Characteristics and immune functions of the endogenous CRISPR-Cas systems in myxobacteria.

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) system widely occurs in prokaryotic organisms to recognize and destruct genetic invaders. Systematic collation and characterization of endogenous CRISPR-Cas systems are conducive to our understanding and potential utilization of this natural genetic machinery. In this study, we screened 39 complete and 692 incomplete genomes of myxobacteria using a combined strategy to dispose of the abridged genome information and revealed at least 19 CRISPR-Cas subtypes, which were distributed with a taxonomic difference and often lost stochastically in intraspecies strains. The cas genes in each subtype were evolutionarily clustered but deeply separated, while most of the CRISPRs were divided into four types based on the motif characteristics of repeat sequences. The spacers recorded in myxobacterial CRISPRs were in high G+C content, matching lots of phages, tiny amounts of plasmids, and, surprisingly, massive organismic genomes. We experimentally demonstrated the immune and self-target immune activities of three endogenous systems in Myxococcus xanthus DK1622 against artificial genetic invaders and revealed the microhomology-mediated end-joining mechanism for the immunity-induced DNA repair but not homology-directed repair. The panoramic view and immune activities imply potential omnipotent immune functions and applications of the endogenous CRISPR-Cas machinery. IMPORTANCE: Serving as an adaptive immune system, clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) empower prokaryotes to fend off the intrusion of external genetic materials. Myxobacteria are a collective of swarming Gram-stain-negative predatory bacteria distinguished by intricate multicellular social behavior. An in-depth analysis of their intrinsic CRISPR-Cas systems is beneficial for our understanding of the survival strategies employed by host cells within their environmental niches. Moreover, the experimental findings presented in this study not only suggest the robust immune functions of CRISPR-Cas in myxobacteria but also their potential applications.

Fe(III)/peroxymonosulfate oxidation system for the degradation of rhein, a toxic component abundance in rhubarb residue.

Rhubarb is widely used in health care, but causing a great amount of rhein-containing herbal residue. Rhein with several toxicities might pollute environment, damage ecology and even hazard human health if left untreated. In this study, the degradation effects of bisulfite- (BS) and peroxymonosulfate- (PMS) based oxidation systems on rhein in rhubarb residue were compared and investigated. The effects of BS and PMS with two valence states of ferric ion (Fe) on the degradation of rhein in rhubarb residue were optimized for the selection of optimal oxidation system. The influences of reaction temperature, reaction time and initial pH on the removal of rhein under the optimal oxidation system were evaluated. The chemical profiles of rhubarb residue with and without oxidation process were compared by UPLC-QTOF-MS/MS, and the degradation effects were investigated by PLS-DA and S plot/OPLS-DA analysis. The results manifested that PMS showed relative higher efficiency than BS on the degradation of rhein. Moreover, Fe(III) promoted the degradation effect of PMS, demonstrated that Fe(III)/PMS is the optimal oxidation system to degrade rhein in rhubarb residue. Further studies indicated that the degradation of rhein by the Fe(III)/PMS oxidation system was accelerated with the prolong of reaction time and the elevation of reaction temperature, and also affected by the initial pH. More importantly, Fe(III)/PMS oxidation system could degrade rhein in rhubarb residue completely under the optimal conditions. In conclusion, Fe(III)/PMS oxidation system is a feasible method to treat rhein in rhubarb residue.

Genome-wide analysis of lipolytic enzymes and characterization of a high-tolerant carboxylesterase from Sorangium cellulosum.

Microorganisms are important sources of lipolytic enzymes with characteristics for wide promising usages in the specific industrial biotechnology. The cellulolytic myxobacterium Sorangium cellulosum is rich of lipolytic enzymes in the genome, but little has been investigated. Here, we discerned 406 potential lipolytic enzymes in 13 sequenced S. cellulosum genomes. These lipolytic enzymes belonged to 12 families, and most are novel with low identities (14-37%) to those reported. We characterized a new carboxylesterase, LipB, from the alkaline-adaptive So0157-2. This enzyme, belonging to family VIII, hydrolyzed glyceryl tributyrate and p-nitrophenyl esters with short chain fatty acids (≤C12), and exhibited the highest activity against p-nitrophenyl butyrate. It retained over 50% of the activities in a broad temperature range (from 20°C to 60°C), alkaline conditions (pH 8.0-9.5), and the enzymatic activity was stable with methanol, ethanol and isopropanol, and stimulated significantly in the presence of 5 mM Ni2+. LipB also exhibited ß-lactamase activity on nitrocefin, but not ampicillin, cefotaxime and imipenem. The bioinformatic analysis and specific enzymatic characteristics indicate that S. cellulosum is a promising resource to explore lipolytic enzymes for industrial adaptations.

Internal Promoters and Their Effects on the Transcription of Operon Genes for Epothilone Production in Myxococcus xanthus.

The biosynthetic genes for secondary metabolites are often clustered into giant operons with no transcription terminator before the end. The long transcripts are frangible and the transcription efficiency declines along with the process. Internal promoters might occur in operons to coordinate the transcription of individual genes, but their effects on the transcription of operon genes and the yield of metabolites have been less investigated. Epothilones are a kind of antitumor polyketides synthesized by seven multifunctional enzymes encoded by a 56-kb operon. In this study, we identified multiple internal promoters in the epothilone operon. We performed CRISPR-dCas9-mediated transcription activation of internal promoters, combined activation of different promoters, and activation in different epothilone-producing M. xanthus strains. We found that activation of internal promoters in the operon was able to promote the gene transcription, but the activation efficiency was distinct from the activation of separate promoters. The transcription of genes in the operon was influenced by not only the starting promoter but also internal promoters of the operon; internal promoters affected the transcription of the following and neighboring upstream/downstream genes. Multiple interferences between internal promoters thus changed the transcriptional profile of operon genes and the production of epothilones. Better activation efficiency for the gene transcription and the epothilone production was obtained in the low epothilone-producing strains. Our results highlight that interactions between promoters in the operon are critical for the gene transcription and the metabolite production efficiency.

Characterization of Constitutive Promoters for the Elicitation of Secondary Metabolites in Myxobacteria.

Genome mining has revealed that myxobacteria contain a myriad of cryptic biosynthetic gene clusters (BGCs). Here, we report the characterization of a panel of myxobacterial promoters with variable strength that are applicable in the engineering of BGCs in myxobacteria. The screened strongest constitutive promoter was used to efficiently enhance the expression of two complex BGCs governing the biosynthesis of myxochromide and DKxanthene in the model myxobacterium Myxococcus xanthus DK1622. We also showcased the combination of promoter engineering and MS2-based spectral networking as an effective strategy to shed light on the previously overlooked chemistry in the family of myxochromide-type lipopeptides. The enriched promoter library substantially expanded the synthetic biology toolkit available for myxobacteria.

Insulin-Like Growth Factor 1 (IGF1) Pathway Member Polymorphisms Are Associated with Risk and Prognosis of Chondrosarcoma.

BACKGROUND The insulin-like growth factor 1 (IGF1) pathway is deeply involved in cell proliferation, including tumorigenesis. Aberrant genetic alterations of IGF1 pathway members were revealed in certain malignancies, including chondrosarcoma (CHS). We proposed that genetic polymorphisms in IGF1 pathways might be associated with susceptibility to tumorigenesis and prognosis of CHS in Chinese populations. MATERIAL AND METHODS We recruited 112 pathologically diagnosed CHS cases and 104 cancer-free controls in this study. There were 5 single-nucleotide polymorphisms of IGF1 pathway members (IGF1R rs2016347, IGF1 rs1520220, IGF1 rs2946834, IGF3BP3 rs2270628, and IGF2 rs4320932) genotyped that subsequently underwent bioinformatic analyses. DNA from validated CHS cases was extracted from frozen blood samples preserved in liquid nitrogen, while DNA from tumor-free controls was extracted from fresh blood. SNP genotyping was conducted by PCR. RESULTS The variant T allele of IGF1R (rs2016347) is potentially correlated with poor outcome in patients with conventional CHS. The GT and TT genotypes of IGF1R rs2016347 predicted statistically significant higher risk of tumor metastasis and higher histological grade of CHS. CONCLUSIONS We hypothesized that IGF1 member polymorphisms are associated with chondrosarcoma. We found that genetic polymorphisms in IGF1 pathway members are associated with elevated risk and poor prognosis of conventional CHS patients in Chinese populations. IGF1R rs2016347 polymorphisms were associated with the risk of lung metastasis of CHS. The IGF1 pathway members do not appear to be involved in the tumorigenesis of CHS.

Effects of transcriptional mode on promoter substitution and tandem engineering for the production of epothilones in Myxococcus xanthus.

Promoter optimization is an economical and effective approach to overexpress heterologous genes and improve the biosynthesis of valuable products. In this study, we swapped the original promoter of the epothilone biosynthetic gene cluster in Myxococcus xanthus with two endogenous strong promoters P pilA and P groEL1 , respectively, which, however, decreased the epothilone production ability. The transcriptional abilities by the two promoters were found to be bloomed in the growth stage but markedly decreased after the growth, whereas the original promoter P epo functioned majorly after the exponential growth stage. Tandem repeat engineering on the original promoter P epo remarkably increased epothilone production. The tandem promoter exerted similar expressional pattern as P epo did in M. xanthus. We demonstrated that differential transcriptional modes markedly affected the efficiency of promoters in controlling the gene expressions for the production of the secondary metabolite epothilones. Our study provides an insight into exploiting powerful promoters to produce valuable secondary metabolites, especially in host with limited known promoters.

Effect of the timing of surgery on the fracture healing process and the expression levels of vascular endothelial growth factor and bone morphogenetic protein-2.

The aim of the present study was to observe the effect of varying the timing of surgery on the fracture healing process and the expression levels of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)-2 in rats. A total of 192 rats underwent closed femur fracture modelling. The rats underwent open reduction and internal fixation surgery 1, 3, 5, 7, 11 and 14 days subsequent to the fracture occurring. Immunohistochemical staining and analysis of the VEGF and BMP-2 expression levels were simultaneously conducted on bone from the fracture site of the rats on various days. The VEGF and BMP-2 expression levels at the fracture sites were higher and were maintained for a longer period of time in the 7- and 11-day surgery groups than in the other surgery groups and the rats that did not undergo surgery. The 5-day surgery group demonstrated a greater intensity in BMP-2 expression compared with the remaining surgery groups; however, no significant differences were identified between 1-day surgery and non surgery groups. In the 3-day surgery group, the expression levels VEGF and BMP-2 were low at each stage of the fracture-healing process and were lower compared with those observed in the non-surgery group. The timing of the surgical procedures affected the VEGF and BMP-2 expression levels at the fracture sites of the experimental rats and, the optimal time for performing surgery was identified to be within the first two weeks. However, surgery may not be conducive to fracture healing if it is performed within the first few days following fracture.

An experimental model of urethral stricture in rabbits using holmium laser under urethroscopic direct visualization.

OBJECTIVE: To establish an experimental rabbit model of urethral stricture using holmium laser under direct urethroscopic visualization. METHODS: Sixteen adult male New Zealand rabbits were divided into equally-sized control and experimental groups. All rabbits underwent retrograde urethrography and transurethral endoscopy with a 7.5 F urethroscope after intramuscular anesthetic injection. We used a holmium:YAG laser to injure the distal urethra in all rabbits in the experimental group under direct visualization. Thirty days after surgery, all animals were evaluated with retrograde urethrography and urethroscopy. The flow rate of the isolated urethras was measured to evaluate urethral stricture formation. RESULTS: One rabbit in the experimental group (12.5%) died of infection 4 days after surgery. Thirty days after surgery, retrograde urethrography and urethroscopy revealed strictures in all seven surviving rabbits (87.5%) in the experimental group. The mean flow rate of the isolated urethras was significantly lower in the experimental group than in the control group. CONCLUSION: A rabbit model of urethral stricture can be successfully established using holmium laser under direct urethroscopic visualization, providing an ideal object for research concerning the pathogenesis and molecular biology of urethral strictures.

Muscle mass loss and intermuscular lipid accumulation were associated with insulin resistance in patients receiving hemodialysis.

BACKGROUND: An accelerated muscle wasting was the pivotal factor for protein-energy wasting in end stage renal disease. However, very few researches have examined the skeletal muscle quantity and quality in clinical patients. This study investigated the muscle morphologic changes by magnetic resonance imaging (MRI) and analyzed the related factors in hemodialysis patients. METHODS: Fifty-eight patients receiving maintenance hemodialysis (HD) were investigated and 28 healthy adults with gender and age matched were used as controls (Control). Anthropometry, cytokine factors, and laboratory data were measured. The muscle and intermuscular adipose tissues (IMAT) were analyzed via a Thigh MRI. The bicep samples were observed after HE staining. Homeostatic model assessment of insulin resistance (HOMA-IR) was measured and their association with muscle wasting was analyzed. RESULTS: HD patients tended to have a lower protein diet, anthropometry data, and serum albumin, but the C reactive protein and interleukin-6 increased significantly. The MRI showed that HD patients had less muscle mass and a lower muscle/total ratio, but the fat/muscle and IMAT was higher when compared to the Control group. The muscle fiber showed atrophy and fat accumulation in the biceps samples come from the HD patients. Moreover, we found that the HD patients presented with a high level of plasma fasting insulin and increased HOMA-IR which negatively correlated with the muscle/total ratio, but positively with the fat/muscle ratio. CONCLUSIONS: Muscle wasting presented early before an obvious malnutrition condition emerged in HD patients. The main morphological change was muscle atrophy along with intermuscular lipid accumulation. Insulin resistance was associated with muscle wasting in dialysis patients.

[A multicenter study of mid urethral sling procedures in treatment of female stress urinary incontinence].

OBJECTIVE: To investigate the clinical efficacy, surgical indications and postoperative complications of mid urethral sling procedures in treatment of female stress urinary incontinence. METHODS: A multicenter clinical trial was conducted from April 2002 to April 2008 in five hospitals, 304 cases of genuine stress urinary incontinence and 8 cases of mixed incontinence were included. TVT procedures were carried out in 134 patients, TVTO procedures in 167 patients, Monarc procedures in 11 patients. Perioperative evaluations included: operating time, bleeding volume, and perioperative complications. Operative efficacy was classified into three categories: cure, improved and failure and evaluated before discharge, 3 months after surgery and then every year. RESULTS: TVT group had longer operating time [(18.5 + or - 9.6) min] and more bleeding volume [(32.2 + or - 12.6) ml] than those in TVTO group [(11.5 + or - 3.1) min, (12.8 + or - 8.5) ml] and in Monarc group [(11.1 + or - 2.6) min, (12.3 + or - 3.5) ml] with P < 0.05. Monarc and TVTO procedures had higher cure rates and improve rates comparing with TVT, but the differences were of no significance. The cure rate (95.7%) in patients with genuine stress incontinence were significantly higher than that in patients with mixed incontinence (37.5%). No significant differences of total intra- and postoperative complications were noted for all of the three procedures. However, bladder injury tended to occur in TVT group and obturator nerve injury and vaginal injury tended to occur in TVTO group. Transient voiding dysfunction and urinary retention were the most common complications. CONCLUSIONS: Mid urethral sling procedures have excellent clinical outcomes in the treatment of female stress urinary incontinence.