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1.
Chembiochem ; : e202400285, 2024 May 16.
Article in English | MEDLINE | ID: mdl-38752893

ABSTRACT

ω-Transaminases (ω-TAs) are attractive biocatalysts asymmetrically catalyzing ketones to chiral amines. However, poor non-native catalytic activity and substrate promiscuity severely hamper its wide application in industrial production. Protein engineering efforts have generally focused on reshaping the substrate-binding pockets of ω-TAs. However, hotspots around the substrate tunnel as well as distant sites outside the pockets may also affect its activity. In this study, the ω-TA from Bacillus megaterium (BmeTA) was selected for engineering. The tunnel mutation Y164F synergy with distant mutation A245T which was acquired through a multiple sequence alignment showed improved soluble expression, a 3.7-fold higher specific activity and a 19.9-fold longer half-life at 45℃. Molecule Dynamics simulation explains the mechanism of improved catalytic activity, enhanced thermostability and improved soluble expression of BmeTAY164F/A245T(2M). Finally, the resting cells of 2M were used for biocatalytic processes. 450 mM of S-methoxyisopropylamine (S-MOIPA) was obtained with an ee value of 97.3% and a conversion rate of 90%, laying the foundation for its industrial production. Mutant 2M was also found to be more advantageous in catalyzing the transamination of various ketones. These results demonstrated that sites that are far away from the active center also play an important role in the redesign of ω-TAs.

2.
Mitochondrial DNA B Resour ; 9(1): 209-213, 2024.
Article in English | MEDLINE | ID: mdl-38298222

ABSTRACT

The mitochondrial genome (mitogenome) of Boulenophrys baishanzuensis (Anura: Megophryidae) was sequenced by the Illumina platform. The assembled circular mitogenome of B. baishanzuensis had a total length of 17,040 bp, with a GC content of 41.25%. It consisted of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes, and a D-loop region. The majority of the PCGs were encoded by the H-strand, while one PCG (nad6) and eight tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser2, tRNA-Glu, and tRNA-Pro) were encoded in the L-strand. Phylogenetic analysis revealed that the newly sequenced species formed a clade with other Boulenophrys species, while the genus Boulenophrys itself formed a sister group with the genus Atympanophrys.

3.
J Pediatr Orthop B ; 33(2): 136-141, 2024 Mar 01.
Article in English | MEDLINE | ID: mdl-37129032

ABSTRACT

To evaluate demographic characteristics and distribution of pediatric supracondylar fractures (SCFs) at a tertiary hospital in South China. A retrospective observational study was conducted on children aged 15 years or younger with a diagnosis of SCFs during the period from January 2016 to December 2018. Patients' medical records and radiographs were retrospectively analyzed for age at the time of injury, sex, site and mechanism of traumatic injury. A total of 760 patients with 761 SCFs were reviewed (453 males, 59.6%, and 307 females, 40.4%). There were 748 extension-type fractures (98.3%) and 13 flexion-type fractures (1.7%). Associated injuries were identified in 30/760 (3.9%) patients: associated fracture ( n  = 15; 2%), nerve injury ( n  = 12; 1.6%), open fracture ( n  = 2; 0.2%) and compartment syndrome ( n  = 1; 0.1%). Age at the time of fracture has a bimodal pattern with a first peak around the age of 1 year and a second peak around the age of 4-5 years. The fractures occurred mostly around 11 a.m. and between 4 and 9 p.m. in the evening. Most fractures occurred at home (50.7%), and falling down (62.2%) was the most frequent mechanism of injury. SCFs occurred most frequently in children aged 1 and 4-5 years, and during daylight hours. In about 96% of cases, these were isolated injuries, and falling down was found to be the most frequent traumatic mechanism. Based on our findings, targeted educational efforts and interventions can be set up in order to prevent the occurrence of SCFs in South China. Level of evidence: III.


Subject(s)
Fractures, Open , Humeral Fractures , Male , Female , Child , Humans , Retrospective Studies , China/epidemiology
4.
J Asian Nat Prod Res ; 25(12): 1217-1222, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37163366

ABSTRACT

A new abietane diterpenoid, 1ß, 11-epoxyabieta-12-hydroxy-8, 11, 13-triene-7-one (1), along with three known compounds (2-4), was isolated from Lycopodium complanatum. Their structures were confirmed by the analysis of 1D, 2D NMR and HRESIMS data, and comparison with previous spectral data. All compounds were tested for inhibitory activities against A549, HepG2 and MCF-7 tumor cell lines. [Figure: see text].


Subject(s)
Antineoplastic Agents, Phytogenic , Lycopodium , Humans , Abietanes/pharmacology , Abietanes/chemistry , Molecular Structure , Lycopodium/chemistry , Cell Line, Tumor , MCF-7 Cells , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/chemistry
5.
Anal Methods ; 15(10): 1315-1322, 2023 Mar 09.
Article in English | MEDLINE | ID: mdl-36802289

ABSTRACT

In this work, a new type of H2S sensor was fabricated by means of drop-coating of an Au/SnO-SnO2 nanosheet material, which was prepared by a one-pot hydrothermal reaction, onto a gold electrode in an alumina ceramic tube with the formation of a thin nanocomposite film. The microstructure and morphology of the nanosheet composites were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). A gas-sensitivity study presented good H2S-sensing performance of such Au/SnO-SnO2 nanosheet composites. At an optimal operating temperature of 240 °C and ambient temperature of 25 °C, the resulting sensor showed a good linear response to H2S in a range of 1.0 to 100 ppm with a low detection limit of 0.7 ppm, and a very fast response-recovery time of 22 s for response and 63 s for recovery, respectively. The sensor was also unaffected by ambient humidity and had good reproducibility and selectivity. When being applied to the monitoring of H2S in an atmospheric environment in a pig farm, the response signal to H2S was only attenuated by 4.69% within 90 days, proving that the sensor had a long and stable service lifetime for continuous running and showing its important practical application prospects.

6.
Chem Biodivers ; 19(10): e202200767, 2022 Oct.
Article in English | MEDLINE | ID: mdl-36098055

ABSTRACT

Two new C21 steroidal glycosides, brapreguanes A and B (1-2) were isolated from 75 % aqueous ethanol extract of Selaginella braunii Baker. Their structures were established by spectroscopic analyses (1D/2D NMR spectra and HR-ESI-MS). The absolute configurations of sugar were elucidated by enzymatic hydrolysis and GCMS analysis. In addition, all compounds were evaluated for the anti-proliferative activities against various human cancer cells in vitro. Compounds exhibited no inhibition to various human cancer cells.


Subject(s)
Selaginellaceae , Humans , Selaginellaceae/chemistry , Molecular Structure , Glycosides/pharmacology , Glycosides/chemistry , Sugars , Ethanol , Plant Extracts
7.
Transl Lung Cancer Res ; 11(5): 832-844, 2022 May.
Article in English | MEDLINE | ID: mdl-35693282

ABSTRACT

Background: A paucity of strategies exist for extensive-stage small cell lung cancer (ES-SCLC) patients who fail the first-line chemotherapy. Apatinib is a tyrosine kinase inhibitor (TKI) that selectively inhibits vascular endothelial growth factor receptor-2 (VEGFR-2), which has been demonstrated to have active anti-tumor activity in ES-SCLC when used only or combined with PD-1 inhibitors or chemotherapy with good tolerance. However, the efficacy and safety of apatinib monotherapy is unclear in second-line or beyond treatment of ES-SCLC. Methods: In this prospective, exploratory, single-arm, multi-center study, eligible patients were aged 18 years or older with histologically confirmed ES-SCLC, and had progressed on, or were intolerant to previous systemic treatment. Patients received apatinib 500 mg (orally qd, every 4 weeks a cycle). The efficacy was assessed after 1 cycle and then every 2 cycles based on computed tomography imaging per the Response Evaluation Criteria in Solid Tumors (RECIST, version 1.1). The primary endpoint was progression-free survival (PFS). The adverse events (AEs) were assessed per the National Cancer Institute Common Terminology Criteria for Adverse Events 4.0 (NCI-CTCAE 4.0). This study is registered in the Chinese Clinical Trial Registry, number ChiCTR-OPC-17013964. Results: From 28 July 2017 to 21 June 2019, 62 patients were screened for eligibility, among whom 57 patients were available for efficacy and safety analysis. The objective response rate (ORR) was 14.3% and disease control rate (DCR) was 79.6%. The median PFS was 5.6 months [95% confidence interval (CI): 3.3-8.0 months] and the median overall survival (OS) was 11.2 months (95% CI: 7.5-24.0 months). Among the participants who received apatinib as second-line treatment, the median PFS and OS were 6.1 months (95% CI: 2.6-7.6 months) and 12.0 months (95% CI: 7.9 months to not reached), respectively. The most common AEs of all grades were anemia (36.8%), hypertension (33.3%), fatigue (31.6%), blood bilirubin increased (22.8%), elevated transaminase (19.3%), and hand-foot syndrome (17.54%). Grade 3 AEs included 2 (3.5%) cases of hypertension and 1 (1.8%) case of fatigue. No grade 4/5 AEs were observed. Conclusions: Apatinib showed encouraging anti-tumor activity in pretreated ES-SCLC patients with tolerable toxicities. Further larger scale studies are warranted to demonstrate the efficacy of apatinib.

8.
Lasers Surg Med ; 54(6): 835-840, 2022 08.
Article in English | MEDLINE | ID: mdl-35395702

ABSTRACT

BACKGROUND: Pyogenic granuloma (PG) is a common vascular neoplasm in children. Data on 595 nm pulsed dye lasers for the treatment of PG in children remain scarce. OBJECTIVE: To summarize the clinical characteristics and to evaluate the effectiveness and safety of the 595 nm pulsed dye laser for the treatment of PG in children. STUDY DESIGN: Retrospective case series. METHODS: A retrospective study was performed on 212 patients treated for PG with a 595 nm pulsed dye laser. SPSS version 19.0 was used for statistical analysis. RESULTS: Among all 212 patients treated, 208 showed complete resolution of the lesion, and 4 dropped out after one treatment due to bleeding. A single treatment was sufficient in 139 (66.8%) patients, while two or three treatments were sufficient in 69 (33.2%) patients. Male patients responded better than female patients (χ2 = 7.603, p =0.006). Lesions in the nonorbital region responded better than those in the orbital region (χ2 = 7.445, p =0.006). The size of the lesion affected the effectiveness, and lesions with smaller diameters (t = -5.776, p <0.01) and heights (t = -10.368, p <0.01) showed better results. COMPLICATIONS AND SIDE EFFECTS: Twelve patients (5.8%) were reported to have local complications and side effects, including edematous erythema, slight bleeding, hyperpigmentation, and hypopigmentation. The edematous erythema and slight bleeding disappeared gradually after several days. The localized pigment changes usually resolved spontaneously and disappeared completely after 6 months. CONCLUSIONS: Our experience confirmed the efficacy and safety of the 595 nm pulsed dye laser for the treatment of PG in children.


Subject(s)
Granuloma, Pyogenic , Lasers, Dye , Child , Erythema , Female , Granuloma, Pyogenic/surgery , Humans , Lasers, Dye/therapeutic use , Male , Retrospective Studies , Treatment Outcome
9.
Pain Physician ; 25(1): E43-E54, 2022 01.
Article in English | MEDLINE | ID: mdl-35051151

ABSTRACT

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a progressive and painful disorder due to impaired blood supply to the femoral head, yet little is known about the effect of ozone therapy in femoral head necrosis. OBJECTIVES: We aimed to evaluate the clinical and radiographic outcomes of ozone therapy in the treatment of ONFH. STUDY DESIGN: Nonrandomized clinical trial. SETTINGS: The study was conducted in a single-center, academic institution. METHODS: A total of 71 patients (107 hip joints) with Association Research Circulation Osseous (ARCO) stage-I, II, III, and IV ONFH were included and assigned to undergo either intraarticular O2-O3 mixture hip injections with ozonated autohemotherapy (ozone therapy group, n = 39, 58 hip joints) or protected weight bearing (control group, n = 32, 49 hip joints). The primary outcomes included the Visual Analog Scale (VAS) for pain intensity and Harris Hip Score (HHS) for hip function. The secondary outcomes included bone marrow edema examination, and conversion to total hip arthroplasty (THA). RESULTS: Ozone therapy effectively improves VAS for pain intensity and HHS during the follow-up period compared to the control group. Ozone therapy showed a significant resolution of bone marrow edema of the femoral head compared to the control group (P < 0.001). Thirteen of the 49 hips (26.53%) in the control group underwent THA, whereas only 6 hips (10.34%) in the ozone therapy group required THA during a 30-month follow-up (P = 0.041). The cumulative analysis revealed a low rate of conversion to THA in the ozone therapy group (logrank test; P = 0.022). LIMITATIONS: The study is limited by a single treatment protocol in addition to the lack of a randomized design. CONCLUSIONS: Ozone therapy was associated with significant pain relief, improvement in hip function, and bone marrow edema resolution that may delay the need for THA in patients affected by ONFH.Institutional Review Board (IRB) approval number: HK2018-10-28.Clinical trials registration number: ChiCTR1900023449.


Subject(s)
Arthroplasty, Replacement, Hip , Femur Head Necrosis , Ozone , Femur Head/surgery , Femur Head Necrosis/complications , Femur Head Necrosis/diagnosis , Femur Head Necrosis/therapy , Humans , Ozone/therapeutic use , Pilot Projects
10.
Nat Prod Res ; 36(3): 772-779, 2022 Feb.
Article in English | MEDLINE | ID: mdl-32762444

ABSTRACT

Four new prenylflavonol glycosides (1-4) along with two known analogues (5-6) were isolated from the leaves of Cyclocarya paliurus for the first time. The structures of these compounds were characterized by comprehensive analysis of 1 D, 2 D NMR, HRESIMS, UV data and enzymatic hydrolysis. In bioassays, compounds 1-4 were evaluated for inhibitory effects on xanthine oxidase (XOD) and effects on the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS) induced RAW264.7 cells. Moreover, compounds 1 and 2 showed outstanding XOD inhibitions with IC50 values of 18.16 ± 3.91 and 37.65 ± 5.67 µM, and exhibited inhibitions against LPS-induced NO production with IC50 values of 80.50 ± 3.09 and 82.28 ± 2.87 µM.


Subject(s)
Juglandaceae , Triterpenes , Glycosides/pharmacology , Plant Leaves , Xanthine Oxidase
11.
Zhonghua Nan Ke Xue ; 27(7): 589-594, 2021 Jul.
Article in Chinese | MEDLINE | ID: mdl-34914224

ABSTRACT

OBJECTIVE: To study the effects of Nur77 on prostate cancer (PCa) cell growth and its potential value in the treatment of PCa. METHODS: We detected the expression of the NUR77 protein in human PCa tissues and cells by Western blot and determined the effects of Nur77 on the proliferation and apoptosis of the PCa cells by flow cytometry. RESULTS: Nur77 and AR were expressed in the human PCa tissue and cells, and overexpressed NUR77 inhibited the proliferation and cell cycle progression of the PCa LNCaP cells. The small-molecule receptor agonists cytosporone B and DIMC of Nur7 significantly suppressed the growth and induced the apoptosis of the PCa LNCaP cells. CONCLUSIONS: Nur77 inhibits the proliferation and induces the apoptosis of PCa cells, and is expected to be a potential molecular target for the treatment of PCa.


Subject(s)
Prostatic Neoplasms , Cell Proliferation , Humans , Male
12.
Front Cell Dev Biol ; 9: 720383, 2021.
Article in English | MEDLINE | ID: mdl-34692679

ABSTRACT

Hearing loss is a serious illness affecting people's normal life enormously. The acoustic properties of a tympanic membrane play an important role in hearing, and highly depend on its geometry, composition, microstructure and connection to the surrounding annulus. While the conical geometry of the tympanic membrane is critical to the sound propagation in the auditory system, it presents significant challenges to the study of the 3D microstructure of the tympanic membrane using traditional 2D imaging techniques. To date, most of our knowledge about the 3D microstructure and composition of tympanic membranes is built from 2D microscopic studies, which precludes an accurate understanding of the 3D microstructure, acoustic behaviors and biology of the tissue. Although the tympanic membrane has been reported to contain elastic fibers, the morphological characteristic of the elastic fibers and the spatial arrangement of the elastic fibers with the predominant collagen fibers have not been shown in images. We have developed a 3D imaging technique for the three-dimensional examination of the microstructure of the full thickness of the tympanic membranes in mice without requiring tissue dehydration and stain. We have also used this imaging technique to study the 3D arrangement of the collagen and elastic fibrillar network with the capillaries and cells in the pars tensa-annulus unit at a status close to the native. The most striking findings in the study are the discovery of the 3D form of the elastic and collagen network, and the close spatial relationships between the elastic fibers and the elongated fibroblasts in the tympanic membranes. The 3D imaging technique has enabled to show the 3D waveform contour of the collagen and elastic scaffold in the conical tympanic membrane. Given the close relationship among the acoustic properties, composition, 3D microstructure and geometry of tympanic membranes, the findings may advance the understanding of the structure-acoustic functionality of the tympanic membrane. The knowledge will also be very helpful in the development of advanced cellular therapeutic technologies and 3D printing techniques to restore damaged tympanic membranes to a status close to the native.

13.
World J Clin Cases ; 9(21): 5900-5908, 2021 Jul 26.
Article in English | MEDLINE | ID: mdl-34368308

ABSTRACT

BACKGROUND: For parturients with paroxysmal uterine contraction pain, rapid analgesia is needed. We used preprocedure ultrasound imaging combined with the palpation technique in epidural analgesia for labor, and evaluated the usefulness of this technique in epidural labor analgesia. AIM: To evaluate the usefulness of preprocedure ultrasound imaging in epidural analgesia for labor. METHODS: In this prospective randomized observational study, 72 parturients were assigned to two groups (combined or palpation group). The target interspace of all parturients was first identified by the palpation technique. Then in the combined group, preprocedure ultrasound imaging was used before epidural puncture. In the palpation group, only the traditional anatomical landmarks technique (palpation technique) was performed. The primary outcome was total duration of the epidural procedure (for the ultrasound group, the duration of the preprocedure ultrasound imaging was included). The secondary outcomes were the number of skin punctures, the success rate at first needle pass, the number of needle passes, the depth from the skin to epidural space, and the complications of the procedure. RESULTS: Total duration of the epidural procedure was similar between the two groups (406.5 ± 92.15 s in the combined group and 380.03 ± 128.2 s in the palpation group; P = 0.318). A significant improvement was demonstrated for epidural puncture and catheterization in the combined group. The number of needle passes was 1.14 in the combined group and 1.72 in the palpation group (P = 0.001). The number of skin puncture sites was 1.20 in the combined group and 1.25 in the palpation group (P = 0.398). The success rate at first needle pass was 88.89% in the combined group and 66.67% in the palpation group (P = 0.045). CONCLUSION: This study demonstrated that the total duration of epidural procedures with preprocedure ultrasound imaging combined with the palpation technique was not longer than the traditional anatomical landmarks technique, which were performed by six experienced anesthesiologists in parturients with normal weights undergoing labor analgesia.

14.
Nat Prod Res ; 35(21): 4018-4024, 2021 Nov.
Article in English | MEDLINE | ID: mdl-32316785

ABSTRACT

Three previously undescribed dammarane triterpenoid glycosides (1-3) along with five known analogues (4-8) were isolated from the leaves of Cyclocarya paliurus. Their structures and configurations were determined on the basis of comprehensive spectroscopic analyses, chemical hydrolysis and DFT GIAO 13C NMR calculation. All the isolates were evaluated cytotoxic activities against seven human cancer cell lines (MCF-7, PC-3, Du145, NCI-H1975, PC-9, SKVO3 and HepG2). Moreover, compound 4 showed a wide spectrum of cytotoxicity against human cancer cells with IC50 values ranging from 11.31 to 29.51 µM.


Subject(s)
Juglandaceae , Triterpenes , Glycosides/pharmacology , Humans , Magnetic Resonance Spectroscopy , Molecular Structure , Plant Leaves , Triterpenes/pharmacology
15.
Food Sci Anim Resour ; 40(4): 563-577, 2020 Jul.
Article in English | MEDLINE | ID: mdl-32734264

ABSTRACT

This study assessed breed differences in fatty acid composition and meat quality of Longissimus thoracis et lumborum (LTL) and semitendinosus (SE) of Angus× Chinese Simmental (AS), Wagyu×Chinese Simmental (WS), and Chinese Simmental (CS). CS (n=9), AS (n=9) and WS (n=9) were randomly selected from a herd of 80 bulls which were fed and managed under similar conditions. Fatty acid profile and meat quality parameters were analyzed in duplicate. Significant breed difference was observed in fatty acid and meat quality profiles. AS exhibited significantly (p<0.05) lower C16:0 and higher C18:1n9c compared with CS. AS breed also had a tendency (p<0.10) to lower total saturated fatty acid (SFA), improve C18:3n3 and total unsaturated fatty acid (UFA) compared with CS. Crossbreed of AS and WS had significantly (p<0.05) improved the lightness, redness, and yellowness of muscles, and lowered cooking loss, pressing loss, and shear force compared with CS. These results indicated that fatty acid composition and meat quality generally differed among breeds, although the differences were not always similar in different tissues. Fatty acid composition, meat color, water holding capacity, and tenderness favored AS over CS. Thus, Angus cattle might be used to improve fatty acid and meat quality profiles of CS, and AS might contain better nutritive value, organoleptic properties, and flavor, and could be potentially developed as an ideal commercial crossbreed.

16.
Article in English | MEDLINE | ID: mdl-32850749

ABSTRACT

Pseudomonas putida (P. putida) KT2440 is a paradigmatic environmental-bacterium that possesses significant potential in synthetic biology, metabolic engineering and biodegradation applications. However, most genome editing methods of P. putida KT2440 depend on heterologous repair proteins and the provision of donor DNA templates, which is laborious and inefficient. In this report, an efficient cytosine base editing system was established by using cytidine deaminase (APOBEC1), enhanced specificity Cas9 nickase (eSpCas9ppD10A) and the uracil DNA glycosylase inhibitor (UGI). This constructed base editor converts C-G into T-A in the absence of DNA strands breaks and donor DNA templates. By introducing a premature stop codon in target spacers, we successfully applied this system for gene inactivation with an efficiency of 25-100% in various Pseudomonas species, including P. putida KT2440, P. aeruginosa PAO1, P. fluorescens Pf-5 and P. entomophila L48. We engineered an eSpCas9ppD10A-NG variant with a NG protospacer adjacent motif to expand base editing candidate sites. By modifying the APOBEC1 domain, we successfully narrowed the editable window to increase gene inactivation efficiency in cytidine-rich spacers. Additionally, multiplex base editing in double and triple loci was achieved with mutation efficiencies of 90-100% and 25-35%, respectively. Taken together, the establishment of a fast, convenient and universal base editing system will accelerate the pace of future research undertaken with P. putida KT2440 and other Pseudomonas species.

17.
Bioorg Chem ; 101: 104018, 2020 08.
Article in English | MEDLINE | ID: mdl-32629277

ABSTRACT

Eight new prenylflavonol glycosides (1-8), along with five known analogues (9-13) were isolated from the n-butanol extract of the dried leaves of Cyclocarya paliurus (family Juglandaceae) for the first time. The structures of these compounds were characterized by comprehensive analysis of 1D, 2D NMR, HRESIMS, UV data and acid hydrolysis. In bioassay, all these thirteen prenylflavonol glycosides exhibited inhibitory effects on xanthine oxidase (XOD) activity. Especially compounds 2 and 7, showed outstanding IC50 values of 31.81 ± 2.20 and 29.71 ± 3.69 µM, respectively.


Subject(s)
Enzyme Inhibitors/pharmacology , Flavonols/pharmacology , Glycosides/pharmacology , Juglandaceae/chemistry , Plant Leaves/chemistry , Xanthine Oxidase/antagonists & inhibitors , Plant Extracts/chemistry , Spectrum Analysis/methods , Structure-Activity Relationship
18.
Appl Biochem Biotechnol ; 192(3): 923-934, 2020 Nov.
Article in English | MEDLINE | ID: mdl-32617841

ABSTRACT

Nicotinate dehydrogenase (NDHase) is a membrane protein with three subunits (ndhS, ndhL, and ndhM), which is difficult to express in a functional form using common hosts such as Escherichia coli, Bacillus subtilis, or Pichia pastoris. Comamonas testosteroni is a suitable microbial chassis for expressing multi-subunit membrane proteins. However, the expression of NDHase in C. testosteroni is extremely low. We have developed a systematic approach to create an efficient protein expression system in C. testosteroni CNB-2 using multi-level N-terminal engineering. We selected a strong promoter for the Mmp1 system that enables control of transcriptional strength in unconventional bacteria. This enhanced the expression of a green fluorescent reporter protein threefold. Following modification of the N-terminal Shine-Dalgarno sequence and rearrangement of amino acid sequence in the starting area of the gene encoding NDHase, enzyme activity increased from 90.6 to 165 U/L. These optimized N-terminal Shine-Dalgarno and amino acid sequences were used to enhance the expression of ndhL subunit and improve the balance expression of three subunits of NDHase, resulting in enzyme activity of 192 U/L that far surpasses the previously reported level. These results highlight a promising strategy for the development of other heterologous expression systems for challenging proteins using unconventional bacteria.


Subject(s)
Comamonas testosteroni/genetics , Genetic Engineering , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/genetics , Genes, Reporter/genetics , Promoter Regions, Genetic/genetics
19.
Kaohsiung J Med Sci ; 36(8): 592-598, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32196963

ABSTRACT

Although micro RNA (miRNA) expression profiles are widely investigated in renal cell carcinoma (RCC), their potential roles for affecting RCC initiation and progression remain largely unknown. Here, we examined the aberrant expression profiles of miRNAs inhuman metastatic RCC tissues based on Gene Expression Omnibus (GSE37989). We further validated them iRNAs expression data in the largest clinical dataset: The Cancer Genome Atlas (TCGA). And cell adhesion and migration abilities and epithelial me senchymal transition (EMT) related proteins were assessed in both normal and tumor RCC cell lines. We suggest that hsa-miR-143 is a potential tumor suppressor in RCC as its down regulation positively correlated with adverse prognosis. Biologically, cell adhesion, migration, and EMT were dramatically inhibited by miR-143. Mechanistically, we found that miR-143 targets ABL proto-oncogene 2 (ABL2), which was also found to be an indicator for poor survival in TCGA database. Our results have important implications in understanding functions of miRNAs in metastatic RCC and will provide a basis for further clinical application.


Subject(s)
Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , MicroRNAs/metabolism , Protein-Tyrosine Kinases/metabolism , Cell Adhesion/genetics , Cell Line, Tumor , Cell Movement/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/genetics , Neoplasm Metastasis , Proto-Oncogene Mas
20.
Spine (Phila Pa 1976) ; 45(17): E1066-E1076, 2020 Sep 01.
Article in English | MEDLINE | ID: mdl-32205688

ABSTRACT

STUDY DESIGN: An experimental animal study of treatment of spinal cord injury (SCI). OBJECTIVE: This report aims to evaluate the in vivo effects of butylphthalide NBP on SCI biology and to explore its potential mechanism. SUMMARY OF BACKGROUND DATA: SCI causes great damage to humans. The inflammatory and reconstructive processes after SCI is regulated by activation of astroglial and microglial cells. Activated microglia/macrophages can be divided into M2 (anti-inflammatory) and M1 (pro-inflammatory) phenotypes. Butylphthalide (3-n-butylphthalide or NBP) treatment can significantly alleviate ischemic brain damage, and further study has confirmed that central neuroprotective effects can be realized by converting M1 polarized microglia/macrophages to the M2 phenotype. Thus far, it remains unknown whether NBP can modulate the transition of macrophages/microglia between the M1 and M2 phenotypes. METHODS: We randomly divided male mice into three groups (sham group, SCI group, SCI+ NBP group). Molecular and histological tests were performed to detect the macrophage/microglia polarization as well as the potential mechanism of NBP in vivo and in vitro. RESULT: It was found that NBP treatment significantly attenuated the motor dysfunction and neuronal apoptosis induced by SCI. Treatment with NBP could also reduce pro-inflammatory cytokine release after SCI and could facilitate macrophage/microglia M2 polarization and inhibit M1 polarization after SCI. To verify the findings in animal experiments, we examined the effect of NBP on BV2 cell polarization, the results showed that NBP treatment could enhance M2 polarization and inhibit M1 polarization, and that M2 polarization occurred in a p38-dependent manner. CONCLUSION: NBP plays an important role in the anti-inflammatory response in SCI via the facilitation of macrophage/microglia M2 polarization as well as the inhibition of macrophage/microglia M1 polarization. The M2 polarization of macrophages/microglia occurs via activation of p38 pathway. LEVEL OF EVIDENCE: 3.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Benzofurans/therapeutic use , Macrophages/metabolism , Microglia/metabolism , Spinal Cord Injuries/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Anti-Inflammatory Agents/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Benzofurans/pharmacology , Cell Polarity/drug effects , Cell Polarity/physiology , Macrophages/drug effects , Male , Mice , Mice, Inbred C57BL , Microglia/drug effects , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Phosphorylation/drug effects , Phosphorylation/physiology , Spinal Cord Injuries/drug therapy , Thoracic Vertebrae/injuries
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