An atypical two-component system, AtcR/AtcK, simultaneously regulates the biosynthesis of multiple secondary metabolites in Streptomyces bingchenggensis.

Streptomyces bingchenggensis is an industrial producer of milbemycins, which are important anthelmintic and insecticidal agents. Two-component systems (TCSs), which are typically situated in the same operon and are composed of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Here, an atypical TCS, AtcR/AtcK, in which the encoding genes (sbi_06838/sbi_06839) are organized in a head-to-head pair, was demonstrated to be indispensable for the biosynthesis of multiple secondary metabolites in S. bingchenggensis. With the null TCS mutants, the production of milbemycin and yellow compound was abolished but nanchangmycin was overproduced. Transcriptional analysis and electrophoretic mobility shift assays showed that AtcR regulated the biosynthesis of these three secondary metabolites by a MilR3-mediated cascade. First, AtcR was activated by phosphorylation from signal-triggered AtcK. Second, the activated AtcR promoted the transcription of milR3. Third, MilR3 specifically activated the transcription of downstream genes from milbemycin and yellow compound biosynthetic gene clusters (BGCs) and nanR4 from the nanchangmycin BGC. Finally, because NanR4 is a specific repressor in the nanchangmycin BGC, activation of MilR3 downstream genes led to the production of yellow compound and milbemycin but inhibited nanchangmycin production. By rewiring the regulatory cascade, two strains were obtained, the yield of nanchangmycin was improved by 45-fold to 6.08 g/L and the production of milbemycin was increased twofold to 1.34 g/L. This work has broadened our knowledge on atypical TCSs and provided practical strategies to engineer strains for the production of secondary metabolites in Streptomyces.IMPORTANCEStreptomyces bingchenggensis is an important industrial strain that produces milbemycins. Two-component systems (TCSs), which consist of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Coupled encoding genes of TCSs are typically situated in the same operon. Here, TCSs with encoding genes situated in separate head-to-head neighbor operons were labeled atypical TCSs. It was found that the atypical TCS AtcR/AtcK played an indispensable role in the biosynthesis of milbemycin, yellow compound, and nanchangmycin in S. bingchenggensis. This atypical TCS regulated the biosynthesis of specialized metabolites in a cascade mediated via a cluster-situated regulator, MilR3. Through rewiring the regulatory pathways, strains were successfully engineered to overproduce milbemycin and nanchangmycin. To the best of our knowledge, this is the first report on atypical TCS, in which the encoding genes of RR and HK were situated in separate head-to-head neighbor operons, involved in secondary metabolism. In addition, data mining showed that atypical TCSs were widely distributed in actinobacteria.

MilR3, a unique SARP family pleiotropic regulator in Streptomyces bingchenggensis.

Streptomyces bingchenggensis is the main industrial producer of milbemycins, which are a group of 16-membered macrocylic lactones with excellent insecticidal activities. In the past several decades, scientists have made great efforts to solve its low productivity. However, a lack of understanding of the regulatory network of milbemycin biosynthesis limited the development of high-producing strains using a regulatory rewiring strategy. SARPs (Streptomyces Antibiotic Regulatory Proteins) family regulators are widely distributed and play key roles in regulating antibiotics production in actinobacteria. In this paper, MilR3 (encoded by sbi_06842) has been screened out for significantly affecting milbemycin production from all the 19 putative SARP family regulators in S. bingchenggensis with the DNase-deactivated Cpf1-based integrative CRISPRi system. Interestingly, milR3 is about 7 Mb away from milbemycin biosynthetic gene cluster and adjacent to a putative type II PKS (the core minimal PKS encoding genes are sbi_06843, sbi_06844, sbi_06845 and sbi_06846) gene cluster, which was proved to be responsible for producing a yellow pigment. The quantitative real-time PCR analysis proved that MilR3 positively affected the transcription of specific genes within milbemycin BGC and those from the type II PKS gene cluster. Unlike previous "small" SARP family regulators that played pathway-specific roles, MilR3 was probably a unique SARP family regulator and played a pleotropic role. MilR3 was an upper level regulator in the MilR3-MilR regulatory cascade. This study first illustrated the co-regulatory role of this unique SARP regulator. This greatly enriches our understanding of SARPs and lay a solid foundation for milbemycin yield enhancement in the near future.

Extended Maxillary Osteotomy Guide: A Design That Allows Manipulation of the Osteotomy Direction on the Posterior and Inner Walls of the Maxilla.

PURPOSE: Design an extended osteotomy guide (EOG) for Le Fort I osteotomy to improve the safety of surgery. MATERIALS AND METHODS: The digital Le Fort I osteotomy guide was designed in MIMICS 23.0. Twenty-eight patients were randomized into 2 groups. Patients in the experimental group used EOG, and patients in the control group used a traditional osteotomy guide (TOG). Virtual designs and actual postoperative outcomes were compared by cone-beam computed tomography. The safety of the operation was confirmed by the accuracy of the osteotomy direction and depth on the inner and posterior walls of the maxilla. RESULTS: All positioning deviations of both osteotomy guides were <0.3 mm (P>0.05). The osteotomy depths on the inner and posterior walls with the EOG and TOG deviated by 0.789±1.179 and 1.811±1.345 mm (P=0.004) and 0.648±0.999 and 1.262±0.942 mm (P=0.030), respectively. The angles of deviation of the osteotomy direction on the inner and posterior walls by the EOG and TOG were 2.025±2.434 and 5.069±2.391 degrees (P<0.001) and 2.772±2.979 and 8.653±4.690 degrees (P<0.001), respectively. CONCLUSIONS: The EOG was more accurate than TOG for manipulating osteotomy direction and depth on the inner and posterior maxillary walls. Thus, EOG could ensure higher surgical safety than TOG.

Oncologic safety of the pedicled submental island flap for reconstruction in oral tongue squamous cell carcinoma: An analysis of 101 cases.

OBJECTIVE: To evaluate whether the pedicle submental island flap (SIF) can be safely used in the oral tongue squamous cell carcinoma (OTSCC) patients with pathologically node-positive (pN+) neck, especially pN+ at level I. METHODS: Retrospectively, 101 OTSCC patients with SIF reconstruction were enrolled. Oncological outcomes included the total locoregional recurrence, the SIF related locoregional recurrence (SRLR) which referred to the local recurrence at flap and ipsilateral neck recurrence at level I, recurrence free survival (RFS), overall survival (OS), and disease specific survival (DSS). RESULTS: Sixty-one patients were pathologically node-negative (pN0) and 40 were pN+. Thirteen patients experienced locoregional recurrence, of which 5 had a SRLR. The pN+ group had a significantly higher locoregional recurrence rate, lower 5-year RFS, OS and DSS than pN0 group (P < 0.05). Patients with pN0 had a significantly higher neck RFS when compared to those with pN+ either at level I (P = 0.005) or at other levels (P < 0.001). However, the neck RFS was similar between the two subgroups of pN+ (P = 0.550). Especially, patients with pN+ at level I had a significantly higher SRLR rate (P = 0.006) compared to those with pN0 at level I. Multivariate analysis showed that pN+ was an unfavorable factor for tumor recurrence and OS. CONCLUSION: Our data did not support the use of SIF in OTSCC patients with pN+ neck at level I due to an significantly increased SRLR rate compared to those with pN0 neck at level I.

N-linoleyltyrosine resisted the growth of non-small cell lung cancer cells via the regulation of CB1 and CB2 involvement of PI3K and ERK pathways.

Background: N-linoleyltyrosine (NITyr), one of the anandamide analogs, exerts activity via the endocannabinoid receptors (CB1 and CB2), which showed anti-tumor effects in various tumors. Therefore, we speculated that NITyr might show anti-non-small cell lung cancer (NSCLC) effects via the CB1 or CB2 receptor. The purpose of the investigation was to reveal the anti-tumor ability of NITyr on A549 cells and its mechanisms. Methods: The viability of A549 cells was measured by MTT assay, and the cell cycle and apoptosis were both examined by flow cytometry; in addition, cell migration was tested by wound healing assay. Apoptosis-related markers were measured by immunofluorescence. The downstream signaling pathways (PI3K, ERK, and JNK) of CB1 or CB2 were examined through Western blotting. The expressions of CB1 and CB2 were detected by immunofluorescence. Finally, the AutoDock software was used to validate the binding affinity between the targets, such as CB1 and CB2, with NITyr. Results: We found that NITyr inhibited cell viability, hindered the cell cycle, resulted in apoptosis, and inhibited migration. The CB1 inhibitor, AM251, and the CB2 inhibitor, AM630, weakened the aforementioned phenomenon. The immunofluorescence assay suggested that NITyr upregulated the expression of CB1 and CB2. Western blot analysis indicated that NITyr upregulated the expression of p-ERK, downregulated the expression of p-PI3K, and did not affect p-JNK expression. In conclusion, NITyr showed a role in inhibiting NSCLC through the activation of CB1 and CB2 receptors involved in PI3K and ERK pathways.

N-linoleyltyrosine ameliorates high-fat diet-induced obesity in C57BL/6 mice via cannabinoid receptor regulation.

Objectives: N-linoleyltyrosine (NITyr) showed mild effects in preclinical studies. The research discussed the effect of NITyr on a high-fat diet (HFD) induced obese (DIO) mice, and preliminarily explored its mechanism. Methods: The DIO mice were established by feeding an HFD for 12 weeks and subsequently administrated orally with NITyr (30, 60 and 100 mg/kg) for four weeks. The indexes of serum and liver samples were determined by ELISA kit. The pathological status of adipose and liver were detected by HE staining. The factors related to energy and lipid metabolism were measured via western blot. Results: NITyr at 60 and 100 mg/kg/day suppressed the weight gain without affecting water and food intake. Accordingly, NITyr reduced adipose weight and the area of individual adipocytes and increased the number of adipocytes. Moreover, NITyr didn't affect the appetite-related indexes such as ghrelin, peptide YY and brain-derived neurotrophic factor. Besides, NITyr didn't affect other organ coefficients except for the liver. Correspondingly, NITyr reduced alanine aminotransferase and aspartate aminotransferase levels, yet didn't influence IL-1ß and TNF-α levels, and the liver injury. The levels of triacylglycerol (TG), total cholesterol (TC), glucose, insulin, adiponectin and leptin in serum were assessed to evaluate the effect of NITyr on glucose and lipid metabolism. NITyr decreased the levels of TG, TC and glucose, and didn't affect insulin, adiponectin and leptin levels. Meanwhile, NITyr up-regulated p-AMPK and the cannabinoid receptor 2 (CB2) expressions, and down-regulated PPAR, FAS and cannabinoid receptor 1 (CB1) expressions.Overall, NITyr suppressed lipid accumulation via improving lipid and glucose metabolism involving CB1 and CB2 receptors.