Crystallization and preliminary X‐ray diffraction analysis of eukaryotic α2‐macroglobulin family members modified by methylamine,proteases and glycosidases

α₂‐Macroglobulin (α₂M) has many functions in vertebrate physiology. To understand the basis of such functions, high‐resolution structural models of its conformations and complexes with interacting partners are required. In an attempt to grow crystals that diffract to high or medium resolution, we isolated native human α₂M (hα₂M) and its counterpart from chicken egg white (ovostatin) from natural sources. We developed specific purification protocols, and modified the purified proteins either by deglycosylation or by conversion to their induced forms. Native proteins yielded macroscopically disordered crystals or crystals only diffracting to very low resolution (>20 Å), respectively. Optimization of native hα₂M crystals by varying chemical conditions was unsuccessful, while dehydration of native ovostatin crystals improved diffraction only slightly (10 Å). Moreover, treatment with several glycosidases hindered crystallization. Both proteins formed spherulites that were unsuitable for X‐ray analysis, owing to a reduction of protein stability or an increase in sample heterogeneity. In contrast, transforming the native proteins to their induced forms by reaction either with methylamine or with peptidases (thermolysin and chymotrypsin) rendered well‐shaped crystals routinely diffracting below 7 Å in a reproducible manner.     

超声引导竖脊肌平面阻滞对胸腔镜肺叶切除患者术后镇痛和细胞免疫功能的影响

目的观察超声引导下竖脊肌平面(ESP)阻滞对胸腔镜下肺叶切除患者术后镇痛和细胞免疫功能的影响。方法选择择期胸腔镜下肺叶切除术患者90例,美国麻醉医师协会(ASA)分级Ⅰ～Ⅲ级。随机分为单纯静脉全麻组(C组)、椎旁神经阻滞+静脉全麻组(P组)和ESP阻滞+静脉全麻组(E组)。E组和P组分别在全麻诱导前行超声引导下ESP阻滞和椎旁神经阻滞,20 min后测定阻滞范围和效果,术毕3组均采用静脉自控镇痛(PCIA)。记录入手术室(T_0)、麻醉诱导前(T_1)、切皮即刻(T_2)和术毕(T_3)3组患者平均动脉压(MAP)和心率(HR);记录术后2 h (T_4)、8 h (T_5)、12 h (T_6)、24 h (T_7)和48 h (T_8)静态数字疼痛评分[NRS(R)]和动态NRS评分[NRS(M)];并于T_0、T_3、T_7和T_8抽取静脉血5 mL,检测血液自然杀伤(NK)细胞数目和Th1/Th2比值;记录术后镇痛泵按压次数、药物输注总量、曲马多给药次数和术后不良反应发生情况。结果与T_0比较,P组患者在T_1时点MAP降低、HR增快(P 0.05),C组患者T_2和T_3时点MAP增高、HR增快(P 0.05);P组和E组患者T_2和T_3时点MAP低于C组、HR慢于C组(P 0.05);C组和E组患者T_1时点MAP高于P组、HR慢于P组(P 0.05);P组和E组患者术后T_4～T_6时点的NRS(R)低于C组(P 0.05),E组患者术后T_7时点NRS (R)低于C组(P 0.05);P组和E组患者术后T_4～T_6时点的NRS (M)低于C组(P 0.05);P组和E组各时点NRS比较,差异无统计学意义(P 0.05)。各组患者在T_3、T_7和T_8时点NK细胞数目、Th1/Th2比值均较T_0降低,E组和P组在T_3、T_7和T_8时点NK细胞数目、Th1/Th2比值高于C组(P 0.05),E组和P组比较,差异无统计学意义(P 0.05);E组和P组术中瑞芬太尼用量、镇痛泵按压次数、药物输注总量和曲马多给药次数均明显少于C组(P 0.05),E组与P组比较,差异无统计学意义。P组术后低血压发生率高于C组和E组(P 0.05)、C组术后恶心呕吐发生率高于P组和E组(P 0.05)。结论超声引导下ESP阻滞,对胸腔镜下肺叶切除患者术后疼痛和免疫功能的影响与椎旁神经阻滞相当,但对患者循环影响更小。     

4型登革病毒NS2A蛋白通过UXT-V1逃避宿主免疫的机制研究

目的探讨4型登革病毒NS2A蛋白通过泛转录表达因子剪接变异体1(UXT-V1)逃避宿主免疫的机制。方法在HEK-293T细胞中,4型登革病毒感染后过表达NS2A蛋白,通过实时荧光定量PCR(qPCR)检测干扰素-β(IFN-β)、白细胞介素-8(IL-8)、干扰素诱导蛋白54(ISG54)的表达量;通过荧光素酶报告实验检测NS2A蛋白对IFN-β与核因子κB(NF-κB)通路的影响;通过免疫共沉淀检测NS2A蛋白与UXT-V1的相互作用;通过核蛋白抽取检测IRF3和p65的核定位。结果4型登革病毒NS2A蛋白与宿主UXT-V1存在相互作用。此外,4型登革病毒NS2A蛋白抑制宿主IFN-β、IL-8和ISG54的mRNA水平,阻断IRF3和p65的核转移,抑制宿主IFN-β与NF-κB通路。结论4型登革病毒NS2A蛋白通过与UXT-V1的相关作用阻断宿主固有免疫关键因子IRF3和p65的核定位,抑制宿主IFN-β与NF-κB通路,最终到达其免疫逃避的目的。     

Regulation of self-ligands for activating natural killer cell receptors

Abstract

Natural killer (NK) cells are able to lyse infected and tumor cells while sparing healthy cells. Recognition of diseased cells by NK cells is governed by several activating and inhibitory receptors. We review numerous pathways that have been implicated in the regulation of self-ligands for activating receptors, including NKG2D, DNAM-1, LFA-1, NKp30, NKp44, NKp46, NKp65, and NKp80 found on NK cells and some T cells. Understanding how the regulation of self-encoded ligand expression is regulated may provide novel avenues for future therapeutic approaches to infections and cancer.     

Cellular and molecular chaperone fusion vaccines: Targeting resistant cancer cell populations

Abstract

Molecular chaperone-based vaccines offer a number of advantages for cancer treatment. We have discussed the deployment of a vaccine prepared by gentle isolation of Hsp70 from tumour dendritic cell fusions (Hsp70 fusion vaccine). The vaccine was highly effective in triggering specific T cell immunity and in the treatment of tumour-bearing mice and the preparation was shown to retain an increased amount of tumour antigens compared to other chaperone-based isolates. This approach has the further advantage that tumour sub-populations could be used to prepare the Hsp70 fusion vaccine. Cellular fusion vaccines were made to specifically target drug-resistant cancer cells and tumour cell populations enriched in ovarian cancer stem cells (CSC). Such vaccines showed enhanced capacity to trigger T cell immunity to these resistant ovarian carcinoma populations. We have discussed the potential of using the cellular and Hsp70 fusion vaccine approaches in therapy of treatment-resistant cancer cells and its deployment in combination with ionising radiation or hyperthermia to enhance the effectiveness of both forms of therapy.     

Animal models for neonatal diseases in humans

The development of vaccines for infants and young children requires the use of animal models at various stages of preclinical development. Animal models are being used to assess the quantity and quality of the immune response, onset and duration of the response, induction of systemic versus local immunity, protection against challenge infection for the assessment of vaccine efficacy, as well as safety and toxicity of the vaccine formulation itself. A variety of animal models are available, each with its own specific advantages and disadvantages. Here, we review the most common animal models for preclinical vaccine development for human infants.     

PDCD2 functions in cancer cell proliferation and predicts relapsed leukemia

PDCD2 is an evolutionarily conserved eukaryotic protein with unknown function. The Drosophila PDCD2 ortholog Zfrp8 has an essential function in fly hematopoiesis. Zfrp8 mutants exhibit marked lymph gland hyperplasia that results from increased proliferation of partially differentiated hemocytes, suggesting Zfrp8 may participate in cell growth. Based on the above observations we have focused on the role of PDCD2 in human cancer cell proliferation and hypothesized that aberrant PDCD2 expression may be characteristic of human malignancies. We report that PDCD2 is highly expressed in human acute leukemia cells as well as in normal hematopoietic progenitors. PDCD2 knockdown in cancer cells impairs their proliferation, but not viability relative to parental cells, supporting the notion that PDCD2 overexpression facilitates cancer cell growth. Prospective analysis of PDCD2 in acute leukemia patients indicates PDCD2 RNA expression correlates with disease status and is a significant predictor of clinical relapse. PDCD2’s role in cell proliferation and its high expression in human malignancies make it an attractive, novel potential molecular target for new anti-cancer therapies.     

Cellular- and micro-dosimetry of heterogeneously distributed tritium

Abstract

Purpose: The assessment of radiotoxicity for heterogeneously distributed tritium should be based on the subcellular dose and relative biological effectiveness (RBE) for cell nucleus. In the present work, geometry-dependent absorbed dose and RBE were calculated using Monte Carlo codes for tritium in the cell, cell surface, cytoplasm, or cell nucleus.

Materials and methods: Penelope (PENetration and Energy LOss of Positrins and Electrons) code was used to calculate the geometry-dependent absorbed dose, lineal energy, and electron fluence spectrum. RBE for the intestinal crypt regeneration was calculated using a lineal energy-dependent biological weighting function. RBE for the induction of DNA double strand breaks was estimated using a nucleotide-level map for clustered DNA lesions of the Monte Carlo damage simulation (MCDS) code.

Results: For a typical cell of 10 μm radius and 5 μm nuclear radius, tritium in the cell nucleus resulted in much higher RBE-weighted absorbed dose than tritium distributed uniformly. Conversely, tritium distributed on the cell surface led to trivial RBE-weighted absorbed dose due to irradiation geometry and great attenuation of beta particles in the cytoplasm. For tritium uniformly distributed in the cell, the RBE-weighted absorbed dose was larger compared to tritium uniformly distributed in the tissue.

Conclusions: Cellular- and micro-dosimetry models were developed for the assessment of heterogeneously distributed tritium.     

Johns Hopkins course: 8th annual Current Topics in Gastrointestinal and Liver Pathology

The 8th annual Current Topics in Gastrointestinal and Liver Pathology course was held on the campus of the Johns Hopkins Medical Institutions in Baltimore, MD, USA, on the 8–9 November 2008. Lectures and hands-on microscopy sessions were provided by the Johns Hopkins Division of Gastrointestinal Pathology faculty and a guest faculty member from the University of Michigan (MI, USA). Key topics included newly recognized staging issues, the application of molecular techniques and practical topics for diagnostic pathologists.