Modified comb-shaped antisense oligonucleotides may secure us against many important viral diseases including AIDS

To inhibit HIV replication and infection, we have designed novel linear single stranded modified antisense nucleic acid oligonucleotides ending with or without chain terminating bases (Which resemble the shape of the comb). They were targeting specifically the HIV-1 clone pNL4-3 strong promoter pre PBS region to stop cDNA synthesis within or before the R region, preventing the viral reverse transcriptase (RT) jumping to the 3' end and continue copying the virus. The main advantages of our comb shaped oligonucleotides are their specificity and extreme protection against resistance by known viral mutations. Promising results were obtained for two 15-mer compounds at one tenth azidothymidine concentration. As a result we claim that when adapted properly, the comb shaped antivirals can be used to target the genomic RNA of a number of serious viruses such as for example Ebola, SARS-CoV-2, Influenza, Dengue, hepatitis C, Chikungunya and Zika as they are all using polymerases to copy their genomic RNA1-8. Their genomic RNA could be destroyed through the human or viral endonucleases instead of the viral RT RNAseH site when their polymerases are stopped at specific sites.

Enhancing glutamine production by optimising the GS-GOGAT pathway in Corynebacterium glutamicum and NH4+-limited fermentation

The GS-GOGAT pathway is a key metabolic pathway of glutamate and glutamine. Optimising this pathway, leading to metabolic flux to glutamine, can increase glutamine production and reduce the production of the by-product glutamate. The NH-limited fermentation process limits the concentration of NH to increase the activity of GS and further increase the yield of glutamine. The GS-GOGAT pathway was optimised by knocking out the GOGAT genes NCgl0181 and NCgl0182 and the glutaminase genes NCgl2395 and NCgl2500 and by integrating a copy of the GS gene glnAbsu from Bacillus subtilis and a copy of the glutamine synthase gene glnAlcb from Lactobacillus acidophilus into the genomic NCgl0182 and NCgl2500 sites. Furthermore, the pXT01 plasmid with the strong promoter tuf was used to overexpress glnAbsu and glnAlcb. To obtain an optimal NH-limited fermentation process, the effects of starting feeding with (NH)SO at different times of fermentation and three (NH)SO feeding strategies on glutamine fermentation were studied, and a NH-limited fermentation process that was the most suitable for glutamine fermentation was determined. After optimising the GS-GOGAT pathway, Corynebacterium glutamicum G-6 was subjected to the NH-limited fermentation process to greatly increase the production of glutamine. The yield of glutamine reached 98.7 g/L, which was 104.8% higher than that in the original strain GM34; the content of glutamate reached 4.5 g/L, which then decreased by 85.2%; the GS activity increased significantly, and the sugar-acid conversion rate reached 41.2%.

Enhancing glutamine production by optimising the GS-GOGAT pathway in Corynebacterium glutamicum and NH4+-limited fermentation

The GS-GOGAT pathway is a key metabolic pathway of glutamate and glutamine. Optimising this pathway, leading to metabolic flux to glutamine, can increase glutamine production and reduce the production of the by-product glutamate. The NH-limited fermentation process limits the concentration of NH to increase the activity of GS and further increase the yield of glutamine. The GS-GOGAT pathway was optimised by knocking out the GOGAT genes NCgl0181 and NCgl0182 and the glutaminase genes NCgl2395 and NCgl2500 and by integrating a copy of the GS gene glnAbsu from Bacillus subtilis and a copy of the glutamine synthase gene glnAlcb from Lactobacillus acidophilus into the genomic NCgl0182 and NCgl2500 sites. Furthermore, the pXT01 plasmid with the strong promoter tuf was used to overexpress glnAbsu and glnAlcb. To obtain an optimal NH-limited fermentation process, the effects of starting feeding with (NH)SO at different times of fermentation and three (NH)SO feeding strategies on glutamine fermentation were studied, and a NH-limited fermentation process that was the most suitable for glutamine fermentation was determined. After optimising the GS-GOGAT pathway, Corynebacterium glutamicum G-6 was subjected to the NH-limited fermentation process to greatly increase the production of glutamine. The yield of glutamine reached 98.7 g/L, which was 104.8% higher than that in the original strain GM34; the content of glutamate reached 4.5 g/L, which then decreased by 85.2%; the GS activity increased significantly, and the sugar-acid conversion rate reached 41.2%.

Systematic identification of a panel of strong promoter regions from Listeria monocytogenes for fine-tuning gene expression

Background Attenuated Listeria monocytogenes (Lm) has been widely used as a vaccine vector in the prevention and treatment of pathogen infection and tumor diseases. In addition, previous studies have proved that the attenuated Lm can protect zebrafish from Vibrio infections, indicating that the attenuated Lm has a good application prospect in the field of aquatic vaccines. However, the limitation mainly lies in the lack of a set of well-characterized natural promoters for the expression of target antigens in attenuated Lm. Results In our study, candidate strong promoters were identified through RNA-seq analysis, and characterized in Lm through enhanced green fluorescent protein (EGFP). Nine native promoters that showed stronger activities than that of the known strong promoter P36 under two tested temperatures (28 and 37 °C) were selected from the set, and P29 with the highest activity was 24-fold greater than P36. Furthermore, we demonstrated that P29 could initiate EGFP expression in ZF4 cells and zebrafish embryos. Conclusions This well-characterized promoter library can be used to fine-tune the expression of different proteins in Lm. The availability of a well-characterized promoter toolbox of Lm is essential for the analysis of yield increase for biotechnology applications.

Managing career plateau: case of Aster Retail, UAE

Purpose The purpose of this paper is to discuss how Aster Retail (AR), UAE, handled career plateau challenge by adopting multiple strategies and earning employee commitment and motivation for business growth. Design/methodology/approach The organization addressed two types of plateaus – structural and content by creating both vertical and lateral opportunities/options for employees, and supporting them with resources to build required capabilities, and managing their career aspirations. The strategies also helped AR to remain true to the organization’s philosophy, “We will treat you well.” Findings The study enunciates how HR initiatives can add value by converting the negative phenomenon of plateau, into an opportunity for employees to grow. Originality/value The study has three contributions: How in a retail organization with strong promoter principles and values, both structural and content plateau are addressed, and linked with business strategies? The study sheds light on how organizational and HR support for career management addresses employee plateau, particularly for solid citizens. makes the employees feel “not plateaued” at all; and in the long run, why and how HR managers should focus more on proactively addressing content plateau than structural plateau.

Targeted Transgene Expression in Rice Using a Callus Strong Promoter for Selectable Marker Gene Control

Precise expression of a transgene in the desired manner is important for plant genetic engineering and gene function deciphering, but it is a challenge to obtain specific transgene expression free from the interference of the constitutive promoters used to express the selectable marker gene, such as the Cauliflower mosaic virus (CaMV) 35S promoter. So, the solutions to avoid these inappropriate regulations are largely demanded. In this study, we report the characterization of a callus strong promoter (CSP1) in rice and its application for accurate transgene expression. Our results indicate that the high expression of the CSP1 promoter in the callus enables efficient selection of hygromycin equivalent to that provided by the CaMV 35S promoter, whereas its expression in other tissues is low. To evaluate possible leaky effects, the expression of a β-glucuronidase reporter driven by six specific promoters involving hormone signaling, pathogen response, cell fate determination, and proliferation was observed in transgenic rice plants generated by CSP1-mediated selection. Distinct β-glucuronidase expression was found consistently in most of the transgenic lines obtained for each promoter. In addition, we applied these specific marker lines to investigate the root cellular responses to exogenous cytokinin and auxin treatment. The results reveal that the root growth inhibition by cytokinin was differently regulated at high and low concentrations. In summary, we have established the feasibility of using callus-specific promoter-dependent selection to mitigate the transgene misexpression in rice. By enabling efficient transformation, rice plants with reliable transgene expression will be easily acquired for broad applications.

Increasing Yield of 2,3,5,6-Tetramethylpyrazine in Baijiu Through Saccharomyces cerevisiae Metabolic Engineering

Baijiu is a traditional distilled beverage in China with a rich variety of aroma substances. 2,3,5,6-tetramethylpyrazine (TTMP) is an important component in Baijiu and has the function of promoting cardiovascular and cerebrovascular health. During the brewing of Baijiu, the microorganisms in jiuqu produce acetoin and then synthesize TTMP, but the yield of TTMP is very low. In this work, 2,3-butanediol dehydrogenase (BDH) coding gene BDH1 and another BDH2 gene were deleted or overexpressed to evaluate the effect on the content of acetoin and TTMP in Saccharomyces cerevisiae. The results showed that the acetoin synthesis of strain α5-D1B2 was significantly enhanced by disrupting BDH1 and overexpressing BDH2, leading to a 2.6-fold increase of TTMP production up to 10.55 mg/L. To further improve the production level of TTMP, the α-acetolactate synthase (ALS) of the pyruvate decomposition pathway was overexpressed to enhance the synthesis of diacetyl. However, replacing the promoter of the ILV2 gene with a strong promoter (PGK1p) to increase the expression level of the ILV2 gene did not result in further increased diacetyl, acetoin and TTMP production. Based on these evidences, we constructed the diploid strains AY-SB1 (ΔBDH1:loxP/ΔBDH1:loxP) and AY-SD1B2 (ΔBDH1:loxP-PGK1p-BDH2-PGK1t/ΔBDH1:loxP-PGK1p-BDH2-PGK1t) to ensure the fermentation performance of the strain is more stable in Baijiu brewing. The concentration of TTMP in AY-SB1 and AY-SD1B2 was 7.58 and 9.47 mg/L, respectively, which represented a 2.3- and 2.87-fold increase compared to the parental strain. This work provides an example for increasing TTMP production in S. cerevisiae by genetic engineering, and highlight a novel method to improve the quality and beneficial health attributes of Baijiu.

Genetic Heterogeneity of KLF1, a Master Regulator of Erythropoiesis, Revealed an Autosomal Recessive Ψβ-Thalassemia and a Very Strong Promoter In Vivo

The Erythroid Kruppel-like Factor 1 or KLF1 is a transcription factor that functioned in the early stage genetic programming of erythroid progenitors to promote physiological γ to β globin gene switching. Indeed, we showed that a truncation mutation (p.K288X) in KLF1 that deleted the DNA binding zinc finger domain resulted in marked KLF1 deficiency and a hematological condition that resembled a hereditary persistence of fetal hemoglobin (HbF) or a β thalassemia. Here, we describe five additional families with the same p.K288X mutation but varied hematological and HbF levels together with genetic and phenotypic data on a 600 data-set from the same Maltese population. The data accounted for a strong promoter embedded within a region of genetic heterogeneity of KLF1 that led to a ψβ thalassemia. Whole genome sequencing on 15 subjects of six families (FamF1 - FamF6) segregating (two) p.K288X frameworks of KLF1 had variable degrees of microcytosis (MCV; 76.1fL -77.4 fL) and HbF levels (HbF 2480 mg/dL - 802mg/dL) due to complex heterozygosity between promoter, coding and truncating mutations in KLF1. Case II-6 of FamF1 with the highest HbF (2480 mg/dL) had 2 promoter and 2 coding mutations in cis and in trans to the p.K288X truncation. Nine (9) KLF1 frameworks (A - I) were derived by transmission disequilibrium analyses of the family data, each assembled from 15 mutations and resulting in 7 genotypes among the families. The p.K288X truncation was found on a second rarer framework. Additional, rarer KLF1 frameworks were found with haploview in the population dataset. The population dataset was made up of 198 β thalassemia heterozygotes and 400 others from the clinic and the biobank and that had no β globin gene mutations, variable blood counts or hemoglobin profiles or both. They were older than 2 years of age, not pregnant and had normal iron levels. The number of KLF1 mutations differed from 0 in the wild-type framework A to 6 in one of the rare frameworks X. Six mutations were in the promoter region and 6 were in the coding region that defined a "KLF1 Variable Region" 5' genomic coordinate 12887183 - 12888273, whereas very few were found in the 3' (genomic coordinate 12884589 - 12884752) that defined the KLF1 "Constant Region" The Constant region has also been evolutionary conserved. It included the zinc finger domain and the proteasome binding site. The genotype - phenotype correlations and the family data were consistent with an autosomal recessive condition that resembled a β thalassemia, thus a ψβ thalassemia. It differed from a silent thalassemia because the β globin gene sequence was wild type. It provided a diagnosis for families with iron resistant microcytosis and borderline hemoglobin phenotypes suitable for counselling in the clinical setting. It was consistent with the observation among the families regarding the high strength of the KLF1 promoter. Multiple "hits" were necessary to suppress the biosynthesis of KLF1 for hemoglobin switching to escape perinatal suppression. The effect of the 6 promoter mutations were confirmed in vitro with native and induced K562 and Hek293T cell lines. Presumably, during normal development, the strong promoter served to rapidly drown KLF1 binding sites with KLF1 molecules to direct progenitors to erythropoiesis with the appropriate adult hemoglobin profile in the perinatal period. The diagnosis of the patients with selected genotypes due to compound and double heterozygosities in promoter and coding sequences shall further permit quantification of differential promoter function in vivo. Figure Disclosures No relevant conflicts of interest to declare.

Questions and a myriad answers: Coming together and drifting apart in the historical sciences

There is no end to the questions you can ask, and no end to the answers you can give. Where then, in this space of endless possibilities, can research begin; and how can researchers be expected to reach any consensus on what are useful question-answer-pairs? This present article recounts the story of Sigfried Giedion and Bruno Zevi. Space, Time and Architecture, a book printed at Harvard University ties the fates of the two Europeans. Giedion is the author, Zevi is a reader surrounded by a transatlantic group of followers. Initially a strong promoter of Giedion's book, Zevi later changed his mind and went on to propose his own, divergent theory of space and architecture. Zevi and Giedion's story of coming together and drifting apart is not unique. We all live in a world in which ideas spread and diversify as people search for questions and a myriad answers.