Engineering a pharmacologically superior form of leptin for the treatment of obesity

Leptin plays a central role in the homeostasis of body weight through its regulatory effects on appetite and energy expenditure, yet in trials as a therapeutic agent for the treatment of obesity in humans it has been disappointing. The poor clinical efficacy of leptin results from its short circulating half-life, low potency and poor solubility, necessitating large and frequent doses to obtain even modest clinical benefit. Engineered Fc-leptin immunofusins, consisting of the Fc fragment of an immunoglobulin gamma chain followed by leptin, exhibit improved pharmacological properties with very consistent and potent biological activities. Furthermore, in extending the circulating half-life of the protein in vivo from a few minutes for leptin to many hours for Fc-leptin, these proteins have the potential to reduce drastically the dosage and frequency of administration required to obtain clinical benefit. The results of this study show that the engineered leptin immunofusins described here have significantly enhanced pharmacological properties in comparison with the recombinant leptin that was used in clinical trials. As such, they could represent an important step towards a therapeutically superior form of leptin if the disappointing performance of leptin in early clinical trials was due to its poor pharmacological properties rather than any conceptual weakness in the strategy of using leptin for the treatment of obesity and its related disorders.     

Current strategies and challenges for the purification of stem cells

During the past decade, stem cell transplant has emerged as a novel therapeutic alternative for several diseases. If therapies are to be implemented in clinical settings, efficient scale‐up of stem cells isolation methodologies would be crucial. A brief, process‐oriented overview of the current most widely used technologies for stem cells separation is presented. This review classifies available methods into three broad categories: (1) isopycnic centrifugation, including density gradient and cell culture; (2) immunochemical, employing immune labeling; and (3) novel, tagless procedures. These groups are further subdivided into more specific techniques, highlighting their advantages and limitations. Particular cases in each category were selected to further compare the purification parameters of recovery, cell viability, purity, process time, and throughput. A particular focus on process scale‐up feasibility and the challenges of this rapidly emerging field are stated. Lastly, likely directions are suggested for the development of new proposals which will make stem cells purification more advantageous and viable for clinical use. Copyright © 2011 Society of Chemical Industry     

The development of a medium for the in vitro expansion of mammalian neural stem cells

A new medium, PPRF-m2, has been developed for the in vitro expansion of murine neural stem cells. Neural stem cells (NSC) are primitive cells that have the capability to give rise to all of the cells that comprise the central nervous system (CNS). PPRF-m2 was developed in part by examining key aspects of a commercial NSC medium used in our laboratory, and includes the basal media combination RPMI/DMEM/F12 in a 1:1:1 volumetric ratio supplemented with a hormone mixture, sodium bicarbonate (21.4 g/L), Hepes (4.3 g/L), glucose (1.75 g/L), glutamine (0.14 g/L), epidermal growth factor (20 μg/L) and basic fibroblast growth factor (10 μg/L). Cells inoculated into PPRF-m2 effectively doubled 4 times in 5 days to achieve a maximum viable cell density of 1.2 × 10⁶ cells/mL in stationary culture. This was approximately 650% higher than the maximum cell density achieved in the commercial NSC medium. In addition, the PPRF-m2 cultures were observed to contain less cellular debris, and the cells were less granular and exhibited a lower degree of attachment. Immunocytochemistry revealed that cells grown in PPRF-m2 retained the ability to generate neurons, astrocytes, and oligodendrocytes.     

Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies

Bispecific single-chain diabodies (scDb) consist of the variableheavy and light chain domains of two antibodies connected bythree linkers. The structure of an scDb in the V_H–V_L orientationis V_HA–linkerA–V_LB–linkerM–V_HB–linkerB–V_LA,with linkers A and B routinely chosen to be 5–6 residuesand linker M 15–20 residues. Here, we applied displayof scDb on filamentous phage to analyse the composition of optimallinker sequences. The three linkers were randomized in lengthand sequence using degenerated triplets coding for only sixhydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly,Ala). Antigen-binding clones were then isolated by one to tworounds of selection on the two different antigens recognizedby the bispecific scDb. Using an scDb directed against carcinoembryonicantigen (CEA) and ß-galactosidase (Gal), we foundthat monomeric scDb had a preferred length of 15 or more aminoacid residues for the middle linker M and of 3–6 residuesfor the linkers A and B. No obvious bias towards a preferredlinker sequence was observed. Reduction of the middle linkerbelow 13 residues led to the formation of dimeric scDb, whichmost likely results from interchain pairing between all theV_H and V_L domains. Dimeric scDb were also formed by fragmentspossessing a long linker M and linkers A and B of 0 or 1 residue.We assume that these dimeric scDb are formed by intrachain pairingof the central variable domains and interchain pairing of theflanking variable domains. Thus, the latter molecules representa novel format of bispecific and tetravalent molecules. Thedescribed strategy allows for the isolation of both optimizedand minimal linker sequences for the assembly of monomeric ordimeric single-chain diabodies.     

Fabrication of PLA/MWCNT/Fe3O4 composite nanofibers for leukemia cancer cells

The efficient delivery of daunorubicin loaded poly (lactic acid) (PLA)/multiwalled carbon nanotubes (MWCNT)/Fe₃O₄ composite nanofibers was investigated. The synthesized nanofibers were characterized using SEM, TEM, and XRD analysis. The proliferation inhibition effect of PLA/MWCNT/Fe₃O₄ nanofibrous scaffolds on leukemia K562 cell lines was investigated. The effect of nanofiber concentration on the daunorubicin delivery in the absence and presence of external magnetic field was also evaluated. The results indicated that the incorporation of daunorubicin into the prepared nanofibrous scaffold under applied magnetic field could have synergistic cytotoxic effect on leukemia cancer cells. The drug release mechanism followed the non-Fickian transport.     

SBR处理酒店废水的工程应用

采用序列间歇式活性污泥工艺（SBR）对酒店废水进行处理,通过对系统工艺参数进行调整,达到最佳出水效果。消毒池出水水质优于生活污水排放标准（GB18918-2002）一级标准中的A类排放标准,COD值30 mg/L～50 mg/L;氨氮质量浓度3 mg/L～5 mg/L;总磷质量浓度0.2 mg/L～0.4 mg/L。     

膜生物反应器技术处理洗浴废水的工程应用

根据洗浴废水的水质水量，设计了生物接触氧化和膜生物反应器相结合的处理工艺(水量为120m^3／d)。通过对工艺相关设计参数的选取和调试运行结果表明，该工艺具有处理效果稳定、抗冲击负荷强以及操作简便等优点。     

木器漆用消泡剂及泡沫的消除对策(续)

４木器漆起泡的原因及防止措施造成木器漆起泡的原因是很多的,并非所有的泡沫都能借助消泡剂消除,应该针对不同情况采取对应措施,再配合适宜的消泡剂消除涂料中的微泡。４１表面活性剂稳定的微泡希望木器漆具有良好的表面状态,使用流平剂是必不可缺少的。上面论述了...     

Evaluation of the Usability of a Low-Cost 3D Printer in a Tissue Engineering Approach for External Ear Reconstruction

The use of alloplastic materials instead of autologous cartilage grafts offers a new perspective in craniofacial reconstructive surgery. Particularly for regenerative approaches, customized implants enable the surgeon to restore the cartilaginous framework of the ear without donor site morbidity. However, high development and production costs of commercially available implants impede clinical translation. For this reason, the usability of a low-cost 3D printer (Ultimaker 2+) as an inhouse-production tool for cheap surgical implants was investigated. The open software architecture of the 3D printer was modified in order to enable printing of biocompatible and biologically degradable polycaprolactone (PCL). Firstly, the printing accuracy and limitations of a PCL implant were compared to reference materials acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA). Then the self-made PCL-scaffold was seeded with adipose-tissue derived stem cells (ASCs), and biocompatibility was compared to a commercially available PCL-scaffold using a cell viability staining (FDA/PI) and a dsDNA quantification assay (PicoGreen). Secondly, porous and solid patient-customized ear constructs were manufactured from mirrored CT-imagining data using a computer-assisted design (CAD) and computer-assisted manufacturing (CAM) approach to evaluate printing accuracy and reproducibility. The results show that printing of a porous PCL scaffolds was possible, with an accuracy equivalent to the reference materials at an edge length of 10 mm and a pore size of 0.67 mm. Cell viability, adhesion, and proliferation of the ASCs were equivalent on self-made and the commercially available PCL-scaffolds. Patient-customized ear constructs could be produced well in solid form and with limited accuracy in porous form from all three thermoplastic materials. Printing dimensions and quality of the modified low-cost 3D printer are sufficient for selected tissue engineering applications, and the manufacturing of personalized ear models for surgical simulation at manufacturing costs of EUR 0.04 per cell culture scaffold and EUR 0.90 (0.56) per solid (porous) ear construct made from PCL. Therefore, in-house production of PCL-based tissue engineering scaffolds and surgical implants should be further investigated to facilitate the use of new materials and 3D printing in daily clinical routine.     

木器漆用消泡剂及泡沫的消除对策

论述了木器漆用消泡剂的种类和破泡机理以及在溶剂型木器漆中泡沫的形成与消除对策。     

Discovery and Characterization of CD12681, a Potent RORγ Inverse Agonist,Preclinical Candidate for the Topical Treatment of Psoriasis

With possible implications in multiple autoimmune diseases, the retinoic acid receptor‐related orphan receptor RORγ has become a sought‐after target in the pharmaceutical industry. Herein are described the efforts to identify a potent RORγ inverse agonist compatible with topical application for the treatment of skin diseases. These efforts culminated in the discovery of N‐(2,4‐dimethylphenyl)‐N‐isobutyl‐2‐oxo‐1‐[(tetrahydro‐2H‐pyran‐4‐yl)methyl]‐2,3‐dihydro‐1H‐benzo[d]imidazole‐5‐sulfonamide (CD12681), a potent inverse agonist with in vivo activity in an IL‐23‐induced mouse skin inflammation model.     

生态组合池污水处理工艺的工程应用与优化

生态组合池工艺去除机理主要是通过生物-生态联合作用去除污水中的污染物。该工艺虽然能使出水指标达到GB 18918—2002《城镇污水处理厂污染物排放标准》一级A标准,但运行中仍会存在一些问题。结合市政和纺织产业园污水处理工程实例,对生态组合池工艺技术进行说明,通过总结项目运行情况,发现在冬季低温季节,特别是进水总氮超标情况下,出水总氮出现超标情况;试运行期进水浓度变化较大时,供气分配量需要手动及时调整;絮凝反应区底部分区,导致絮凝剂投加点后段富磷污泥不能及时排出,占用稳定区容积等问题。针对以上问题,提出增加混合液回流管道、优化布水系统和供气系统、增设钢筋混凝土结构沉淀池、预埋排泥管道并预留定期排泥端口、减少稳定区深度等措施。     

含氰含铬电镀废水的分类处理工程实例

桂林市某机械配件厂,根据电镀生产废水特征,对含铬、含氰、综合、含碱废水进行分类收集,并对含铬和含氰废水进行分类预处理,最后采用化学中和法使处理后废水达标排放.处理过程采用自动化控制,经调试运行表明该电镀生产废水处理工艺运行稳定,处理效果良好,主要污染物去除率均可达98%以上,处理后出水水质符合<污水综合排放标准>(GB 8978-1996)一级标准.     

Graphene nanogrids for selective and fast osteogenic differentiation of human mesenchymal stem cells

Graphene nanogrids (fabricated by graphene nanoribbons obtained through oxidative unzipping of multi-walled carbon nanotubes) were used as two-dimensional selective templates for accelerated differentiation of human mesenchymal stem cells (hMSCs), isolated from umbilical cord blood, into osteogenic lineage. The biocompatible and hydrophilic graphene nanogrids showed high actin cytoskeleton proliferations coinciding with patterns of the nanogrids. The amounts of proliferations were found slightly better than proliferation on hydrophilic graphene oxide (GO) sheets, and significantly higher than non-uniform proliferations on hydrophobic reduced graphene oxide (rGO) sheets and polydimethylsiloxane substrate. In the presence of chemical inducers, the reduced graphene oxide nanoribbon (rGONR) grid showed a highly accelerated osteogenic differentiation of the hMSCs (a patterned differentiation) in short time of 7 days in which the amount of the osteogenesis was ∼2.2 folds greater than the differentiation (a uniform differentiation) on the rGO sheets. We found that although in the absence of any chemical inducers the graphene nanogrids showed slight patterned osteogenic differentiations, the graphene sheets could not present any differentiation. Therefore, the highly accelerated differentiation on the rGONR grid was assigned to both its excellent capability in adsorption of the chemical inducers and physical stresses induced by the surface topographic features of the nanogrids.     

Engineering a Carbonyl Reductase for Scalable Preparation of (S)-3-Cyclopentyl-3-hydroxypropanenitrile,the Key Building Block of Ruxolitinib

(S)-3-Cyclopentyl-3-hydroxypropanenitrile is the key precursor for the synthesis of ruxolitinib. The bioreduction of 3-cyclopentyl-3-ketopropanenitrile ( 1 a ) offers an attractive method to access this important compound. A carbonyl reductase (PhADH) from Paraburkholderia hospita catalyzed the reduction of 1 a giving the (S)-alcohol ( 1 b ) with 85 % ee. Rational engineering of PhADH resulted in a double mutant H93C/A139L, which enhanced the enantioselectivity from 85 % to >98 %, as well as a 6.3-fold improvement in the specific activity. The bioreduction of 1 a was performed at 200 g/L (1.5 M) substrate concentration, leading to isolation of (S)- 1 b in 91 % yield. Similarly, using this mutant enzyme, 3-cyclohexyl-3-ketopropanenitrile ( 2 a ) and 3-phenyl-3-ketopropanenitrile ( 3 a ) were reduced at high concentration affording the corresponding alcohols in >99 % ee, and 90 % and 92 % yield, respectively. The results showed that the variant H93C/A139L was a powerful biocatalyst for reduction of β-substituted-β-ketonitriles.     

3kt/aPTFE工程制冷压缩机的选型及应用

比较各种制冷压缩机的特点,阐述制冷压缩机选型思路,介绍国内第一套一机五工况、最低温度-50℃的离心式制冷压缩机组的工艺特点及其使用、技改情况。     

Proof of a lower bound formula for the expected reward in the levin-robbins sequential elimination procedure

We prove the general validity of a lower bound formula conjectured by Levin and Robbins to give a lower bound for the expected reward in a sequential elimination procedure designed to identify the best of c binomial populations.     

Integrated in Silico and Experimental Approach towards the Design of a Novel Recombinant Protein Containing an Anti-HER2 scFv

Biological therapies, such as recombinant proteins, are nowadays amongst the most promising approaches towards precision medicine. One of the most innovative methodologies currently available aimed at improving the production yield of recombinant proteins with minimization of costs relies on the combination of in silico studies to predict and deepen the understanding of the modified proteins with an experimental approach. The work described herein aims at the design and production of a biomimetic vector containing the single-chain variable domain fragment (scFv) of an anti-HER2 antibody fragment as a targeting motif fused with HIV gp41. Molecular modeling and docking studies were performed to develop the recombinant protein sequence. Subsequently, the DNA plasmid was produced and HEK-293T cells were transfected to evaluate the designed vector. The obtained results demonstrated that the plasmid construction is robust and can be expressed in the selected cell line. The multidisciplinary integrated in silico and experimental strategy adopted for the construction of a recombinant protein which can be used in HER2+-targeted therapy paves the way towards the production of other therapeutic proteins in a more cost-effective way.