity of TLGST while Ca²⁺, Cu²⁺, Fe²⁺ and Zn²⁺ inhibited it. TLGST was inhibited by cumene hydroperoxide, p-hydroxymercuribenzoate, lithocholic acid, hematin, triphenyltin chloride, p-chloromercuribenzoic acid (pCMB), N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), iodoacetamide, EDTA and quercetin. pCMB inhibited TLGST competitively with Ki value of 0.3 mM.

Conclusions: These findings will help to understand the various physiologic conditions of ticks and targeting TLGST could be significant tool for development of prospective vaccines against ticks as a bio-control strategy to overcome the rapid grows in pesticide-resistant tick populations.

Background: Bletilla species are endangered terrestrial orchids used in natural skin care formulas in Asia for a long history. In order to explore the bioactivity potential of Bletilla species as a cosmetic ingredient in a sustainable resource manner, the callus of Bletilla formosana (Hayata) Schltr. was established and extracted by an eco-friendly supercritical fluid CO₂ extraction (SFE-CO₂) method. The intracellular reactive oxygen species (ROS) scavenging activity and antioxidation-related gene expression of the callus extract were evaluated in both Hs68 fibroblast cells and HaCaT keratinocytes. The melanogenesis-inhibitory effect was investigated in B16F10 melanoma cells and in an in vivo zebrafish model.

Results: The calli of B. formosana were propagated for 10-15 generations with a consistent yellow friable appearance and then subjected to SFE-CO₂ extraction to obtain a yellow pasty extract. Obvious intracellular ROS scavenging activity of the extract was detected in both Hs68 and HaCaT cells with 64.30 ± 8.27% and 32.50 ± 4.05% reduction at the concentration of 250 μg/mL. Moreover, marked expression levels of heme oxygenase-1 (HO-1) and (NAD(P)H) quinone oxidoreductase-1 (NQO1) genes were detected after 6-h and 24-h treatments. These results indicate the cellular antioxidative activity of B. formosana callus extract was probably activated via the nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 signaling pathway. Melanogenesis-inhibitory effect of the extract was observed in α-MSH stimuli-inducing B16F10 cells with 28.46% inhibition of intracellular melanin content at the concentration of 50 μg/ml. The effect was confirmed with in vivo zebrafish embryos that showed a relative pigmentation density of 80.27 ± 7.98% at the concentration of 100 μg/mL without toxicity.

Conclusion: Our results shed light on a sustainable utilization of Bletilla species as a potential ingredient for skin.

Background: Saccharomyces cerevisiae (S. cerevisiae) has been demonstrated in vitro to sensitize several breast cancer cell lines and to be a safe, non-toxic drug with anti-skin cancer action in mice. Furthermore, plasmonic photothermal treatment using gold nanorods has been authorized as a novel method for in vitro and in vivo cancer therapy.

Results: When compared to tumor-free rats, the treatment with S. cerevisiae conjugated to gold nanospheres (GNSs) lowered Bcl-2 levels while increasing FasL, Bax, cytochrome c, and caspases 8, 9, and 3 levels. Histopathological results showed changes reflecting the ability of nanogold conjugated heat-killed yeast to induce apoptosis is greater than heat-killed yeast alone as the nanogold conjugated with heat-killed yeast showed no tumor, no hyperplasia, no granulation tissue formation, no ulceration, and no suppuration. Nanogold conjugated with heat-killed yeast-treated breast cancer group displayed normal levels of ALT and AST, indicating relatively healthy hepatic cells.

Conclusion: Our results proved that nanogold conjugated heat-killed yeast can initiate apoptosis and can be used as a safe non-invasive method for breast cancer treatment more effectively than the yeast alone. This, in turn, gives us new insight and a future hope for the first time that breast cancer can be treated by non-invasive, simple, safe, and naturally originated method and achieves a hopeful treatment and a novel method for in vivo cancer therapy.