Repeated-Batch and Continuous Production of L-Lactic Acid by Rhizopus oryzae Immobilized in Calcium Alginat Beads:ReactorPerformance and Kinetic Model

Repeated-batch and continuous production of L-lactic acid by immobilized Rhizopusoryzae with calcium alginate entrapment method in a three-phase fluidized-bed bioreactor was stud-ied.The operation conditions were optimized.The productivity based on total reactor volume wasabout 3 times higher than that with free cells in a traditional stirred tank bioreactor.A mathemat-ical model was proposed and the model predictions were in good agreement with the experimentaldat.

Synthesis and Characterization of Chitosan-g-poly- (D, L-lactic acid) Copolymer

Biodegradable chitosan-g-poly (D, L-lactic acid) copolymers were prepared via two methods. (1) The lactide was grafted onto hydroxyl groups of chitosan by using macromolecular initiator sodium of trimethylsilyl-chitosan, (2) poly (D,L-lactic acid)(PLA) with low molecular weight can be linked to the amino group by coupling activated PLA to trimethylsilyl-chitosan. Two graft copolymers had hydrophilic-hydrophobic character and can be applied as carriers for drug delivery.

Saikosaponin D improves nonalcoholic fatty liver disease via gut microbiota-bile acid metabolism pathway

Non-alcoholic fatty liver disease(NAFLD)is the main cause of chronic liver disease worldwide.Bupleurum is widely used in the treatment of non-alcoholic fatty liver,and saikosaponin D(SSD)is one of the main active components of Bupleurum.The purpose of this study was to investigate the efficacy of SSD in the treatment of NAFLD and to explore the mechanism of SSD in the improvement of NAFLD based on“gut-liver axis”.Our results showed that SSD dose-dependently alleviated high fat diet-induced weight gain in mice,improved insulin sensitivity,and also reduced liver lipid accumulation and injury-related biomarkers aspartate aminotransferase(AST)and alanine aminotransferase(ALT).Further exploration found that SSD inhibited the mRNA expression levels of farnesoid X receptor(Fxr),small heterodimer partner(Shp),recombinant fibroblast growth factor 15(Fgf15)and apical sodium dependent bile acid transporter(Asbt)in the intestine,suggesting that SSD improved liver lipid metabolism by inhibiting intestinal FXR signaling.SSD can significantly reduce the gut microbiota associated with bile salt hydrolase(BSH)expression,such as Clostridium.Decreased BSH expression reduced the ratio of unconjugated to conjugated bile acids,thereby inhibiting the intestinal FXR.These data demonstrated that SSD ameliorated NAFLD potentially through the gut microbiota-bile acidintestinal FXR pathway and suggested that SSD is a promising therapeutic agent for the treatment of NAFLD.

Microwave induced mutagenesis of Lactobacillus casei subsp. rhamnosus for enhancing L-lactic acid production

Mutagenesis of Lactobacillus casei subsp, rhamnosus Xl-12 after low power microwave irradiation was investigated. Under a microwave power of 400 W and irradiation length of 3 min, a mutated strain W4-3-9 with high-yield L-lactic acid was obtained by screening. Compared with the starting strain X1-12, the L-lactic acid production of W4-3-9 was increased by 58.0% at a concentration of 115.8 g/L. The strain maintained the capability of producing a high L-lactic acid level after 10 generations. Cell surface morphology and DNA structures of parental and mutated strains were observed by atomic force microscopy （ AFM ）. Amplified fragment length polymorphism （AFLP） analysis suggested the difference in AFLP band pattern between the mutated and non-mutated strains. Sequencing and BLAST analysis revealed that the catalytic site of lactate dehydrogenase （DHL） was changed due to the microwave induced mutation.

Preparation of Poly ( L-lactic acid ) / Vermiculites Composites by in situ Copolymerization of Lactic Acid and Hydroxylic Vermiculites

L-lactic acid (L-LA) based copolymer/hydroxylation vermiculites composites (PLLA-co-bis A/HVMTs) were prepared by in situ reaction among L-LA, adipic acid, and hydroxylation lamellar vermiculites (HVMTs) using bisphenol-A epoxy resin as chain extending agent. HVMTs were obtained by sulfuric acid-leaching of lamellar vermiculites (VMTs). The effects of sulfuric acid leaching on the VMTs structure were characterized by X-ray diffraction (XRD), 29Si magic-angle spinning nuclear magnetic resonance(29Si NMR), Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FE-SEM), and thermogravimetric analysis (TGA). FTIR, FE-SEM, and TGA were used to characterize the reaction activity of HVMTs. The results indicated that VMTs with increased hydroxyl groups had been successfully obtained and could react with -COOH of the reaction system. The amount of L-LA based copolymer grafted on the surface of HVMTs was more than 22%. The onset decomposition temperature of L-LA based copolymer grafted on the surface of HVMTs was 30℃ higher than that of free L-LA based copolymer.

L-LACTIC ACID FERMENTATION IN A ROTATING-DISC CONTACTOR WITH SIMULTANEOUS PRODUCT SEPARATION BY ION-EXCHANGE

A rotating disk contactor（RDC）was designed to perform L-lactic acid fermentation with afilamentous fungi,Rhizopus oryzae,which was immobilized on the surfaces of the rotating discs.Thebioreactor was operated using repeated-batch method as well as continuous feeding method.Ananionic resin,D354,slightly basic in nature and of high selectivity and capacity was chosen for lacticacid separation.A coupled process of L-lactic acid fermentation and ion-exchange separation wasevaluated experimentally.The results indicated that the pH value of the fermentation broth could bemaintained at about 3-3.5 without any addition of alkali.The conversion ratio of glucose to L-lacticacid was about 0.7 g·g-1 and the fermentation rate reached as high as 62.5 g·h-1·m-2.

Synthesis of the Biomimetic Polymer: Aliphatic Diamine and RGDS Modified Poly(d,l-lactic acid)

A novel poly（d, /-lactic acid） （PDLLA） based biomimetic polymer was synthesized by grafting maleic anhydride, butanediamine and arg-gly-asp-ser （RGDS） peptides onto the backbone of PDLLA, aiming to overcome the acidity and auto-accelerating degradation of PDLLA during degradation and to improve its biospecificity and biocompatibility. The synthetic copolymer was characterized by FTIR, ^13C NMR and amino acid analyzer （AAA）.

Effect of cis-9,trans-11-conjugated linoleic acid on cell cycle of gastric adenocarcinoma cell line(SGC-7901)

AIM: To determine the effect of cis -9, trans -11-conjugated linoleic acid (c9, t11-CLA) on the cell cycle of gastric cancer cells (SGC-7901) and its possible mechanism in inhibition cancer growth. METHODS: Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A, B(1), D(1), p16(ink4a) and p21(cip/waf1) of SGC-7901 cells which were treated with various c9, t11-CLA concentrations (25, 50, 100 and 200 micromol.L(-1))of c 9, t 11-CLA for 24 and 48h, with a negative control (0.1% ethane). RESULTS: The cell growth and DNA synthesis of SGC-7901 cells were inhibited by c9, t11-CLA.SGC-7901 cells. Eight day after treatment with various concentrations of c9, t11-CLA mentioned above, the inhibition rates were 5.92%, 20.15%, 75.61% and 82.44%, respectively and inhibitory effect of c9, t11-CLA on DNA synthesis (except for 25 micromol.L, 24h) showed significantly less (3)H-TdR incorporation than that in the negative controls (P【0.05 and P【0.01). Immunocytochemical staining demonstrated that SGC-7901 cells preincubated in media supplemented with different c9, t11-CLA concentrations at various times significantly decreased the expressions of PCNA (the expression rates were 7.2-3.0%, 24h and 9.1-0.9% at 48h, respectively), Cyclin A (11.0-2.3%, 24h and 8.5-0.5%,48h), B(1) (4.8-1.8% at 24h and 5.5-0.6% at 48h)and D(1) (3.6-1.4% at 24h and 3.7%-0 at 48h) as compared with those in the negative controls(the expressions of PCNA, Cyclin A, B(1) and D(1) were 6.5% at 24h and 9.0% at 48h, 4.2% at 24h and 5.1% at 48h, 9.5% at 24h and 6.0% at 48h,respectively)(P【0.01), whereas the expressions of P16(ink4a) and P21(cip/waf1), cyclin-dependent kinases inhibitors(CDKI), were increased. CONCLUSION: The cell growth and proliferation of SGC-7901 cell is inhibited by c9, t11-CLA via blocking the cell cycle, with reduced expressions of cyclin A,B(1) and D(1) and enhanced expressions of CDKI(P16(ink4a) and p21(cip/waf1)).

Fabrication and Characterization of Poly Lactic Acid Scaffolds by Fused Deposition Modeling for Bone Tissue Engineering

Three-dimensional porous poly-lactic acid(PLA) scaffold was fabricated using fused deposition modeling(FDM) method including 30%, 50% and 70% nominal porosity. Study of phases in initial polymeric material and printed scaffolds was done by X-ray diffraction(XRD), and no significant phase difference was observed due to the manufacturing process, and the poly-lactic acid retains its crystalline properties. The results of the mechanical properties evaluation by the compression test show that the mechanical properties of the scaffold have decreased significantly with increasing the porosity of scaffold. The microstructure of scaffolds were studied by scanning electron microscope(SEM), showing that the pores had a regular arrangement and their morphology changed with porosity change. The mechanical properties of the poly-lactic acid scaffolds printed using fused deposition modeling, can be adapted to the surrounding tissue, by porosity change.

3D printed lactic acid bacteria hydrogel:cell release kinetics and stability

In this study,a new type of 3 D printed living biological hydrogel was developed by integrating lactic acid bacteria(LAB)into biocompatible and non-toxic polymer materials.Interestingly,the living materials loaded with LAB can be freeze-dried and reused for more than 100 times.The bio-hydrogel can be used to co-culture different LAB and keep its fermentation performance stable in long-term use.The release kinetics model and response surface method were used to simulate and optimize the bacteria release mode in the bio-hydrogel.The results show that the release of bacteria from hydrogel is regulated by the coupling of Fickian diffusion and polymer swelling.The stability of LAB hydrogel was evaluated by reuse experiments.The images of confocal microscopy and scanning electron microscope showed that the bacteria with high cell viability were distributed in the hydrogel and intact structure of the living hydrogel was maintained after 100 times of reuse as yoghurt starter.In conclusion,the 3 D printed LAB bio-hydrogel developed in this study has the advantage of reuse and sustainability,which is expected to open up a new way for the preparation of food culture starter.

3D printing of personalized polylactic acid scaffold laden with GelMA/autologous auricle cartilage to promote ear reconstruction

At present,the clinical reconstruction of the auricle usually adopts the strategy of taking autologous costal cartilage.This method has great trauma to patients,poor plasticity and inaccurate shaping.Three-dimensional(3D)printing technology has made a great breakthrough in the clinical application of orthopedic implants.This study explored the combination of 3D printing and tissue engineering to precisely reconstruct the auricle.First,a polylactic acid(PLA)polymer scaffold with a precisely customized patient appearance was fabricated,and then auricle cartilage fragments were loaded into the 3D-printed porous PLA scaffold to promote auricle reconstruction.In vitro,gelatin methacrylamide(GelMA)hydrogels loaded with different sizes of rabbit ear cartilage fragments were studied to assess the regenerative activity of various autologous cartilage fragments.In vivo,rat ear cartilage fragments were placed in an accurately designed porous PLA polymer ear scaffold to promote auricle reconstruction.The results indicated that the chondrocytes in the cartilage fragments could maintain the morphological phenotype in vitro.After three months of implantation observation,it was conducive to promoting the subsequent regeneration of cartilage in vivo.The autologous cartilage fragments combined with 3D printing technology show promising potential in auricle reconstruction.

Transplantation of Nogo-66 receptor gene-silenced cells in a poly(D,L-lactic-co-glycolic acid) scaffold for the treatment of spinal cord injury

Inhibition of neurite growth, which is in large part mediated by the Nogo-66 receptor, affects neural regeneration following bone marrow mesenchymal stem cell transplantation. The tissue engineering scaffold poly（D,L-lactide-co-glycolic acid） has good histocompatibility and can promote the growth of regenerating nerve fibers. The present study used small interfering RNA to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells and Schwann cells, which were subsequently transplanted with poly（D,L-lactide-co-glycolic acid） into the spinal cord lesion regions in rats. Simultaneously, rats treated with scaffold only were taken as the control group. Hematoxylin-eosin staining and immunohistochemistry revealed that at 4 weeks after transplantation, rats had good motor function of the hind limb after treatment with Nogo-66 receptor gene-silenced ceils prus the poly（O,L-lactide-co-glycolic acid） scaffold compared with rats treated with scaffold only, and the number of bone marrow mesenchymal stem cells and neuron-like cells was also increased. At 8 weeks after transplantation, horseradish peroxidase tracing and transmission electron microscopy showed a large number of unmyelinated and myelinated nerve fibers, as well as intact regenerating axonal myelin sheath following spinal cord hemisection injury. These experimental findings indicate that transplantation of Nogo-66 receptor gene-silenced bone marrow mesenchymal stem cells and Schwann cells plus a poly（D,L-lactide-co-glycolic acid） scaffold can significantly enhance axonal regeneration of spinal cord neurons and improve motor function of the extremities in rats following spinal cord injury.

Composite Biomaterials Based on Poly(L-Lactic Acid)and Functionalized Cellulose Nanocrystals

The biocomposite films were prepared from poly(L-lactic acid)and cellulose nanocrystals.To improve interfacial compatibility of hydrophilic cellulose nanocrystals with hydrophobic matrix polymer as well as to provide the osteoconductive properties,cellulose was functionalized with poly(glutamic acid).The modified cellulose nanocrystals were better distributed and less aggregated within the matrix,which was testified by scanning electron,optical and polarized light microscopy.According to mechanical tests,composites filled with nanocrystals modified with PGlu demonstrated higher values of Young’s modulus,elongation at break and tensile strength.Incubation of composite materials in model buffer solutions for 30 weeks followed with staining of Ca^(2+)deposits with Alizarin Red S assay testified better mineralization of materials containing PGlu-modified cellulose nanocrystals as filler.As the result of in vivo experiment,the developed composite materials showed less level of inflammation in comparison with pure polymer matrix and composites filled with non-functionalized cellulose nanocrystals.

Multi-porous electroactive poly(L-lactic acid)/ polypyrrole composite micro/nano fibrous scaffolds promote neurite outgrowth in PC12 cells

In this study, poly（L-lactic acid）/ammonium persulfate doped-polypyrrole composite fibrous scaffolds with moderate conductivity were produced by combining electrospinning with in situ polymerization. PC12 cells were cultured on these fibrous scaffolds and their growth following electrical stimulation （0-20.0 μA stimulus intensity, for 1-4 days） was observed using inverted light microscopy, and scanning electron microscopy coupled with the MTT cell viability test. The results demonstrated that the poly（L-lactic acid）/ammonium persulfate doped-polypyrrole fibrous scaffold was a dual multi-porous micro/nano fibrous scaffold. An electrical stimulation with a current intensity 5.0- 10.0 μAfor about 2 days enhanced neuronal growth and neurite outgrowth, while a high current intensity （over 15.0 μA） suppressed them. These results indicate that electrical stimulation with a moderate current intensity for an optimum time frame can promote neuronal growth and neurite outgrowth in an intensity- and time-dependent manner.

Effects of Calcium and Magnesium on Phosphorus Availability in Ferralsols and Rice Production in Forest Zones of Côte d’Ivoire

Phosphorus bioavailability has long been a recurring problem in tropical acid soils. A pot experiment was carried out during three (3) successive rice production cycles at Adiopodoumé to evaluate the response of the NERICA 5 rice accession to various doses of calcium, magnesium and phosphorous. The experiment was conducted using a randomized split-plot design. The interactive effects of calcium carbonate (0, 25, 50 and 75 kg·Ca·ha-1) and magnesium sulfate (0, 25, 50 and 75 kg·Mg·ha-1) and Togo natural phosphate (0, 25, 50 and 75 kg·P·ha-1) were determined at each production cycle. The results showed that single-dose natural phosphate supplementation for three cropping cycles resulted in an average enrichment of around 2 mg·P·kg-1 after each trial following its continuous dissolution, with an increase in DSP (33.31% to 70.52%). The study revealed one strategy for managing and enhancing native P with cations and another for exogenous P: there would be a synergy of Ca/Mg on native P, whereas an antagonism would characterize the two parameters in phosphate fertilization.

Gambogic acid induces apoptosis and inhibits colorectal tumor growth via mitochondrial pathways

AIM: To investigate the effect of gambogic acid(GA) on apoptosis in the HT-29 human colon cancer cell line. METHODS: H-29 cells were used for in vitro experiments in this study. Relative cell viability was assessed using MTT assays. Cell apoptosis was detected by terminal deoxynucleotidyl transferase d UTP nick end labeling and Hoechst 33342 staining, and quantified by flow cytometry. Cellular ultrastructure was observed by transmission electron microscopy. Real-time PCR and Western blot analyses were used to evaluate gene and protein expression levels. For in vivo experiments, BALB/c nude mice received subcutaneous injections of HT-29 cells in the right armpit. When well-established xenografts were palpable with a tumor size of 75 mm3, mice were randomly assigned to a vehicle(negative) control, positive control or GA treatment group(n = 6 each). The animals in the treatment group received one of three dosages of GA(in saline; 5, 10 or 20 mg/kg) via the caudal vein twice weekly, whereas animals in the negative and positive control groups were given equal volumes of 0.9% saline or 10 mg/kg docetaxel, respectively, via the caudal vein once weekly. RESULTS: The cell viability assay showed that GA inhibited proliferation of HT-29 cells in a dose- and time-dependent manner after treatment with GA(0.00, 0.31, 0.62, 1.25, 2.50, 5.00 or 10.00 μmol/L) for 24, 48 or 72 h. After 48 h, the percentage of apoptotic cells in cells treated with 0.00, 1.25, 2.50 and 5.00 μmol/L GA was 1.4% ± 0.3%, 9.8% ± 1.2%, 25.7% ± 3.3% and 49.3% ± 5.8%, respectively. Ultrastructural analysis of HT-29 cells treated for 48 h with 2.5μmol/L GA revealed apoptotic bodies and condensed and fragmented nuclei. Levels of caspase-8,-9 and-3 m RNAs were significantly increased after treatment with GA(1.25, 2.50 or 5.00 μmol/L) for 48 h(P < 0.05 for all). Protein levels of apoptosis-related factors Fas, Fas L, FADD, cytochrome c, and Apaf-1 were increased in GA-treated cells, whereas levels of pro-caspase-8,-9 and-3 were significantly decreased(P < 0.05 for all). Furthermore, GA significantly and dose-dependently inhibited the growth of HT-29 tumors in a mouse xenograft model(P < 0.05).CONCLUSION: GA inhibits HT-29 proliferation via induction of apoptosis. The anti-cancer effects are likely mediated by death receptor(extrinsic) and mitochondrial(intrinsic) pathways.

Estrogen affects neuropathic pain through upregulating N-methyl-D-aspartate acid receptor 1 expression in the dorsal root ganglion of rats

Estrogen affects the generation and transmission of neuropathic pain,but the specific regulatory mechanism is still unclear.Activation of the N-methyl-D-aspartate acid receptor 1（NMDAR1） plays an important role in the production and maintenance of hyperalgesia and allodynia.The present study was conducted to determine whether a relationship exists between estrogen and NMDAR1 in peripheral nerve pain.A chronic sciatic nerve constriction injury model of chronic neuropathic pain was established in rats.These rats were then subcutaneously injected with 17β-estradiol,the NMDAR1 antagonist D（-）-2-amino-5-phosphonopentanoic acid（AP-5）,or both once daily for 15 days.Compared with injured drug na？ve rats,rats with chronic sciatic nerve injury that were administered estradiol showed a lower paw withdrawal mechanical threshold and a shorter paw withdrawal thermal latency,indicating increased sensitivity to mechanical and thermal pain.Estrogen administration was also associated with increased expression of NMDAR1 immunoreactivity（as assessed by immunohistochemistry） and protein（as determined by western blot assay） in spinal dorsal root ganglia.This 17β-estradiol-induced increase in NMDAR1 expression was blocked by co-administration with AP-5,whereas AP-5 alone did not affect NMDAR1 expression.These results suggest that 17β-estradiol administration significantly reduced mechanical and thermal pain thresholds in rats with chronic constriction of the sciatic nerve,and that the mechanism for this increased sensitivity may be related to the upregulation of NMDAR1 expression in dorsal root ganglia.

社区H型高血压患者药物基因作用靶点多态性分布及叶酸联合维生素D干预效果研究

背景H型高血压严重影响着人们的健康及生活质量,目前临床上治疗高血压主要根据患者症状和临床经验选择药物,降压效果不理想,急需探寻降压药物基因分布的多态性,为高血压患者进行个体化用药指导。目的探讨济南市社区H型高血压药物作用靶点基因多态性分布及叶酸联合维生素D的干预作用,为该地区开展高血压医防融合精准医疗提供参考依据。方法2020年6月—2022年6月随机抽取山东省济南市槐荫区20家街道办事处社区卫生服务中心200例血压控制不佳的高血压患者为研究对象,治疗前首先进行5类常用抗高血压药物[利尿剂、β受体阻滞剂、血管紧张素转化酶抑制剂(ACEI)、钙离子通道抑制剂(CCB)、血管紧张素Ⅱ受体拮抗剂(ARB)]相关高血压个体化用药基因位点的基因多态性检测。将患者随机分为基因导向治疗组(基因组)与基因导向协同叶酸、维生素D治疗组(基因导向组),每组100例。基因组根据检测的高血压基因作用位点的特点调整用药;基因导向组在基因组治疗方案的基础上同时服用叶酸、维生素D。干预初始(M0)、干预3个月(M3)、干预6个月(M6)时采集患者晨间未服用降压药物情况下坐位收缩压和舒张压。记录患者患病情况、不良反应发生情况、脑卒中发生情况,进行基因测序,检测血清同型半胱氨酸(Hcy)浓度。采用Pearson相关性分析或Spearman秩相关分析探究性别、年龄、收缩压、舒张压与Hcy的相关性。结果研究对象性别(r_(s)=-0.463)、收缩压(r=0.181)、舒张压(r=0.188)与Hcy水平有相关性(P<0.05)。5类抗高血压药物基因作用靶点中,与药物代谢酶基因多态性位点相关的分别是CYP3A5(A6986G)、CYP2C9(c.1075A>C)、CYP2D6(c.100C>T),与药物作用靶点敏感性基因多态性位点相关的是ADRB1、ACEI(I/D)、AGTR1、NPPA。基因组A6986G:CYP3A5*1/*1(AA)、ACEI(I/D):D/D、c.100 C>T:CYP2D6*1/*1(CC)患者M3、M6舒张压低于M0,A6986G:CYP3A5a1/a3(AG)、ADRB1 c.1165 G>C:GG、c.1075 A>C:CYP2C9*1/*3(AC)、c.1075 A>C:CYP2C9*3/*3(CC)患者M6舒张压低于M0,A6986G:CYP3A5*3/*3(GG)、ADRB1 c.1165 G>C:CC、ACEI(I/D):I/I、c.1075 A>C:CYP2C9*1/*1(AA)、AGTR1 c.1166 A>C:AA、NPPA T2238C:TT、c.100 C>T:CYP2D6*10/*10(TT)患者M3、M6收缩压、舒张压低于M0,ADRB1 c.1165 G>C:GC、ACEI(I/D):I/D、c.100 C>T:CYP2D6*1/*10(CT)患者M6收缩压低于M0,M3、M6舒张压低于M0,差异有统计学意义(P<0.05)。Hcy水平组间比较结果显示,M3、M6基因导向组Hcy水平低于基因组,差异有统计学意义(P<0.05)。组内比较结果显示,基因组M6 Hcy水平低于M0,基因导向组M3、M6 Hcy水平低于M0,M6 Hcy水平低于M3,差异有统计学意义(P<0.05)。收缩压、舒张压组间比较结果显示,M3、M6基因导向组收缩压、舒张压低于基因组,差异有统计学意义(P<0.05)。组内比较结果显示,基因组、基因导向组M6收缩压、舒张压低于M0,M6收缩压低于M3,差异有统计学意义(P<0.05)。结论社区H型高血压患者中存在高血压药物相关基因多态性的表达差异,个体化用药效果显著;叶酸联合维生素D协同治疗更能显著降低H型高血压水平。