Identification of RimR2 as a positive pathway-specific regulator of rimocidin biosynthesis in Streptomyces rimosus M527.

BACKGROUND: Streoptomyces rimosus M527 is a producer of the polyene macrolide rimocidin which shows activity against various plant pathogenic fungi. Notably, the regulatory mechanisms underlying rimocidin biosynthesis are yet to be elucidated. RESULTS: In this study, using domain structure and amino acid alignment and phylogenetic tree construction, rimR2, which located in the rimocidin biosynthetic gene cluster, was first found and identified as a larger ATP-binding regulators of the LuxR family (LAL) subfamily regulator. The rimR2 deletion and complementation assays were conducted to explore its role. Mutant M527-ΔrimR2 lost its ability to produce rimocidin. Complementation of M527-ΔrimR2 restored rimocidin production. The five recombinant strains, M527-ER, M527-KR, M527-21R, M527-57R, and M527-NR, were constructed by overexpressing rimR2 gene using the promoters permE*, kasOp*, SPL21, SPL57, and its native promoter, respectively, to improve rimocidin production. M527-KR, M527-NR, and M527-ER exhibited 81.8%, 68.1%, and 54.5% more rimocidin production, respectively, than the wild-type (WT) strain, while recombinant strains M527-21R and M527-57R exhibited no obvious differences in rimocidin production compared with the WT strain. RT-PCR assays revealed that the transcriptional levels of the rim genes were consistent with the changes in rimocidin production in the recombinant strains. Using electrophoretic mobility shift assays, we confirmed that RimR2 can bind to the promoter regions of rimA and rimC. CONCLUSION: A LAL regulator RimR2 was identified as a positive specific-pathway regulator of rimocidin biosynthesis in M527. RimR2 regulates the rimocidin biosynthesis by influencing the transcriptional levels of rim genes and binding to the promoter regions of rimA and rimC.

Effects of S-adenosylmethionine on production of secondary metabolites in Streptomycesdiastatochromogenes 1628.

Streptomyces are famous for their ability to synthesize a large number of bioactive compounds as secondary metabolites containing antibiotics, enzyme inhibitors, and other small molecules with potential physiological activity (Niu et al., 2016; Song et al., 2019; Yin et al., 2019). Secondary metabolites are produced by a multi-step reaction of a primary metabolite as a precursor (Liu et al., 2013; Li et al., 2021). Therefore, it is of great research significance to increase the overall synthesis level of antibiotics by increasing the amount of synthesis of precursors.

Cloning and Overexpression of the Toy Cluster for Titer Improvement of Toyocamycin in Streptomyces diastatochromogenes.

The nucleoside antibiotic toyocamycin (TM) is a potential fungicide that can control plant diseases, and it has become an attractive target for research. Streptomyces diastatochromogenes 1628, a TM-producing strain, was isolated by our laboratory and was considered to be a potent industrial producer of TM. Recently, the putative TM biosynthetic gene cluster (toy cluster) in S. diastatochromogenes 1628 was found by genome sequencing. In this study, the role of toy cluster for TM biosynthesis in S. diastatochromogenes 1628 was investigated by heterologous expression, deletion, and complementation. The extract of the recombinant strain S. albusJ1074-TC harboring a copy of toy cluster produced TM as shown by HPLC analysis. The Δcluster mutant completely lost its ability to produce TM. TM production in the complemented strain was restored to a level comparable to that of the wild-type strain. These results confirmed that the toy cluster is responsible for TM biosynthesis. Moreover, the introduction of an extra copy of the toy cluster into S. diastatochromogenes 1628 led to onefold increase in TM production (312.9 mg/l vs. 152.1 mg/l) as well as the transcription of all toy genes. The toy gene cluster was engineered in which the native promoter of toyA gene, toyM gene, toyBD operon, and toyEI operon was, respectively, replaced by permE ∗ or SPL57. To further improve TM production, the engineered toy gene cluster was, respectively, introduced and overexpressed in S. diastatochromogenes 1628 to generate recombinant strains S. diastatochromogenes 1628-EC and 1628-SC. After 84 h, S. diastatochromogenes 1628-EC and 1628-SC produced 456.5 mg/l and 638.9 mg/l TM, respectively, which is an increase of 2- and 3.2-fold compared with the wild-type strain.

[Keyhole Approach Endoscopic Surgery versus Stereotactic Aspiration plus Urokinase in Treating Basal Ganglia Hypertensive Intracerebral Hemorrhage].

Objective To compare the short-and long-term effect of two minimal invasive surgical therapies including keyhole approach endoscopic surgery(KAES)and stereotactic aspiration plus urokinase(SAU)in treating basal ganglia hypertensive intracerebral hemorrhage(hICH). Methods The clinical data of 117 hICH patients(63 received KAES and 54 received SAU)were retrospectively analyzed.The operation time,blood loss during surgery,and drainage time were compared between two groups.The residual hematoma volume,hematoma clearance rate(HCR),Glasgow coma scale(GCS)score,and National Institute of Health Stroke Scale(NIHSS)score were recorded at baseline and in the ultra-early stage,early stage,and sub-early stage after surgery.The 30-day mortality and serious adverse events were assessed and the 6-month modified Rankin scale(mRS)score was rated.Results Baseline data showed no significant difference between these two groups.Compared with the SAU group,the KAES group had significantly longer operation time,more intraoperative blood loss,and shorter drainage time(all P<0.001).In the ultra-early stage after surgery,HCR was significantly higher in the KAES group(P<0.001),whereas in the early and sub-early stage,HCR showed no significant differences(all P>0.05).In the ultra-early and early stage,the GCS and NIHSS scores showed no significant differences between two groups(all P>0.05),whereas in the sub-early stage,the NIHSS score was better in the SAU group(P=0.034).The 30-day mortality and incidences of serious adverse events showed no significant difference(all P>0.05).The good recovery(mRS≤3)at 6-months follow-up showed no significant difference between the two groups(P=0.413).Conclusions Both KAES and SAU are safe and effective in treating basal ganglia hICH.In the ultra-early stage after surgery,KAES achieves better residual hematoma volume and HCR,and patients undergoing SAU quickly catch up.The short-and long-term effectiveness of SAU is comparable or even superior to KAES.

Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527.

An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10-5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE* commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in ß-glucuronidase (GUS) activity compared with the control promoter permE*. Promoter SPL-57 showed activity comparable to that of permE*. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.

Modified minimally invasive transforaminal lumbar interbody fusion using a trans-multifidus approach: a safe and effective alternative to open-TLIF.

BACKGROUND: Application of minimally invasive transforaminal lumbar interbody fusion (MI-TLIF) is limited by long fluoroscopy time and a steep learning curve. Herein, MI-TLIF was modified using a trans-multifidus approach, assisted by microscope, termed MMI-TLIF, and the clinical outcomes of MMI-TLIF and open-TLIF were compared. METHODS: Forty-nine patients treated with MMI-TLIF were matched with 49 subjects who underwent open-TLIF. Patients were assessed using the North American Spine Society Score (NASS), Oswestry Disability Index (ODI), Short Form-36 (SF-36), and Visual Analogue Score (VAS) before surgery and during follow-up (6 months and 2 years). The four-type Bridwell anterior fusion grading system was used to evaluate fusion rates at 2 years. RESULTS: The median fluoroscopic time did not differ significantly between the MMI-TLIF and open-TLIF groups. MMI-TLIF surgery took significantly longer than open-TLIF (91.3 vs. 82.5 min; P < 0.05). Meanwhile, MMI-TLIF patients lost significantly less blood than open-TLIF patients (75.3 vs. 215.2 ml; P < 0.05), and MMI-TLIF patients were hospitalized for less long than open-TLIF patients (3.7 vs. 6.9 days; P < 0.05) and reported less pain, faster ambulation, and lower morphine intake than open-TLIF patients (all P < 0.05). The NASS, ODI, VAS, and SF-36 scores were significantly improved 6 months and 2 years postsurgery in both groups, compared with preoperative values, and similar values were obtained for both groups. Finally, fusion rates were similar in MMI-TLIF and open-TLIF patients. CONCLUSIONS: Overall, these findings strongly suggest the superiority of MMI-TLIF to open-TLIF. Therefore, MMI-TLIF could be a safe and effective alternative to MI-TLIF and open-TLIF.

Gene expression analysis in response to osmotic stimuli in the intervertebral disc with DNA microarray.

BACKGROUND: Intervertebral disc (IVD) cells experience a broad range of physicochemical stimuli under physiologic conditions, including alterations in their osmotic environment. At present, the molecular mechanisms underlying osmotic regulation in IVD cells are poorly understood. This study aims to screen genes affected by changes in osmotic pressure in cells of subjects aged 29 to 63 years old, with top-scoring pair (TSP) method. METHODS: Gene expression data set GSE1648 was downloaded from Gene Expression Omnibus database, including four hyper-osmotic stimuli samples, four iso-osmotic stimuli samples, and three hypo-osmotic stimuli samples. A novel, simple method, referred to as the TSP, was used in this study. Through this method, there was no need to perform data normalization and transformation before data analysis. RESULTS: A total of five pairs of genes ((CYP2A6, FNTB), (PRPF8, TARDBP), (RPS5, OAZ1), (SLC25A3, NPM1) and (CBX3, SRSF9)) were selected based on the TSP method. We inferred that all these genes might play important roles in response to osmotic stimuli and age in IVD cells. Additionally, hyper-osmotic and iso-osmotic stimuli conditions were adverse factors for IVD cells. CONCLUSIONS: We anticipate that our results will provide new thoughts and methods for the study of IVD disease.

[Effectiveness of minimally invasive transforaminal lumbar interbody fusion assisted with microscope in treatment of lumbar degenerative disease].

OBJECTIVE: To investigate the effectiveness of minimally invasive transforaminal lumbar interbody fusion (TLIF) assisted with microscope for lumbar degenerative disease. METHODS: Retrospective analysis was made on the clinical data of 82 patients with lumbar degenerative disease (minimally invasive group) undergoing minimally invasive TLIF assisted with microscope between January 2010 and June 2011, which was compared with those of 76 patients (traditional group) undergoing traditional open TLIF. There was no significant difference in age, gender, disease duration, disease type, lesion level, preoperative visual analogue scale (VAS), and preoperative Oswestry disability index (ODI) between 2 groups (P > 0.05). The perioperative related parameters, radiography index, and effectiveness were documented and compared. RESULTS: There was no significant difference in operation time and intraoperative radiological exposure time between 2 groups (P > 0.05), but intraoperative blood loss and postoperative drainage volume in the minimally invasive group were significantly less than those in the traditional group (P < 0.05). Dural tear occurred in 2 patients of the traditional group. Superficial infection of incision occurred in 1 case in each group, respectively; and primary healing of incision was obtained in the other patients. All patients were followed up 12-28 months (mean, 18 months). No failure of internal fixation occurred. Radiological analysis showed that the bone graft fusion rate was 96.1% (73/76) in the traditional group and 95.1% (78/82) in the minimally invasive group at last follow-up, showing no significant difference (chi2 = 0.012 2, P = 0.912 0). The postoperative ODI and VAS score were significantly improved when compared with preoperative ones in 2 groups (P < 0.05); the ODI of the minimally invasive group were significantly better than those of the traditional group at 3 months (t = -11.941 1, P = 0.000 0), and the VAS score of the minimally invasive group was significantly lower than that of the traditional group at 1 day and 3 months (P < 0.05); but no significant difference was found in ODI and VAS score between 2 groups at 1 year and last follow-up (P > 0.05). CONCLUSION: Minimally invasive TLIF is an effective method to treat lumbar degenerative disease. This procedure is safe and reliable because it has less injury, less blood loss, and milder pain than the traditional open TLIF, and the short-term effectiveness is comparable in 2 procedures.

[Instrumented slip reduction combined with 360 degrees circumferential fusion and restoration of laminae for adult isthmic spondylolisthesis].

OBJECTIVE: To evaluate the mid-term clinical outcome of instrumented slip reduction combined with 360 degrees circumferential fusion and restoration laminae for symptomatic adult isthmic spondylolisthesis. METHODS: Between October 2004 and March 2008, 44 patients with symptomatic isthmic spondylolisthesis underwent instrumented slip reduction combined with 360 degrees circumferential fusion and restoration laminae. There were 15 males and 29 females with an average age of 38.4 years (range, 28-45 years). The disease duration was 14 months to 7 years (38 months on average). The affected vertebrae was L4-5 in 18 patients and L5, S1 in 26 patients. According to Meyerding's grade for spondylolisthesis, 28 cases were rated as grade II and 16 as grade III. The visual analogue scale (VAS), Oswestry disability index (ODI), and the short form 36 health survey (SF-36) scores were evaluated before operation and at last follow-up; the radiographical outcome was evaluated by measuring slipping percentage, heights of intervertebral space and foramen, and fusion rate. RESULTS; All patients were followed up 20-60 months (42 months on average). The VAS, ODI, and SF-36 scores were all significantly improved at last follow-up when compared with those before operation (P < 0.05). According to Morelos criteria, the clinical results were excellent in 32 patients, good in 9, and fair in 3; the excellent and good rate was 93.2%. The preoperative average percentage of slip was 47.5%, which was improved to 2.6% 3 days after operation; the total average reduction rate was 97.4%, and it was maintained at last followup. The heights of intervertebral space and foramen were all improved significantly after operation (P < 0.05), and there was no significant difference between at 3 days after operation and at last follow-up (P > 0.05). X-ray and CT showed bony fusion 1 year after operation in all patients with a fusion rate of 100%. Complications included pain at donor site of iliac bone in 4 cases, superficial infection in 2 cases, dural tear in 1 case, and degeneration of adjacent vertebrae in 2 cases; no nerve root injury, pseudoarthrosis, failure of internal fixation, and acquired spinal canal stenosis occurred. CONCLUSION; Instrumented slip reduction combined with 360 degrees circumferential fusion and restoration laminae is a reliable procedure for adult isthmic spondylolisthesis with satisfactory mid-term results, a high fusion rate and low complication rate. The long-term outcomes should be verified by follow-up in the future.