Influence of vacuoles with gas–liquid inclusions on the thermobaric destruction conditions of natural quartz under dynamic heating in an RF-TICP torch system

In the present work,the turbulent mixing process of a polydisperse quartz particle flow with a plasma stream generated by a radio-frequency(RF)inductively coupled plasma torch was numerically studied.The thermobaric stress in the quartz particles under dynamic heating in a heterogeneous plasma flow was determined by a two-stage approximation approach.The effect of the presence of vacuoles in natural quartz on the particle thermobaric destruction conditions was studied.It was found that the equivalent thermal and baric stresses in quartz particles may significantly increase in the presence of vacuoles within a small gas volume fraction.The influence of the regime and energetic working conditions of an RF inductively coupled plasma torch system on the particle thermobaric destruction conditions was examined,and a recommendation was given to promote the degree of thermobaric destruction of quartz particles,which is of substantial importance for improving the overall enrichment efficiency of quartz concentrates.

Vacuoles of Candida yeast as a specialized niche for Helicobacter pylori

Helicobacter pylori(H.pylori)are resistant to hostile gastric environments and antibiotic therapy,reflecting the possibility that they are protected by an ecological niche,such as inside the vacuoles of human epithelial and immune cells.Candida yeast may also provide such an alternative niche,as fluorescently labeled H.pylori were observed as fast-moving and viable bacterium-like bodies inside the vacuoles of gastric,oral,vaginal and foodborne Candida yeasts.In addition,H.pylori-specific genes and proteins were detected in samples extracted from these yeasts.The H.pylori present within these yeasts produce peroxiredoxin and thiol peroxidase,providing the ability to detoxify oxygen metabolites formed in immune cells.Furthermore,these bacteria produce urease and VacA,two virulence determinants of H.pylori that influence phago-lysosome fusion and bacterial survival in macrophages.Microscopic observations of H.pylori cells in new generations of yeasts along with amplification of H.pylori-specific genes from consecutive generations indicate that new yeasts can inherit the intracellular H.pylori as part of their vacuolar content.Accordingly,it is proposed that yeast vacuoles serve as a sophisticated niche that protects H.pylori against the environmental stresses and provides essential nutrients,including ergosterol,for its growth and multiplication.This intracellular establishment inside the yeast vacuole likely occurred long ago,leading to the adaptation of H.pylori to persist in phagocytic cells.The presence of these bacteria within yeasts,including foodborne yeasts,along with the vertical transmission of yeasts from mother to neonate,provide explanations for the persistence and propagation of H.pylori in the human population.This Topic Highlight reviews and discusses recent evidence regarding the evolutionary adaptation of H.pylori to thrive in host cell vacuoles.

Vacuole import and degradation pathway: Insights into a specialized autophagy pathway

Glucose deprivation induces the synthesis of pivotagluconeogenic enzymes such as fructose-1,6-bisphos-phatase, malate dehydrogenase, phosphoenolpyruvatecarboxykinase and isocitrate lyase in Saccharomycescerevisiae. However, following glucose replenishment,these gluconeogenic enzymes are inactivated and de-graded. Studies have characterized the mechanismsby which these enzymes are inactivated in response toglucose. The site of degradation of these proteins hasalso been ascertained to be dependent on the dura-tion of starvation. Glucose replenishment of short-termstarved cells results in these proteins being degradedin the proteasome. In contrast, addition of glucose tocells starved for a prolonged period results in theseproteins being degraded in the vacuole. In the vacuoledependent pathway, these proteins are sequestered inspecialized vesicles termed vacuole import and degra-dation (Vid). These vesicles converge with the endo-cytic pathway and deliver their cargo to the vacuolefor degradation. Recent studies have identified thatinternalization, as mediated by actin polymerization, isessential for delivery of cargo proteins to the vacuolefor degradation. In addition, components of the targetof rapamycin complex 1 interact with cargo proteins during glucose starvation. Furthermore, Tor1p dissoci-ates from cargo proteins following glucose replenish-ment. Future studies will be needed to elaborate on the importance of internalization at the plasma membrane and the subsequent import of cargo proteins into Vid vesicles in the vacuole dependent degradation pathway.

Glucose alleviates cadmium toxicity by increasing cadmium fi xation in root cell wall and sequestration into vacuole in Arabidopsis

Glucose (Glu) is involved in not only plant physiological and developmental events but also plant responses to abiotic stresses. Here, we found that the exogenous Glu improved root and shoot growth, reduced shoot cadmium (Cd) concentration, and rescued Cd-induced chlorosis in Arabidopsis thaliana (Columbia ecotype, Col-0) under Cd stressed conditions. Glucose increased Cd retained in the roots, thus reducing its translocation from root to shoot significantly. The most Cd retained in the roots was found in the hemicellulose 1. Glucose combined with Cd (Glu t Cd) treatment did not affect the content of pectin and its binding capacity of Cd while it increased the content of hemicelluloses 1 and the amount of Cd retained in it significantly. Furthermore, Leadmium Green staining indicated that more Cd was compartmented into vacuoles in Glu t Cd treatment compared with Cd treatment alone, which was in accordance with the significant upregulation of the expression of tonoplast-localized metal transporter genes, suggesting that com-partmentation of Cd into vacuoles also contributes to the Glu-alleviated Cd toxicity. Taken together, we demonstrated that Glu-alleviated Cd toxicity is mediated through increas-ing Cd fixation in the root cell wall and sequestration into the vacuoles.

Vacuole Integrity Maintained by DUF300 Proteins Is Required for Brassinosteroid Signaling Regulation

Brassinosteroid （BR） hormone signaling controls multiple processes during plant growth and development and is initiated at the plasma membrane through the receptor kinase BRASSINOSTEROID INSENSITIVE1 （BRI1） together with co-receptors such as BRI1-ASSOClATED RECEPTOR KINASE1 （BAK1）. BRI1 abun- dance is regulated by endosomal recycling and vacuolar targeting, but the role of vacuole-related proteins in BR receptor dynamics and BR responses remains elusive. Here, we show that the absence of two DUF300 domain-containing tonoplast proteins, LAZARUS1 （LAZl） and LAZl HOMOLOG1 （LAZlH1）, causes vacuole morphology defects, growth inhibition, and constitutive activation of BR signaling. Intriguingly, tonoplast accumulation of BAK1 was substantially increased and appeared causally linked to enhanced BRI1 trafficking and degradation in lazl lazlhl plants. Since unrelated vacuole mutants exhibited normal BR responses, our findings indicate that DUF300 proteins play distinct roles in the regulation of BR signaling by maintaining vacuole integrity required to balance subcellular BAK1 pools and BR receptor distribution.

Structure and Function of TPC1 Vacuole SV Channel Gains Shape

Plants and animals in endosomes operate TPCI/SV-type cation channels. All plants harbor at least one TPC1 gene. Although the encoded SV channel was firstly discovered in the plant vacuole membrane two decades ago, its biological function has remained enigmatic. Recently, the structure of a plant TPC1/SV channel protein was determined. Insights into the 3D topology has now guided site-directed mutation ap- proaches, enabling structure-function analyses of TPC1/SV channels to shed new light on earlier findings. Fou2 plants carrying a hyperactive mutant form of TPC1 develop wounding stress phenotypes. Recent studies with fou2 and mutants that lack functional TPC1 have revealed atypical features in local and long-distance stress signaling, providing new access to the previously mysterious biology of this vacuolar cation channel type in planta.

Turnover of diacylglycerol kinase 4 by cytoplasmic acidification induces vacuole morphological change and nuclear DNA degradation in the early stage of pear self-incompatibility response

Pear has an S-RNase-based gametophytic selfincompatibility(SI)system.Nuclear DNA degradation is a typical feature of incompatible pollen tube death,and is among the many physiological functions of vacuoles.However,the specific changes that occur in vacuoles,as well as the associated regulatory mechanism in pear SI,are currently unclear.Although research in tobacco has shown that decreased activity of diacylglycerol kinase(DGK)results in the morphological change of pollen tube vacuole,whether DGK regulates the pollen tube vacuole of tree plants and whether it occurs in SI response,is currently unclear.We found that DGK activity is essential for pear pollen tube growth,and DGK4 regulates pollen tube vacuole morphology following its high expression and deposition at the tip and shank edge of the pollen tube of pear.Specifically,incompatible S-RNase may induce cytoplasmic acidification of the pollen tube by inhibiting V-ATPase V0 domain a1 subunit gene expression as early as 30 min after treatment,when the pollen tube is still alive.Cytoplasmic acidification induced by incompatible S-RNase results in reduced DGK4 abundance and deposition,leading to morphological change of the vacuole and fragmentation of nuclear DNA,which indicates that DGK4 is a key factor in pear SI response.

Homotypic Vacuole Fusion Requires VTI11 and Is Regulated by Phosphoinositides

Most plant cells contain a large central vacuole that is essential to maintain cellular turgor. We report a new mutant allele of VTI11 that implicates the SNARE protein VTI11 in homotypic fusion of protein storage and lytic vacuoles. Fusion of the multiple vacuoles present in vtill mutants could be induced by treatment with Wortmannin and LY294002, which are inhibitors of Phosphatidylinositol 3-Kinase （PI3K）. We provide evidence that Phosphatidylinositol 3-Phosphate （Ptdlns（3）P） regulates vacuole fusion in vtill mutants, and that fusion of these vacuoles requires intact microtubules and actin filaments. Finally, we show that Wortmannin also induced the fusion of guard cell vacuoles in fava beans, where vacuoles are naturally fragmented after ABA-induced stomata closure. These results suggest a ubiquitous role of phosphoinositides in vacuole fusion, both during the development of the large central vacuole and during the dynamic vacuole remodeling that occurs as part of stomata movements.

V-ATPase,ScNhxlp and Yeast Vacuole Fusion

Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos. It is a central cellular reaction that plays important roles in signal transduction, protein sorting and subcellular compartmentation. Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summarized in this article. It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na＋/H＋ antiporter ScNhxlp are key components of the vacuole fusion machinery in yeast. Yeast ScNhxlp regulates vacuole fusion by controlling the luminal pH. V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast. Fission defects are epistatic to fusion defects. Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast, the fusion reaction does not need the transport activity but requires the physical presence of the proton pump. V0, the membrane-integral sector of the V-ATPase, forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the V0 trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.

Protein Domains Involved in Assembly in the Endoplasmic Reticulum Promote Vacuolar Delivery when Fused to Secretory GFP, Indicating a Protein Quality Control Pathway for Degradation in the Plant Vacuole

The correct folding and assembly of newly synthesized secretory proteins are monitored by the protein quality control system of the endoplasmic reticulum （ER）. Through interactions with chaperones such as the binding protein （BiP） and other folding helpers, quality control favors productive folding and sorts for degradation defective proteins. A major route for quality control degradation identified in yeast, plants, and animals is constituted by retrotranslocation from the ER to the cytosol and subsequent disposal by the ubiquitin/proteasome system, but alternative routes involving the vacuole have been identified in yeast. In this study, we have studied the destiny of sGFP418, a fusion between a secretory form of GFP and a domain of the vacuolar protein phaseolin that is involved in the correct assembly of phaseolin and in BiP recognition of unassembled subunits. We show that sGFP418, despite lacking the phaseolin vacuolar sorting signal, is delivered to the vacuole and fragmented, in a process that is inhibited by the secretory traffic inhibitor brefeldin A. Moreover, a fusion between GFP and a domain of the maize storage protein γ-zein involved in zein polymerization also undergoes post-translational fragmentation similar to that of sGFP418. These results show that defective secretory proteins with permanently exposed sequences normally involved in oligomerization can be delivered to the vacuole by secretory traffic. This strongly suggests the existence of a plant vacuolar sorting mechanism devoted to the disposal of defective secretory proteins.

Simplification of vacuole structure during plant cell death triggered by culture filtrates of Erwinia carotovora

Vacuoles are suggested to play crucial roles in plant defense-related cell death. During programmed cell death, previous live cell imaging studies have observed vacuoles to become simpler in structure and have implicated this simplification as a prelude to the vacuole＇s rupture and consequent lysis of the plasma membrane. Here, we examined dynamics of the vacuole in cell cycle-synchronized tobacco BY-2 （Nicotiana tabacum L. cv. Bright Yellow 2） cells during ceil death induced by application of culture filtrates of Erwinia carotovora. The filtrate induced death in about 90% of the cells by 24 h. Prior to cell death, vacuole shape simplified and endoplasmic actin filaments disassembled; however, the vacuoles did not rupture until after plasma membrane integrity was lost. Instead of facilitating rupture, the simplification of vacuole structure might play a role in the retrieval of membrane components needed for defense-related cell death.

Maize ZmVPP5 is a truncated Vacuole H^＋-PPase that confers hypersensitivity to salt stress

In plants, Vacuole H^＋-PPases（VPPs） are important Aproton pumps and encoded by multiple genes. In addition to full-length VPPs, several truncated forms are expressed, but their biological functions are unknown. In this study, we functionally characterized maize vacuole H^＋-PPase 5（ZmVPP5）, a truncated VPP in the maize genome. Although ZmVPP5 shares high sequence similarity with ZmVPP1 ZmVPP5 lacks the complete structure of the conserved proton transport and the inorganic pyrophosphatase-related domain. Phylogenetic analysis suggests that ZmVPP5 might be derived from an incomplete gene duplication event. ZmVPP5 is expressed in multiple tissues, and ZmVPP5 was detected in the plasma membrane, vacuole membrane and nuclei of maize cells. The overexpression of ZmVPP5 in yeast cells caused a hypersensitivity to salt stress. Transgenic maize lines with overexpressed ZmVPP5 also exhibited the salt hypersensitivity phenotype. A yeast two-hybrid analysis identified the ZmBag6 like protein as a putative ZmVPP5-interacting protein. The results of bimolecular luminescence complementation（Bi LC）assay suggest an interaction between ZmBag6-like protein and ZmVPP5 in vivo. Overall, this study suggests that ZmVPP5 might act as a VPP antagonist and participate in the cellular response to salt stress. Our study of ZmVPP5 has expanded the understanding of the origin and functions of truncated forms of plant VPPs.

Multiple functions of the vacuole in plant growth and fruit quality

Vacuoles are organelles in plant cells that play pivotal roles in growth and developmental regulation.The main functions of vacuoles include maintaining cell acidity and turgor pressure,regulating the storage and transport of substances,controlling the transport and localization of key proteins through the endocytic and lysosomal-vacuolar transport pathways,and responding to biotic and abiotic stresses.Further,proteins localized either in the tonoplast(vacuolar membrane)or inside the vacuole lumen are critical for fruit quality.In this review,we summarize and discuss some of the emerging functions and regulatory mechanisms associated with plant vacuoles,including vacuole biogenesis,vacuole functions in plant growth and development,fruit quality,and plant-microbe interaction,as well as some innovative research technology that has driven advances in the field.Together,the functions of plant vacuoles are important for plant growth and fruit quality.The investigation of vacuole functions in plants is of great scientific significance and has potential applications in agriculture.

Oculopharyngeal Weakness, Hypophrenia, Deafness, and Impaired Vision： A Novel Autosomal Dominant Myopathy with Rimmed Vacuoles

Background： Myopathies with rimnled vacuoles are a heterogeneous group of muscle disorders with progressive muscle weakness and varied clinical manifestations but similar features in muscle biopsies. Here, we describe a novel autosomal dominant myopathy with rimmed vacuoles in a large family with 11 patients of three generations affected. Methods： A clinical study including family history, obstetric, pediatric, and development history was recorded. Clinical examinations including physical examination, electromyography （EMG）, serum creatine kinase （CK）, bone X-rays, and brain magnetic resonance imaging （MRI） were performed in this family. Open muscle biopsies were performed on the proband and his mother. To find the causative gene, the whole-exome sequencing was carried out. Results： Disease onset was from adolescence to adulthood, but the affected patients of the third generation presented an earlier onset and more severe clinical manifestations than the older generations. Clinical features were characterized as dysarthria, dysphagia, external ophthalmoplegia, limb weakness, hypophrenia, deafness, and impaired vision. However, not every patient manifested all symptoms. Serum CK was mildly elevated and EMG indicated a myopathic pattern. Brain MRI showed cerebellum and brain stem mildly atrophy. Rimmed vacuoles and inclusion bodies were observed in muscle biopsy. The whole-exome sequencing was performed, but the causative gene has not been found. Conclusions： We reported a novel autosomal dominant myopathy with rimmed vacuoles characterized by dysarthria, dysphagia, external ophthalmoplegia, limb weakness, hypophrenia, deafness, and impaired vision, but the causative gene has not been tbund and needs further study.

Tracking tonoplast protein behaviors in intact vacuoles

Subject Code:C02With the support by the National Natural Science Foundation of China,the research team led by Prof.Lin Jinxing(林金星)at the College of Biological Sciences&Biotechnology,Beijing Forestry University,overcame the limitations of existing techniques and expanded the study of protein characteristics from the plasma membrane to the vacuole membranes.This study was published in Molecular Plant(2016,DOI:

Impact of Helicobacter pylori virulence markers on clinical outcomes in adult populations

BACKGROUND In recent years,associations between specific virulence markers of Helicobacter pylori(H.pylori)and gastrointestinal disorders have been suggested.AIM To investigate the presence of virulence factors including vacuolating cytotoxin A genotypes(s1m1,s1m2,s2m1,and s2m2),cytotoxin-associated gene A(CagA),and urease activity in H.pylori strains isolated from Arab and Jewish populations in northern Israel and to assess associations between these factors and patients’demographics and clinical outcomes.METHODS Patients(n=108)who underwent gastroscopy at the Baruch Padeh Medical Center,Poriya due to symptomatic gastroduodenal pathologies as part of H.pylori diagnosis were enrolled in the study.Gastric biopsy specimens were collected from the antrum of the stomach.Clinical condition was assessed by clinical pathology tests.Bacteria were isolated on modified BD Helicobacter Agar(BD Diagnostics,Sparks,MD,United States).Bacterial DNA was extracted,and PCR was performed to detect CagA and vacuolating cytotoxin A genes.Urease activity was assessed using a rapid urease test.RESULTS A significant correlation was found between disease severity and patient ethnicity(P=0.002).A significant correlation was found between CagA presence and the s1m1 genotype(P=0.02),which is considered the most virulent genotype.Further,a higher level of urease activity was associated with isolates originating from the Jewish population.Moreover,higher urease activity levels were measured among CagA-/s1m1 and CagA-/s2m2 isolates.CONCLUSION Our study highlights the importance of incorporating molecular methods for detection of virulence markers of H.pylori in order to tailor optimal treatments for each patient.Further investigation should be performed regarding associations between H.pylori virulence factors and ethnicity.

Restoration of Mitochondrial Structure and Function within Helicobacter pylori VacA Intoxicated Cells

The Helicobacter pylori vacuolating cytotoxin (VacA) is an intracellular, mitochondrial-targeting exotoxin that rapidly causes mitochondrial dysfunction and fragmentation. Although VacA targeting of mitochondria has been reported to alter overall cellular metabolism, there is little known about the consequences of extended exposure to the toxin. Here, we describe studies to address this gap in knowledge, which have revealed that mitochondrial dysfunction and fragmentation are followed by a time-dependent recovery of mitochondrial structure, mitochondrial transmembrane potential, and cellular ATP levels. Cells exposed to VacA also initially demonstrated a reduction in oxidative phosphorylation, as well as increase in compensatory aerobic glycolysis. These metabolic alterations were reversed in cells with limited toxin exposure, congruent with the recovery of mitochondrial transmembrane potential and the absence of cytochrome c release from the mitochondria. Taken together, these results are consistent with a model that mitochondrial structure and function are restored in VacA-intoxicated cells.

Microtubule Structure and Male Sterility in a Gene-Cytoplasmic Male Sterile Line of Rice, Zhen Shan 97A

Histological changes that occur during microsporogenesis are documented in a gene-cytoplasmic male sterile rice ( Oryza saliva L.) line, Zhen Shan 97A, its maintainer line, Zhen Shan 97B, and the restorer line, Ce64 of a Mine hybrid rice production system. In the restorer line, Ce64, the developing microsporocytes have dense cytoplasm and a distinct set of circumferential microtubules around the nucleus. Successive cytokinesis results in the formation of tetrads. The microtubules within the cells of tetrads and microspores radiate from the surface of the nucleus towards the outer edge of the cytoplasm. Subsequent pollen development is normal. During the course of microspore formation tubulin speckles can be found in the cytoplasm. The general pattern of development and microtubule organization in the maintainer lined Zhen Shan 97B, is similar to Ce64, except that a few more tubulin speckles appear during microspore formation. In the case of the mate sterile line, Zhen Shan 97A, a number of abnormalities can be discerned during early microsporogenesis. These include vacuoles forming within the developing microsporocyte and faintly stained microtubules with no defined distribution pattern. Prominent tubulin speckles are common within the cytoplasm. For those microsporocytes that undergo meiosis, no defined organizational patterns of microtubules can be found within the tetrad. All microspores abort soon after. Abnormalities and defects in microtubule organization observed in Zhen Shan 97A showed that complex interactions between the cytoplasm and the nucleus began at very early stage of microsporocyte development.

The Role of Vacuolar Processing Enzyme

The classification, characters and maturation methods of VPEs were sum- marized, and its regulation function to vacuolar was also analyzed. Furthermore, effects of the enzyme in vacuolar-mediated plant defense mechanism were discussed to point out that VPEs were divided into 3 subfamilies via autocatalytic mature including seed-type VPE, vegetative-type VPE and new-type VPE. Especially, seed- type VPE mediated the process of storage protein, while vegetative-type VPE and new-type VPE regulated and controlled programmed death of plant cells.

Physiological functions of Atg6/Beclin 1： a unique autophagy-related protein

The most striking morphological feature of eukaryotic cells is the presence of various membrane-enclosed compartments. These compartments, including organelles and transient transport intermediates, are not static. Rather, dynamic exchange of proteins and membrane is needed to maintain cellular homeostasis. One of the most dramatic examples of membrane mobilization is seen during the process ofmacroautophagy. Macroautophagy is the primary cellular pathway for degradation of long-lived proteins and organelles. In response to environmental cues, such as starvation or other types of stress, the cell produces a unique membrane structure, the phagophore. The phagophore sequesters cytoplasm as it forms a double-membrane cytosolic vesicle, an autophagosome. Upon completion, the autophagosome fuses with a lysosome or a vacuole in yeast, which delivers hydrolases that break down the inner autophagosome membrane along with its cargo, and the resulting macromolecules are released back into the cytosol for reuse. Autophagy is therefore a recycling process, allowing cells to survive periods of nutrient limitation; however, it has a wider physiological role, participating in development and aging, and also in protection against pathogen invasion, cancer and certain neurodegenerative diseases. In many cases, the role ofautophagy is identified through studies of an autophagy-related protein, Atg6/Beclin 1. This protein is part of a lipid kinase complex, and recent studies suggest that it plays a central role in coordinating the cytoprotective function ofautophagy and in opposing the cellular death process of apoptosis. Here, we summarize our current knowledge ofAtg6/Beclin 1 in different model organisms and its unique function in the cell.