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Author(s):  
Faezeh Abbaszadeh ◽  
Narges Eslami ◽  
Parisa Shiri Aghbash ◽  
Hamed Ebrahimzadeh Leylabadlo ◽  
Hossein Bannazadeh Baghi

: Viral respiratory infections are a leading cause of illness and mortality in all age groups worldwide. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) has spread throughout the world, igniting the twenty-first century’s deadliest pandemic. Research has shown that phages, which are bacterial viruses, can help treat viral infections with the effect on the immune system and their antiviral activity. Phages have specific activity and affect only the target without any side effects on other parts of the human body. Human phage-related diseases have not been reported yet; therefore, phages can be a very safe treatment, especially in many viral infections. The results of clinical studies have a promising future regarding the use of phages. It is possible that the phages display technique aided in the production of SARS-CoV-2 specific antibodies against its viral protein, which prevented the virus from binding or replicating and preventing secondary microbial infections, which have been linked to many patient deaths. Furthermore, an effective antiviral vaccine can be produced by using the same technique. Given the growing number of coronaviruses cases around the world, in the present paper, we review the possible mechanisms of phages against the COVID-19 disease and the method that may be a solution to eliminate the virus.


2021 ◽  
Vol 1 ◽  
Author(s):  
Eun Young Hong ◽  
Sun-Gu Lee ◽  
Hyungdon Yun ◽  
Byung-Gee Kim

Agmatine, involved in various modulatory actions in cellular mechanisms, is produced from arginine (Arg) by decarboxylation reaction using arginine decarboxylase (ADC, EC 4.1.1.19). The major obstacle of using wild-type Escherichia coli ADC (ADCes) in agmatine production is its sharp activity loss and instability at alkaline pH. Here, to overcome this problem, a new disulfide bond was rationally introduced in the decameric interface region of the enzyme. Among the mutants generated, W16C/D43C increased both thermostability and activity. The half-life (T1/2) of W16C/D43C at pH 8.0 and 60°C was 560 min, which was 280-fold longer than that of the wild-type, and the specific activity at pH 8.0 also increased 2.1-fold. Site-saturation mutagenesis was subsequently performed at the active site residues of ADCes using the disulfide-bond mutant (W16C/D43C) as a template. The best variant W16C/D43C/I258A displayed a 4.4-fold increase in the catalytic efficiency when compared with the wild-type. The final mutant (W16C/D43C/I258A) was successfully applied to in vitro synthesis of agmatine with an improved yield and productivity (>89.0% yield based on 100 mM of Arg within 5  h).


Author(s):  
Cecilia Oliver ◽  
German Martinez

AbstractMeiosis is a specialized cell division that is key for reproduction and genetic diversity in sexually reproducing plants. Recently, different RNA silencing pathways have been proposed to carry a specific activity during meiosis, but the pathways involved during this process remain unclear. Here, we explored the subcellular localization of different ARGONAUTE (AGO) proteins, the main effectors of RNA silencing, during male meiosis in Arabidopsis thaliana using immunolocalizations with commercially available antibodies. We detected the presence of AGO proteins associated with posttranscriptional gene silencing (AGO1, 2, and 5) in the cytoplasm and the nucleus, while AGOs associated with transcriptional gene silencing (AGO4 and 9) localized exclusively in the nucleus. These results indicate that the localization of different AGOs correlates with their predicted roles at the transcriptional and posttranscriptional levels and provide an overview of their timing and potential role during meiosis.


2021 ◽  
Vol 25 (7) ◽  
pp. 1163-1166
Author(s):  
J.N. Ozioko ◽  
B.O. Ezema ◽  
K.O. Omeje ◽  
S.O.O. Eze

Peroxidase was extracted from leaves of oil palm tree with 0.01M phosphate buffer pH 7.0. It was partially purified using 70% ammonium sulphate ((NH4)2SO4) precipitation. This resulted in peroxidase with activity of (26U/ml) and specific activity of 35.8U/mg. Effect of heat on the activity of peroxidase was studied at temperature of 323-363°K. After gel filtration on sephadex G100, the peroxidase activity increased to 27U/ml, with specific activity of 55U/mg .The overall purification fold was 4 with 51.9% enzyme recovery. The peroxidase partially purified from leaves of oil palm tree showed pH and temperature optima of 5.0 and 50°C respectively. High pH and temperature stabilities of pH 5.0 to pH 9.0 and 50°C to 70°C were obtained respectively. Also, the activation energy (Ea) of the reaction was - 21.616kj/mol. The free energy changes (ΔG) were 96008.64, 96315.59, 97901.63, 94132.33 and 97146.75kj/mol at 323,333,343,353 and 363°K respectively. It was observed that the D-values were decreasing with increasing temperature with a Z-value of 0.044. The enthalpy results suggest that the reaction was exothermic, non-spontaneous and reversible.


Catalysts ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1410
Author(s):  
Anastasia Sedova ◽  
Lenka Rucká ◽  
Pavla Bojarová ◽  
Michaela Glozlová ◽  
Petr Novotný ◽  
...  

Industries such as mining, cokemaking, (petro)chemical and electroplating produce effluents that contain free cyanide (fCN = HCN + CN−). Currently, fCN is mainly removed by (physico)chemical methods or by biotreatment with activated sludge. Cyanide hydratases (CynHs) (EC 4.2.1.66), which convert fCN to the much less toxic formamide, have been considered for a mild approach to wastewater decyanation. However, few data are available to evaluate the application potential of CynHs. In this study, we used a new CynH from Exidia glandulosa (protein KZV92691.1 designated NitEg by us), which was overproduced in Escherichia coli. The purified NitEg was highly active for fCN with 784 U/mg protein, kcat 927/s and kcat/KM 42/s/mM. It exhibited optimal activities at pH approximately 6–9 and 40–45 °C. It was quite stable in this pH range, and retained approximately 40% activity at 37 °C after 1 day. Silver and copper ions (1 mM) decreased its activity by 30–40%. The removal of 98–100% fCN was achieved for 0.6–100 mM fCN. Moreover, thiocyanate, sulfide, ammonia or phenol added in amounts typical of industrial effluents did not significantly reduce the fCN conversion, while electroplating effluents may need to be diluted due to high fCN and metal content. The ease of preparation of NitEg, its high specific activity, robustness and long shelf life make it a promising biocatalyst for the detoxification of fCN.


PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259837
Author(s):  
Zora Novakova ◽  
Daria Khuntsaria ◽  
Marketa Gresova ◽  
Jana Mikesova ◽  
Barbora Havlinova ◽  
...  

Human protoporphyrinogen oxidase IX (hPPO) is an oxygen-dependent enzyme catalyzing the penultimate step in the heme biosynthesis pathway. Mutations in the enzyme are linked to variegate porphyria, an autosomal dominant metabolic disease. Here we investigated eukaryotic cells as alternative systems for heterologous expression of hPPO, as the use of a traditional bacterial-based system failed to produce several clinically relevant hPPO variants. Using bacterially-produced hPPO, we first analyzed the impact of N-terminal tags and various detergent on hPPO yield, and specific activity. Next, the established protocol was used to compare hPPO constructs heterologously expressed in mammalian HEK293T17 and insect Hi5 cells with prokaryotic overexpression. By attaching various fusion partners at the N- and C-termini of hPPO we also evaluated the influence of the size and positioning of fusion partners on expression levels, specific activity, and intracellular targeting of hPPO fusions in mammalian cells. Overall, our results suggest that while enzymatically active hPPO can be heterologously produced in eukaryotic systems, the limited availability of the intracellular FAD co-factor likely negatively influences yields of a correctly folded protein making thus the E.coli a system of choice for recombinant hPPO overproduction. At the same time, PPO overexpression in eukaryotic cells might be preferrable in cases when the effects of post-translational modifications (absent in bacteria) on target protein functions are studied.


Foods ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2819
Author(s):  
Huibing Chi ◽  
Meirong Chen ◽  
Linshu Jiao ◽  
Zhaoxin Lu ◽  
Xiaomei Bie ◽  
...  

L-asparaginase (E.C.3.5.1.1) is a well-known agent that prevents the formation of acrylamide both in the food industry and against childhood acute lymphoblastic leukemia in clinical settings. The disadvantages of L-asparaginase, which restrict its industrial application, include its narrow range of pH stability and low thermostability. In this study, a novel L-asparaginase from Mycobacterium gordonae (GmASNase) was cloned and expressed in Escherichia coli BL21 (DE3). GmASNase was found to be a tetramer with a monomeric size of 32 kDa, sharing only 32% structural identity with Helicobacter pylori L-asparaginases in the Protein Data Bank database. The purified GmASNase had the highest specific activity of 486.65 IU mg−1 at pH 9.0 and 50 °C. In addition, GmASNase possessed superior properties in terms of stability at a wide pH range of 5.0–11.0 and activity at temperatures below 40 °C. Moreover, GmASNase displayed high substrate specificity towards L-asparagine with Km, kcat, and kcat/Km values of 6.025 mM, 11,864.71 min−1, and 1969.25 mM−1min−1, respectively. To evaluate its ability to mitigate acrylamide, GmASNase was used to treat potato chips prior to frying, where the acrylamide content decreased by 65.09% compared with the untreated control. These results suggest that GmASNase is a potential candidate for applications in the food industry.


AAPS Open ◽  
2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Martin R. Edelmann ◽  
Christophe Husser ◽  
Martina B. Duschmalé ◽  
Guy Fischer ◽  
Claudia Senn ◽  
...  

AbstractA novel approach to tritium-labeled antisense oligonucleotides (ASO) was established by conjugating N-succinimidyl propionate, as well as maleimide-derivatives, to the 3′-end of ASOs targeting metastasis-associated lung adenocarcinoma transcript 1 (Malat1) containing amino- or sulfhydryl-linkers. In vitro stability and Malat1 RNA reduction studies demonstrated that N-ethylmaleimide (NEM) could be used as a stable tag while maintaining the desired target interaction. The corresponding radioactive label conjugation using [3H]-NEM resulted in tritium-labeled ASOs with a high molar specific activity of up to 17 Ci/mmol. Single-dose in vivo studies in mice were carried out to compare [3H]-ASOs with their unlabeled counterpart ASOs, with and without conjugation to N-acetylgalactosamine (GalNAc), for tissue and plasma concentrations time profiles. Despite the structural modification of the labeled ASOs, in vitro target interaction and in vivo pharmacokinetic behaviors were similar to that of the unlabeled ASOs. In conclusion, this new method provides a powerful technique for fast and safe access to tritium-labeled oligonucleotides, e.g., for pharmacokinetic, mass balance, or autoradiography studies. Graphical abstract


2021 ◽  
Author(s):  
Drishtant Singh ◽  
Samiksha . ◽  
Seema Madhumal Thayil ◽  
Satwinder Kaur Sohal ◽  
Anup Kumar Kesavan

Abstract An expression system based on the cry gene regulatory elements was constructed. The Terminator region of cry gene from B. thuringiensis subsp. kurstaki HD-1 was cloned in pSG1151 plasmid downstream to gfpmut1. The promoter region of the cry gene was amplified to give three different reading frames. The Promoter region of cry gene was cloned in pSG1151T plasmid upstream to gfpmut1. The expression of GFP under the promoter/terminator expression system was evaluated by checking the expression of gfpmut1 under the same promoter. The GFP content of pSG1151 and three constructs; pDSA1, pDSA2 and pDSA3 were compared by fluorescence spectroscopy. The fluorescent intensity of pSG1151 and pDSA1 were compared at time interval of 6 hours upto 72 hours. Both the samples showed detectable fluorescence that increased with time up to 12 hours, but the increase in the fluorescence of pDSA1 was 3 times higher as compared to pSG1151. A cold peptidase gene was cloned under the control of the cry promoter. The transformed E.coli DH5α colonies were patched on skim milk agar plates and the clones of pSG1151CP and pDSA1CP were compared on the basis of zone of clearance. The zone of clearance of pDSA1CP was much higher as compared to that of pSG1151CP. The cell-free supernatant of Bacillus sp. S1DI 10 and recombinant pDSA1CP collected at different time points was assayed for the specific activity of the extracellular protease. At 72 hours the protease activity in pDSA1CP was 2.7 fold higher compared to that of wild Bacillus sp. S1DI 10.


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