Platelet-rich plasma promotes bone formation, restrains adipogenesis and accelerates vascularization to relieve steroids-induced osteonecrosis of the femoral head.

Steroid-associated necrosis of the femoral head (SANFH) is one of the most common and refractory chronic diseases with increasing incidence. The typical pathological changes of SANFH include decreased osteogenic differentiation, enhanced intramedullary adipocytes deposition and impaired osseous circulation. In this study, we investigated the effects and potential mechanisms of Platelet-rich plasma (PRP) on SANFH. Sixty Sprague-Dawley rats were randomly divided into the control, PRP donor, model, and PRP groups. Compared to the model group, PRP treatment significantly increased the hemorheological indexes and serum levels of bone gla-protein (BGP) and vascular endothelial growth factor (VEGF), while decreased the levels of triglyceride (TG) and total cholesterol (TC). Meanwhile, Micro-CT and histopathological stain (Hematoxylin-eosin and Alcian blue-hematoxylin/orange G staining) were performed on the femoral head for morphological and histopathological evaluation, indicating that bone trabecular microstructure and bone mineral density (BMD) were significantly improved after PRP treatment. Immunohistochemical analysis revealed that PRP remarkably up-regulated the expression of osteogenic markers including ß-catenin and alkaline phosphatase (ALP), angiogenic markers containing VEGF and platelet endothelial cell adhesion molecule-1 (CD31), while down-regulated adipogenic markers involving fatty acid-binding protein (FABP-4), and peroxisome proliferator-activated receptor gamma (PPAR-γ) in SANFH rat models. In summary, for the first time, PRP was demonstrated to prevent the development of SANFH through stimulating bone formation and vascularization as well as retarding adipogenesis.

Incidence and risk factors for retrograde type A dissection and stent graft-induced new entry after thoracic endovascular aortic repair.

OBJECTIVE: Stent graft (SG)-induced new entry (SINE) and retrograde type A dissection (RTAD) are serious device-related complications occurring after thoracic endovascular aortic repair (TEVAR) for Stanford type B aortic dissection (TBAD) and may lead to endograft-related complications including retrograde dissection and death. The purpose of this study was to investigate the incidence and risk factors for the development of RTAD and SINE after TEVAR for TBAD and to identify the complications associated with this. METHODS: From April 2005 to October 2013, there were 997 patients who underwent TEVAR for TBAD; 852 were followed up (0-6 years; mean, 2.6 years), and 59 SINEs developed in 53 patients. The oversizing ratio and incidence of RTAD and SINE were compared between proximal bare stent (PBS) and non-PBS groups and RTAD and SINE and non-RTAD and non-SINE groups. The baseline characteristics and SG configurational factors potentially affecting both RTAD and distal SINE were analyzed. RESULTS: There was no significant difference between PBS and non-PBS groups in the incidence of RTAD. A greater oversizing ratio was related to a higher distal SINE rate. SINE was seen more frequently in smokers and in patients with hypertension, Marfan syndrome, and TEVAR in the chronic phase and less frequently in complicated dissection cases. Device-related factors for SINE were SG with a connecting bar and SG length <165 mm. The SG length <165 mm increased the overall proximal and distal SINE incidence in multivariate analysis. CONCLUSIONS: The presence of a PBS is not associated with a higher RTAD rate, whereas the use of an SG with a connecting bar and length <165 mm increases the risk of RTAD and SINE after TEVAR.

[Autophagy and hypoxic ischemic brain injuries].

Autophagy is a highly evolutionarily conserved physiological mechanism of organism, including several stages such as autophagosomes formation, the fusion of lysosomes and autophagosomes, and autophagosomes degradation. In physiological conditions, autophagy is responsible for clearing the spoiled organelles and long-lived proteins to maintain the homeostasis of cells and organism. Meanwhile, autophagy is also involved in the formation and development of diseases, but the mechanism has not been confirmed yet. The relationship between autophagy and hypoxic ischemic brain injuries represented by stroke is a research hotpot in recent years, but there is no clear conclusion about autophagy's role and mechanism in hypoxic ischemic brain injuries. We reviewed the activation, function and mechanism of autophagy in hypoxic ischemic brain injuries, in order to provide some perspectives on these researches.

A strategy for enhanced circular DNA construction efficiency based on DNA cyclization after microbial transformation.

BACKGROUND: With the rapid development of synthetic biology, the demand for assembling multiple DNA (genes) fragments into a large circular DNA structure in one step has dramatically increased. However, for constructions of most circular DNA, there are two contradictions in the ligation/assembly and transformation steps. The ligation/assembly consists of two different reactions: 1) the ligation/assembly between any two pieces of a linear form DNA; 2) the cyclization (or self-ligation) of a single linear form DNA. The first contradiction is that the bimolecular ligation/assembly requires a higher DNA concentration while the cyclization favors a lower one; the second contradiction is that a successful transformation of a ligation/assembly product requires a relatively high DNA concentration again. This study is the first attempt to use linear plasmid and Cyclization After Transformation (CAT) strategy to neutralize those contradictions systematically. RESULTS: The linear assembly combined with CAT method was demonstrated to increase the overall construction efficiency by 3-4 times for both the traditional ligation and for the new in vitro recombination-based assembly methods including recombinant DNA, Golden Gate, SLIC (Sequence and Ligation Independent Cloning) and Gibson Isothermal Assembly. Finally, the linear assembly combined with CAT method was successfully applied to assemble a pathway of 7 gene fragments responsible for synthesizing precorrin 3A which is an important intermediate in VB12 production. CONCLUSION: The linear assembly combined with CAT strategy method can be regarded as a general strategy to enhance the efficiency of most existing circular DNA construction technologies and could be used in construction of a metabolic pathway consisting of multiple genes.

[Microenvironments induce iPSCs and BMSCs into neuron-like cells--Reelin's regulative role in cell differentiation and polarization].

The present study was aimed to investigate how the induced pluripotent stem cells (iPSCs) and bone marrow mesenchymal stem cells (BMSCs) differentiate into neuron-like cells under the induction of hippocampal microenvironments and Reelin's regulation. iPSCs or BMSCs were co-cultured with WT (wild type) or genotypic hippocampal slice and cerebral homogenate supernatant, then the stem cells' differentiation under the induction of hippocampal environment was observed by using immunofluorescence technique. In the meantime, stem cells were co-cultured with hippocampal slice and cerebral conditioned medium of reeler (Reelin deletion) mouse respectively. The results showed that both adhesive iPSCs and BMSCs on WT hippocampal slice exhibited lamination of double "C" shape with high density on granular and pyramidal layers. The stem cells could differentiate into neuron-like cells with obvious polarization on WT hippocampal slice. In pyramidal cell layer, the differentiated neuron-like cells were oriented vertically with similar shapes of pyramidal cell in vivo, and the cells within molecule layer were arranged horizontally. In addition, adhesive iPSCs and BMSCs could differentiate into Nestin positive neural stem cells and NeuN positive neurons, respectively, under WT hippocampal microenvironment. On the other hand, under induction of hippocampal microenvironment of reeler mouse, iPSCs and BMSCs differentiation could also be seen, but their lamination was in disorder, and cell polarization was irregular. Moreover, differentiation and polarization of the iPSCs and BMSCs were delayed. These results suggest both iPSCs and BMSCs can differentiate into neuron-like cells under the induction of hippocampal microenvironments. Reelin is involved in the regulation of neuronal differentiation and cell polarization. Without Reelin, the cellular lamination and polarization appear irregular, and the stem cells' differentiation is delayed.

[Whole Process Analysis and Fate Behavior of Microplastics in Urban Wastewater Treatment Plants, Including their Occurrence Forms, Components, and Removal Efficiency].

Microplastics are among the most difficult new pollutants to remove in wastewater treatment plants. In order to explore the occurrence form, size distribution, composition, removal efficiency, migration law, and fate behavior characteristics of microplastic particles in sewage plants, taking a sewage treatment plant in Hohhot as an example, a total of 17 sampling sites were set up. The LAS X software counted the shape, abundance, and size of microplastics and conducted a full-process analysis. The results showed that： fibrous microplastics had the highest abundance and widest distribution and were the main form of existence, accounting for 61.8% of the total abundance； the size of microplastics ranged mainly between 0 and 1.00 mm, and among the four sizes, the abundance of microplastics 0.25 to 0.50 mm in China was the highest, accounting for 32.9%. Among the eight types of plastic components detected, polyester substances （PET, PBT）, cellulose, and polypropylene （PP） were the main components, accounting for 25%, 21%, and 17%, respectively. The influent abundance of the sewage plant was （73 ±5） n·L-1, the effluent abundance was （14 ±2） n·L-1, and the overall removal rate was （80.8 ±12.1）%. Among the three treatment stages of the sewage plant, only the primary treatment played a role in removal, and the abundance of microplastics surged in the secondary treatment. Different structures playing a major role in the removal of microplastics were fine grids （49.2 ±7.4）% and secondary sedimentation tanks （92.4 ±13.9）%. Microplastics mainly existed in the form of fibers, fragments, and films. The proportion of fibers was approximately 70%, and the size of fragments was mainly concentrated between 0.50 and 5.00 mm. Most fragments were in the range of 5.00 mm, accounting for 50%, making them the main form apart from fibrous. The film-like size was mostly concentrated in the range of less than 0.50 mm, accounting for more than 10%. Therefore, improving the removal of small-sized fibrous and film-like microplastics and large-sized fragmented microplastic particles can effectively reduce the pollution risk of microplastics in the environment caused by sewage plant drainage.

Patterns of femoropopliteal recurrence after routine and selective stenting endoluminal therapy.

OBJECTIVE: This study determined the incidence and characteristics of recurrent disease after femoropopliteal angioplasty, following either selective or routine stenting of diseased site(s). METHODS: Retrospective analysis of a prospectively maintained database for femoropopliteal interventions from June 2003 to July 2010 was performed. Interventions during this period were from a single institution, followed up at 1, 3, and 6 months after initial intervention and on a semiannual basis thereafter with clinical examinations and duplex ultrasound imaging. Two groups were identified: those with routine stenting (RS; routine stenting for all diseased areas) and those with selective stenting (SS; selective stenting for only segments which exhibited compromised flow from residual stenosis or significant dissection). Patients who developed recurrent symptoms (claudication, rest pain), a decrease in ankle-brachial index (ABI) (>0.2), or duplex documentation of a significant (>80%) recurrent stenosis underwent reintervention. Patient demographics, comorbidities, Trans-Atlantic Inter-Society Consensus (TASC) II classification, runoff, and degree of calcification (none, mild, moderate, severe) at initial intervention were recorded. The time to reintervention and recurrence pattern were recorded for both groups. RESULTS: During the study period, 746 endovascular interventions in 477 patients were performed. Total reintervention rate, including bypass, amputation, and asymptomatic occlusion after initial intervention, was 36.48% (group SS, 42.9%; group RS, 33.1%; P=.04). Of all initial interventions, 182 endovascular reinterventions in 165 patients for recurrent femoropopliteal disease were identified (group SS, 70; group RS, 95). No differences were noted among the groups in gender, comorbidities, initial TASC II classification, run off, calcification scores, or statin or clopidogrel use, or both. Time to recurrence was similar in the RS and SS groups. TASC II classification, runoff score, and degree of calcification were similar between the two groups. Although not statistically significant, analysis of recurrence pattern demonstrated de novo stenosis was more common in the SS group (50.0% vs 34.7%; P=.06). CONCLUSIONS: This single-center retrospective study found a significant difference in the incidence of recurrence requiring reintervention between patients treated with selective and routine stenting for femoropopliteal disease. Analysis of endovascular reinterventions, however, reveals no significant difference in recurrence time or recurrence pattern between the two groups. No significant differences were identified in time to recurrence, TASC II classification, runoff, and calcification of endovascular reinterventions between the two groups' end points. Additional prospective studies to evaluate the roles of routine and selective stenting in symptomatic femoropopliteal peripheral arterial disease and to investigate recurrence lesion characteristics and the patency of multiple endovascular interventions between these two groups are needed.

Trimethoxy-benzaldehyde levofloxacin hydrazone inducing the growth arrest and apoptosis of human hepatocarcinoma cells.

BACKGROUND: In order to search for new structural modification strategies on fluoroquinolones, we have designed and synthesized a series of fluoroquinolone derivatives by linking various hydrazine compounds to the C-3 carboxyl group of levofloxacin and assessed their anticancer activities. Several novel levofloxacin derivatives displayed potent cytotoxicity against the tested cancer cell lines in vitro. In the present study, we investigated the effect of 1-Cyclopropyl-6-fluoro-4-oxo-7- piperazin-1, 4-dihydro- quinoline- 3-carboxylic acid benzo [1,3] dioxol-5- ylmethylene- hydrazide (QNT11) on the apoptosis of human hepatocarcinoma cells in vitro. METHODS: The inhibition effects of QNT11 on cell proliferation were examined by MTT assay. Cell apoptosis was determined by TUNEL and DNA agarose gel electrophoresis method. The topoisomerase ΙΙ activity was measured by agarose gel electrophoresis using Plasmid pBR322 DNA as the substrate. Cell cycle progression was analyzed using flow cytometry in conjunction with ethanol fixation and propidium iodide staining. Mitochondrial membrane potential (△ψm) was measured by high content screening image system. The caspase-9, caspase-8, caspase-3, Bcl-2, Bax, CDK1, Cyclin B1and cytochrome c protein expressions were detected by Western blot analysis. RESULTS: QNT11 showed selective cytotoxicity against Hep3B, SMMC-7721, MCF-7 and HCT-8 cell lines with IC50 values of 2.21 µM, 2.38 µM, 3.17 µM and 2.79 µM, respectively. In contrast, QNT11 had weak cytotoxicity against mouse bone marrow mesenchymal stem cells (BMSCs) with IC50 value of 7.46 µM. Treatment of Hep3B cells with different concentrations of QNT11 increased the percentage of the apoptosis cells significantly, and agarose gel electrophoresis revealed the ladder DNA bands typical of apoptotic cells, with a decrease in the mitochondrial membrane potential. Compared to the control group, QNT11 could influence the DNA topoisomerase IIactivity and inhibit the religation of DNA strands, thus keeping the DNA in fragments. There was a significant increase of cytochrome c in the cytosol after 24 h of treatment with QNT11 and a decrease in the mitochondrial compartment. Observed changes in cell cycle distribution by QNT11 treated might be caused by insufficient preparation for G2/M transition. In addition, QNT11 increased the protein expression of Bax, caspase-9, caspase-8, caspase-3, as well as the cleaved activated forms of caspase-9, caspase-8 and caspase-3 significantly, whereas the expression of Bcl-2 decreased. CONCLUSIONS: Our results showed that QNT11 as a fluoroquinolone derivative exerted potent and selectively anticancer activity through the mechanism of eukaryotic topoisomerase II poisoning. The growth inhibition was in large part mediated via apoptosis-associated mitochondrial dysfunction and regulation of Bcl-2 signaling pathways.

[Endovascular repair for isolated iliac artery aneurysm].

OBJECTIVE: To evaluate the short and middle-term efficacies of endovascular repair for isolated iliac artery aneurysms (IIAAs). METHODS: Retrospective analyses were performed for the clinical and follow-up data of 27 patients undergoing endovascular repair at Department of Vascular Surgery, Affiliated Zhongshan Hospital, Fudan University from January 2008 to December 2012. For asymptomatic aneurysms, repair criterion was a diameter over 3 cm while under 3 cm for symptomatic aneurysms. RESULTS: There were 19 asymptomatic and 8 symptomatic cases. And 25 selective and 2 emergent operations were performed. The interventions included endovascular stent grafts (n = 17), coil embolization (n = 1), both stent grafts and coils (n = 7) and hybrid operations (n = 2). Technical success was achieved in all cases. No delayed healing or infection of inguinal access sites occurred. In-stent thrombosis could be observed in 2 cases. Buttock claudication was observed in one case. Sexual dysfunction occurred in three cases. Endoleaks were confirmed by intraoperative digital subtraction angiography in three cases. There was no occurrence of colorectal ischemia or postoperative aneurysmal rupture. Patients were followed up for a mean period of 33 ± 18 months. One patient died of recurrent renal cell carcinoma. CONCLUSION: Endovascular repair of isolated iliac artery aneurysms provides good short and middle-term patency. It may become a first-choice for treating isolated iliac artery aneurysms in the future.

3D printed PEKK bone analogs with internal porosity and surface modification for mandibular reconstruction: An in vivo rabbit model study.

Polyetheretherketone (PEEK) and its derivative polyetherketoneketone (PEKK) have been used as implant materials for spinal fusing and enjoyed their success for many years because of their mechanical properties similar to bone and their chemical inertness. The osseointegration of PEEKs is datable. Our strategy was to use custom-designed and 3D printed bone analogs with an optimized structure design and a modified PEKK surface to augment bone regeneration for mandibular reconstruction. Those bone analogs had internal porosities and a bioactive titanium oxide surface coating to promote osseointegration between native bone and PEKK analogs. Our workflow was 3D modeling, bone analog designing, structural optimization, mechanical analysis via finite element modeling, 3D printing of bone analogs and subsequently, an in vivo rabbit model study on mandibular reconstruction and histology evaluation. Our results showed the finite element analysis validated that the porous PEKK analogs provided a mechanical-sound structure for functional loadings. The bone analogs offered a perfect replacement for segmented bones in the terms of shape, form and volume for surgical reconstruction. The in vivo results showed that bioactive titanium oxide coating enhanced new bone in-growth into the porous PEKK analogs. We have validated our new approach in surgical mandibular reconstruction and we believe our strategy has a significant potential to improve mechanical and biological outcomes for patients who require mandibular reconstruction procedures.

[Risk Assessment of Heavy Metals in Dust Fall Around Reservoirs in Typical Ecologically Fragile Areas and Traceability Based on APCS-MLR Model].

To assess the health risk status and pollution sources of heavy metals in the atmosphere of ecologically vulnerable areas, the surrounding area of Dahekou Reservoir in Xilingol League was selected as the study area. From 2021 to 2022, 12 monitoring points for atmospheric dust fall were collected for a period of one year. A total of 144 samples were collected to determine the contents of eight types of heavy metals, namely Cr, Ni, Pb, Cu, Zn, Mn, As, and Cd. The potential ecological index (Eri) and health risk assessment model were used to assess the risk level of atmospheric heavy metals on ecological security and human health. The analysis of enrichment factors, principal components, and the model of absolute principal component multiple linear regression (APCS-MLR) receptor were used to analyze the sources of heavy metal pollution qualitatively in the atmosphere of the study area. The results showed that:① the mean value of the comprehensive potential ecological risk of heavy metals in the annual atmospheric dust fall in the study area was at a high ecological risk, and only the Cd value was at a very high risk level among the heavy metals, whereas the remaining were at a slight risk. ② The results of the health risk showed that intake by hand, mouth, and skin contact were the main exposure routes, which led to non-carcinogenic and carcinogenic risks. Children were under non-carcinogenic and acceptable carcinogenic risks in different months. During those months, the main source of the risks was As. ③ Through enrichment factor analysis, principal component analysis, and APCS-MLR receptor model calculation, the results revealed that the proportion of wind-blown sources was the largest, accounting for 37.82%, and the contribution rates of coal combustion and traffic sources to Cu, Cd, Pb, and Zn were 73.01%, 40.22%, 70.31%, and 32.82%, respectively. The contribution rate of mining activities to As was 42.59%, while that of industrial sources of Cd was 22.01%; the contributions of other human activity sources of Cd, As, Pb, and Zn were 21.12%, 34.40%, 23.04%, and 32.15%, respectively.

Mid-term outcomes of microfragmented adipose tissue plus arthroscopic surgery for knee osteoarthritis: A randomized, active-control, multicenter clinical trial.

BACKGROUND: Osteoarthritis (OA) is the most prevalent form of degenerative whole-joint disease. Before the final option of knee replacement, arthroscopic surgery was the most widely used joint-preserving surgical treatment. Emerging regenerative therapies, such as those involving platelet-rich plasma, mesenchymal stem cells, and microfragmented adipose tissue (MFAT), have been pushed to the forefront of treatment to prevent the progression of OA. Currently, MFAT has been successfully applied to treat different types of orthopedic diseases. AIM: To assess the efficacy and safety of MFAT with arthroscopic surgery in patients with knee OA (KOA). METHODS: A randomized, multicenter study was conducted between June 2017 and November 2022 in 10 hospitals in Zhejiang, China. Overall, 302 patients diagnosed with KOA (Kellgren-Lawrence grades 2-3) were randomized to the MFAT group (n = 151, were administered MFAT following arthroscopic surgery), or the control group (n = 151, were administered hyaluronic acid following arthroscopic surgery). The study outcomes were changes in the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, the visual analog scale (VAS) score, the Lequesne index score, the Whole-Organ Magnetic Resonance Imaging Score (WORMS), and safety over a 24-mo period from baseline. RESULTS: The changes in the WOMAC score (including the three subscale scores), VAS pain score, and Lequesne index score at the 24-mo mark were significantly different in the MFAT and control groups, as well as when comparing values at the posttreatment visit and those at baseline (P < 0.001). The MFAT group consistently demonstrated significant decreases in the WOMAC pain scores and VAS scores at all follow-ups compared to the control group (P < 0.05). Furthermore, the WOMAC stiffness score, WOMAC function score, and Lequesne index score differed significantly between the groups at 12 and 24 mo (P < 0.05). However, no significant between-group differences were observed in the WORMS at 24 mo (P = 0.367). No serious adverse events occurred in both groups. CONCLUSION: The MFAT injection combined with arthroscopic surgery treatment group showed better mid-term clinical outcomes compared to the control group, suggesting its efficacy as a therapeutic approach for patients with KOA.

Enhanced co-production of hydrogen and poly-(R)-3-hydroxybutyrate by recombinant PHB producing E. coli over-expressing hydrogenase 3 and acetyl-CoA synthetase.

Recombinant Escherichia coli was constructed for co-production of hydrogen and polyhydroxybutyrate (PHB) due to its rapid growth and convenience of genetic manipulation. In particular, anaerobic metabolic pathways dedicated to co-production of hydrogen and PHB were established due to the advantages of directing fluxes away from toxic compounds such as formate and acetate to useful products. Here, recombinant E. coli expressing hydrogenase 3 and/or acetyl-CoA synthetase showed improved PHB and hydrogen production when grown with or without acetate as a carbon source. When hydrogenase 3 was over-expressed, hydrogen yield was increased from 14 to 153 mmol H(2)/mol glucose in a mineral salt (MS) medium with glucose as carbon source, accompanied by an increased PHB yield from 0.55 to 5.34 mg PHB/g glucose in MS medium with glucose and acetate as carbon source.

Production and characterization of poly(3-hydroxypropionate-co-4-hydroxybutyrate) with fully controllable structures by recombinant Escherichia coli containing an engineered pathway.

Copolyesters of 3-hydroxypropionate (3HP) and 4-hydroxybutyrate (4HB), abbreviated as P(3HP-co-4HB), was synthesized by Escherichia coli harboring a synthetic pathway consisting of five heterologous genes including orfZ encoding 4-hydroxybutyrate-coenzyme A transferase from Clostridium kluyveri, pcs' encoding the ACS domain of tri-functional propionyl-CoA ligase (PCS) from Chloroflexus aurantiacus, dhaT and aldD encoding dehydratase and aldehyde dehydrogenase from Pseudomonas putida KT2442, and phaC1 encoding PHA synthase from Ralstonia eutropha. When grown on mixtures of 1,3-propanediol (PDO) and 1,4-butanediol (BDO), compositions of 4HB in microbial P(3HP-co-4HB) were controllable ranging from 12 mol% to 82 mol% depending on PDO/BDO ratios. Nuclear magnetic resonance (NMR) spectra clearly indicated the polymers were random copolymers of 3HP and 4HB. Their mechanical and thermal properties showed obvious changes depending on the monomer ratios. Morphologically, P(3HP-co-4HB) films only became fully transparent when monomer 4HB content was around 67 mol%. For the first time, P(3HP-co-4HB) with adjustable monomer ratios were produced and characterized.

Production of copolyesters of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates by E. coli containing an optimized PHA synthase gene.

BACKGROUND: Microbial polyhydroxyalkanoates (PHA) are biopolyesters consisting of diverse monomers. PHA synthase PhaC2Ps cloned from Pseudomonas stutzeri 1317 is able to polymerize short-chain-length (scl) 3-hydroxybutyrate (3HB) monomers and medium-chain-length (mcl) 3-hydroxyalkanoates (3HA) with carbon chain lengths ranging from C6 to C12. However, the scl and mcl PHA production in Escherichia coli expressing PhaC2Ps is limited with very low PHA yield. RESULTS: To improve the production of PHA with a wide range of monomer compositions in E. coli, a series of optimization strategies were applied on the PHA synthase PhaC2Ps. Codon optimization of the gene and mRNA stabilization with a hairpin structure were conducted and the function of the optimized PHA synthase was tested in E. coli. The transcript was more stable after the hairpin structure was introduced, and western blot analysis showed that both codon optimization and hairpin introduction increased the protein expression level. Compared with the wild type PhaC2Ps, the optimized PhaC2Ps increased poly-3-hydroxybutyrate (PHB) production by approximately 16-fold to 30% of the cell dry weight. When grown on dodecanoate, the recombinant E. coli harboring the optimized gene phaC2PsO with a hairpin structure in the 5' untranslated region was able to synthesize 4-fold more PHA consisting of 3HB and medium-chain-length 3HA compared to the recombinant harboring the wild type phaC2Ps. CONCLUSIONS: The levels of both PHB and scl-mcl PHA in E. coli were significantly increased by series of optimization strategies applied on PHA synthase PhaC2Ps. These results indicate that strategies including codon optimization and mRNA stabilization are useful for heterologous PHA synthase expression and therefore enhance PHA production.

Hyperproduction of poly(4-hydroxybutyrate) from glucose by recombinant Escherichia coli.

BACKGROUND: Poly(4-hydroxybutyrate) [poly(4HB)] is a strong thermoplastic biomaterial with remarkable mechanical properties, biocompatibility and biodegradability. However, it is generally synthesized when 4-hydroxybutyrate (4HB) structurally related substrates such as γ-butyrolactone, 4-hydroxybutyrate or 1,4-butanediol (1,4-BD) are provided as precursor which are much more expensive than glucose. At present, high production cost is a big obstacle for large scale production of poly(4HB). RESULTS: Recombinant Escherichia coli strain was constructed to achieve hyperproduction of poly(4-hydroxybutyrate) [poly(4HB)] using glucose as a sole carbon source. An engineering pathway was established in E. coli containing genes encoding succinate degradation of Clostridium kluyveri and PHB synthase of Ralstonia eutropha. Native succinate semialdehyde dehydrogenase genes sad and gabD in E. coli were both inactivated to enhance the carbon flux to poly(4HB) biosynthesis. Four PHA binding proteins (PhaP or phasins) including PhaP1, PhaP2, PhaP3 and PhaP4 from R. eutropha were heterologously expressed in the recombinant E. coli, respectively, leading to different levels of improvement in poly(4HB) production. Among them PhaP1 exhibited the highest capability for enhanced polymer synthesis. The recombinant E. coli produced 5.5 g L(-1) cell dry weight containing 35.4% poly(4HB) using glucose as a sole carbon source in a 48 h shake flask growth. In a 6-L fermentor study, 11.5 g L(-1) cell dry weight containing 68.2% poly(4HB) was obtained after 52 h of cultivation. This was the highest poly(4HB) yield using glucose as a sole carbon source reported so far. Poly(4HB) was structurally confirmed by gas chromatographic (GC) as well as (1)H and (13)C NMR studies. CONCLUSIONS: Significant level of poly(4HB) biosynthesis from glucose can be achieved in sad and gabD genes deficient strain of E. coli JM109 harboring an engineering pathway encoding succinate degradation genes and PHB synthase gene, together with expression of four PHA binding proteins PhaP or phasins, respectively. Over 68% poly(4HB) was produced in a fed-batch fermentation process, demonstrating the feasibility for enhanced poly(4HB) production using the recombinant strain for future cost effective commercial development.

Piperonal ciprofloxacin hydrazone induces growth arrest and apoptosis of human hepatocarcinoma SMMC-7721 cells.

AIM: To investigate the cytotoxic effects of piperonal ciprofloxacin hydrazone (QNT4), a novel antibacterial fluoroquinolone derivative, against human hepatocarcinoma SMMC-7721 cells. METHODS: Human hepatocarcinoma cells (SMMC-7721), human breast adenocarcinoma cells (MCF-7) and human colon adenocarcinoma cells (HCT-8) were tested. The effects of QNT4 on cell proliferation were examined using MTT assay. Cell apoptosis was determined using Hoechst 33258 fluorescence staining, TUNEL assay and agarose gel electrophoresis. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. Mitochondrial membrane potential (Δψm) was measured using a high content screening imaging system. Protein expression of caspase-9, caspase-8, caspase-3, p53, Bcl-2, Bax, and cytochrome c was detected with Western blot analysis. RESULTS: Treatment with QNT4 (0.625-10 µmol/L) potently inhibited the proliferation of the cancer cells in time- and dose-dependent manners (the IC(50) value at 24 h in SMMC-7721 cells, MCF-7 cells and HCT-8 cells was 2.956±0.024, 3.710±0.027, and 3.694±0.030 µmol/L, respectively). Treatment of SMMC-7721 cells with QNT4 (0.2146, 2.964, and 4.600 µmol/L) for 24 h dose-dependently increased the percentage of apoptotic cells, elicited characteristic DNA "ladder" bands, and decreased the mitochondrial membrane potential. QNT4 dose-dependently increased topoisomerase II-mediated DNA breaks while inhibiting DNA relegation, thus keeping the DNA in fragments. Treatment of SMMC-7721 cells with QNT4 significantly increased cytochrome c in the cytosol, and decreased cytochrome c in the mitochondrial compartment. QNT4 (3-7.39 µmol/L) significantly increased the protein expression of p53, Bax, caspase-9, caspase-3, and the cleaved activated forms of caspase-9 and caspase-3 in SMMC-7721 cells. In contrast, the expression of Bcl-2 was decreased, while caspase-8 had no significant change. CONCLUSION: QNT4 induced the apoptosis of SMMC-7721 cells via inhibiting topoisomerase II activity and modulating mitochondrial-dependent pathways.

Updated systematic review and meta-analysis of randomized clinical trials comparing carotid artery stenting and carotid endarterectomy in the treatment of carotid stenosis.

BACKGROUND: To compare carotid artery stenting (CAS) versus carotid endarterectomy (CEA) in the treatment of carotid stenosis, including two recently published, large, prospective, randomized trials of these therapies. METHODS: We searched electronic databases for prospective, randomized, controlled trials involving carotid stenosis patients who underwent CAS or CEA, focusing on studies published in 1995 to 2010. Primary outcomes were death, stroke, and myocardial infarction. RESULTS: Thirteen trials containing 7,501 patients were analyzed, and odds ratios (ORs) were calculated for CAS versus CEA. The risk of stroke or death within 30 days was higher after CAS than CEA (OR = 1.57; 95% confidence interval [CI] = 1.11-2.22), especially in previously symptomatic patients (OR = 1.89; 95% CI = 1.48-2.41). However, the risk of stroke or death within 1 year was comparable (OR = 1.12; 95% CI = 0.55-2.30). In a subgroup analysis, the risk of death and disabling stroke at 30 days did not differ significantly between CEA and CAS (death: OR = 1.43; 95% CI = 0.85-2.40; disabling stroke: OR = 1.28; 95% CI = 0.89-1.83), whereas the rate of nondisabling stroke within 30 days was much higher in the CAS group (OR = 1.87; 95% CI = 1.40-2.50). The risks of myocardial infarction within 30 days and 1 year were significantly less for CAS. CONCLUSION: CAS is inferior to CEA with regard to the incidence of stroke or death for periprocedural outcomes, especially in symptomatic patients. However, CAS was associated with a lower incidence of myocardial infarction. These procedures may be considered complementary rather than competing modes of therapy, each of which can be optimized with careful patient selection.

[The development of a type II endoleak domesticated pig model with continuous pressure monitor].

OBJECTIVE: To develop a type II endoleak porcine model with continuous pressure monitor by the strain-gauge pressure transducer and the patch. METHODS: Nine tamed porcine was randomized as the experiment group of 6 domesticated pigs and the control group of 3 domesticated pigs. When the abdominal aortic aneurysm (AAA) was created, the 2(nd) and 3(rd) pair of infrarenal lumber arteries were preserved in the experiment group, while ligated in the control group. The stent-graft was implanted in the endovascular repair. CT angiography was performed to seek endoleak, and the angiography was performed to excluded the type I and type III endoleak. The pressure in the excluded AAA monitored by the strain-gauge pressure transducer was recorded and compared in both groups to evaluated the effect of type II endoleak in the experiment group. RESULTS: The AAA porcine model with pressure monitor were successfully developed in all 9 porcine. The endoleak and the retrograde flow of the lumber arteries were confirmed by CT angiography in the experiment group. The ratio of the average pressure after the procedure to before the procedure was higher in the experiment group than the control group (U = 0.000, P = 0.020). The ratio of the pulse pressure after the procedure to the average pressure before the procedure was higher in the experiment group than the control group (U = 0.000, P = 0.020). CONCLUSION: It is feasible to develop type II endoleak domesticated pig model by preserving the lumber arteries for the retrograde flow, and the AAA model with pressure monitor by the strain-gauge pressure transducer and the patch.

Production of 3-hydroxypropionate homopolymer and poly(3-hydroxypropionate-co-4-hydroxybutyrate) copolymer by recombinant Escherichia coli.

Conversion of 3-hydroxypropionate (3HP) from 1,3-propanediol (PDO) was improved by expressing dehydratase gene (dhaT) and aldehyde dehydrogenase gene (aldD) of Pseudomonas putida KT2442 under the promoter of phaCAB operon from Ralstonia eutropha H16. Expression of these genes in Aeromonas hydrophila 4AK4 produced up to 21 g/L 3HP in a fermentation process. To synthesize homopolymer poly(3-hydroxypropionate) (P3HP), and copolymer poly(3-hydroxypropionate-co-3-hydroxybutyrate) (P3HP4HB), dhaT and aldD were expressed in E. coli together with the phaC1 gene encoding polyhydroxyalkanoate (PHA) synthase gene of Ralstonia eutropha, and pcs' gene encoding the ACS domain of the tri-functional propionyl-CoA ligase (PCS) of Chloroflexus aurantiacus. Up to 92 wt% P3HP and 42 wt% P3HP4HB were produced by the recombinant Escherichia coli grown on PDO and a mixture of PDO+1,4-butanediol (BD), respectively.