In Silico Design of an Oseltamivir Derivative with Increased Affinity against Wild-Type and Mutant Variants of Neuraminidase and Hemagglutinin of Influenza A H1N1 Virus

Antiviral resistance has turned into a world concern nowadays. Influenza A H1N1 emerged as a problem at the world level due to the neuraminidase (NA) mutations. The NA mutants conferred resistance to oseltamivir and zanamivir. Several efforts were conducted to develop better anti-influenza A H1N1 drugs. Our research group combined in silico methods to create a compound derived from oseltamivir to be tested in vitro against influenza A H1N1. Here we show the results of a new compound derived from oseltamivir but with specific chemical modifications, with significant affinity either on NA (in silico and in vitro assays) or HA (in silico) from influenza A H1N1 strain. We include docking and molecular dynamics (MD) simulations of the oseltamivir derivative at the binding site onto NA and HA of influenza A H1N1. Additionally, the biological experimental results show that oseltamivir derivative decreases the lytic-plaque formation on viral susceptibility assays, and it does not show cytotoxicity. Finally, oseltamivir derivative assayed on viral NA showed a concentration-dependent inhibition behavior at nM, depicting a high affinity of the compound for the enzyme, corroborated with the MD simulations results, placing our designed oseltamivir derivative as a potential antiviral against influenza A H1N1.     

An optimized live bacterial delivery vehicle safely and efficaciously delivers bacterially transcribed therapeutic nucleic acids

There is an unmet need for delivery platforms that realize the full potential of next-generation nucleic acid therapeutics. The in vivo usefulness of current delivery systems is limited by numerous weaknesses, including poor targeting specificity, inefficient access to target cell cytoplasm, immune activation, off-target effects, small therapeutic windows, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we characterize the safety and efficacy of a delivery platform comprising engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli SVC1) for intracellular cargo delivery. SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to allow escape of their cargo from the phagosome, and to have minimal immunogenicity. We describe SVC1's ability to deliver short hairpin RNA (shRNA), localized SVC1 administration to various tissues, and its minimal immunogenicity. To validate the therapeutic potential of SVC1, we used it to deliver influenza-targeting antiviral shRNAs to respiratory tissues in vivo. These data are the first to establish the safety and efficacy of this bacteria-based delivery platform for use in multiple tissue types and as an antiviral in the mammalian respiratory tract. We expect that this optimized delivery platform will enable a variety of advanced therapeutic approaches.     

人源诱导多能干细胞体外定向分化为功能性肝细胞的方法研究

摘要 目的：研究开发一种简易、快速在体外使多能诱导干细胞（induced pluripotent stem cells,iPSCs）定向分化为功能性肝样细胞的培养方法。方法：根据正常肝细胞在体内的发育规律,设计简化诱导方法使iPS细胞定向分化为内胚层细胞,应用qPCR和流式细胞术鉴定其纯度后进一步诱导分化为肝样细胞,并通过qPCR、ELISA、免疫荧光等技术鉴定肝细胞的性状和功能。结果：iPS细胞诱导7天后, OCT4和NANOG的表达水平显著下降,内胚层细胞相关基因CXCR4、FOXA2和HNF4A表达水平明显升高。内胚层细胞继续诱导培养15天后,肝细胞特异性标志基因ALB、TDO2、RBP4、G6PC和肝药酶基因CYPs等显著上调,同时产生高水平的白蛋白和尿素;PAS糖原染色为阳性,能主动摄取和释放吲哚菁绿,证实诱导成的肝样细胞具备正常肝细胞的部分功能。结论：该诱导方案能够在体外使iPS细胞遵循正常肝脏发育通路简易、高效地分化为功能性肝细胞。本研究为大量获得iPS来源的肝细胞及其在细胞疗法和药筛模型中的运用提供了可能性。     

A greenhouse technique to screen pigeonpea for resistance to Heterodera cajani*

Heterodera cajani is an important nematode pest of pigeonpea in India and a simple and reliable greenhouse procedure has been developed to screen pigeonpea genotypes for resistance to it. In pot experiments, white cysts of H. cajani were counted on the roots of the susceptible genotype ICPL 87 at 15, 30 and 45 days after seedling emergence in soils infested with different levels of H. cajani. The seedlings were rated for the number of white cysts per root system on a one (highly resistant, no cysts) to nine (highly susceptible, more than 30 cysts) scale. White cysts were not easy to see on wet roots but were clearly visible on slightly dried roots. Cyst counts and ratings were more uniform when roots of 30 day old seedlings were evaluated than when 15 or 45 day old seedlings were examined. Effects of different H. cajani infestation levels on the ratings were not significant although the use of higher inoculum densities (16 to 27 eggs and juveniles/cm³ soil) was effective in reducing variability. This procedure was used to screen 60 pigeonpea genotypes and all of them were rated seven or nine. Ten accessions of Atylosia spp. and Rhynchosia spp. were rated three.     

Search for substrate-based inhibitors fitting the S2' space of malarial aspartic protease plasmepsin II.

Plasmepsin (Plm) has been identified as an important target for the development of new antimalarial drugs, since its inhibition leads to the starvation of Plasmodium falciparum. A series of substrate-based dipeptide-type Plm II inhibitors containing the hydroxymethylcarbonyl isostere as a transition-state mimic were synthesized. The general design principle was provision of a conformationally restrained hydroxyl group (corresponding to the set residue at the P2' position in native substrates) and a bulky unit to fit the S2' pocket.     

Submitochondrial particles asin vitro biosensors of heavy metal toxicity

The effects on mitochondrial respiratory parameters of heavy metals, such as Cu, Ni, Pb, Cd, Zn, Ag, Hg, were recorded by using thein vitro response of submitochondrial particles (SMP) from beef heart mitochondria.The toxicity of these elements was estimated by determining their effects on the energy-coupled reverse electron transfer (RET), which is induced by ATP and succinate at first site level of the respiratory chain in SMP.The RET rate was easily monitored by recording spectrophotometrically at 340 nm the production of NADH, arising from the reduction of exogenous NAD⁺ by RET.The toxicity values were expressed as the toxicant molar concentration which decreases the rate of reduction of NAD⁺ to an extent of 50 percent (EC50). The toxicity increased in the following order: Ni²⁺2+2+< Cd²⁺2+2++.The SMP data were compared with the toxicity values obtained from a variety of biological systems currently used for toxicity testing. The results obtained demonstrate that the SMP test generally provides a good estimate of metal toxicity for several fish and invertebrate species. This is demonstrated by the statistical parameters obtained in the regression analysis. The broadened 95% confidence intervals and, in particular, the poor correlations obtained for some aquatic organisms can be ascribed to the more complex metabolic interactions and competing toxic pathways in aquatic organisms, when compared to SMP.     

Isolation of a phospholipase A1-producing microorganism

A bacterium which has phospholipase A₁ activity was isolated from soil. It is aerobic, motile, oxidase-negative and has flagella. The G+C content of the DNA was 58.1 mol%. The major isoprenoids of cell was were Q-8 and MK-8. The main cellular fatty acids were saturated straight chain (n-16) and cyclic (17:) fatty acids. Based on its morphological, physiological and chemotaxonomic characteristics, this organism was placed in the genusSerratia. Nutritional factors affecting enzyme production were explored. Xylose and ammonium sulfate were the best carbon and nitrogen sources, respectively. Ferrous ions exerted a considerable positive effect on enzyme production. The optimal pH and temperature for phospholipase A₁ production were 7.0 and 30°C, respectively.     

一例智力低下患者7q~ 标记染色体的来源鉴定

以人类染色体显微切割、PCR技术构建的现有人类染色体特异性和染色体区带特异性探针池作为绘画探针,采用正向染色体绘画技术,结合染色体筛查方法,查明了一例7q~ 标记染色体患者的染色体附加片段来源于3q26→3qter。确定该患者的核型为46,XX,-7, der(7)t(7;3)(7pter→7q32::3q26→3qter)。应用这个策略,能够快速有效地鉴定标记染色体的来源。     

Interlaboratory Reproducibility In Reporting Inadequate Cervical Smears—A Multicentre Multinational Study

A set of 300 vaginal smears was interpreted by 13 cytologists from six European laboratories, who were requested to report inadequate and suboptimal smears. the set had been appropriately seeded to reach approximately 10% inadequate and 20% suboptimal smear frequency. According to the majority report, 230 smears were classified as adequate (76.7%), 43 as inadequate (14.3%), and 27 as suboptimal (9.0%). Agreement with the majority report ranged from 52% to 91% (average 78%). Kappa statistics for reporting inadequate smears showed a high level of agreement for five cytologists, and fair to good agreement for eight. In contrast, K statistics for reporting suboptimal smears showed fair to good agreement with the majority report only in five instances, whereas agreement was poor for eight cytologists. ‘Inadequate smear’rates may be used to compare the quality of smears received in different laboratories, as there is a high level of agreement among cytologists as to what constitutes an inadequate smear. However, this is not true for ‘suboptimal smear’ rates, and more precise reporting criteria must be defined and tested if an intermediate category is to be retained to report poor quality smears: more precise reporting criteria must be defined and tested if an intermediate category is to be retained to report poor quality smears. Une série de 300 frottis cervico-vaginaux a été examinee par 13 cytologistes appartenant à 6 laboratoires européens auxquels il avait été demandé de signaler les frottis inadéquats et sub-optimaux. Cette série de frottis a été constituée de façon à atteindre approximativement une fréquence de 10% de frottis inadéquats et de 20% de frottis sub-optimaux. Si I'on considère les réponses faites par la majorité des cytologistes, 230 frottis ont été classés adéquats (76,7%), 43 classés inadéquats (14,3%), et 27 comme sub-optimaux (9,0%). La concordance avec les réponses de la majorité des cytologistes varie de 52%à 91% (moyenne 78%). Concernant le signalement des frottis inadéquats, le test Kappa est excellent pour 5 cytologistes et moyen à bon pour les 8 autres. Au contraire, pour le signalement des frottis sub-optimaux, le test Kappa montre un accord moyen à bon avec les réponses de la majorité seulement dans 5 cas alors que I'agrément est mauvais pour 8 cytologistes. La fréquence des frottis inadéquats peut être utilisée pour comparer la qualité de prélèvement du frottis dans différents laboratoires, alors que les comparaisons de la fréquence des frottis sub-optimaux sont discutables, du fait d'un biais de diagnostic évident. Des critéres plus précis pour le signalement des frottis sub-optimaux doivent étre définis et testés si cette catégorie doit être retenue pour rapporter les frottis de mauvaise qualité. 300 Abstriche wurden durch 13 Zytologen aus 6 europäischen Laboratorien hinsichtlich der Kriterien ‘unbrauchbar’und ‘suboptimal’beurteilt. Die Sammlung enthielt etwa 10% unbrauchbare und 20% suboptimale Präparate. Mehrheitlich wurden 230 Abstriche als auswertbar (76,7%), 43 als unbrauchbar (14,3%) und 27 als suboptimal (9,0%) bezeichnet. Die Statistik ergab hinsichtlich der unbrauchbaren Abstriche eine ausgezeichnete Ûbereinstimmung für 5 und eine gute Ûbereinstim-mung für 8 Zytologen. Demgegenüber zeigte sich hinsichtlich der Beurteilung suboptimal nur 5mal eine gute und 8mal eine schlechte Ûbereinstimmung. Damit ist die Kategorie suboptimal ungeeignet zum Vergleich und bedarf einer genaueren Definition wenn sie für die Qualitätskontrolle eingesetzt werden soll.     

Identification of New Oligodendrocyte- and Myelin-Specific Genes by a Differential Screening Approach

Abstract: We have isolated several new genes that are specifically expressed by oligodendrocytes in the CNS. This was achieved by differential screening of a rat spinal cord cDNA library with probes derived from normal and from oligodendrocyte-free spinal cord mRNAs. Four of these genes are exclusively expressed by oligodendrocytes: Three of these are not related to known genes, whereas one encodes the myelin oligodendrocyte glycoprotein (MOG). Four other genes are expressed by oligodendrocytes as well as by Schwann cells. One gene codes for apolipoprotein D, which is thought to be involved in lipid metabolism. A second cDNA sequence codes for the recently identified galactosylceramide-synthesizing enzyme UDP-galactose:ceramide galactosyl-transferase. The third gene encodes a small protein with four putative transmembrane domains that is related to a T-lymphocyte-specific membrane protein, MAL. The fourth gene encodes the rat homologue of the stearyl-CoA-desaturase 2 (SCD2) gene, which is specifically expressed in the nervous system and involved in the synthesis and regulation of long-chain unsaturated fatty acids essential for myelination. Finally, we found that a member of the β-tubulin family is highly expressed in oligodendrocytes as well as neurons. The identification of several new proteins that may play a role in myelin synthesis and sheath formation will lead to new insight into this complex mechanism.