INPP5E regulates CD3ζ enrichment at the immune synapse by phosphoinositide distribution control.

The immune synapse, a highly organized structure formed at the interface between T lymphocytes and antigen-presenting cells (APCs), is essential for T cell activation and the adaptive immune response. It has been shown that this interface shares similarities with the primary cilium, a sensory organelle in eukaryotic cells, although the roles of ciliary proteins on the immune synapse remain elusive. Here, we find that inositol polyphosphate-5-phosphatase E (INPP5E), a cilium-enriched protein responsible for regulating phosphoinositide localization, is enriched at the immune synapse in Jurkat T-cells during superantigen-mediated conjugation or antibody-mediated crosslinking of TCR complexes, and forms a complex with CD3ζ, ZAP-70, and Lck. Silencing INPP5E in Jurkat T-cells impairs the polarized distribution of CD3ζ at the immune synapse and correlates with a failure of PI(4,5)P2 clearance at the center of the synapse. Moreover, INPP5E silencing decreases proximal TCR signaling, including phosphorylation of CD3ζ and ZAP-70, and ultimately attenuates IL-2 secretion. Our results suggest that INPP5E is a new player in phosphoinositide manipulation at the synapse, controlling the TCR signaling cascade.

Rab34 is necessary for early stages of intracellular ciliogenesis.

Primary cilia are sensory organelles present on most vertebrate cells and are critical for development and health. Ciliary dysfunction is associated with a large class of human pathologies collectively known as ciliopathies. These include cystic kidneys, blindness, obesity, skeletal malformations, and other organ anomalies. Using a proximity biotinylation with Ift27 as bait, we identified the small guanosine triphosphatase (GTPase) Rab34 as a ciliary protein. Rab34 localizes to the centrosomes near the mother centriole, the axoneme of developed cilia, and highly dynamic tubule structures in the centrosomal region. Rab34 is required for cilia formation in fibroblasts, where we find that Rab34 loss blocks ciliogenesis at an early step of ciliary vesicle formation. In inner medullary collecting duct (IMCD3) epithelial cells, the requirement is more complex, with Rab34 needed in cells grown at low density but becoming less important as cell density increases. Ciliogenesis can proceed by an internal pathway where cilia form in the cytoplasm before being displayed on the ciliary surface or cilia can assemble by an external pathway where the centriole docks on the plasma membrane before ciliary assembly. Fibroblasts are thought to use the internal pathway, although IMCD3 cells are thought to use the external pathway. However, we find that IMCD3 cells can use the internal assembly pathway and significant numbers of internally assembling cilia are observed in low-density cells. Together, our work indicates that Rab34 is required for internal assembly of cilia, but not for cilia built on the cell surface.

The Possibilities of Using Ultrasonically Activated Streams to Reduce the Risk of Foodborne Infection from Salad.

In this study, we investigated the effects of an ultrasonically activated stream (UAS) on the removal of microbial contaminants from spinach leaves. The microbial loads on samples cleaned with and without UAS were enumerated using the cell culture method and compared against unwashed samples on day 0 and day 6 after cleaning. The effects of UAS cleaning on leaf quality were also examined through both macroscopic and microscopic inspection, as well as measurement of the electrolyte leakage rate. Results showed that the microbial load on samples cleaned with UAS for 2 min was significantly lower on day 6 after cleaning than on those treated without ultrasound. Comparison between the cleaning effects of UAS for 40 s versus 2 min indicated that a cleaning duration of 2 min allowed sufficient time for UAS to disaggregate and detach the microbial contamination more effectively. In this case, the induction of bacteria into a viable but non-culturable state does not affect the shelf-life test results as much as it does with a 40 s clean. UAS cleaning for 2 min did not produce significant surface damage, which can affect overall leaf quality. These findings highlight the potential of UAS systems in the salad industry to improve the microbiological quality and shelf life of salads.

Improving livestock feed safety and infection prevention: Removal of bacterial contaminants from hay using cold water, bubbles and ultrasound.

The ingestion of contaminated hay is detrimental to livestock wellbeing. In this study, the feasibility of using an ultrasonically activated stream (UAS) to clean bacterial contamination from hay was investigated. Hay samples were stained with SYTO-9 nucleic acid stain for the in-situ visualization of microbes on the surface using an episcopic differential interference contrast microscope coupled with epi-fluorescence. The total microbial load per sample was calculated by measuring the mean percentage area of SYTO-9 positive staining. The cleaning efficacy was evaluated by comparing the total microbial coverage before and after cleaning. The cleaning performance between an UAS and a non UAS were compared and results have shown that an exposure of 60 s to an UAS demonstrated an 87.94 ± 2.22% removal of the bacterial contaminants, exceeding that of non UAS (21.85 ± 13.63% removal). UAS is capable of removing bacterial contaminants without the use of antimicrobial agents, therefore its cleaning mechanism can potentially prevent infection and reduce antimicrobial resistance. The cleaning mechanism of UAS can be adapted for the development of a new hay cleaning strategy for effective removal of bacterial contaminant to improve feed safety.

Super-resolution microscopy reveals coupling between mammalian centriole subdistal appendages and distal appendages.

Subdistal appendages (sDAPs) are centriolar elements that are observed proximal to the distal appendages (DAPs) in vertebrates. Despite the obvious presence of sDAPs, structural and functional understanding of them remains elusive. Here, by combining super-resolved localization analysis and CRISPR-Cas9 genetic perturbation, we find that although DAPs and sDAPs are primarily responsible for distinct functions in ciliogenesis and microtubule anchoring, respectively, the presence of one element actually affects the positioning of the other. Specifically, we find dual layers of both ODF2 and CEP89, where their localizations are differentially regulated by DAP and sDAP integrity. DAP depletion relaxes longitudinal occupancy of sDAP protein ninein to cover the DAP region, implying a role of DAPs in sDAP positioning. Removing sDAPs alter the distal border of centrosomal γ-tubulins, illustrating a new role of sDAPs. Together, our results provide an architectural framework for sDAPs that sheds light on functional understanding, surprisingly revealing coupling between DAPs and sDAPs.

Differential requirements for the EF-hand domains of human centrin 2 in primary ciliogenesis and nucleotide excision repair.

Centrin 2 is a small conserved calcium-binding protein that localizes to the centriolar distal lumen in human cells. It is required for efficient primary ciliogenesis and nucleotide excision repair (NER). Centrin 2 forms part of the xeroderma pigmentosum group C protein complex. To explore how centrin 2 contributes to these distinct processes, we mutated the four calcium-binding EF-hand domains of human centrin 2. Centrin 2 in which all four EF-hands had been mutated to ablate calcium binding (4DA mutant) was capable of supporting in vitro NER and was as effective as the wild-type protein in rescuing the UV sensitivity of centrin 2-null cells. However, we found that mutation of any of the EF-hand domains impaired primary ciliogenesis in human TERT-RPE1 cells to the same extent as deletion of centrin 2. Phenotypic analysis of the 4DA mutant revealed defects in centrosome localization, centriole satellite assembly, ciliary assembly and function and in interactions with POC5 and SFI1. These observations indicate that centrin 2 requires calcium-binding capacity for its primary ciliogenesis functions, but not for NER, and suggest that these functions require centrin 2 to be capable of forming complexes with partner proteins.This article has an associated First Person interview with the first author of the paper.

Differential Proteomics Reveals Discrete Functions of Proteins Interacting with Hypo- versus Hyper-phosphorylated NS5A of the Hepatitis C Virus.

Protein phosphorylation is a reversible post-translational modification that regulates many biological processes in almost all living forms. In the case of the hepatitis C virus (HCV), the nonstructural protein 5A (NS5A) is believed to transit between hypo- and hyper-phosphorylated forms that interact with host proteins to execute different functions; however, little was known about the proteins that bind either form of NS5A. Here, we generated two high-quality antibodies specific to serine 235 nonphosphorylated hypo- vs serine 235 phosphorylated (pS235) hyper-phosphorylated form of NS5A and for the first time segregated these two forms of NS5A plus their interacting proteins for dimethyl-labeling based proteomics. We identified 629 proteins, of which 238 were quantified in three replicates. Bioinformatics showed 46 proteins that preferentially bind hypo-phosphorylated NS5A are involved in antiviral response and another 46 proteins that bind pS235 hyper-phosphorylated NS5A are involved in liver cancer progression. We further identified a DNA-dependent kinase (DNA-PK) that binds hypo-phosphorylated NS5A. Inhibition of DNA-PK with an inhibitor or via gene-specific knockdown significantly reduced S232 phosphorylation and NS5A hyper-phosphorylation. Because S232 phosphorylation initiates sequential S232/S235/S238 phosphorylation leading to NS5A hyper-phosphorylation, we identified a new protein kinase that regulates a delicate balance of NS5A between hypo- and hyper-phosphorylation states, respectively, involved in host antiviral responses and liver cancer progression.

Single-particle tracking localization microscopy reveals nonaxonemal dynamics of intraflagellar transport proteins at the base of mammalian primary cilia.

Primary cilia play a vital role in cellular sensing and signaling. An essential component of ciliogenesis is intraflagellar transport (IFT), which is involved in IFT protein recruitment, axonemal engagement of IFT protein complexes, and so on. The mechanistic understanding of these processes at the ciliary base was largely missing, because it is challenging to observe the motion of IFT proteins in this crowded region using conventional microscopy. Here, we report short-trajectory tracking of IFT proteins at the base of mammalian primary cilia by optimizing single-particle tracking photoactivated localization microscopy for IFT88-mEOS4b in live human retinal pigment epithelial cells. Intriguingly, we found that mobile IFT proteins "switched gears" multiple times from the distal appendages (DAPs) to the ciliary compartment (CC), moving slowly in the DAPs, relatively fast in the proximal transition zone (TZ), slowly again in the distal TZ, and then much faster in the CC. They could travel through the space between the DAPs and the axoneme without following DAP structures. We further revealed that BBS2 and IFT88 were highly populated at the distal TZ, a potential assembly site. Together, our live-cell single-particle tracking revealed region-dependent slowdown of IFT proteins at the ciliary base, shedding light on staged control of ciliary homeostasis.

Two separate functions of NME3 critical for cell survival underlie a neurodegenerative disorder.

We report a patient who presented with congenital hypotonia, hypoventilation, and cerebellar histopathological alterations. Exome analysis revealed a homozygous mutation in the initiation codon of the NME3 gene, which encodes an NDP kinase. The initiation-codon mutation leads to deficiency in NME3 protein expression. NME3 is a mitochondrial outer-membrane protein capable of interacting with MFN1/2, and its depletion causes dysfunction in mitochondrial dynamics. Consistently, the patient's fibroblasts were characterized by a slow rate of mitochondrial dynamics, which was reversed by expression of wild-type or catalytic-dead NME3. Moreover, glucose starvation caused mitochondrial fragmentation and cell death in the patient's cells. The expression of wild-type and catalytic-dead but not oligomerization-attenuated NME3 restored mitochondrial elongation. However, only wild-type NME3 sustained ATP production and viability. Thus, the separate functions of NME3 in mitochondrial fusion and NDP kinase cooperate in metabolic adaptation for cell survival in response to glucose starvation. Given the critical role of mitochondrial dynamics and energy requirements in neuronal development, the homozygous mutation in NME3 is linked to a fatal mitochondrial neurodegenerative disorder.

Super-resolution architecture of mammalian centriole distal appendages reveals distinct blade and matrix functional components.

Distal appendages (DAPs) are nanoscale, pinwheel-like structures protruding from the distal end of the centriole that mediate membrane docking during ciliogenesis, marking the cilia base around the ciliary gate. Here we determine a super-resolved multiplex of 16 centriole-distal-end components. Surprisingly, rather than pinwheels, intact DAPs exhibit a cone-shaped architecture with components filling the space between each pinwheel blade, a new structural element we term the distal appendage matrix (DAM). Specifically, CEP83, CEP89, SCLT1, and CEP164 form the backbone of pinwheel blades, with CEP83 confined at the root and CEP164 extending to the tip near the membrane-docking site. By contrast, FBF1 marks the distal end of the DAM near the ciliary membrane. Strikingly, unlike CEP164, which is essential for ciliogenesis, FBF1 is required for ciliary gating of transmembrane proteins, revealing DAPs as an essential component of the ciliary gate. Our findings redefine both the structure and function of DAPs.

STED and STORM Superresolution Imaging of Primary Cilia.

The characteristic lengths of molecular arrangement in primary cilia are below the diffraction limit of light, challenging structural and functional studies of ciliary proteins. Superresolution microscopy can reach up to a 20 nm resolution, significantly improving the ability to map molecules in primary cilia. Here we describe detailed experimental procedure of STED microscopy imaging and dSTORM imaging, two of the most powerful superresolution imaging techniques. Specifically, we emphasize the use of these two methods on imaging proteins in primary cilia.

Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication.

The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein.

The reciprocal interaction between obesity and obstructive sleep apnoea.

Obesity is a significant risk factor in the pathogenesis of obstructive sleep apnoea (OSA) altering airway anatomy and collapsibility, and respiratory control. The association between obesity and OSA has led to an increasing focus on the role of weight loss as a potential treatment for OSA. To date, most discussion of obesity and OSA assumes a one-way cause and effect relationship, with obesity contributing to the pathogenesis of OSA. However, OSA itself may contribute to the development of obesity. OSA has a potential role in the development and reinforcement of obesity via changes to energy expenditure during sleep and wake periods, dietary habits, the neurohormonal mechanisms that control satiety and hunger, and sleep duration arising from fragmented sleep. Thus, there is emerging evidence that OSA itself feeds back into a complex mechanism that leads either to the development or reinforcement of the obese state. Whilst current evidence does not confirm that treatment of OSA directly influences weight loss, it does suggest that the potential role OSA plays in obesity and weight loss deserves further research.

Use sagittal reconstruction CT for making decisions regarding the surgical strategy for cervical ossification of the posterior longitudinal ligament / 中华外科杂志

<p><b>OBJECTIVE</b>Use sagittal reconstruction CT to verify the surgical strategy for cervical ossification of the posterior longitudinal ligament (OPLL).</p><p><b>METHODS</b>A retrospective study of 161 patients (106 males and 55 females) who had undergone surgery for OPLL from July 2007 to November 2010 was performed. The mean age at surgery was 54.5 years (range from 26 to 77 years). The mean follow-up period was 28 months (12 - 54 months). There were 40 patients accept anterior approach surgeries (anterior group) which include 14 cases of anterior cervical corpectomy and fusion and 26 cases of anterior cervical discectomy and fusion. There were 120 patients accept posterior approach surgeries (posterior group) which was spinous process-splitting laminoplasty for cervical myelopathy using coralline hydroxyapatite. One patient accepted combined anterior and posterior approach. According to the sagittal reconstruction CT, the main reason for spinal cord compression was cervical disc herniation in anterior group, and OPLL in posterior group. The level of spinal cord compression was 1 to 2 levels in anterior group, and 1 to 5 levels in posterior group with a major of 2 to 4 levels. As the classification of OPLL, segmental type and circumscribed type were major of segmental type in anterior group and all of the four types were in posterior group, the distribution of each type was average. The patients of posterior group were classified into two groups according to the modified K-line classification, and clinical results were compared between the two groups. The modified K-line was defined as a line that connects the midpoints of the spinal canal at C(2) and C(7) on sagittal CT myelography. Compression to the spinal cord did not exceed the K-line in the modified K-line(+) group and did exceed it in the modified K-line(-) group. Clinical data were compared using t-test or χ(2) test. Correlation analysis was used to determine the relationships of C(2)-C(7) angulation between sagittal reconstruction CT and neutral position X-ray.</p><p><b>RESULTS</b>The patient of anterior group had better recovery rate of the JOA score (72% ± 27%) than the posterior group (59% ± 35%) at the latest follow-up (t = 2.238, P = 0.027). In posterior group, the patients of modified K-line(+) group had better recovery rate of the JOA score (63% ± 37%) than the K-line(-) group (49% ± 30%) at the latest follow up (t = 2.150, P = 0.034). The C(2)-C(7) angulation on sagittal reconstruction CT was 11° ± 9° which has significantly correlated with the C(2)-C(7) angulation on neutral position X-ray which was 10° ± 10° (r = 0.947, P < 0.01).</p><p><b>CONCLUSIONS</b>Considering the selection of surgical approach, it should be combined with the main clinical diagnosis for spinal cord compression, the level of compression, the classification of OPLL and the kyphotic alignment of the cervical spine. The modified K-line is a simple and practical tool for making decisions regarding the surgical strategy for cervical OPLL patients.</p>

Endovascular treatment of gastroduodenal artery aneurysm.

Gastroduodenal artery aneurysms are rare. Common causes include blunt trauma, pancreatitis, infection, autoimmune disorders, vascular intervention and surgery. We report 2 patients with gastroduodenal artery aneurysms, the first being an idiopathic true aneurysm and the next, a pseudoaneurysm resulting from pancreatitis. Diagnoses were made by computed tomography scans with successful embolization of both patients. Treatment of gastroduodenal artery aneurysms includes surgery, endovascular techniques or observation. Embolization is a feasible option for gastroduodenal artery aneurysms and pseudoaneurysms.

[The role of local immune response in gastric mucosa in the protection induced by Helicobacter pylori vaccine with chitosan as adjuvant].

OBJECTIVE: To study the immunological protection of Helicobacter pylori (H. pylori) vaccine with chitosan as adjuvant and it's mechanism. METHODS: One-grade female BALB/c mice were randomly divided into nine groups and immunized by (1) PBS alone, (2) chitosan solution alone, (3) chitosan particles alone, (4) H. pylori antigen alone, (5) H. pylori antigen plus chitosan solution, (6) H. pylori antigen plus chitosan particles, (7) H. pylori antigen plus CT, (8) H. pylori antigen plus chitosan solution and cholera toxin (CT), (9) H. pylori antigen plus chitosan particles and CT orally respectively once a week for four weeks. At 4 weeks after the last immunization, these mice were challenged by alive H. pylori (1 x 10(9)/ml) twice at two days intervals. At 4 weeks after the last challenge, these mice were all killed and Gastric mucosa were embedded in paraffin, sectioned and assayed with Giemsa stain. The other gastric mucosa were used to quantitatively culture H. pylori. An ELISA was used to detect anti-H. pylori IgA in saliva and gastric mucosa and a quantitative ELISA was used to detect IL-2, IL-4, IL-10 content in gastric mucosa, and SP immunohistochemical method was used to detect secretory immunoglobulin A (sIgA) in gastric mucosa. RESULTS: (1) In the groups with chitosan as adjuvant, 60% mice could achieve immunological protection, which was according to that with CT as adjuvant (58.33%), and was significantly higher than H. pylori antigen alone and other groups without H. pylori antigen (P < 0.01 or P < 0.05). While the rates of protection in the groups with chitosan plus CT as adjuvant were 84.62%, 85.71% and the H. pylori colonization score in it was significantly lower than that in the groups with CT as adjuvant and without adjuvants (P < 0.01 or P < 0.05). (2) the labeling index for sIgA-positive lumen of glands and special anti-H. pylori IgA levels in gastric mucosa in the groups with chitosan as an adjuvant had no difference with those in the group with CT as an adjuvant (P > 0.05) and were significantly higher than those in non-adjuvant groups, while those in the groups with chitosan plus CT were significantly higher than those in the group with CT as an adjuvant (P < 0.01 or P < 0.05). (3) Before challenge, the content of IL-2 in gastric mucosa were no different among different groups (P > 0.05). After challenge the content of IL-2 were significantly higher in the groups with adjuvant than those in the control group (P < 0.01 or P < 0.05), Moreover, those in the groups with antigen after challenge were significantly higher than those before challenge (P < 0.05). (4) Before challenge, the content of IL-10 in gastric mucosa were significantly higher in the groups with chitosan as adjuvant than those in the control group and the group without adjuvant (P < 0.01 or P < 0.05). After challenge, the content of IL-10 were no different among different groups (P > 0.05). Moreover, those in the groups with adjuvant after challenge were significantly lower than those before challenge (P < 0.01). (5) Before challenge, the content of IL-4 in gastric mucosa were significantly higher in the groups with chitosan as adjuvant than those in the control group and the group without adjuvant (P < 0.05), After challenge, the content of IL-4 were significantly higher in the groups with chitosan particles as an adjuvant than those in the group with CT as an adjuvant (P < 0.05), and those in the group with chitosan solution as an adjuvant were significantly higher than those in control group, non-adjuvant group and the groups with CT (P < 0.01 or P < 0.05), Moreover, those in the groups with adjuvant after challenge were significantly lower than those before challenge (P < 0.01 or P < 0.05). CONCLUSIONS: (1) H. pylori vaccine with chitosan as adjuvant could protect against H. pylori infection, this suggested that chitosan could be a mucosa adjuvant of H. pylori vaccine, and it could effectively elicit special humoral immune response of systemic and local mucosa, which might be one of its protective mechanism. (2) H. pylori vaccine with chitosan as adjuvant may induce both Th1 and Th2 type immune response, and after challenge it could reverse the inhibition of Th2 induced by H. pylori infection and recover the Th1/Th2 imbalance. which might contribute to the immune protection against H. pylori.