Recombinant Gene Research Articles

Oral Delivery of miR146a Conjugated to Cerium Oxide Nanoparticles Improves an Established T Cell-Mediated Experimental Colitis in Mice

Background: Dysregulated inflammation and oxidative stress are strongly implicated in the pathogenesis of inflammatory bowel disease. We have developed a novel therapeutic that targets inflammation and oxidative stress. It is comprised of microRNA-146a (miR146a)-loaded cerium oxide nanoparticles (CNPs) (CNP-miR146a). We hypothesized that oral delivery of CNP-miR146a would reduce colonic inflammation in a mouse model of established, chronic, T cell-mediated colitis. Methods: The stability of CNP-miR146a and mucosal delivery was assessed in vitro with simulated gastrointestinal fluid and in vivo after oral gavage by quantitative real-time RT-PCR. The efficacy of orally administered CNP-miR146a was tested in mice with established colitis using the model of adoptive naïve T-cell transfer in recombinant activating gene 2 knockout (Rag2−/−) mice. Measured outcomes included histopathology; CD45+ immune cell infiltration; oxidative DNA damage (tissue 8-hydroxy-2′-deoxyguanosine; 8-OHdG); expression of IL-6 and TNF mRNA and protein, and flow cytometry analysis of lamina propria Th1 and Th17 cell populations. Results: miR146a expression remained stable in simulated gastric and intestinal conditions. miR146a expression increased in the intestines of mice six hours following oral gavage of CNP-miR146a. Oral delivery of CNP-miR146a in mice with colitis was associated with reduced inflammation and oxidative stress in the proximal and distal colons as evidenced by histopathology scoring, reduced immune cell infiltration, reduced IL-6 and TNF expression, and decreased populations of CD4+Tbet+IFNg+ Th1, CD4+RorgT+IL17+ Th17, as well as pathogenic double positive IFNg+IL17+ T cells. Conclusions: CNP-miR146a represents a novel orally available therapeutic with high potential to advance into clinical trials.

A Comprehensive Review of Pfu DNA Polymerase: Extraction, Production and Applications

Pfu DNA Polymerase, a thermostable enzyme derived from the hyperthermophilic archaeon Pyrococcus furiosus, is widely recognized for its high fidelity and strong processivity. Its 3'-5' exonuclease activity makes it indispensable for the correct amplification of both short and complex DNA strands. These biochemical properties of Pfu DNA Polymerase have encouraged considerable advancements in its extraction and production methods. This review covers some of the traditional approaches for purification, including protein purification and affinity chromatography, and updates on recent advances in recombinant gene expression, automated systems of production, and membrane-based technologies. New ways of engineering enzymes have recently been developed, such as CRISPR-Cas9-mediated gene optimization, which raised the bar on extraction efficiency to meet emerging demand. Once challenging, Pfu DNA Polymerase production has been significantly streamlined through recombinant expression in E. coli both at a laboratory and commercial scale. The optimization techniques that are involved with IPTG concentration and Response Surface Methodology have increased the yield by up to 30%. Auto-induction means even higher biomass outputs are allowed. Today, applications of Pfu DNA Polymerase span from standard PCR up to advanced clinical diagnostics in the fields of molecular biology, forensic analysis, clinical microbiology, and biotechnology.

Homologous recombination deficiency in pancreatic neuroendocrine tumors.

Aim: Pancreatic Neuroendocrine tumors (pNETs) are a heterogeneous group of neoplasms whose tumor biology is still little known. Thanks to next-generation sequencing, pathogenic mutations in base-excision-repair MUTYH gene and homologous recombination genes CHEK2 and BRCA2 seem to have a role in the development of pNETs.Research design & methods: We assumed that Homologous Recombination Deficiency (HRD) could be a critical pathogenetic mechanism for pNETs. We evaluated the HR status in a case series of 33 patients diagnosed with pNET at the Modena Cancer Center using the AmoyDX HRD Focus assay.Results: The AmoyDx test did not identify any HRD-positive patients (median GSS equal to 1.1, positive score: >50), and no pathogenic BRCA variants were detected. However, thanks to the SNP analysis, a consistent number of partial or complete single-copy deletions or duplications in several chromosomes.Conclusion: The AmoyDX HRD focus assay performed well on pancreatic samples, despite being originally designed for ovarian cancer and used on samples stored for over a year. Larger studies are needed to further assess the role of HRD assays in pNETs research.

Abstract 4124039: Aging-Associated Protein Medin Induces Human Coronary Artery Endothelial Proinflammatory and Prothrombotic Activation

Background: Age is the most important risk factor for coronary artery disease (CAD), independent of traditional risk factors such as hypertension, diabetes (DM), hyperlipidemia and smoking, through poorly understood mechanisms. Medin, a cleavage product of MFGE8 protein that forms the most common human amyloid, accumulates in the vasculature with age, including the coronary arteries. Although medin amyloid has been implicated in various diseases such as Alzheimer’s disease, vascular dementia and aortic aneurysms, its role in CAD remains unknown. Medin was shown to induce changes characteristic of vascular aging, such as endothelial and smooth muscle dysfunction in human donor cerebral arteries. Medin also induced proinflammatory activation of human brain microvascular endothelial cells through NFκB activation . Aims: To determine the effects of medin on pro-inflammatory and prothrombotic activation of human coronary artery endothelial cells (HCAECs). Methods: Primary HCAECs (passages 6-8) were exposed for 20 hours to physiologic doses of recombinant medin (0.5, 1 and 5 µM) and gene expression of pro-inflammatory proteins (IL-6, IL-8, IL-1b and intercellular adhesion molecule (ICAM)-1), prothrombotic protein (plasminogen activator inhibitor (PAI)-1) and anticoagulant membrane protein thrombomodulin were quantified using rtPCR and compared. Separately, HCAECs were exposed to medin (0.5, 1 and 5 µM) with or without small molecule specific inhibitor of NFκB (RO106-9920, 10 uM) for 20 hours and protein expression of IL-6 in conditioned media was measured using ELISA. Results: Medin caused dose-dependent increases in gene expressions of pro-inflammatory cytokines IL-6, IL-8, IL-1b, ICAM-1 (Figure 1 A-D). Medin caused a dose-dependent increase in PAI-1 and a dose-dependent decrease in thrombomodulin (Fig. 1 E-F). Co-treatment with RO106-9920 prevented medin-induced increase in IL-6 protein expression (Fig. 1G). Conclusions: Medin induces pro-inflammatory and prothrombotic activation of human coronary artery endothelial cells. The proinflammatory activation is likely NFκB-dependent. Medin is a promising potential novel mediator of CAD and vascular aging.

An In Vitro RNA Editing-Based Reporter Assay for Transcriptional Activity of Therapeutic Gene in Gene Therapy Products.

The expression of therapeutic genes is critical for the efficacy of gene therapy products. However, existing methods such as immunological analysis at the protein level or reverse-transcription PCR at the RNA level are unable to accurately quantify the expression activity of the target gene. Herein, an in vitro RNA editing-based reporter assay was developed to detect specific mRNA. The designed sensor RNA could specifically identify the target mRNA, and the reporter gene was activated in a dose-dependent manner because of RNA editing mediated by endogenous adenosine deaminases acting on RNA. Of note, all sensors that targeted different regions, including the gene of interest, tag sequence, and 3' untranslated region, showed a dose-dependent response pattern. The sensor reporter assay, which was used for quantifying the transcriptional activity of recombinant adeno-associated virus-based gene therapy products, revealed excellent performance in terms of assay specificity, precision (inter-assay relative standard deviation < 15%), accuracy (90-115% recovery), and linearity (R2 > 0.99). The reporter assay could also be employed for other gene therapy vectors, including mRNA and recombinant lentivirus. Thus, a robust and reliable platform was developed for assessing the transcriptional activity of therapeutic genes, thereby offering a powerful tool for the quality control of gene therapy products.

Link Between Individual Codon Frequencies and Protein Expression: Going Beyond Codon Adaptation Index.

An important role of a particular synonymous codon composition of a gene in its expression level is well known. There are a number of algorithms optimizing codon usage of recombinant genes to maximize their expression in host cells. Nevertheless, the underlying mechanism remains unsolved and is of significant relevance. In the realm of modern biotechnology, directing protein production to a specific level is crucial for metabolic engineering, genome rewriting and a growing number of other applications. In this study, we propose two new simple statistical and empirical methods for predicting the protein expression level from the nucleotide sequence of the corresponding gene: Codon Expression Index Score (CEIS) and Codon Productivity Score (CPS). Both of these methods are based on the influence of each individual codon in the gene on the overall expression level of the encoded protein and the frequencies of isoacceptors in the species. Our predictions achieve a correlation level of up to r = 0.7 with experimentally measured quantitative proteome data of Escherichia coli, which is superior to any previously proposed methods. Our work helps understand how codons determine protein abundances. Based on these methods, it is possible to design proteins optimized for expression in a particular organism.

Establishing the green algae Chlamydomonas incerta as a platform for recombinant protein production.

Chlamydomonas incerta, a genetically close relative of the model green alga Chlamydomonas reinhardtii, shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii, this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta's capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii, C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii. For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii, as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incerta's potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.

The decrease in Rad51 and DNA ligase IV nuclear protein expression in Msh2 knockdown HC11 cells induced the low CRISPR/Cas9-mediated knock-in efficiency at the β-casein gene locus.

Successful gene editing technology is crucial in molecular biology and related fields. An essential part of an efficient knock-in system is increasing homologous recombination (HR) efficiency in the double-strand break (DSB) repair pathways. Interestingly, HR is closely related to the DNA mismatch repair (MMR) pathway, whereby MMR-related gene Msh2 recognizes a mismatch of nucleotides in recombinant intermediates or gene conversion formed during HR. This study aimed to investigate how the knockdown of Msh2 affects HR-mediated knock-in efficiency at the mouse β-casein locus. Therefore, we investigated the effect of inhibiting Msh2 expression on the expression of the HR-related gene Rad51 and the key enzyme DNA ligase IV involved in non-homologous end joining (NHEJ). The knock-in vector targeting the mouse β-casein gene locus, programmed guide RNA, and Msh2 siRNA expression vector were co-transfected in HC11 cells, or only the Msh2 siRNA expression vector was transfected. Knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA and protein expression of Msh2, HR-related gene Rad51, and NHEJ-related gene DNA ligase IV were evaluated by quantitative reverse transcription PCR (RT-qPCR) and Western blot analysis. The knock-in vector efficiency at the mouse β-casein gene locus significantly decreased upon Msh2 knockdown in HC11 mouse mammary epithelial cells (HC11 cell). Additionally, the knockdown of the DNA MMR-related gene Msh2 protein significantly downregulated the nuclear protein expression of the HR-related Rad51 and NHEJ-related DNA ligase IV genes. The decreased Msh2 protein expression in the nucleus downregulated the Rad51 and ligase IV protein expressions. Consequently, reduced Rad51 expression results in decreased knock-in efficiency.

Development of engineered IL-36γ-hypersecreting Lactococcus lactis to improve the intestinal environment

Interleukin (IL) 36 is a member of the IL-1-like proinflammatory cytokine family that has a protective role in mucosal immunity. We hypothesized that mucosal delivery of IL-36γ to the intestine would be a very effective way to prevent intestinal diseases. Here, we genetically engineered a lactic acid bacterium, Lactococcus lactis, to produce recombinant mouse IL-36γ (rmIL-36γ). Western blotting and enzyme-linked immunosorbent assay results showed that the engineered strain (NZ-IL36γ) produced and hypersecreted the designed rmIL-36γ in the presence of nisin, which induces the expression of the recombinant gene. We administered NZ-IL36γ to mice via oral gavage, and collected the ruminal contents and rectal tissues. Colony PCR using primers specific for NZ-IL36γ, and enzyme-linked immunosorbent assay to measure the rmIL-36γ concentrations of the ruminal contents showed that NZ-IL36γ colonized the mouse intestines and secreted rmIL-36γ. A microbiota analysis revealed increased abundances of bacteria of the genera Acetatifactor, Eubacterium, Monoglobus, and Roseburia in the mouse intestines. Real-time quantitative PCR of the whole colon showed increased Muc2 expression. An in vitro assay using murine colorectal epithelial cells and human colonic cells showed that purified rmIL-36γ promoted Muc2 gene expression. Taken together, these data suggest that NZ-IL36γ may be an effective and attractive tool for delivering rmIL-36γ to improve the intestinal environment.

Global prevalence of Mycobacterium massiliense strains with recombinant rpoB genes (Rec-Mas) horizontally transferred from Mycobacterium abscessus: two major types, dominant circulating clone 7 and MLST ST46 sequence type.

Horizontal gene transfer (HGT) events play a pivotal role in the evolution of Mycobacterium abscessus into dominant circulating clones (DCCs), which is capable of causing patient-to-patient transmission. In particular, HGT of the rpoB gene between strains of different subspecies of M. abscessus could also compromise differentiation between strains of M. abscessus. Here, for the first time, using 1,786 M. abscessus genome sequences, we evaluated the global prevalence of M. abscessus strains subjected to rpoB HGT. We found a greater prevalence of M. abscessus subjected to rpoB HGT than to those subjected to hsp65 HGT, which is mainly due to two Rec-mas clones, dominant circulating clone 7 and ST46, which are responsible for dissemination between non-CF patients in Asia. Our data highlight the importance of rpoB HGT in the evolution of M. abscessus, particularly Mycobacterium massiliense, into virulent DCC clones.

Immunogenicity of Brucella Trivalent Immunogen-Containing Polyethyleneimine Nanostructure Targeted with LPS in a Mouse Model.

Brucella is a facultative intracellular gram-negative coccobacillus. It is nonsporulating and reproduced in macrophage phagosomes. The use of nanostructures as drug and vaccine carriers has recently received attention due to their ability to control the release profile and protect the drug molecules. This study presents a suitable nano-polyethyleneimine formulation to be used as an immunoadjuvant and LPS along with trivalent candidate antigens of TF, BP26, and omp31 to selectively stimulate the immune response. After designing and evaluating the immunogenic structure by databases and bioinformatics software, recombinant protein cloning and gene expression were performed in Escherichia coli BL21 bacteria. This protein was extracted from the cultured cells, purified by Ni-NTA column. After placing the antigen inside the polyethyleneimine nanostructure, various properties of the nanoparticles, including their size, zeta potential, and retention rate for injection and inhalation of mice, diffusion efficacy, and antigen binding evaluation were evaluated. Mice were treated with different groups of antigens and nanoparticles on days 0, 10, 24, and 38. Two weeks after the last injection, the level of cytokines were investigated in spleen cells, including IFN-γ, IL-4, and IL-12. The serum concentration of IgG2a and IgG1 antibodies were also assessed. The response was consistent with significant production of IgG1, IgG2a, IFN-γ21, IL-12, and IL-4 compared to the controls (P < 0.05). Compared to the positive and negative control groups, recombinant protein and nanoparticles showed a good response in subsequent injections with live bacterial strains. The present study also revealed the potential of the developed recombinant protein as a candidate in the design and manufacture of subunit vaccines against Brucella species. This protein stimulates cellular and humoral immune responses compared to the positive control groups. These findings can be useful in the prevention and control of brucellosis and pave the way for further research by researchers around the world.

Escherichia coli Reporter Strains Allow for the In Vivo Evaluation of Recombinant Elongation Factor Protein (EF-P)

Background: Elongation factor protein (EF-P) in bacteria helps ribosomes to incorporate contiguous proline residues (xPro) into proteins. In this way, EF-P rescues ribosomes from stalling at these xPro motifs. Whereas EF-P deficiency is lethal for some species, others show reduced virulence or generally lower growth rates, such as Escherichia coli (E. coli). EF-P needs to be post-translationally modified to gain full functionality. Methods: We constructed E. coli K-12 mutant strains with deletion of the serA gene leading to an auxotrophy for L-serine. Then, we engineered a 6xPro motif in the recombinant serA gene, which was then chromosomally inserted under its native promoter. Furthermore, mutant strains which were deleted for efp and/or epmA (encoding the EF-P modification protein EpmA) were engineered. Results: Δefp, ΔepmA, and Δefp/ΔepmA double mutants showed already significantly reduced growth rates in minimal media. ΔserA derivatives of these strains were complemented by the wt serA gene but not by 6xPro-serA. ΔserA mutants with intact efp were complemented by all serA-constructs. Chromosomal expression of the recombinant efp gene from E. coli or from the pathogen, Staphylococcus aureus (S. aureus), restored growth, even without epmA expression. Conclusions: We provide a novel synthetic reporter system for in vivo evaluation of EF-P deficiency. In addition, we demonstrated that both EF-P-E. coli and EF-P-S. aureus restored the growth of a 6xPro-serA: Δefp, ΔepmA strain, which is evidence that modification of EF-P might be dispensable for rescuing of ribosomes stalled during translation of proline repeats.

Evolution of the correlated genomic variation landscape across a divergence continuum in the genus Castanopsis.

The heterogeneous landscape of genomic variation has been well documented in population genomic studies. However, disentangling the intricate interplay of evolutionary forces influencing the genetic variation landscape over time remains challenging. In this study, we assembled a chromosome-level genome for Castanopsis eyrei and sequenced the whole genomes of 276 individuals from 12 Castanopsis species, spanning a broad divergence continuum. We found highly correlated genomic variation landscapes across these species. Furthermore, variations in genetic diversity and differentiation along the genome were strongly associated with recombination rates and gene density. These results suggest that long-term linked selection and conserved genomic features have contributed to the formation of a common genomic variation landscape. By examining how correlations between population summary statistics change throughout the species divergence continuum, we determined that background selection alone does not fully explain the observed patterns of genomic variation; the effects of recurrent selective sweeps must be considered. We further revealed that extensive gene flow has significantly influenced patterns of genomic variation in Castanopsis species. The estimated admixture proportion correlated positively with recombination rate and negatively with gene density, supporting a scenario of selection against gene flow. Additionally, putative introgression regions exhibited strong signals of positive selection, an enrichment of functional genes, and reduced genetic burdens, indicating that adaptive introgression has played a role in shaping the genomes of hybridizing species. This study provides insights into how different evolutionary forces have interacted in driving the evolution of the genomic variation landscape.

Affibody-based molecular probe 99mTc-(HE)3ZHER2:V2 for non-invasive HER2 detection in ovarian and breast cancer xenografts.

This study aimed to assess the biodistribution and bioactivity of the affibody molecular probe 99mTc-(HE)3ZHER2:V2, prepared by genetic recombination, and to investigate its potential for targeted human epidermal growth factor receptor 2 (HER2) imaging in SKOV3 ovarian cancer and MDA-MB-361 breast cancer xenografts. Affibody molecules were generated through genetic recombination. The radiochemical purity of the 99mTc-labeled HER2 affibody was determined using reverse phase high performance liquid chromatography (RP-HPLC). Evaluation of HER2 affinity in SKOV3 ovarian cancer cells and MDA-MB-361 breast cancer cells (HER2-positive) was conducted by calculating equilibrium dissociation constants. Biodistribution of the 99mTc-labeled affibody molecular probe was assessed in Balb/c mice bearing SKOV3 tumors. Tumor targeting specificity was evaluated in Balb/c mice using SKOV3, MDA-MB-361, and AT-3 (HER2-negative) xenografts. Affibody (HE)3ZHER2:V2, generated through recombinant gene expression, was successfully labeled with 99mTc, achieving a radiochemical purity of (96.0 ± 1.7)% (n = 3) as determined by RP-HPLC. This molecular probe exhibited specific binding to HER2-positive SKOV3 cells, demonstrating intense radioactive uptake. Biodistribution analysis showed rapid accumulation of 99mTc-(HE)3ZHER2:V2 in HER2-positive tumors post-administration, primarily clearing through the urinary system. Single-photon emission computed tomography imaging conducted 1-3 h after intravenous injection of 99mTc-(HE)3ZHER2:V2 into HER2-positive SKOV3 and MDA-MB-361 nude mouse models confirmed targeted uptake of the molecular probe by the tumors. The molecular probe 99mTc-(HE)3ZHER2:V2 developed in this study effectively targets HER2 for imaging HER2-positive SKOV3 and MDA-MB-361 xenografts in vivo. It exhibits rapid blood clearance without evident toxic effects, suggesting its potential as a valuable marker for detecting HER2 expression in tumor cells.

A marker-free genetic manipulation method for Glaesserella parasuis strains developed by alternately culturing transformants at 37°C and 30°C

BackgroundGlaesserella parasuis (G. parasuis) is the causative agent of Glässer’s disease, which causes significant economic losses in the swine industry. However, research on the pathogenesis of G. parasuis has been hampered by the lack of a simple and efficient marker-free knockout system.ResultsIn this study, a marker-free knockout system was developed for G. parasuis using a temperature-sensitive vector. By alternating the incubation of transformants at 30°C and 37°C, we optimized the screening process for this system. The system was successfully applied to knockout the KanR cassette from JS0135ΔnanH::KanR, achieving a knockout efficiency of 90% in the final round of screening. To confirm that temperature variation was a key factor, we proceeded with knocking out the nanH and apd genes in the CF7066 strain. The knockout efficiency reached up to 100%, with the shortest screening time being only four days. The knockout of the nanH gene resulted in a significant reduction in the growth vitality of the strains, while the knockout of the apd gene led to an approximate 56% improvement in the adhesion rate. Additionally, we observed that the expression of recombinant genes in transformants was higher at 30℃ than at 37℃, with the recC gene being upregulated approximately 7-fold. In contrast, there was almost no difference in the expression of recombinant genes between 30℃ and 37℃ in the wild-type strains. This discrepancy was likely due to an elevated copy number of target plasmids at 30℃, which may have resulted in the enhanced expression of recombinant genes.ConclusionsIn conclusion, this newly developed gene knockout system for G. parasuis presents a valuable tool for advancing research on this organism.

Gene editing by SSB/CRISPR-Cas9 ribonucleoprotein in bacteria

The application of CRISPR-Cas9 ribonucleoprotein (RNP) for gene editing is commonly used in plants and animals, but its application in bacteria has not been reported. In this study, we employed DNA single-strand binding protein (SSB) to construct an SSB/CRISPR-Cas9 RNP-editing system for non-homologous recombination and homologous recombination gene editing of the upp gene in bacteria. The RNP targeting the upp gene, along with SSB, was introduced into the protoplasts of Escherichia coli, Pseudomonas, and Bacillus subtilis. Transformants were obtained on plates containing 5-fluorouracil (5-FU) with gene editing efficiencies (percentage of transformants relative to the number of protoplasts) of 9.75 %, 5.02 %, and 8.37 %, respectively, and sequencing analysis confirmed 100 % non-homologous recombination. When RNP, SSB, and a 100-nucleotide single-stranded oligodeoxynucleotide (ssODN) donor were introduced into the protoplasts of these bacteria, transformants were obtained with editing efficiencies of 45.11 %, 30.13 %, and 27.18 %, respectively, and sequencing confirmed 100 % homologous recombination knockout of the upp gene. Additionally, introducing RNP, SSB, and a 100 base-pair double-stranded oligodeoxynucleotide (dsODN) donor containing a tetracycline resistance gene (tetR-dsODN) resulted in transformants on 5-FU plates with editing efficiencies of 35.94 %, 22.46 %, and 19.08 %, respectively, with sequencing confirming 100 % homologous recombination replacement of the upp gene with tetR. These results demonstrate that the SSB/CRISPR-Cas9 RNP system can efficiently, simply, and rapidly edit bacterial genomes without the need for plasmids. This study is the first to report the use of RNP-based gene editing in bacteria.

Approach to the child and adolescent with Adrenal Insufficiency.

The management of adrenal insufficiency is challenging, and the overall goals of treatment are to prevent life-threatening adrenal crises, to optimize linear growth, to control androgen levels without overdosing in subjects with congenital adrenal hyperplasia (CAH) and to improve quality of life in affected individuals. Standard glucocorticoid formulations fail to replicate the circadian rhythm of cortisol and control the adrenal androgen production driven by adrenocorticotropic hormone. In order to personalize and tailor glucocorticoid therapy and to improve patient outcomes, new pharmacological strategies have been developed that best mimic physiological cortisol secretion. Novel therapeutic approaches in the management of adrenal insufficiency include new ways to deliver circadian cortisol replacement as well as various adjunctive therapies to reduce androgen production and/or androgen action/effects. Preclinical studies are exploring the role of restorative cell-based therapies, and a first recombinant adeno-associated virus-based gene therapy is also being developed in humans with CAH. In this article, we present three illustrative cases of adrenal insufficiency with different underlying etiologies and times of presentation. Diagnostic and management processes are discussed with emphasis on treatment and outcomes. We have also provided the most up-to-date evidence for the tailored management of children and adolescents with adrenal insufficiency.

Multicenter non-interventional study of prevalence of homologous recombination gene mutations and approaches to treatment of metastatic castration-resistant prostate cancer in Russia (ADAM)

Multicenter non-interventional study of prevalence of homologous recombination gene mutations and approaches to treatment of metastatic castration-resistant prostate cancer in Russia (ADAM)

Sucrose as an electron source for cofactor regeneration in recombinant Escherichia coli expressing invertase and a Baeyer Villiger monooxygenase

BackgroundThe large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer–Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L−1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon.ResultsHerein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer–Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW−1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW− 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm.ConclusionsResults show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.

Deficiency of Adenosine Deaminase 2

Adenosine deaminase 2 (ADA2) deficiency is an autosomal recessively inherited autoinflammatory disorder caused by loss-of-function mutations in the ADA2 gene. Although the pathogenesis involves the triggering of a proinflammatory cascade due to increased production of inflammatory cytokines such as tumor necrosis factor (TNF)-α and dysregulation of neutrophil extracellular trap formation resulting from an excess accumulation of extracellular adenosine, the pathogenetic mechanism still needs further clarification due to the broad clinical spectrum. In addition to the initially described vasculitis-related symptoms, hematological, immunological, and autoinflammatory symptoms are now well recognized. The diagnosis is made by demonstration of pathogenic variants of ADA2 with biallelic loss of function and identification of low plasma ADA2 catalytic activity. Currently, TNF-α inhibitors are the treatment of choice for controlling vasculitis manifestations and preventing strokes. However, in patients presenting with severe hematologic findings, TNF-α inhibitors are not the treatment of choice and hematopoietic stem cell transplantation has been shown to be successful in selected cases. Recombinant ADA2 protein and gene therapy are promising treatment modalities for the future. In conclusion, ADA2 deficiency has a broad phenotype and should be considered in the differential diagnosis of different clinical situations. In this review, we summarize the disease manifestations of ADA2 deficiency and available treatment options.