Bridging PCR: An Efficient and Reliable Scheme Implemented for Genome-Walking.

The efficacy of the available genome-walking methods is restricted by low specificity, high background, or composite operations. We herein conceived bridging PCR, an efficient genome-walking approach. Three primers with random sequences, inner walker primer (IWP), bridging primer (BP), and outer walker primer (OWP), are involved in bridging PCR. The BP is fabricated by splicing OWP to the 5'-end of IWP's 5'-part. A bridging PCR set is constituted by three rounds of amplification reactions, sequentially performed by IWP, BP plus OWP, and OWP, respectively pairing with three nested sequence-specific primers (SSP). A non-target product arising from IWP alone undergoes end-lengthening mediated by BP. This modified non-target product is a preferentially formed hairpin between the lengthened ends, instead of binding with shorter OWP. Meanwhile, a non-target product, triggered by SSP alone or SSP plus IWP, is removed by nested SSP. As a result, only the target DNA is accumulated. The efficacy of bridging PCR was validated by walking the gadA/R genes of Levilactobacillus brevis CD0817 and the hyg gene of rice.

Semi-Site-Specific Primer PCR: A Simple but Reliable Genome-Walking Tool.

Genome-walking has been frequently applied to molecular biology and related areas. Herein, a simple but reliable genome-walking technique, termed semi-site-specific primer PCR (3SP-PCR), is presented. The key to 3SP-PCR is the use of a semi-site-specific primer in secondary PCR that partially overlaps its corresponding primary site-specific primer. A 3SP-PCR set comprises two rounds of nested amplification reactions. In each round of reaction, any primer is allowed to partially anneal to the DNA template once only in the single relaxed-stringency cycle, creating a pool of single-stranded DNAs. The target single-stranded DNA can be converted into a double-stranded molecule directed by the site-specific primer, and thus can be exponentially amplified by the subsequent high-stringency cycles. The non-target one cannot be converted into a double-strand due to the lack of a perfect binding site to any primer, and thus fails to be amplified. We validated the 3SP-PCR method by using it to probe the unknown DNA regions of rice hygromycin genes and Levilactobacillus brevis CD0817 glutamic acid decarboxylase genes.

PhoP- and GlnR-mediated regulation of metK transcription and its impact upon S-adenosyl-methionine biosynthesis in Saccharopolyspora erythraea.

BACKGROUND: Erythromycin A (Er A) has a broad antibacterial effect and is a source of erythromycin derivatives. Methylation of erythromycin C (Er C), catalyzed by S-adenosyl-methionine (SAM)-dependent O-methyltransferase EryG, is the key final step in Er A biosynthesis. Er A biosynthesis, including EryG production, is regulated by the phosphate response factor PhoP and the nitrogen response factor GlnR. However, the regulatory effect of these proteins upon S-adenosyl-methionine synthetase (MetK) production is unknown. RESULTS: In this study, we used bioinformatics approaches to identify metK (SACE_3900), which codes for S-adenosyl-methionine synthetase (MetK). Electrophoretic mobility shift assays (EMSAs) revealed that PhoP and GlnR directly interact with the promoter of metK, and quantitative PCR (RT-qPCR) confirmed that each protein positively regulated metK transcription. Moreover, intracellular SAM was increased upon overexpression of either phoP or glnR under phosphate or nitrogen limited conditions, respectively. Finally, both the production of Er A and the transformation ratio from Er C to Er A increased upon phoP overexpression, but surprisingly, not upon glnR overexpression. CONCLUSIONS: Manipulating the phosphate and nitrogen response factors, PhoP and GlnR provides a novel strategy for increasing the yield of SAM and the production of Er A in Saccharopolyspora erythraea .

Sodium-Ion-Free Fermentative Production of GABA with Levilactobacillus brevis CD0817.

Gamma-aminobutyric acid (GABA) has positive effects on many physiological processes. Lactic acid bacterial production of GABA is a future trend. This study aimed to produce a sodium-ion-free GABA fermentation process for Levilactobacillus brevis CD0817. In this fermentation, both the seed and fermentation media used L-glutamic acid instead of monosodium L-glutamate as the substrate. We optimized the key factors influencing GABA formation, adopting Erlenmeyer flask fermentation. The optimized values of the key factors of glucose, yeast extract, Tween 80, manganese ion, and fermentation temperature were 10 g/L, 35 g/L, 1.5 g/L, 0.2 mM, and 30 °C, respectively. Based on the optimized data, a sodium-ion-free GABA fermentation process was developed using a 10-L fermenter. During the fermentation, L-glutamic acid powder was continuously dissolved to supply substrate and to provide the acidic environment essential for GABA synthesis. The current bioprocess accumulated GABA at up to 331 ± 8.3 g/L after 48 h. The productivity of GABA was 6.9 g/L/h and the molar conversion rate of the substrate was 98.1%. These findings demonstrate that the proposed method is promising in the fermentative preparation of GABA by lactic acid bacteria.

Protocol to access unknown flanking DNA sequences using Wristwatch-PCR for genome-walking.

Here we describe a protocol for wristwatch PCR, an approach based on wristwatch-like structure formed between walking primers to obtain unknown flanks. We specify the criteria for designing wristwatch primers and gene-specific primers. We detail how to set wristwatch primer permutations to obtain personalized walking outcomes and improve walking efficiency. We describe experimental procedures for isolating a DNA of interest using three rounds of nested wristwatch PCR as well as the subsequent steps for DNA purification, cloning, and sequencing. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).1.

DAR-PCR: a new tool for efficient retrieval of unknown flanking genomic DNA.

Various PCR-based genome-walking methods have been developed to acquire unknown flanking DNA sequences. However, the specificity and efficacy levels, and the operational processes, of the available methods are unsatisfactory. This work proposes a novel walking approach, termed differential annealing-mediated racket PCR (DAR-PCR). The key to DAR-PCR is the use of primer-mediated intra-strand annealing (ISA). An ISA primer consists of a 5' root homologous to the known sequence and a heterologous 3' bud. In the single low-stringency cycle, the ISA primer anneals to a site on an unknown region and extends towards the sequence-specific primer (SSP) 1 site, thereby forming a target single-stranded DNA bound by the SSP1 complement and the ISA primer. In the subsequent more stringent cycles, its complementary strand is accumulated, owing to the differential annealing between the moderate-stringency ISA primer and the high-stringency SSP1. The accumulation of this strand provides an opportunity for ISA mediated by the ISA primer root. A loop-back extension subsequent to ISA occurs, creating a racket-like DNA with the known region positioned at both ends of the unknown sequence. This DNA is exponentially amplified during the secondary PCR driven by an SSP pair inner to SSP1. DAR-PCR was validated as an efficient walking method by determining unknown flanking sequences in Lactobacillus brevis and Oryza sativa.

Fusion primer driven racket PCR: A novel tool for genome walking.

The limitations of the current genome-walking strategies include strong background and cumbersome experimental processes. Herein, we report a genome-walking method, fusion primer-driven racket PCR (FPR-PCR), for the reliable retrieval of unknown flanking DNA sequences. Four sequence-specific primers (SSP1, SSP2, SSP3, and SSP4) were sequentially selected from known DNA (5'→3') to perform FPR-PCR. SSP3 is the fragment that mediates intra-strand annealing (FISA). The FISA fragment is attached to the 5' end of SSP1, generating a fusion primer. FPR-PCR comprises two rounds of amplification reactions. The single-fusion primary FPR-PCR begins with the selective synthesis of the target first strand, then allows the primer to partially anneal to some place(s) on the unknown region of this strand, producing the target second strand. Afterward, a new first strand is synthesized using the second strand as the template. The 3' end of this new first strand undergoes intra-strand annealing to the FISA site, followed by the formation of a racket-like DNA by a loop-back extension. This racket-like DNA is exponentially amplified in the secondary FPR-PCR performed using SSP2 and SSP4. We validated this FPR-PCR method by identifying the unknown flanks of Lactobacillus brevis CD0817 glutamic acid decarboxylase genes and the rice hygromycin gene.

Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy.

Genome walking is a method used to retrieve unknown flanking DNA. Here, we reported wristwatch (WW) PCR, an efficient genome walking technique mediated by WW primers (WWPs). WWPs feature 5'- and 3'-overlap and a heterologous interval. Therefore, a wristwatch-like structure can be formed between WWPs under relatively low temperatures. Each WW-PCR set is composed of three nested (primary, secondary, and tertiary) PCRs individually performed by three WWPs. The WWP is arbitrarily annealed somewhere on the genome in the one low-stringency cycle of the primary PCR, or directionally to the previous WWP site in one reduced-stringency cycle of the secondary/tertiary PCR, producing a pool of single-stranded DNAs (ssDNAs). A target ssDNA incorporates a gene-specific primer (GSP) complementary at the 3'-end and the WWP at the 5'-end and thus can be exponentially amplified in the next high-stringency cycles. Nevertheless, a non-target ssDNA cannot be amplified as it lacks a perfect binding site for any primers. The practicability of the WW-PCR was validated by successfully accessing unknown regions flanking Lactobacillus brevis CD0817 glutamate decarboxylase gene and the hygromycin gene of rice. The WW-PCR is an attractive alternative to the existing genome walking techniques.

RegX3-Mediated Regulation of Methylcitrate Cycle in Mycobacterium smegmatis.

Mycobacterium tuberculosis is a global human pathogen that infects macrophages and can establish a latent infection. Emerging evidence has established the nutrients metabolism as a key point to study the pathogenesis of M. tuberculosis and host immunity. It was reported that fatty acids and cholesterol are the major nutrient sources of M. tuberculosis in the period of infection. However, the mechanism by which M. tuberculosis utilizes lipids for maintaining life activities in nutrient-deficiency macrophages is poorly understood. Mycobacterium smegmatis is fast-growing and generally used to study its pathogenic counterpart, M. tuberculosis. In this work, we found that the phosphate sensing regulator RegX3 of M. smegmatis is required for its growing on propionate and surviving in macrophages. We further demonstrated that the expression of prpR and related genes (prpDBC) in methylcitrate cycle could be enhanced by RegX3 in response to the phosphate-starvation condition. The binding sites of the promoter region of prpR for RegX3 and PrpR were investigated. In addition, cell morphology assay showed that RegX3 is responsible for cell morphological elongation, thus promoting the proliferation and survival of M. smegmatis in macrophages. Taken together, our findings revealed a novel transcriptional regulation mechanism of RegX3 on propionate metabolism, and uncovered that the nutrients-sensing regulatory system puts bacteria at metabolic steady state by altering cell morphology. More importantly, since we observed that M. tuberculosis RegX3 also binds to the prpR operon in vitro, the RegX3-mediated regulation might be general in M. tuberculosis and other mycobacteria for nutrient sensing and environmental adaptation.

[Ross procedure in treatment of aortic disease: clinical experience of 15 cases].

OBJECTIVE: To summarize the clinical experience of Ross procedure in treatment of aorta valve diseases. METHODS: The clinical data of 15 patients with aorta valve diseases, 12 men and 3 women, aged 30 +/- 14, including 13 cases of congenital aorta valve disease, 1 case of senile degenerative aortic valve disease, and 1 case of subacte bacterial endocarditis complicated by aortic stenosis (AS), with the heart function (NYHA) of class II in 11 cases and class III in 4 cases, underwent Ross procedure from October 1994 to September 2002 in Anzhen Hospital. Before operation, ultrasound cardiography showed moderate to severe AS and/or aortic insufficiency (AI) with an average aortic valve annulus diameter (AVD) of 2.4 +/- 0.4 cm and normal pulmonary valve. Operation was performed on all patients under cardiopulmonary bypass and moderate hypothermia. The operation procedure was as follows: (1) to take off the auto-pulmonary artery valve; (2) to remove the dysfunctional aortic valve and auto-transplant the pulmonary valve on the aortic root; and (3) to put a pulmonary homograft so as to re-establish on the right ventricular outflow tract. RESULTS: The perieoperative mortality is 0. After the operation, the mean pressure gradient of aortic valve was in the normal limitation (7.23 +/- 0.14 mm Hg), the left ventricular diastolic diameter decreased significantly (P < 0.001), the left ventricular ejection fraction was 0.48 +/- 0.22, and the heart function (NYHA) was at the classes I - II in all the patients. All cases received follow-up of 1 - 9 years, their heart function was all in Class I, and the function of their aortic and pulmonary valves remained well. CONCLUSION: Ross procedure is a kind of effective alterative operation in treating patients with aortic valve disease.

A novel surgical procedure: scaffold-pulmonary autograft transplantation.

Mitral valve-related operations are easy to perform and show good results, but to prevent severe thromboembolism or a high ratio of prosthetic valve destruction by tissue, lifetime anticoagulant therapy is essential after the operation. Thus, identifying a new type of surgical procedure and prosthetic valve to cure mitral valve diseases is necessary. Pulmonary valve autograft transplantation (Ross II) with the "top hat" transplantation technique was first reported by Ross DN to cure mitral disease. Because the "top hat" procedure has some shortcomings, we designed the scaffold-pulmonary autograft transplantation procedure and performed animal experiments to confirm the feasibility and effectiveness of the procedure. A total of 13 minipigs, weighing 20-25 kg, were employed as experimental animals to undergo scaffold-pulmonary autograft valve transplantation in our surgical animal lab. The surgical procedure was performed under hypothermic general anaesthesia and extracorporeal circulation (or cardiopulmonary bypass, CPB). Briefly, the chest cave was opened through the left intercostal, the pulmonary valve autograft was harvested during on-pump beating heart, and the pulmonary valve autograft was mounted in a self-made pulmonary valve scaffold and transferred to the mitral valve annulus without removing the mitral instruments. Finally, the outflow tract of the right ventricle was re-established with a pig pulmonary homograft. After finishing data collection, all animals were executed 1 hour after removal from the CPB. For the 13 minipigs that underwent the operation, the CPB time was 182.4 ± 23.4 min. Two of the thirteen cases died of bleeding during the operation and of a post-operative pulmonary embolism, and the remaining eleven survived for one hour. The pressure of the left atrium did not increase significantly (P = 1.00), and the ultrasonic cardiograph (UCG) showed good function of the new mitral valves, with mean ejection fraction (EF) values of 63.6%. The mitral valve orifice areas were 1.10 ± 0.13 cm(2) (pre-operation) and 1.01 ± 0.08 cm(2) (post-operation) (P = 0.013). The function and structure of the new mitral valves were normal. We preliminarily consider scaffold-pulmonary autograft valve transplantation to be a new alternative to cure mitral valve disease, but advanced chronic animal experiments will be needed to confirm the long-term results of the operation. The results showed it could be a new alternative to cure mitral valve disease.