Phosphoantigen/IL2 Expansion and Differentiation of Vγ2Vδ2 T Cells Increase Resistance to Tuberculosis in Nonhuman Primates

Dominant Vγ2Vδ2 T-cell subset exist only in primates, and recognize phosphoantigen from selected pathogens including M. tuberculosis(Mtb). In vivo function of Vγ2Vδ2 T cells in tuberculosis remains unknown. We conducted mechanistic studies to determine whether earlier expansion/differentiation of Vγ2Vδ2 T cells during Mtb infection could increase immune resistance to tuberculosis in macaques. Phosphoantigen/IL-2 administration specifically induced major expansion and pulmonary trafficking/accumulation of phosphoantigen-specific Vγ2Vδ2 T cells, significantly reduced Mtb burdens and attenuated tuberculosis lesions in lung tissues compared to saline/BSA or IL-2 controls. Expanded Vγ2Vδ2 T cells differentiated into multifunctional effector subpopulations capable of producing anti-TB cytokines IFNγ, perforin and granulysin, and co-producing perforin/granulysin in lung tissue. Mechanistically, perforin/granulysin-producing Vγ2Vδ2 T cells limited intracellular Mtb growth, and macaque granulysin had Mtb-bactericidal effect, and inhibited intracellular Mtb in presence of perforin. Furthermore, phosphoantigen/IL2-expanded Vγ2Vδ2 T effector cells produced IL-12, and their expansion/differentiation led to enhanced pulmonary responses of peptide-specific CD4+/CD8+ Th1-like cells. These results provide first in vivo evidence implicating that early expansion/differentiation of Vγ2Vδ2 T effector cells during Mtb infection increases resistance to tuberculosis. Thus, data support a rationale for conducting further studies of the γδ T-cell-targeted treatment of established TB, which might ultimately help explore single or adjunctive phosphoantigen expansion of Vγ2Vδ2 T-cell subset as intervention of MDR-tuberculosis or HIV-related tuberculosis

A flow cytometer based protocol for quantitative analysis of bloom-forming cyanobacteria (Microcystis) in lake sediments

A quantitative protocol for the rapid analysis of Microcystis cells and colonies in lake sediment was developed using a modified flow cytometer, the CytoSense. For cell enumeration, diluted sediment samples containing Microcystis were processed with sonication to disintegrate colonies into single cells. An optimized procedure suggested that 5 mg dw (dry weight)/mL dilution combined with 200 W x 2 min sonication yielded the highest counting efficiency. Under the optimized determination conditions, the quantification limit of this protocol was 3.3x10(4) cells/g dw. For colony analysis, Microcystis were isolated from the sediment by filtration. Colony lengths measured by flow cytometry were similar to those measured by microscopy for the size range of one single cell to almost 400 mu m in length. Moreover, the relationship between colony size and cell number was determined for three Microcystis species, including Microcystis flos-aquae, M. aeruginosa and M. wessenbergii. Regression formulas were used to calculate the cell numbers in different-sized colonies. The developed protocol was applied to field sediment samples from Lake Taihu. The results indicated the potential and applicability of flow cytometry as a tool for the rapid analysis of benthic Microcystis. This study provided a new capability for the high frequency monitoring of benthic overwintering and population dynamics of this bloom-forming cyanobacterium.A quantitative protocol for the rapid analysis of Microcystis cells and colonies in lake sediment was developed using a modified flow cytometer, the CytoSense. For cell enumeration, diluted sediment samples containing Microcystis were processed with sonication to disintegrate colonies into single cells. An optimized procedure suggested that 5 mg dw (dry weight)/mL dilution combined with 200 W x 2 min sonication yielded the highest counting efficiency. Under the optimized determination conditions, the quantification limit of this protocol was 3.3x10(4) cells/g dw. For colony analysis, Microcystis were isolated from the sediment by filtration. Colony lengths measured by flow cytometry were similar to those measured by microscopy for the size range of one single cell to almost 400 mu m in length. Moreover, the relationship between colony size and cell number was determined for three Microcystis species, including Microcystis flos-aquae, M. aeruginosa and M. wessenbergii. Regression formulas were used to calculate the cell numbers in different-sized colonies. The developed protocol was applied to field sediment samples from Lake Taihu. The results indicated the potential and applicability of flow cytometry as a tool for the rapid analysis of benthic Microcystis. This study provided a new capability for the high frequency monitoring of benthic overwintering and population dynamics of this bloom-forming cyanobacterium

A Novel RNF139 Mutation in Hemangioblastomas: Case Report

Hemangioblastomas (HBs) are classified as grade I tumors with uncertain origin according to the World Health Organization's classification system. HBs are characterized by rich mesenchymal cells and abundant capillaries. It has been shown that tumorigenesis of HBs depends on mutational inactivation of Von Hippel-Lindau (VHL) tumor suppressor gene. Therefore, the majority of patients will undergo VHL single gene test, and sequencing scheme is rarely used in clinic. In this study, we described a girl and her father successively found to have HBs within half a year. The results of next-generation sequencing (NGS) and Sanger sequencing analysis showed that both of them carried heterozygous mutation of RNF139 p.Q650R. This mutation was interpreted as Pathogenic variation based on the American College of Medical Genetics and Genomics (ACMG) guideline. Sanger sequencing was performed with other family members. No mutation on rs118184842 locus of RNF139 gene was found in the samples from the girl's mother, uncle and aunt. This report supports that the novel mutation of RNF139 p.Q650R probably serve as a key role in HBs progression

DR*W201/P65 Tetramer Visualization of Epitope-Specific CD4 T-Cell during M. tuberculosis Infection and Its Resting Memory Pool after BCG Vaccination

In vivo kinetics and frequencies of epitope-specific CD4 T cells in lymphoid compartments during M. tuberculosis infection and their resting memory pool after BCG vaccination remain unknown.Macaque DR*W201 tetramer loaded with Ag85B peptide 65 was developed to directly measure epitope-specific CD4 T cells in blood and tissues form macaques after M. tuberculosis infection or BCG vaccination via direct staining and tetramer-enriched approach. The tetramer-based enrichment approach showed that P65 epitope-specific CD4 T cells emerged at mean frequencies of approximately 500 and approximately 4500 per 10(7) PBL at days 28 and 42, respectively, and at day 63 increased further to approximately 22,000/10(7) PBL after M. tuberculosis infection. Direct tetramer staining showed that the tetramer-bound P65-specific T cells constituted about 0.2-0.3% of CD4 T cells in PBL, lymph nodes, spleens, and lungs at day 63 post-infection. 10-fold expansion of these tetramer-bound epitope-specific CD4 T cells was seen after the P65 peptide stimulation of PBL and tissue lymphocytes. The tetramer-based enrichment approach detected BCG-elicited resting memory P65-specific CD4 T cells at a mean frequency of 2,700 per 10(7) PBL.Our work represents the first elucidation of in vivo kinetics and frequencies for tetramer-bound epitope-specific CD4 T cells in the blood, lymphoid tissues and lungs over times after M. tuberculosis infection, and BCG immunization

Bovine Herpesvirus Type 1 (BHV-1) UL49.5 Luminal Domain Residues 30 to 32 Are Critical for MHC-I Down-Regulation in Virus-Infected Cells

Bovine herpesvirus type 1 (BHV-1) UL49.5 inhibits transporter associated with antigen processing (TAP) and down-regulates cell-surface expression of major histocompatibility complex (MHC) class I molecules to promote immune evasion. We have constructed a BHV-1 UL49.5 cytoplasmic tail (CT) null and several UL49.5 luminal domain mutants in the backbone of wild-type BHV-1 or BHV-1 UL49.5 CT- null viruses and determined their relative TAP mediated peptide transport inhibition and MHC-1 down-regulation properties compared with BHV-1 wt. Based on our results, the UL49.5 luminal domain residues 30–32 and UL49.5 CT residues, together, promote efficient TAP inhibition and MHC-I down-regulation functions. In vitro, BHV-1 UL49.5 Δ30–32 CT-null virus growth property was similar to that of BHV-1 wt and like the wt UL49.5, the mutant UL49.5 was incorporated in the virion envelope and it formed a complex with gM in the infected cells

Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK

葡萄糖是生物中最基本、最主要的营养物质，它不仅是机体能量的主要来源，也是生物质合成的主要原料。因此，葡萄糖的水平对于生物体是极其重要的。然而，在生活中，体内葡萄糖水平的波动是十分常见的，这是因为我们不可能每时每刻都在摄入葡萄糖：睡一大觉、剧烈运动几个小时或者太忙了没时间吃饭，都会引起葡萄糖水平的显著下降。这时，机体能够触发一套有效的过程应对这类“不利情况”，其中最为关键的就是激活“代谢的核心调节”——AMPK。在葡萄糖水平下降时，被激活的AMPK能够迅速启动脂肪、蛋白质的分解代谢，关闭它们的合成代谢，从而起到维持机体的能量和物质代谢的平衡，弥补机体因葡萄糖不足引起的胁迫压力。那么，机体如何感受葡萄糖水平下降，并“传递”给AMPK使其激活呢？林圣彩教授课题组的这项研究正是发现了生理状态下机体感受葡萄糖水平的机制。通过研究他们发现，无论在不含葡萄糖的细胞培养条件下，还是在饥饿的低血糖的动物体内，都不能观测到AMP水平的上升，这充分说明了机体有一套尚不为人知的、独立于AMP的感应葡萄糖水平的机制。在进一步的研究中他们揭示了这一完整过程：葡萄糖水平下降将引起的葡萄糖代谢中间物——果糖1,6-二磷酸（fructose-1,6-bisphosphate）水平的下降，该过程进一步地被糖酵解通路上的代谢酶——醛缩酶（aldolase）感应，因为醛缩酶正是将含有6个碳原子的果糖1,6-二磷酸裂解成三碳糖的酶，一旦醛缩酶“吃不到”由葡萄糖衍生的果糖1,6-二磷酸，它便“翻脸”，传递给也正是林圣彩教授课题组先前发现的溶酶体途径进而激活AMPK。该过程完全不涉及AMP水平，即能量水平的变化，是一条全新的、完全建立在实际的生理情况上的通路。林圣彩教授进一步地把葡萄糖水平总结为一种“状态信号”，以区别于传统的“能量信号”。据悉，该葡萄糖感知通路的发现对开发用于治疗肥胖症，乃至延长寿命的药物具有深远的意义。【Abstract】The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metabolism. Glucose deprivation activates AMP-activated protein kinase (AMPK)1, but it is unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK2, 3, 4, 5. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex containing at least v-ATPase, ragulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation6, 7. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addition of FBP disrupts the association of axin and LKB1 with v-ATPase and ragulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.D.G.H. was supported by an Investigator Award from the Wellcome Trust (097726) and a Programme Grant from Cancer Research UK (C37030/A15101). S.-C.L. was supported by grants from the National Key Research and Development Project of China (2016YFA0502001) and the National Natural Science Foundation of China (#31430094, #31690101, #31571214, #31601152 and #J1310027)

The Lysosomal v-ATPase-Ragulator Complex Is a Common Activator for AMPK and mTORC1, Acting as a Switch between Catabolism and Anabolism

林圣彩教授课题组长期致力于细胞信号转导的研究。近年来，该课题组潜心研究，不断攻关，取得了一系列重大成果，如揭示细胞如何应对生长因子缺乏的内在机理，发现了细胞自噬“路线图”、还发现了细胞如何感应“饥饿”信号AMP的信号传导通路等。其中，“发现细胞自噬‘路线图’”成果曾登上《科学》杂志，并入选2012年度“中国科学十大进展”。AMPK and mTOR play principal roles in governing metabolic programs; however, mechanisms underlying the coordination of the two inversely regulated kinases remain unclear. In this study we found, most surprisingly, that the late endosomal/lysosomal protein complex v-ATPase-Ragulator, essential for activation of mTORC1, is also required for AMPK activation. We also uncovered that AMPK is a residential protein of late endosome/lysosome. Under glucose starvation, the v-ATPase-Ragulator complex is accessible to AXIN/LKB1 for AMPK activation. Concurrently, the guanine nucleotide exchange factor (GEF) activity of Ragulator toward RAG is inhibited by AXIN, causing dissociation from endosome and inactivation of mTORC1. We have thus revealed that the v-ATPase-Ragulator complex is also an initiating sensor for energy stress and meanwhile serves as an endosomal docking site for LKB1-mediated AMPK activation by forming the v-ATPase-Ragulator-AXIN/LKB1-AMPK complex, thereby providing a switch between catabolism and anabolism. Our current study also emphasizes a general role of late endosome/lysosome in controlling metabolic programs

Stability Analysis and Stability Enhancement Based on Virtual Harmonic Resistance for Meshed DC Distributed Power Systems with Constant Power Loads

This paper addresses the stability issue of the meshed DC distributed power systems (DPS) with constant power loads (CPLs) and proposes a stability enhancement method based on virtual harmonic resistance. In previous researches, the network dynamics of the meshed DC DPS are often ignored, which affects the derivation of the equivalent system impendence. In addition, few of them have considered the meshed DC DPS including multiple sources with voltage-controlled converters and CPLs. To tackle the aforementioned challenge, this paper mainly makes the following efforts. The component connection method (CCM) is employed and expanded to derive the stability criterion of the meshed DC DPS with CPLs. This stability criterion can be simplified to relate only with the network node admittance matrix, the output impendences of the sources, and the input admittances of the CPLs. A virtual harmonic resistance through the second-order generalized integrator (SOGI) is added in the source with the voltage-controlled converter to lower the peak of the source output impendence, which can enhance the stability of the meshed DC DPS. The effectiveness of the proposed stability criterion and stability enhancement method are verified by nonlinear dynamic simulations

Bovine herpesvirus 1 (BHV-1) envelope protein gE subcellular trafficking is contributed by two separate YXXL/Φ motifs within the cytoplasmic tail which together promote efficient virus cell-to-cell spread

Bovine herpesvirus envelope glycoprotein E (gE) and, in particular, the gE cytoplasmic tail (CT) is a virulence determinant in cattle. Also, the gE CT contributes to virus cell-to-cell spread and anterograde neuronal transport. In this study, our goal was to map the gE CT sub-domains that contribute to virus cell-to-cell spread property. A panel of gE-CT specific mutant viruses was constructed and characterized, in vitro, with respect to their plaque phenotypes, gE recycling and gE basolateral membrane targeting. The results revealed that disruption of the tyrosine-based motifs, YTSL and YTVV, individually produced smaller plaque phenotypes than the wild type. However, they were slightly larger than the gE CT-null virus plaques. The Y467A mutation affected the gE endocytosis, gE trans-Golgi network (TGN) recycling, and gE virion incorporation properties. However, the Y563A mutation affected only the gE basolateral cell-surface redistribution function. Notably, the simultaneous Y467A/Y563A mutations produced gE CT-null virus-like plaque phenotypes

An Improved DDV Algorithm for the Retrieval of Aerosol Optical Depth From NOAA/AVHRR Data

Aerosol Optical Depth (AOD) is one of the important parameters to characterize the physical properties of the atmospheric aerosol, which is used to describe the extinction characteristics of the aerosol, and also to calculate the aerosol content, to assess the degree of air pollution and to study aerosol climate effect. To study the historical change of aerosol in long-time series, the advanced very high resolution radiometer (AVHRR) data earliest used for aerosol research was used in this study. Due to the lack of shortwave infrared (SWIR) (center at 2.13 mu m) of the sensor, the relationship between the blue and red bands with SWIR cannot be provided, and the visible band used to calculate the normalized difference vegetation index (NDVI) contains the wavelength range of red and green, it is very difficult to calculate the accurate land surface reflectance (LSR). Therefore, based on the Dense Dark Vegetation algorithm (DDV), we propose to introduce mature MODIS vegetation index products (MYD13) to correct AVHRR NDVI, to support the estimation of AVHRR LSR, determine the relationship between corrected AVHRR NDVI and visible band LSR, and to carry out aerosol retrieval. The results show that about 63% of the data are within the error line, and there is a consistent distribution trend in the inter-comparison validation with MODIS aerosol products (MYD04)