Cabbage and fermented vegetables : From death rate heterogeneity in countries to candidates for mitigation strategies of severe COVID-19

Large differences in COVID-19 death rates exist between countries and between regions of the same country. Some very low death rate countries such as Eastern Asia, Central Europe, or the Balkans have a common feature of eating large quantities of fermented foods. Although biases exist when examining ecological studies, fermented vegetables or cabbage have been associated with low death rates in European countries. SARS-CoV-2 binds to its receptor, the angiotensin-converting enzyme 2 (ACE2). As a result of SARS-CoV-2 binding, ACE2 downregulation enhances the angiotensin II receptor type 1 (AT(1)R) axis associated with oxidative stress. This leads to insulin resistance as well as lung and endothelial damage, two severe outcomes of COVID-19. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the most potent antioxidant in humans and can block in particular the AT(1)R axis. Cabbage contains precursors of sulforaphane, the most active natural activator of Nrf2. Fermented vegetables contain many lactobacilli, which are also potent Nrf2 activators. Three examples are: kimchi in Korea, westernized foods, and the slum paradox. It is proposed that fermented cabbage is a proof-of-concept of dietary manipulations that may enhance Nrf2-associated antioxidant effects, helpful in mitigating COVID-19 severity.Peer reviewe

Nrf2-interacting nutrients and COVID-19 : time for research to develop adaptation strategies

There are large between- and within-country variations in COVID-19 death rates. Some very low death rate settings such as Eastern Asia, Central Europe, the Balkans and Africa have a common feature of eating large quantities of fermented foods whose intake is associated with the activation of the Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) anti-oxidant transcription factor. There are many Nrf2-interacting nutrients (berberine, curcumin, epigallocatechin gallate, genistein, quercetin, resveratrol, sulforaphane) that all act similarly to reduce insulin resistance, endothelial damage, lung injury and cytokine storm. They also act on the same mechanisms (mTOR: Mammalian target of rapamycin, PPAR gamma:Peroxisome proliferator-activated receptor, NF kappa B: Nuclear factor kappa B, ERK: Extracellular signal-regulated kinases and eIF2 alpha:Elongation initiation factor 2 alpha). They may as a result be important in mitigating the severity of COVID-19, acting through the endoplasmic reticulum stress or ACE-Angiotensin-II-AT(1)R axis (AT(1)R) pathway. Many Nrf2-interacting nutrients are also interacting with TRPA1 and/or TRPV1. Interestingly, geographical areas with very low COVID-19 mortality are those with the lowest prevalence of obesity (Sub-Saharan Africa and Asia). It is tempting to propose that Nrf2-interacting foods and nutrients can re-balance insulin resistance and have a significant effect on COVID-19 severity. It is therefore possible that the intake of these foods may restore an optimal natural balance for the Nrf2 pathway and may be of interest in the mitigation of COVID-19 severity

THE CROSSOVER LINKER. MECHANISMS AND APPLICATIONS IN GENE MODIFICATION

We have developed a novel method for mutating DNA sequences, based on site-specific, in vivo, recombination, known as the crossover linker method. A typical crossover linker contains; (i) a single-stranded overhang for an initial cohesive-end ligation with one terminus of a linearized plasmid, (ii) a mid-section carrying modified sequence information, and (iii) a "homology-searching" sequence at the other end, that is similar to a specific region in the opposite terminus of the plasmid. Following transformation of an E. coli host with a plasmid/linker complex, intramolecular recombination between the homologous regions of the resultant intermediate completes the circularization of the plasmid, with concomitant integration of the linker. Crossover linking is performed on double-stranded DNA and can be used to create deletions andinsertions, as well as to perform site-specific mutagenesis. Both single- and double-stranded linkers with "homology searching" region as short as 5 nucleotides can be used for gene modification. Deletions of over 1000 bp have been achieved using "homology searching" regions of approx. 20 nucleotides in length. In this article, the effectiveness, limitations and mechanism of this process are discussed with emphasis on the application of the crossoverlinker to the manipulation of protein-encoding sequences

Specific degenerate codons enhanced selective expression of human parathyroid hormone in Escherichia coli

Specific degenerate codons in the amino-terminal region of a synthetic human parathyroid hormone (PTH) gene exerted dramatic effects on both products and yield of expression of this 84-amino acid polypeptide in Escherichia coli. With adenine-rich degenerate codons constituting the PTH-(1-5) region, intact PTH has been expressed as the only PTH product at 6.5 mg/liter. In contrast, with guanine-rich degenerate codons, the predominent product was analogue PTH-(8-84). Use of cytosine- or thymine-rich degenerate codons generated only a small amount of immunoreactive product (0.2 mg/l). With the amino terminal region reconstituted with adenine-rich degenerate codons, the mid and carboxyl regions of the synthetic gene were also reconstructed to imitate the E. coli-favored codon degeneracy. Expression yielded the intact PTH at 20 mg/liter. Gel electrophoresis and Western blots, with antibodies specific to the amino or carboxyl terminus of PTH, indicated only a single PTH-related polypeptide, with the same mobility as a synthetic intact PTH sample. Amino acid sequencing, composition analysis, mass spectrometry, and the adenylate cyclase bioassays confirmed the purified product as the processed intact PTH.Peer reviewed: YesNRC publication: Ye

Expression of Trichoderma reesei and Trichoderma viride xylanases in Escherichia coli

Synthetic genes encoding the 190 amino acid Trichoderma reesei xylanase II (TrX) and the closely related Trichoderma viride xylanases have been synthesized in a two-step procedure. Initially, a partial gene encoding amino acids 92-190 was constructed in fusion with the N-terminal half of the Bacillus circulans xylanase (BcX). The remaining BcX gene sequence was replaced during the assembly of the coding sequence for amino acids 1-91. Expression of the synthetic genes in Escherichia coli yielded recombinant xylanases with specific activity generally identical with the natural TrX. However, the recombinant TrX showed thermostability and temperature optimum lower than those of the natural TrX, thus indicating that the posttranslational modifications of the latter in its fungal host are essential to its greater stability. A mutation N19K further decreased the thermostability of the recombinant TrX.NRC publication: Ye