Abstract mRNA vaccine was approved clinically in 2020. Future development includes delivering mRNA to dendritic cells (DCs) specifically to improve effectiveness and avoid off-target cytotoxicity. Here, we developed virus-like particles (VLPs) as a DC tropic mRNA vaccine vector and showed the prophylactic effects in both SARS-CoV-2 and HSV-1 infection models. The VLP mRNA vaccine elicited strong cytotoxic T cell immunity and durable antibody response with the spike-specific antibodies that lasted for more than 9 months. Importantly, we were able to target mRNA to DCs by pseudotyping VLP with engineered Sindbis virus glycoprotein and found the DC-targeting mRNA vaccine significantly enhanced the titer of antigen-specific IgG, protecting the hACE-2 mice from SARS-CoV-2 infection. Additionally, we showed DC-targeted mRNA vaccine also protected mice from HSV-1 infection when co-delivering the gB and gD mRNA. Thus, the VLP may serve as an in situ DC vaccine and accelerate the further development of mRNA vaccines.
Abstract Background Betaine, an osmoprotective compatible solute, has been used to improve l -threonine production in engineered Escherichia coli l -threonine producer. Betaine supplementation upregulates the expression of zwf encoding glucose-6-phosphate dehydrogenase, leading to the increase of NADPH, which is beneficial for l -threonine production. In E. coli , betaine can be taken through ProP encoded by proP or ProVWX encoded by proVWX. ProP is a H + -osmolyte symporter, whereas ProVWX is an ABC transporter. ProP and ProVWX mediate osmotic stress protection by transporting zwitterionic osmolytes, including glycine betaine. Betaine can also be synthesized in E. coli by enzymes encoded by betABIT . However, the influence of ProP, ProVWX and betABIT on l -threonine production in E. coli has not been investigated. Results In this study, the influence of ProP, ProVWX and betABIT on l -threonine production in E. coli has been investigated. Addition of betaine slightly improved the growth of the l -threonine producing E. coli strain TWF001 as well as the l -threonine production. Deletion of betABIT retarded the growth of TWF001 and slightly decreased the l -threonine production. However, deletion of proP or/and proVWX significantly increased the l -threonine production. When proP was deleted, the l -threonine production increased 33.3%; when proVWX was deleted, the l -threonine production increased 40.0%. When both proP and proVWX were deleted, the resulting strain TSW003 produced 23.5 g/l l -threonine after 36 h flask cultivation. The genes betABIT , proC , fadR , crr and ptsG were individually deleted from TSW003, and it was found that further absence of either crr (TWS008) or ptsG (TWS009) improved l -threonine production. TSW008 produced 24.9 g/l l -threonine after 36 h flask cultivation with a yield of 0.62 g/g glucose and a productivity of 0.69 g/l/h. TSW009 produced 26 g/l l -threonine after 48 h flask cultivation with a yield of 0.65 g/g glucose and a productivity of 0.54 g/l/h, which is 116% increase compared to the control TWF001. Conclusions In this study, l -threonine-producing E. coli strains TSW008 and TSW009 with high l -threonine productivity were developed by regulating the intracellular osmotic pressure. This strategy could be used to improve the production of other products in microorganisms.
Escherichia coli is an important strain for L-threonine production. Genetic switch is a ubiquitous regulatory tool for gene expression in prokaryotic cells. To sense and regulate intracellular or extracellular chemicals, bacteria evolve a variety of transcription factors. The key enzymes required for L-threonine biosynthesis in E. coli are encoded by the thr operon. The thr operon could coordinate expression of these genes when L-threonine is in short supply in the cell.The thrL leader regulatory elements were applied to regulate the expression of genes iclR, arcA, cpxR, gadE, fadR and pykF, while the threonine-activating promoters PcysH, PcysJ and PcysD were applied to regulate the expression of gene aspC, resulting in the increase of L-threonine production in an L-threonine producing E. coli strain TWF001. Firstly, different parts of the regulator thrL were inserted in the iclR regulator region in TWF001, and the best resulting strain TWF063 produced 16.34 g L-threonine from 40 g glucose after 30 h cultivation. Secondly, the gene aspC following different threonine-activating promoters was inserted into the chromosome of TWF063, and the best resulting strain TWF066 produced 17.56 g L-threonine from 40 g glucose after 30 h cultivation. Thirdly, the effect of expression regulation of arcA, cpxR, gadE, pykF and fadR was individually investigated on L-threonine production in TWF001. Finally, using TWF066 as the starting strain, the expression of genes arcA, cpxR, gadE, pykF and fadR was regulated individually or in combination to obtain the best strain for L-threonine production. The resulting strain TWF083, in which the expression of seven genes (iclR, aspC, arcA, cpxR, gadE, pykF, fadR and aspC) was regulated, produced 18.76 g L-threonine from 30 g glucose, 26.50 g L-threonine from 40 g glucose, or 26.93 g L-threonine from 50 g glucose after 30 h cultivation. In 48 h fed-batch fermentation, TWF083 could produce 116.62 g/L L-threonine with a yield of 0.486 g/g glucose and productivity of 2.43 g/L/h.The genetic engineering through the expression regulation of key genes is a better strategy than simple deletion of these genes to improve L-threonine production in E. coli. This strategy has little effect on the intracellular metabolism in the early stage of the growth but could increase L-threonine biosynthesis in the late stage.
This study aimed to explore the novel classification of inpatients with new-onset diabetes in Eastern China by the cluster-based classification method and compare the clinical characteristics among the different subgroups.A total of 1017 Inpatients with new-onset diabetes of five hospitals in Eastern China were included in the study. Clustering analysis was used to cluster the data into five subgroups according to six basic variables. The differences in clinical characteristics, treatments, and the prevalence of diabetes-related diseases among the five subgroups were analyzed by multiple groups comparisons and pairwise comparisons. The risk of diabetes-related diseases in the five subgroups was compared by calculating odd ratio (OR). P value < 0.05 was considered significant.Five subgroups were obtained by clustering analysis with the highest proportion of patients with severe insulin-deficient diabetes (SIDD) 451 (44.35%), followed by patients with mild age-related diabetes (MARD) 236 (23.21%), patients with mild obesity-related diabetes (MOD) 207 (20.35%), patients with severe insulin-resistant diabetes (SIRD) 81 (7.96%), and patients with severe autoimmune diabetes (SAID) 42 (4.13%). Five subtypes had their own unique characteristics and treatments. The prevalence and risk of diabetes-related complications and comorbidities were also significantly different among the five subtypes. Diabetic kidney disease (DKD) was the most common in SIRD group. Patients in SIDD, SIRD, and MARD groups were more likely to develop cardiovascular disease (CVD) and/or stroke, diabetic peripheral vascular disease (DPVD), and diabetic distal symmetric polyneuropathy (DSPN). The prevalence and risk of metabolic syndrome (MS) were the highest in MOD and SIRD groups. Patients in SAID group had the highest prevalence and risk of diabetic ketoacidosis (DKA). Patients with MOD were more likely to develop non-alcoholic fatty liver disease (NAFLD).The inpatients with new-onset diabetes in Eastern China had the unique clustering distribution. The clinical characteristics, treatments, and diabetes-related complications and comorbidities of the five subgroups were different, which may provide the basis for precise treatments of diabetes.
Abstract Escherichia coli is generally used as model bacteria to define microbial cell factories for many products and to investigate regulation mechanisms. E. coli exhibits phospholipids, lipopolysaccharides, colanic acid, flagella and type I fimbriae on the outer membrane which is a self-protective barrier and closely related to cellular morphology, growth, phenotypes and stress adaptation. However, these outer membrane associated molecules could also lead to potential contamination and insecurity for fermentation products and consume lots of nutrients and energy sources. Therefore, understanding critical insights of these membrane associated molecules is necessary for building better microbial producers. Here the biosynthesis, function, influences, and current membrane engineering applications of these outer membrane associated molecules were reviewed from the perspective of synthetic biology, and the potential and effective engineering strategies on the outer membrane to improve fermentation features for microbial cell factories were suggested.
Francisella tularensis subsp. novicida U112 phospholipids, extracted without hydrolysis, consist mainly of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and two lipid A species, designated A1 and A2. These lipid A species, present in a ratio of 7:1, comprise 15% of the total phospholipids, as judged by 32Pi labeling. Although lipopolysaccharide is detectable in F. tularensis subsp. novicida U112, less than 5% of the total lipid A is covalently linked to it. A1 and A2 were analyzed by electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry, gas chromatography/mass spectrometry, and NMR spectroscopy. Both compounds are disaccharides of glucosamine, acylated with primary 3-hydroxystearoyl chains at positions 2, 3, and 2' and a secondary palmitoyl residue at position 2'. Minor isobaric species and some lipid A molecules containing a 3-hydroxypalmitoyl chain in place of 3-hydroxystearate are also present. The 4'- and 3'-positions of A1 and A2 are not derivatized, and 3-deoxy-d-manno-octulosonic acid (Kdo) is not detectable. The 1-phosphate groups of both A1 and A2 are modified with an α-linked galactosamine residue, as shown by NMR spectroscopy and gas chromatography/mass spectrometry. An α-linked glucose moiety is attached to the 6'-position of A2. The lipid A released by mild acid hydrolysis of F. tularensis subsp. novicida lipopolysaccharide consists solely of component A1. F. tularensis subsp. novicida mutants lacking the arnT gene do not contain a galactosamine residue on their lipid A. Formation of free lipid A in F. tularensis subsp. novicida might be initiated by an unusual Kdo hydrolase present in the membranes of this organism.
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