Single Membrane Structure Research Articles

Noncanonical autophagy is a new strategy to inhibit HSV-1 through STING1 activation

ABSTRACT STING1 (stimulator of interferon response cGAMP interactor 1) plays an essential role in immune responses for virus inhibition via inducing the production of type I interferon, inflammatory factors and macroautophagy/autophagy. In this study, we found that STING1 activation could induce not only canonical autophagy but also non-canonical autophagy (NCA) which is independent of the ULK1 or BECN1 complexes to form MAP1LC3/LC3-positive structures. Whether STING1-induced NCA has similar characters and physiological functions to canonical autophagy is totally unknown. Different from canonical autophagy, NCA could increase single-membrane structures and failed to degrade long-lived proteins, and could be strongly suppressed by interrupting vacuolar-type H+-translocating ATPase (V-ATPase) activity. Importantly, STING1-induced NCA could effectively inhibit DNA virus HSV-1 in cell model. Moreover, STING1 [1–340], a STING1 mutant lacking immunity and inflammatory response due to deletion of the tail end of STING1, could degrade virus through NCA alone, suggesting that the antiviral effect of activated STING1 could be separately mediated by inherent immunity, canonical autophagy, and NCA. In addition, the translocation and dimerization of STING1 do not rely on its immunity function and autophagy pathway. Similar to canonical autophagy, LC3-positive structures of NCA induced by STING1 could finally fuse with lysosomes, and the degradation of HSV-1 could be reverted by inhibition of lysosome function, suggesting that the elimination of DNA virus via NCA still requires the lysosome pathway. Collectively, we proved that besides its classical immunity function and canonical autophagy pathway, STING1-induced NCA is also an efficient antiviral pathway for the host cell. Abbreviations: ATG: autophagy related; Baf: bafilomycin A1; CASM: conjugation of LC3 to a single membrane; CGAS: cyclic GMP-AMP synthase; cGAMP: cyclic GMP-AMP; CQ: chloroquine; CTD: C-terminal domain; CTT: C-terminal tail; ER: endoplasmic reticulum; ERGIC: ER-Golgi intermediate compartment; HSV-1: herpes simplex virus 1; IRF3: interferon regulatory factor 3; IFNs: interferons; LAMP1: lysosomal associated membrane protein 1; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; RB1CC1/FIP200: RB1 inducible coiled-coil 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; TGOLN2/TGN46: trans-golgi network protein 2; ULK1: unc-51 like autophagy activating kinase 1; V-ATPase: vacuolar-type H+-translocating ATPase; VSV: vesicular stomatitis virus

Analytical model of vibro-acoustic coupling between the membrane loaded with concentrated masses and the acoustic cavity

In this paper, sound radiation and sound transmission characteristics of a membrane-cavity system loaded with concentrated masses are analyzed. The Fourier cosine series function with additional terms is used as the mode function of membrane to model the single membrane structure, which is suitable for arbitrary boundary conditions. The concentrated masses are regarded as the concentrated mass points without considering their sizes, and the coupling structure of membrane and mass blocks is constructed. The structure-sound field fully coupled model is also constructed by using the energy principle and Rayleigh–Ritz method. Several numerical examples computed by finite element method are used for verification. The influence of the added mass blocks on the membrane modes and acoustic properties of the coupling system is discussed, including membrane parameters, mass block arrangement, quantity and mass, etc. Also, the mass blocks are dispersed for analysis. Results show that dispersing the mass can significantly increase the frequency of the coupling mode between the membrane and concentrated masses and better suppress the vibration. But increasing dispersion points does not further enhance vibration suppression. Instead, it expands the area where the membrane vibrates violently.

Hypoxia Induced Changes of Exosome Cargo and Subsequent Biological Effects.

Exosomes are small extracellular vesicles that are secreted by almost all types of cells and exist in almost all extracellular spaces. As an important mediator of intercellular communication, exosomes encapsulate the miRNA, lncRNA, cirRNA, mRNA, cytokine, enzyme, lipid, and other components from the cytoplasm into its closed single membrane structure and transfer them to recipient units in an autocrine, paracrine, or endocrine manner. Hypoxia is a state of low oxygen tension and is involved in many pathological processes. Hypoxia influences the size, quantity, and expression of exosome cargos. Exosomes derived from hypoxic tumor cells transfer genetics, proteins, and lipids to the recipient units to exert pleiotropic effects. Different donor cells produce different cargo contents, target different recipient units and lead to different biological effects. Hypoxic exosomes derived from tumor cells uptaken by normoxic tumor cells lead to promoted proliferation, migration, and invasion; uptaken by extracellular space or liver lead to promoted metastasis; uptaken by endothelial cells lead to promoted angiogenesis; uptaken by immune cells lead to promoted macrophage polarization and changed tumor immune microenvironment. In addition to various types of tumors, hypoxic exosomes also participate in the development of diseases in the cardiovascular system, neuron system, respiratory system, hematology system, endocrine system, urinary system, reproduction system, and skeletomuscular system. Understanding the special characteristics of hypoxic exosomes provide new insight into elaborating the pathogenesis of hypoxia related disease. This review summarizes hypoxia induced cargo changes and the biological effects of hypoxic exosomes in tumors and non-malignant diseases in different systems.

Ultrastructure evidence for vesicles and double-membrane structures involved in cytoplasmic elimination during spermiogenesis in large yellow croaker, Larimichthys crocea (Teleostei, Perciformes, Scienidae)

Spermatids eliminate excess cytoplasm to form streamlined sperm during spermiogenesis, which mechanism is insufficiently elucidated in fish. In this study, we investigated the cytoplasmic elimination procedure in spermatid during spermiogenesis in the large yellow croaker (Larimichthys crocea) using transmission electron microscopy. The early spermatid is subrotund with a centrally located nucleus. With further development, nucleus polarizes into one side of the cell while the cytoplasm with numerous vesicles near the membrane migrates to the caudal region. Furthermore, exocytosis-like structures were detected in middle spermatid. In late spermatid, the vesicles are reduced and rarely observed. These findings indicate that vesicles may be involved in cytoplasmic elimination possibly via exocytosis. In the later spermatid, a double-membrane, autophagosome-like structure envelopes the cytoplasm, which may develop into a single-membrane structure, and gets discarded from the cell as a residual body from the caudal region. This suggests its potential functions in the formation of residual body and cytoplasmic elimination. Overall, our results revealed that polarized development of spermatid causes polarized distribution of cytoplasm necessary for cytoplasmic elimination. Moreover, they provide ultrastructure evidence for vesicles and double-membrane structures involved in discarding spermatid cytoplasm in large yellow croaker, thus offering novel insights into cytoplasmic elimination during spermiogenesis in fish.

Alternative careers at the autophagy factory.

During autophagy, proteins of the ATG8 family are conjugated to phosphatidylethanolamine (PE) in double-membrane structures called phagophores. Now, Durgan et al. have found that, during the non-canonical process of conjugating ATG8 proteins to single-membrane structures, ATG8 can be ligated to phosphatidylserine. Here, I discuss the potential consequences of their findings.

Distinct functions of ATG16L1 isoforms in membrane binding and LC3B lipidation in autophagy-related processes.

Covalent modification of LC3 and GABARAP proteins to phosphatidylethanolamine in the double-membrane phagophore is a key event in the early phase of macroautophagy, but can also occur on single-membrane structures. In both cases this involves transfer of LC3/GABARAP from ATG3 to phosphatidylethanolamine at the target membrane. Here we have purified the full-length human ATG12-5-ATG16L1 complex and show its essential role in LC3B/GABARAP lipidation in vitro. We have identified two functionally distinct membrane-binding regions in ATG16L1. An N-terminal membrane-binding amphipathic helix is required for LC3B lipidation under all conditions tested. By contrast, the C-terminal membrane-binding region is dispensable for canonical autophagy but essential for VPS34-independent LC3B lipidation at perturbed endosomes. We further show that the ATG16L1 C-terminus can compensate for WIPI2 depletion to sustain lipidation during starvation. This C-terminal membrane-binding region is present only in the β-isoform of ATG16L1, showing that ATG16L1 isoforms mechanistically distinguish between different LC3B lipidation mechanisms under different cellular conditions.

A nuclear membrane-derived structure associated with Atg8 is involved in the sequestration of selective cargo, the Cvt complex, during autophagosome formation in yeast

ABSTRACTMacroautophagy (hereafter autophagy) is a conserved intracellular degradation mechanism required for cell survival. A double-membrane structure, the phagophore, is generated to sequester cytosolic cargos destined for degradation in the vacuole. The mechanism involved in the biogenesis of the phagophore is still an open question. We focused on 4 autophagy-related (Atg) proteins (Atg2, Atg9, Atg14, and Atg18), which are involved in the formation of the phagophore in order to gain a more complete understanding of the membrane dynamics that occur during formation of the autophagosome. The corresponding mutants, while defective in autophagy, nonetheless generate the membrane-bound form of Atg8, allowing us to use this protein as a marker for the nascent autophagosome precursor membrane. Using electron microscopy (EM), we discovered in these atg mutants a novel single-membrane structure (~120 to 150 nm in size). Electron tomography revealed that this structure originates from a part of the nuclear membrane, and we have named it the alphasome. Our data suggest that the alphasome is associated with Atg8, and sequesters selective cargo, the Cvt complex, during autophagy.Abbreviations: 3D: three-dimensional; AB: autophagic body; AP: autophagosome; Atg: autophagy-related; Cvt: cytoplasm-to-vacuole targeting; EM: electron microscopy; IEM: immunoelectron microscopy; L: lipid droplet; N: nucleus; NM: nuclear membrane; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor aminopeptidase I; rER: rough endoplasmic reticulum; TEM: transmission electron microscopy; V: vacuole; VLP: virus-like particle;

Endoplasmic reticulum-targeted two-photon turn-on fluorescent probe for nitroreductase in tumor cells and tissues

Hypoxia conditions could increase the activity of intracellular nitroreductase (NTR) and lead to many malignant diseases. Therefore, monitoring the activity of NTR is of great significance to study the related diseases. Organelles play crucial roles in the metabolism of living cells. In these organelles, the endoplasmic reticulum (ER) possesses single membrane structure, and it is the largest organelle in the cell. ER performs the synthesis, processing and modification of proteins and lipid, stabilizing the intracellular Ca2+ concentration and other physiological functions in living cells. Therefore, it is of great significance to develop ER-target probes in living system. Toward this goal, a new endoplasmic reticulum-targeted two-photon fluorescence turn-on NTR probe Na-NTR-ER is designed and synthesized. Probe Na-NTR-ER has been proved to display high sensitivity (36 ng/mL) and selectivity to NTR. Particularly, probe Na-NTR-ER has been successfully applied for the monitoring of NTR in ER with a high the Pearson's colocalization coefficient as 0.90 in HeLa cells and cancerous mouse tissues up to the depth of 100 μm with significant fluorescence signals.

The Origin, Dynamic Morphology, and PI4P-Independent Formation of Encephalomyocarditis Virus Replication Organelles

ABSTRACTPicornaviruses induce dramatic rearrangements of endomembranes in the cells that they infect to produce dedicated platforms for viral replication. These structures, termed replication organelles (ROs), have been well characterized for the Enterovirus genus of the Picornaviridae. However, it is unknown whether the diverse RO morphologies associated with enterovirus infection are conserved among other picornaviruses. Here, we use serial electron tomography at different stages of infection to assess the three-dimensional architecture of ROs induced by encephalomyocarditis virus (EMCV), a member of the Cardiovirus genus of the family of picornaviruses that is distantly related. Ultrastructural analyses revealed connections between early single-membrane EMCV ROs and the endoplasmic reticulum (ER), establishing the ER as a likely donor organelle for their formation. These early single-membrane ROs appear to transform into double-membrane vesicles (DMVs) as infection progresses. Both single- and double-membrane structures were found to support viral RNA synthesis, and progeny viruses accumulated in close proximity, suggesting a spatial association between RNA synthesis and virus assembly. Further, we explored the role of phosphatidylinositol 4-phosphate (PI4P), a critical host factor for both enterovirus and cardiovirus replication that has been recently found to expedite enterovirus RO formation rather than being strictly required. By exploiting an EMCV escape mutant, we found that low-PI4P conditions could also be overcome for the formation of cardiovirus ROs. Collectively, our data show that despite differences in the membrane source, there are striking similarities in the biogenesis, morphology, and transformation of cardiovirus and enterovirus ROs, which may well extend to other picornaviruses.

A Hybrid Boundary Element Model for Simulation and Optimization of Large Piezoelectric Micromachined Ultrasonic Transducer Arrays.

A hybrid boundary element model is proposed for the simulation of large piezoelectric micromachined ultrasonic transducer (PMUT) arrays in immersion. Multiphysics finite element method (FEM) simulation of a single-membrane structure is used to determine stiffness and piezoelectrically induced actuation loading of the membranes. To simulate the arrays of membranes in immersion, a boundary element method is employed, wherein membrane structures are modeled by a surface mesh that is coupled mechanically by mass, stiffness, and damping matrices, and acoustically by a mutual impedance matrix. A multilevel fast multipole algorithm speeds up computation time and reduces memory usage, enabling the simulation of thousands of membranes in a reasonable time. The model is validated with FEM for a small 3 3 matrix array for both square and circular membrane geometries. Two practical optimization examples of large PMUT arrays are demonstrated: membrane spacing of a 7 7 matrix array with circular membranes, and material choice and top electrode coverage of a 32-element linear array with 640 circular membranes. In addition, a simple analytical approach to electrode optimization based on normal mode theory is verified.

Validation of a hybrid boundary and finite element method for the simulation of large membrane-based piezoelectric transducer arrays in immersion

Membrane-based piezoelectric ultrasonic transducers, especially piezoelectric micromachined ultrasonic transducers (PMUTs), are a promising technology for the realization of large transducer arrays for use in integrated imaging, sensing, and actuation where a broadband response is desirable. In this work, a hybrid boundary and finite element method is proposed for the transmit simulation of large PMUT arrays in immersion. Finite element software (COMSOL) readily handles the simulation of single membrane structures, from which static deformation (stiffness) and harmonic displacement data is extracted. A boundary element formulation based on these inputs handles the membrane-to-membrane acoustic cross-coupling through the calculation of a mutual impedance matrix. For arrays consisting of hundreds of membranes or more, the problem of quadratic storage and cubic time complexity for boundary element is avoided by employing a multi-level fast multipole algorithm (Shieh et al., IEEE Trans. UFFC, 63, 1967-1979). We validate this hybrid method for common membrane geometries, including square and circular membranes with varying degrees of electrode coverage.

Autophagy: A brief overview in perspective of dermatology

Autophagy, literally meaning "self-eating," is an intracellular catabolic process of delivering cytosol and/or its specific content to the lysosomes for degradation.The resulting macromolecular constituents are recycled and utilized again by the cells. Basal level autophagy plays an important role in cellular homeostasis through the elimination of the old or damaged organelles, as well as aggregated intracellular proteins. Autophagy refers to sequestration of intact organelles along with a portion of cytosol, into a double-or multi-membrane structure known as phagophore, which elongates, and after closure, forms a vesicular structure known as the autophagosome. Subsequently, the mature autophagosome fuses with a lysosome, thereby forming a single membrane structure, an autolysosome. Autophagy plays a critical role in inflammation, autoimmunity and cellular differentiation. Skin serves as the first line of defense against a variety of environmental insults and autophagy is thought to be a form of an endogenous defense mechanism against such environmental derangements. Autophagy has been linked with keratinocyte differentiation and melanocyte survival, as well as with the pathogenesis of diverse skin disorders including systemic lupus erythematosus, systemic sclerosis, psoriasis, vitiligo, infectious skin diseases and cancer. Autophagy has been one of the most studied phenomena in cell biology and pathophysiology, and given its broad clinical implications, has become a major target for drug discovery. The last decade has seen a substantial upsurge in autophagy-related research and publications; still, the dermatology literature appears to be less initiated. Autophagy will probably change our understanding of dermatological disorders/medicines. Hence, a basic knowledge of autophagy is a prerequisite to understand the developments in the field of autophagy-related research.

ZKSCAN3 Is a Master Transcriptional Repressor of Autophagy

Autophagy constitutes a major cell-protective mechanism that eliminates damaged components and maintains energy homeostasis via recycling nutrients under normal/stressed conditions. Although the core components of autophagy have been well studied, regulation of autophagy at the transcriptional level is poorly understood. Herein, we establish ZKSCAN3, a zinc finger family DNA-binding protein, as a transcriptional repressor of autophagy. Silencing of ZKSCAN3 induced autophagy and increased lysosome biogenesis. Importantly, we show that ZKSCAN3 represses transcription of a large gene set (>60) integral to, or regulatory for, autophagy and lysosome biogenesis/function and that a subset of these genes, including Map1lC3b and Wipi2, represent direct targets. Interestingly, ZKSCAN3 and TFEB are oppositely regulated by starvation and in turn oppositely regulate lysosomal biogenesis and autophagy, suggesting that they act in conjunction. Altogether, our study uncovers an autophagy master switch regulating the expression of a transcriptional network of genes integral to autophagy and lysosome biogenesis/function.

Complex Dynamic Development of Poliovirus Membranous Replication Complexes

Replication of all positive-strand RNA viruses is intimately associated with membranes. Here we utilize electron tomography and other methods to investigate the remodeling of membranes in poliovirus-infected cells. We found that the viral replication structures previously described as "vesicles" are in fact convoluted, branching chambers with complex and dynamic morphology. They are likely to originate from cis-Golgi membranes and are represented during the early stages of infection by single-walled connecting and branching tubular compartments. These early viral organelles gradually transform into double-membrane structures by extension of membranous walls and/or collapsing of the luminal cavity of the single-membrane structures. As the double-membrane regions develop, they enclose cytoplasmic material. At this stage, a continuous membranous structure may have double- and single-walled membrane morphology at adjacent cross-sections. In the late stages of the replication cycle, the structures are represented mostly by double-membrane vesicles. Viral replication proteins, double-stranded RNA species, and actively replicating RNA are associated with both double- and single-membrane structures. However, the exponential phase of viral RNA synthesis occurs when single-membrane formations are predominant in the cell. It has been shown previously that replication complexes of some other positive-strand RNA viruses form on membrane invaginations, which result from negative membrane curvature. Our data show that the remodeling of cellular membranes in poliovirus-infected cells produces structures with positive curvature of membranes. Thus, it is likely that there is a fundamental divergence in the requirements for the supporting cellular membrane-shaping machinery among different groups of positive-strand RNA viruses.

The Transformation of Enterovirus Replication Structures: a Three-Dimensional Study of Single- and Double-Membrane Compartments

ABSTRACTAll positive-strand RNA viruses induce membrane structures in their host cells which are thought to serve as suitable microenvironments for viral RNA synthesis. The structures induced by enteroviruses, which are members of the family Picornaviridae, have so far been described as either single- or double-membrane vesicles (DMVs). Aside from the number of delimiting membranes, their exact architecture has also remained elusive due to the limitations of conventional electron microscopy. In this study, we used electron tomography (ET) to solve the three-dimensional (3-D) ultrastructure of these compartments. At different time points postinfection, coxsackievirus B3-infected cells were high-pressure frozen and freeze-substituted for ET analysis. The tomograms showed that during the exponential phase of viral RNA synthesis, closed smooth single-membrane tubules constituted the predominant virus-induced membrane structure, with a minor proportion of DMVs that were either closed or connected to the cytosol in a vase-like configuration. As infection progressed, the DMV number steadily increased, while the tubular single-membrane structures gradually disappeared. Late in infection, complex multilamellar structures, previously unreported, became apparent in the cytoplasm. Serial tomography disclosed that their basic unit is a DMV, which is enwrapped by one or multiple cisternae. ET also revealed striking intermediate structures that strongly support the conversion of single-membrane tubules into double-membrane and multilamellar structures by a process of membrane apposition, enwrapping, and fusion. Collectively, our work unravels the sequential appearance of distinct enterovirus-induced replication structures, elucidates their detailed 3-D architecture, and provides the basis for a model for their transformation during the course of infection.

Specific Behavior of Intracellular Streptococcus pyogenes That Has Undergone Autophagic Degradation Is Associated with Bacterial Streptolysin O and Host Small G Proteins Rab5 and Rab7

Streptococcus pyogenes (group A streptococcus (GAS)) is a pathogen that invades non-phagocytic host cells, and causes a variety of acute infections such as pharyngitis. Our group previously reported that intracellular GAS is effectively degraded by the host-cell autophagic machinery, and that a cholesterol-dependent cytolysin, streptolysin O (SLO), is associated with bacterial escape from endosomes in epithelial cells. However, the details of both the intracellular behavior of GAS and the process leading to its autophagic degradation remain unknown. In this study, we found that two host small G proteins, Rab5 and Rab7, were associated with the pathway of autophagosome formation and the fate of intracellular GAS. Rab5 was involved in bacterial invasion and endosome fusion. Rab7 was clearly multifunctional, with roles in bacterial invasion, endosome maturation, and autophagosome formation. In addition, this study showed that the bacterial cytolysin SLO supported the escape of GAS into the cytoplasm from endosomes, and surprisingly, a SLO-deficient mutant of GAS was viable longer than the wild-type strain although it failed to escape the endosomes. This intracellular behavior of GAS is unique and distinct from that of other types of bacterial invaders. Our results provide a new picture of GAS infection and host-cell responses in epithelial cells.

Cytoplasmic vacuolization responses to cytopathic bovine viral diarrhoea virus

Bovine Viral Diarrhea Virus (BVDV) is a positive sense, single-stranded RNA virus which exhibits two biotypes in standard cell culture systems. The cytopathic strains of this virus (cpBVDV) induce dramatic cytoplasmic vacuolization in cell cultures, while infection with the non-cytopathic (NCP-BVDV) strains produces no overt changes in the host cells. Our results show that extensive cytoplasmic vacuolization is the earliest morphological change in response to cpBVDV infection in MDBK cells. Cells with extensive vacuolization showed no co-existing chromatin condensation, caspase activation, or loss of membrane integrity. In addition, the caspase inhibitor (zVAD-fmk), although improving cell viability of infected cells from 6.7 ± 2.2% to 18.8 ± 2.2%, did not prevent vacuolization. On the ultrastructural level, the virus-induced cytoplasmic vacuoles are single membrane structures containing organelles and cellular debris, which appear capable of fusing with other vacuoles and engulfing surrounding cytoplasmic materials. LysoTracker Red which marks lysosomes did not stain the virus-induced cytoplasmic vacuoles. In addition, this lysosomal dye could be observed in the cytoplasm of vacuolized cells, suggesting a lysosomal abnormality. Our data demonstrate that cpBVDV induced a novel cell death pathway in MDBK cells that is primarily associated with lysosomal dysfunction and the formation of phagocytic cytoplasmic vacuoles, and this mode of cell death is different from apoptosis and necrosis.

Autophagy in MHC Class II Presentation: Sampling from Within

MHC class II molecules usually present exogenous antigens, but peptidome analyses have also identified many antigens from cytosolic or nuclear sources. In this issue of Immunity, Schmid et al. show that MHC class II molecules can present these through autophagosomes.

THE EXPRESSION OF THE VACUOLE MEMBRANE PROTEIN 1 (VMP1) IS NECESSARY AND SUFFICIENT TO TRIGGER AUTOPHAGY IN PANCREATIC CELLS

Autophagy, the process by which a cell recycles cytoplasm and disposes excess or defective organelles, has been described as a non-apoptotic programmed cell death. By morphological studies, autophagy has been linked to disease process in acute pancreatitis. We have previously characterized a novel membrane protein, whose expression is induced in acinar cell early during acute pancreatitis, correlates with vacuole formation and precedes cell death. In order to study the role of VMP1, we proposed the hypothesis that the expression of VMP1 triggers autophagy in pancreatic acinar cells. Autophagic processes were assayed using AR4-2J pancreatic acinar cells. Polyclonal rabbit antibody was developed using a VMP1-specific peptide antigen. VMP1/V5 and VMP1/EGFP expression plasmids and VMP-RNAi were constructed. Electron microscopy studies showed that VMP1-induced vacuoles have ultraestructural features of autophagy. Autophagosomes, a double membrane structure containing cytoplasmic material, as well as autolysosomes, single membrane structure containing cytoplasmic components at various stages of degradation, were observed in VMP1 expressing cells. VMP1-induced vacuoles were stained with monodansylcadaverine, a fluorescent tracer for autophagy. LC3, a specific marker for autophagic membranes, co-localized with VMP1 transfected cells. These results strongly demonstrate that VMP1 expression induces autophagy. In other series of experiments, AR4-2J cells were treated with rapamycin, a drug that induces autophagy, and VMP1 expression was demonstrated by RT-PCR and Western blot analysis. Finally, RNAi silencing of VMP1 expression prevented autophagy induced by rapamycin demonstrating that VMP1 expression is required for autophagy. Our results demonstrate that VMP1 expression is necessary and sufficient to trigger autophagy. VMP1 expression is an early molecular event that may promote autophagy and could be involved in the regulation of acinar cell death during acute pancreatitis pathogenesis.

Fabry–Perot cavity chemical sensors by silicon micromachining techniques

Micromachined Fabry–Perot microcavity structures filled with polymeric layers composed of poly(3-dodecylthiophene) have been fabricated and studied for use as chemical sensors. The polymer-filled microcavity devices show reversible sensing behavior in response to the exposure of molecular iodine. Here, the chemical dosing results in a dramatic change in the fraction of transmitted light which passes through the microcavity structure (up to 50% at 633 nm). Importantly, the Fabry–Perot microcavity structure produces a significantly larger change in transmitted light intensity compared to a single membrane structure.