Expression of SEAP (secreted alkaline phosphatase) by baculovirus mediated transduction of HEK 293 cells in a hollow fiber bioreactor system

A BacMam baculovirus was designed in our laboratory to express the reporter protein secreted alkaline phosphatase (SEAP) driven by the immediate early promoter of human cytomegalovirus promoter (CMV). In vitro tests have been carried out using this recombinant baculovirus to study the secreted protein in two cell lines and under various culture conditions. The transductions were carried out on two commonly used mammalian cell lines namely the human embryonic kidney (HEK 293A) and Chinese hamster ovary (CHO-K1). Initial studies clearly demonstrated that the transient expression of SEAP was at least 10-fold higher in the HEK 293 cells than the CHO cells under equivalent experimental conditions. Factorial design experiments were done to study the effect of different parameters such as cell density, MOI, and the histone deacetylase inhibitor, trichostatin A concentration. The multiplicity of infection (MOI) and the cell density were found to have the most impact on the process. The enhancer trichostatin A also showed some positive effect. The production of secreted protein in a batch reactor was studied using the Wave disposable bioreactor system. A semi-continuous perfusion process was developed to extend the period of gene expression in mammalian cells using a hollow fiber bioreactor system (HFBR). The growth of cells and viability in both systems was monitored by offline analyses of metabolites. The expression of recombinant protein could be maintained over an extended period of time up to 30 days in the HFBR.     

Synthesis and evaluation of novel dipeptidyl benzoyloxymethyl ketones as caspase inhibitors

We describe novel peptide-based caspase inhibitors. Potent and comparatively selective compounds containing a dipeptide scaffold and a substituted oxymethyl ketone as a warhead were developed. The newly synthesized compounds were tested for inhibition in in vitro enzymatic assays of caspases-1, -3, -6, -8, and -9. The benzyloxycarbonyl-phenylglycyl-aspartyl benzoyloxymethyl ketone (Z-Phg-Asp-CH2OCO-Ph, coded as HU44) was the most potent inhibitor of caspase-1 and caspase-3. Of several analogs of HU44 that were made, the beta-Asp methyl ester (2) is an effective inhibitor against caspase-3 and caspase-8, and less effective against caspase-1. These compounds did not inhibit caspase-6 and caspase-9 significantly.     

Des chambres de pupation de Dermestidae (Insecta : Coleoptera) sur un os de mammifère tertiaire (phosphorites du Quercy) : implications taphonomiques et paléoenvironnementales

The larvae of some species of Dermestidae (Coleoptera) are able to bore the bones of a carrion for pupation. Several pupal chambers have been observed on an unciform belonging to a fossil rhinocerotid (Mesaceratherium sp.) from the late Oligocene/earliest Miocene of the phosphorites of Quercy (SW France). Such an unusual observation demonstrates the occurrence of dermestids in the entomofauna of the phosphorites of Quercy, and suggests at least the existence of paleoclimatic episodes with dry seasons, during a period usually considered as a relatively humid one. This is the earliest occurrence of such ichnofossils on a fossil mammal bone.     

平板式光生物反应器中紫球藻培养条件的优化

研究了平板式光生物反应器中紫球藻(Porphyridium cruentumNaegeli)的培养条件,运用均匀设计法对光照强度、通气速率、装液量、接种密度以及pH等影响紫球藻生长的因素进行优化,获得了在平板式光生物反应器中培养紫球藻的最佳条件:光照强度10 000 lx、通气速率350 L.h-1、装液量6 L、藻细胞接种密度1.1×106mL-1、pH9.0。在最佳条件下藻体的生物量产率和生物量产量分别达到0.431 g.L-1.d-1和3.240 g.L-1,最大生长速率达0.652 g.L-1.d-1,胞外多糖含量高达0.665 g.L-1。另外,在培养过程中隔天补充培养液有利于紫球藻生物量的增加和胞外多糖的产生。     

高密高异质性城市街区景观对心理健康影响评价及循证优化设计

高密高异质性城市环境不仅影响空间审美感受,也影响着城市居民的心理健康。实证研究表明通过引入绿色自然、优化空间设计可缓解这种负面影响。本文结合已有的神经认知学和医学研究,将风景园林学科核心价值之一环境愉悦体验提升到公共健康高度。我们利用神经生物测量等一系列先进技术量化环境体验,并在医学和神经认知学已有实证证据的基础上进一步进行健康影响评价,提出基于健康影响评价的循证设计方法。具体包括测量高密高异质性街道中不同环境空间要素组合对人的健康影响,分析识别具体案例中最容易诱发压力和认知负荷的空间要素,针对性地提出设计干预策略并依托增强现实/虚拟现实技术进行绩效预估。研究整合了技术量化环境体验和环境神经健康评价这2个密切联系的学术前沿,其成果可以直接指导具体案例的循证设计优化。     

A new approach to the design of uniquely folded thermally stable proteins

A new computer program (CORE) is described that predicts core hydrophobic sequences of predetermined target protein structures. A novel scoring function is employed, which for the first time incorporates parameters directly correlated to free energies of unfolding (deltaGu), melting temperatures (Tm), and cooperativity. Metropolis-driven simulated annealing and low-temperature Monte Carlo sampling are used to optimize this score, generating sequences predicted to yield uniquely folded, stable proteins with cooperative unfolding transitions. The hydrophobic core residues of four natural proteins were predicted using CORE with the backbone structure and solvent exposed residues as input. In the two smaller proteins tested (Gbeta1, 11 core amino acids; 434 cro, 10 core amino acids), the native sequence was regenerated as well as the sequence of known thermally stable variants that exhibit cooperative denaturation transitions. Previously designed sequences of variants with lower thermal stability and weaker cooperativity were not predicted. In the two larger proteins tested (myoglobin, 32 core amino acids; methionine aminopeptidase, 63 core amino acids), sequences with corresponding side-chain conformations remarkably similar to that of native were predicted.     

El déficit de vitamina B12 y ácido fólico en la población mayor de 65 años: estudio descriptivo

Introduction

The aim of this study is to establish the prevalence of vitamin B12 and folic acid deficiency in the population of 65 years and over in semi-urban and rural area, as well as investigating the risk factors, and the determining factors of this deficiency in this population and its relationship with the prevalence of concomitant cardiovascular and cognitive diseases.

Materials and methods

A cross-sectrionall study conducted in the Geriatrics Outpatients in the period between 2008 and 2010. Demographic, clinical and laboratory were collected at 3 different times.A bivariate analysis was performed with lack of vitamin B12 and folic acid, as the outcome variables.

Results

Vitamin B12 defieciency was found in 16.5% of the sample, and no folic acid deficiency. A strong association was found with vitamin B12 deficiency and cardiac and cerebrovascular diseases, vascular risk factors and drugs administered in prevention of cardiac events and ischaemic stroke.

Conclusions

There was a higher prevalence of vitamin B12 deficiency in the elderly population in the catchment area of Medina del Campo compared to that found in the literature, but not so with the isolated deficiency of folic acid.     

An apterous scelionid wasp in mid-Cretaceous Burmese amber (Hymenoptera: Scelionidae)

A remarkably specialized parasitoid wasp of the family Scelionidae (Platygastroidea) is described and figured from mid-Cretaceous (Cenomanian) amber of the Hukawng Valley in northern Myanmar. Geoscelio mckellari Engel and Huang, gen. et sp. nov., is unique for its combination of a compact body, 12 antennal flagellomeres, a 1-2-2 tibial spur formula, a distinct malar sulcus, deeply impressed notauli, complete reduction of the wings, and basal crenulae on the metasomal terga and sterna, among many other features, and is placed within a separate tribe, Geoscelionini Engel and Huang, trib. nov. This is the first flightless species of Platygastroidea known from the Mesozoic, and its affinities with other Mesozoic and extant lineages are discussed.     

New approaches to high-throughput structure characterization of SH3 complexes: the example of Myosin-3 and Myosin-5 SH3 domains from S. cerevisiae

SH3 domains are small protein modules that are involved in protein-protein interactions in several essential metabolic pathways. The availability of the complete genome and the limited number of clearly identifiable SH3 domains make the yeast Saccharomyces cerevisae an ideal proteomic-based model system to investigate the structural rules dictating the SH3-mediated protein interactions and to develop new tools to assist these studies. In the present work, we have determined the solution structure of the SH3 domain from Myo3 and modeled by homology that of the highly homologous Myo5, two myosins implicated in actin polymerization. We have then implemented an integrated approach that makes use of experimental and computational methods to characterize their binding properties. While accommodating their targets in the classical groove, the two domains have selectivity in both orientation and sequence specificity of the target peptides. From our study, we propose a consensus sequence that may provide a useful guideline to identify new natural partners and suggest a strategy of more general applicability that may be of use in other structural proteomic studies.     

High yield lipase-catalyzed synthesis of Engkabang fat esters for the cosmetic industry

Engkabang fat esters were produced via alcoholysis reaction between Engkabang fat and oleyl alcohol, catalyzed by Lipozyme RM IM. The reaction was carried out in a 500 ml Stirred tank reactor using heptane and hexane as solvents. Response surface methodology (RSM) based on a four-factor-five-level Central composite design (CCD) was applied to evaluate the effects of synthesis parameters, namely temperature, substrate molar ratio (oleyl alcohol: Engkabang fat), enzyme amount and impeller speed. The optimum yields of 96.2% and 91.4% were obtained for heptane and hexane at the optimum temperature of 53.9 °C, impeller speeds of 309.5 and 309.0 rpm, enzyme amounts of 4.82 and 5.65 g and substrate molar ratios of 2.94 and 3.39:1, respectively. The actual yields obtained compared well with the predicted values of 100.0% and 91.5%, respectively. Meanwhile, the properties of the esters show that they are suitable to be used as ingredient for cosmetic applications.