Proton-Detected Solid-State NMR of the Cell-Free Synthesized α-Helical Transmembrane Protein NS4B from Hepatitis C Virus

Proton-detected 100 kHz magic-angle-spinning (MAS) solid-state NMR is an emerging analysis method for proteins with only hundreds of microgram quantities, and thus allows structural investigation of eukaryotic membrane proteins. This is the case for the cell-free synthesized hepatitis C virus (HCV) nonstructural membrane protein 4B (NS4B). We demonstrate NS4B sample optimization using fast reconstitution schemes that enable lipid-environment screening directly by NMR. 2D spectra and relaxation properties guide the choice of the best sample preparation to record 2D ¹H-detected ¹H,¹⁵N and 3D ¹H,¹³C,¹⁵N correlation experiments with linewidths and sensitivity suitable to initiate sequential assignments. Amino-acid-selectively labeled NS4B can be readily obtained using cell-free synthesis, opening the door to combinatorial labeling approaches which should enable structural studies.     

Generation of New Artificial Metalloproteins by Cofactor Modification of Native Hemoproteins

Heme can be removed from a number of native hemoproteins, thus forming corresponding apoproteins, each of which provides a site for binding of a metal complex. In one example, myoglobin, an O₂ storage protein, can be reconstituted with iron porphycene to dramatically enhance the O₂ affinity. Although it is known that myoglobin has poor enzymatic activity, the insertion of iron corrole or iron porphycene into apomyoglobin increases its H₂O₂-dependent peroxidase/peroxygenase activities. Furthermore, reconstitution with manganese porphycene promotes hydroxylation of an inert C H bond. It is also of interest to insert a non-porphyrinoid complex into an apoprotein. A cavity of apocytochrome c has been found to bind a diiron carbonyl complex, serving as a functional model of diiron hydrogenase. Aponitrobindin has a rigid β-barrel structure that provides an excellent cavity for covalently anchoring a metal complex. A rhodium complex embedded in the cavity of genetically modified nitrobindin has been found to promote stereoselective polymerization of phenylacetylene.     

Reconstituted versus dry alfalfa hay in starter feed diets of Holstein dairy calves: Effects on feed intake,feeding and chewing behavior,feed preference,and health criteria

We investigated the effect of reconstitution of alfalfa hay on feeding and chewing behavior, sorting activity, and health status using 20 neonate Holstein male calves (3 d of age; 40.3 ± 1.30 kg of body weight) that were assigned randomly to 2 treatments, a starter feed containing either 10% dry (AH) or reconstituted alfalfa hay (RAH), each consisting of 10 calves. Alfalfa hay was reconstituted with water 24 h before feeding to achieve a theoretical dry mater (DM) content of 20%. Both starter diets had the same ingredients and nutrient compositions but differed in their DM content (90.9 vs. 66.6% DM for AH vs. RAH, respectively). Calves were weaned on d 50 and remained in the study until d 70. Reconstitution of alfalfa hay increased the percentage of particles retained on 8- and 1.18-mm sieves, but reduced feed materials retained on the bottom pan. Feeding RAH tended to increase meal frequency (preweaning) and increased meal duration (pre- and postweaning), and thereby increased time devoted to eating without affecting nutrient intake. Calves fed RAH tended to have lower eating rate during the pre- and postweaning periods. Tendencies to concurrent increase in rumination frequency and rumination duration in calves fed RAH increased time devoted to ruminating during the preweaning period; however, a tendency to longer rumination duration did not affect ruminating time during the postweaning period. Calves fed RAH spent less time resting and standing and more time drinking during the postweaning period. Non-nutritive oral behaviors were not affected by treatment during the pre- and postweaning period. Feeding RAH decreased sorting of particles retained on 8-mm sieve compared with AH; however, calves in both treatment groups sorted for particles retained on 8- and 1.18-mm sieves and against the feed materials retained on the bottom pan. Crude protein and neutral detergent fiber intakes of particles retained on the both 8- and 1.18-mm sieves increased in calves fed RAH versus AH, with no changes in intake of nutrients retained on the bottom pan. Calves fed AH were more susceptible to develop diarrhea (odds ratio = 2.02) and pneumonia (odds ratio = 4.74) and thereby had lower chances of being treated. We found no difference between treatment groups for frequency and medication days of diarrhea; however, calves fed RAH experienced fewer days with diarrhea. Furthermore, frequency and number of days with pneumonia and administration of medication were greater for calves fed AH compared with calves fed RAH. Blood concentrations of total protein and globulin tended to be higher in calves fed RAH compared with calves fed AH. Overall, feeding RAH increased time devoted to eating by increasing meal frequency and meal duration but did not affect feed intake due to lower eating rate. Calves in both treatment groups exhibited feed sorting at the extent to which they balanced intake of nutrients and met their nutritional needs. Calves, in general, were healthy, but it seems that feeding a diet containing reconstituted alfalfa hay has a potential health-promoting effect, possibly through reducing diet dustiness and improving immune status.     

实施企业再造提升企业实力--浅析国有建筑业企业的"企业再造"

介绍国有建筑业企业“企业再造”的时代背景和基本内涵,以及“企业再造”的实践应用,探索以管理创新为重点,以业务流程重组为起点“企业再造”过程,实现企业无缝管理,培育高度适应性的国有建筑业企业。     

Ovine Hair Follicle Stem Cells Derived from Single Vibrissae Reconstitute Haired Skin

Hair follicle stem cells (HFSCs) possess fascinating self-renewal capacity and multipotency, which play important roles in mammalian hair growth and skin wound repair. Although HFSCs from other mammalian species have been obtained, the characteristics of ovine HFSCs, as well as the methods to isolate them have not been well addressed. Here, we report an efficient strategy to obtain multipotent ovine HFSCs. Through microdissection and organ culture, we obtained keratinocytes that grew from the bulge area of vibrissa hair follicles, and even abundant keratinocytes were harvested from a single hair follicle. These bulge-derived keratinocytes are highly positive for Krt15, Krt14, Tp63, Krt19 and Itga6; in addition to their strong proliferation abilities in vitro, these keratinocytes formed new epidermis, hair follicles and sebaceous glands in skin reconstitution experiments, showing that these are HFSCs from the bulge outer root sheath. Taken together, we developed an efficient in vitro system to enrich ovine HFSCs, providing enough HFSCs for the investigations about the ovine hair cycle, aiming to promote wool production in the future.     

Adaptive Immune Responses in a Multiple Sclerosis Patient with Acute Varicella-Zoster Virus Reactivation during Treatment with Fingolimod

Fingolimod, an oral sphingosine 1-phosphate (S1P) receptor modulator, is approved for the treatment of relapsing forms of multiple sclerosis (MS). The interference with S1P signaling leads to retention particularly of chemokine receptor-7 (CCR7) expressing T cells in lymph nodes. The immunological basis of varicella zoster virus (VZV) infections during fingolimod treatment is unclear. Here, we studied the dynamics of systemic and intrathecal immune responses associated with symptomatic VZV reactivation including cessation of fingolimod and initiation of antiviral therapy. Key features in peripheral blood were an about two-fold increase of VZV-specific IgG at diagnosis of VZV reactivation as compared to the previous months, a relative enrichment of effector CD4+ T cells (36% versus mean 12% in controls), and an accelerated reconstitution of absolute lymphocytes counts including a normalized CD4+/CD8+ ratio and reappearance of CCR7+ T cells. In cerebrospinal fluid (CSF) the lymphocytic pleocytosis and CD4+/CD8+ ratios at diagnosis of reactivation and after nine days of fingolimod discontinuation remained unchanged. During this time CCR7+ T cells were not observed in CSF. Further research into fingolimod-associated VZV reactivation and immune reconstitution is mandatory to prevent morbidity and mortality associated with this potentially life-threatening condition.     

Substituting corn silage with reconstituted forage or nonforage fiber sources in the starter feed diets of Holstein calves: Effects on intake,meal pattern,sorting, and health

We examined the effect of replacing corn silage (CS) with reconstituted alfalfa hay (RAH) or reconstituted beet pulp (RBP) in the starter diets on feeding behavior, sorting, and health criteria using 54 neonatal female Holstein calves that were assigned randomly to 3 groups receiving starter diets containing CS (10% on a dry matter basis), RAH, or RBP. The starter diets had the same nutrient composition and moisture level but differed in particle size distribution. Calves fed CS tended to have a lower intermeal interval compared with calves fed RAH before weaning; however, meal frequency and duration, eating rate, and meal size were not affected by treatment before and after weaning, which resulted in no changes in feed intake and time spent eating. Rumination frequency was higher for calves fed CS than for calves fed RAH or RBP after weaning. Feeding RBP decreased rumination duration compared with CS and RAH after weaning; however, calves fed RBP had a higher rumination bout interval compared with calves fed CS. Calves fed CS spent more time ruminating compared with calves fed RAH or RBP after weaning. Feeding CS tended to decrease and decreased time devoted to standing and lying, respectively, compared with calves fed RBP; however, calves fed RBP tended to spend more time on nonnutritive oral behaviors compared with calves fed RAH. Calves fed CS sorted against particles retained on the 8-mm sieve of the Penn State Particle Separator (PSPS) and for particles retained on the 1.18-mm sieve of the PSPS. Feeding RAH increased sorting for particles retained on the 8- and 1.18-mm sieves of the PSPS. Calves fed RBP sorted only for particles retained on the 1.18-mm sieve of the PSPS. Calves fed RBP tended to be more susceptible to developing pneumonia compared with calves fed CS or RAH; however, frequency and duration of diarrhea and pneumonia or number of days needed to medicate the diseases were unchanged across treatment groups. Initial (d 3) blood total protein concentration was similar (6.51 g/dL) across treatment groups. Overall, replacing CS with RAH or RBP did not affect time devoted to eating and feed intake due to no significant changes in meal size or intermeal interval before and after weaning. Calves showed feed sorting at the extent to which they balanced intake of nutrients and met their nutritional needs. Calves in general were healthy; therefore, CS, RAH, or RBP can be used interchangeably based on availability and competitive feed price.     

质谱成像数据图像重构软件

在质谱成像分析中,将质谱原始数据重构成图像数据是重要的处理过程,当前缺乏专用的向导式软件来完成此功能,为此,开发出一种名为imgGenerate的软件系统。imgGenerate能够一步步引导用户依据质谱原始数据特点设定m/z范围、精度以及数据类型等参数,重构为科学图像Analyze 7.5格式数据。重构后的数据文件可再通过BioMap或IMSview软件进行图像显示处理。应用imgGenerate成功地将大鼠小脑切片和字迹质谱成像分析的原始质谱数据重构成图像数据。应用测试表明,imgGenerate具有向导式、功能全面、专用化、实用等特点。