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Prehensile tails are defined as having the ability to grasp objects and are commonly used as a fifth appendage during arboreal locomotion. Despite the independent evolution of tail prehensility in numerous mammalian genera, data relating muscle structure, physiology, and function of prehensile tails are largely incomplete. Didelphid marsupials make an excellent model to relate myosin heavy chain (MHC) isoform fiber type with structure/function of caudal muscles, as all opossums have a prehensile tail and tail use varies between arboreal and terrestrial forms. Expanding on our previous work in the Virginia opossum, this study tests the hypothesis that arboreal and terrestrial opossums differentially express faster versus slower MHC isoforms, respectively. MHC isoform expression and percent fiber type distribution were determined in the flexor caudae longus (FCL) muscle of Caluromys derbianus (arboreal) and Monodelphis domestica (terrestrial), using a combination of gel electrophoresis and immunohistochemistry analyses. C. derbianus expresses three MHC isoforms (1, 2A, 2X) that are distributed (mean percentage) as 8.2% MHC‐1, 2.6% 1/2A, and 89.2% 2A/X hybrid fibers. M. domestica also expresses MHC‐1, 2A, and 2X, in addition to the 2B isoform, distributed as 17.0% MHC‐1, 1.3% 1/2A, 9.0% 2A, 75.2% 2A/X, and 0.3% 2X/B hybrid fibers. The distribution of similar isoform fiber types differed significantly between species (P < 0.001). Although not statistically significant, C. derbianus was observed to have larger cross‐sectional area (CSA) for each corresponding fiber type along with a greater amount of extra‐cellular matrix. An overall faster fiber type composition (and larger fibers) in the tail of an arboreal specialist supports our hypothesis, and correlates with higher muscle force required for tail hanging and arboreal maneuvering on terminal substrates. Conversely, a broader distribution of highly oxidative fibers in the caudal musculature is well suited for tail nest building/remodeling behaviors of terrestrial opossums. Anat Rec, 297:1364–1376, 2014. © 2014 Wiley Periodicals, Inc.     

Orphan chemoattractant receptor GPR15 mediates dendritic epidermal T‐cell recruitment to the skin

Homing of murine dendritic epidermal T cells (DETCs) from the thymus to the skin is regulated by specific trafficking receptors during late embryogenesis. Once in the epidermis, Vγ3δ1 TCR DETCs are maintained through self‐renewal and participate in wound healing. GPR15 is an orphan G protein‐linked chemoattractant receptor involved in the recruitment of regulatory T cells to the colon. Here we show that GPR15 is highly expressed on fetal thymic DETC precursors and on recently recruited DETCs, and mediates the earliest seeding of the epidermis, which occurs at the time of establishment of skin barrier function. DETCs in GPR15^?/? mice remain low at birth, but later participation of CCR10 and CCR4 in DETC homing allows DETCs to reach near normal levels in adult skin. Our findings establish a role for GPR15 in skin lymphocyte homing and suggest that it may contribute to lymphocyte subset targeting to diverse epithelial sites.     

Three-dimensional measurement: the accuracy and precision of the reflex metrograph

The Reflex Metrograph is an optical plotter which is linked directly to a microcomputer and allows direct three-dimensional measurements of irregular shaped objects up to 300 mm maximum dimension without contacting the object. This study shows that it is possible to generate reproducible results with an operator measurement error of less than 0.2 mm for linear distances on objects up to 200 mm maximum dimension. The Reflex Metrograph tends to undermeasure by 0.67% or by up to 2.00 mm per 300 mm and is very slightly less accurate in the vertical plane. The potential use of this measuring instrument is discussed.     

PSNAV2.0-TNFα重组质粒的构建及其在体外的表达

目的:在前期微囊化基因工程细胞制备平台的基础上,构建分泌型人肿瘤坏死因子α的真核表达载体PSNAV2.0-TNFα重组质粒,并鉴定其蛋白的体外瞬时表达,为进一步利用该基因进行微囊化细胞移植治疗和改善疾病奠定基础。方法:实验于2006-06/2007-05在解放军总医院老年医学研究所细胞生物学实验室完成。①以含有人肿瘤坏死因子αcDNA序列的质粒为模板,通过PCR扩增获得人肿瘤坏死因子α基因片段;将其定向插入真核表达载体PSNAV2.0中,获得重组质粒PSNAV2.0-TNFα。采用SalⅠ和EcoRⅠ双酶切法、PCR法及插入片段序列测定法鉴定该质粒。②利用阳离子脂质体介导法,将其转染到人胚胎肾细胞HEK-293细胞中,构建可持续分泌人肿瘤坏死因子α的基因工程细胞,采用RT-PCR法和Western blot法检测转染细胞培养上清液中人肿瘤坏死因子蛋白的体外瞬时表达。结果:①通过SalⅠ和EcoRⅠ双酶切、PCR及测序鉴定证明:在HEK-293中插入片段正确。②采用RT-PCR和Western blot法检测表明HEK-293细胞培养上清中有人肿瘤坏死因子α蛋白,Mr17000。结论:成功构建了重组质粒PSNAV2.0-TNFα真核表达载体,转染HEK-293细胞后可有效分泌人肿瘤坏死因子α蛋白,并能分泌到细胞外。     

人羊膜间充质细胞具有分化成软骨及成骨细胞的潜能

目的:人羊膜间充质细胞具有比骨髓间充质干细胞更强的扩增能力和免疫原性低等优势。建立体外适宜的诱导培养条件,观察人羊膜间充质细胞定向分化为软骨细胞和成骨细胞的能力。方法:实验于2005-09/2006-12在贵州省细胞工程重点实验室完成。①材料来源:经产妇知情同意,无菌采集健康足月分娩新生儿胎盘6份,实验经医院医学伦理委员会批准。②实验方法:采用机械法剥离羊膜组织,二步酶消化法分离收获人羊膜间充质细胞,按2.2×10~8L~(-1)密度接种,传至第1～2代用于诱导分化实验。向软骨细胞诱导分化时,人羊膜间充质细胞按3×10~8L~(-1)密度接种,诱导培养液为含体积分数0.01的胎牛血清、10 mg/L转化生长因β1、100 nmol/L地塞米松、50 mg/L抗坏血酸、1%培养基添加物。向成骨细胞诱导分化时,人羊膜间充质细胞按6×10~7L~(-1)密度接种,诱导培养液为含体积分数0.1的胎牛血清、100 nmol/L地塞米松、50 mg/L抗坏血酸、5 mmol/Lβ-甘油磷酸。③实验评估:原代细胞用流式细胞仪分析表型,免疫细胞化学染色进行波形蛋白表达鉴定。分别于体外诱导第7,14,21,28天采用免疫细胞化学法检测软骨特异性Ⅱ型胶原的表达,细胞化学法检测蛋白聚糖的表达,钙-钴法检测成骨细胞特异性碱性磷酸酶的表达,茜素红S检测钙盐沉积情况。结果:①免疫组化与表型特征:人羊膜间充质细胞高表达间充质干细胞表面标志CD29、CD44和间充质细胞标志波形蛋白。②向软骨细胞诱导分化:诱导14 d后,人羊膜间充质细胞由长梭型逐渐变为多角形,可检测到Ⅱ型胶原蛋白表达及软骨细胞特异性细胞外基质蛋白聚糖。③向成骨细胞诱导分化:诱导21 d后,可观察到人羊膜间充质细胞的胞浆内有碱性磷酸酶表达,且可见钙盐沉积。结论:人羊膜间充质细胞具有分化成软骨细胞和成骨细胞的特性,可作为骨及软骨组织工程种子细胞的新来源。     

Synthesis and biologic evaluation of 64Cu-labeled rhenium-cyclized alpha-MSH peptide analog using a cross-bridged cyclam chelator.

Early detection of cutaneous melanoma is essential, as prognosis with metastatic melanoma is poor. Previous studies showed that (64)Cu-DOTA-ReCCMSH(Arg(11)) (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid), a cyclic analog of alpha-melanocyte-stimulating hormone (alpha-MSH), has the potential for the detection of malignant melanoma using PET. However, (64)Cu-DOTA-ReCCMSH(Arg(11)) demonstrated high background in nontarget tissues due to the in vivo instability of the Cu-DOTA moiety. CBTE2A (CBTE2A is 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane) has been shown to be a more stable copper chelate with improved in vivo stability, resulting in an improvement in clearance from nontarget tissues. The goal of this study was to conjugate CBTE2A to the alpha-MSH targeting ReCCMSH(Arg(11)) peptide for labeling to (64)Cu and to investigate whether the increased metal-chelate stability with CBTE2A would improve imaging quality. METHODS: The recyclized peptide CBTE2A-ReCCMSH(Arg(11)) was synthesized using a solid-phase peptide synthesizer followed by rhenium cyclization. In vivo characteristics of (64)Cu-CBTE2A-ReCCMSH(Arg(11)) were examined with small-animal PET and acute biodistribution studies in B16/F1 tumor-bearing mice. RESULTS: Biodistribution studies showed high and rapid receptor-mediated tumor uptake with values similar to those reported for (64)Cu- and (86)Y-labeled DOTA-ReCCMSH(Arg(11)). Nontarget organ concentration for (64)Cu-CBTE2A-ReCCMSH(Arg(11)) was considerably lower than that of the (64)Cu-DOTA analog, resulting in significantly higher tumor-to-nontarget tissue ratios. Compared with (86)Y-DOTA-ReCCMSH(Arg(11)), (64)Cu-CBTE2A-ReCCMSH(Arg(11)) demonstrated increased tumor retention and kidney clearance. Small-animal PET images showed that the tumor could be clearly visualized at all time points (0.5-24 h). CONCLUSION: Our data suggest the superior stability of the (64)Cu-CBTE2A moiety compared with (64)Cu-DOTA, making (64)Cu-CBTE2A-ReCCMSH(Arg(11)) an ideal candidate for the PET of malignant melanoma.     

Molecular Organisation of Tick-Borne Encephalitis Virus

Tick-borne encephalitis virus (TBEV) is a pathogenic, enveloped, positive-stranded RNA virus in the family Flaviviridae. Structural studies of flavivirus virions have primarily focused on mosquito-borne species, with only one cryo-electron microscopy (cryo-EM) structure of a tick-borne species published. Here, we present a 3.3 Å cryo-EM structure of the TBEV virion of the Kuutsalo-14 isolate, confirming the overall organisation of the virus. We observe conformational switching of the peripheral and transmembrane helices of M protein, which can explain the quasi-equivalent packing of the viral proteins and highlights their importance in stabilising membrane protein arrangement in the virion. The residues responsible for M protein interactions are highly conserved in TBEV but not in the structurally studied Hypr strain, nor in mosquito-borne flaviviruses. These interactions may compensate for the lower number of hydrogen bonds between E proteins in TBEV compared to the mosquito-borne flaviviruses. The structure reveals two lipids bound in the E protein which are important for virus assembly. The lipid pockets are comparable to those recently described in mosquito-borne Zika, Spondweni, Dengue, and Usutu viruses. Our results thus advance the understanding of tick-borne flavivirus architecture and virion-stabilising interactions.