Peer Review #1 of "Characterization of a Mn-SOD from the desert beetle Microdera punctipennis and its increased resistance to cold stress in E. coli cells (v0.2)"

Insects have developed a complex network of enzymatic antioxidant systems for handling reactive oxygen species (ROS) generated during stress. Superoxide dismutases (SODs) play a determinant role in balancing ROS in insect. However, studies devoted to SODs functions in insects under cold stress are limited. In the present study, we attempted to identify and characterize a mitochondrial manganese SOD (mMn-SOD) from the desert beetle Micordera punctipennis (denoted as MpmMn-SOD) and explore its protective effects on bacteria cells under cold stress. MpmMn-SOD is composed of 202 amino acids with conserved domains required for metal ions binding and enzyme activity. RT-qPCR experiments revealed that the expression of MpmMn-SOD was ubiquitous but tissue-specific and was induced by cold stress. An E. coli (BL21) system was applied to study the function of MpmMn-SOD. The MpmMn-SOD gene was cloned into the prokaryotic expression vector pET-32a to generate a recombinant plasmid pET-32a(MpmMn-SOD). After transformation of the plasmid into E. coli BL21, the fusion protein Trx-His-MpmMn-SOD was overexpressed and identified by SDS-PAGE and Western blotting. Antioxidant activity assay showed that the death zones of the transformed bacteria BL21 (pET32a-mMn-SOD) were smaller in diameter than the control bacteria BL21 (pET32a). Survival curves under −4 °C showed that BL21 (pET32a-mMn-SOD) had significant enhanced cold resistance compared to BL21 (pET32a). Its SOD activity under −4 °C had a significant negative correlation (r = − 0.995) with superoxide anion O2•− content. Accordingly, under cold stress BL21 (pET32a-mMn-SOD) had lower electric conductivity and malondialdehyde (MDA) content than BL21 (pET32a). Taken together, our results showed that cold stress stimulated the expression of MpmMn-SOD in M. punctipennis. The E. coli cells that overexpress MpmMn-SOD increase their resistance to cold stress by scavenging ROS, and mitigate potential cell damage caused by ROS under cold conditions.

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Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

10.26434/chemrxiv.7725002.v1 ◽

2019 ◽

Author(s):

Priya Prakash ◽

Travis Lantz ◽

Krupal P. Jethava ◽

Gaurav Chopra

Keyword(s):

Amyloid Beta ◽

Western Blots ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Set Up ◽

Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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Antimicrobial Resistance and Virulence Characterization of E. Coli Isolated from Subclinical Mastitic Sheep and Goats

Benha Veterinary Medical Journal ◽

10.21608/bvmj.2018.44977 ◽

2018 ◽

Vol 34 (3) ◽

pp. 267-278

Author(s):

Ashraf A. Abd El-Tawab ◽

Mohamed G. Aggour ◽

Fatma I. El- Hofy ◽

Marwa M. Y. El- Mesalami

Keyword(s):

Antimicrobial Resistance ◽

Sheep And Goats ◽

E Coli

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Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽

10.1099/mic.0.28753-0 ◽

2006 ◽

Vol 152 (7) ◽

pp. 2129-2135 ◽

Cited By ~ 17

Author(s):

Taku Oshima ◽

Francis Biville

Keyword(s):

Functional Characterization ◽

Anaerobic Growth ◽

Functional Identification ◽

New Members ◽

E Coli ◽

Inner Membrane Protein ◽

Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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