Vacuole Formation Research Articles

Prevalence of Blastocystis sp. in freshwater fishes, poultry, and caprine from Penang, Malaysia.

Blastocystis is an enigmatic intestinal protist of humans and many animals. There is growing interest in its potential as an enteric pathogen, and the possible role of domestic and in-contact animals as reservoirs for human infection. The purpose of this study was to determine the prevalence of Blastocystis infection in fishes, poultry, and caprine in Penang, Malaysia. A total of 353 faecal samples/intestinal contents, consisting of 123 intestinal contents from freshwater fishes, 96 intestinal contents of commercially barn-reared chickens, 84 intestinal contents of barn-reared quails, and 50 faecal samples of caprine (29 from meat goats and 21 from dairy goats) were collected. Faecal sample/intestinal content from each animal was subjected to in-vitro cultivation method using Jones' medium supplemented with 10% horse serum. The respective colonization frequencies for dairy and meat goats were 47.6% and 31.0%, whereas 26.1% was recorded for chickens and 16.7% for quails. None of the freshwater fishes were found infected with Blastocystis. The organism was most commonly seen as spherically shaped vacuolated forms and cell diameter was significantly larger in poultry than in caprine. For further studies, molecular characterization of Blastocystis in poultry and livestock animals in the study area is highly recommended.

Comparative transcriptomic analysis reveals different host cell responses to Singapore grouper iridovirus and red-spotted grouper nervous necrosis virus

Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) are important pathogens that cause high mortality and heavy economic losses in grouper aquaculture. Interestingly, SGIV infection in grouper cells induces paraptosis-like cell death, while RGNNV infection induces autophagy and necrosis characterized morphologically by vacuolation of lysosome. Here, a comparative transcriptomic analysis was carried out to identify the different molecular events during SGIV and RGNNV infection in grouper spleen (EAGS) cells. The functional enrichment analysis of DEGs suggested that several signaling pathways were involved in CPE progression and host immune response against SGIV or RGNNV. Most of DEGs featured in the KEGG “lysosome pathway” were up-regulated in RGNNV-infected cells, indicating that RGNNV induced lysosomal vacuolization and autophagy might be due to the disturbance of lysosomal function. More than 100 DEGs in cytoskeleton pathway and mitogen-activated protein kinase (MAPK) signal pathway were identified during SGIV infection, providing additional evidence for the roles of cytoskeleton remodeling in cell rounding during CPE progression and MAPK signaling in SGIV induced cell death. Of note, consistent with changes at the transcriptional levels, the post-translational modifications of MAPK signaling-related proteins were also detected during RGNNV infection, and the inhibitors of extracellular signal-regulated kinase (ERK) and p38 MAPK significantly suppressed viral replication and virus induced vacuoles formation. Moreover, the majority of DEGs in interferon and inflammation signaling were obviously up-regulated during RGNNV infection, but down-regulated during SGIV infection, suggesting that SGIV and RGNNV differently manipulated host immune response in vitro. In addition, purine and pyrimidine metabolism pathways were also differently regulated in SGIV and RGNNV-infection cells. Taken together, our data will provide new insights into understanding the potential mechanisms underlying different host cell responses against fish DNA and RNA virus.

Losartan Attenuates Radiation-Induced Damage on Testes and Accelerates Tubular Regeneration.

Male germ cells are particularly susceptible to radiation; infertility being a common consequence after radiotherapy as it impairs spermatogenesis. This study aimed to test whether treatment with losartan (LOS), a selective antagonist of angiotensin II receptor subtype 1 (AT1R), can prevent or attenuate the acute and long-term radiation-induced damage to testes. Wistar rats were randomly distributed into six groups, three of which were studied on day 2 after irradiation: control (CTRL 2), irradiated non-treated (IR 2), and irradiated and treated with LOS (IRLOS 2); and three other groups that were studied on day 60 after irradiation: control (CTRL 60), irradiated non-treated (IR 60), and irradiated and treated with LOS (IRLOS 60). Seven consecutive days before and on the day of irradiation with 2.5 Gy directly administered in the scrotum, the animals were treated with LOS (34 mg/kg/two times/day). This treatment was continued 2 or 60 days after irradiation. The sperm quality was assessed from epididymis cauda. In addition, the testes were submitted to histopathological and morphometric-stereological analysis as well as the proliferating cell nuclear antigen (PCNA) quantification. Serum FSH and LH and plasma testosterone levels were also determined. The data obtained 2 days after the irradiation showed germ cell apoptosis, formation of vacuoles in the seminiferous epithelium, sloughing of germ cells into the lumen, and retention and phagocytosis of step-19 spermatids in Sertoli basal cytoplasm. The treatment with LOS in this period did not prevent or attenuate a radio-induced damage to the testes, illustrating that this drug does not protect against apoptosis derived from direct effects of radiation. On the other hand, 60 days after exposure, the data evidenced the deleterious effects of ionizing radiation on the testes as decreasing of testicular, epididymal, and seminal vesicle masses; tubular atrophy; reduction of cellular proliferation; and loss of germ cells. LOS was able to prevent some of those deleterious effects, promoting improvements in seminal vesicle mass, sperm vitality, plasma testosterone levels, vacuole number, and cell proliferation. In conclusion, inhibition of the AngII/AT1R axis by LOS is effective in protecting the indirect/delayed radiation damage resulting from oxidative stress established in the tissue.

Vitamin C Ameliorates the Cefepime-Induced Changes of Liver Enzymes, Histopathology and Proinflammatory Cytokines in Male Albino Rats

Cefepime belongs to the fourth generation cephalosporin family of antibiotics, it showed side effects such as nephrotoxicity and neurotoxicity. However little reports showed its hepatotoxic effects. The aim of the current study was to evaluate cefepime-induced hepatotoxicity and its associated histopathological changes in the liver. Also, it aimed to examine the effects of cefepime treatment on the proinflammatory cytokines as well as the protective and recovering roles of vitamin C against the cefepime-induced effects. . Blood samples were collected and the liver enzymes in serum ALT, AST and Total Bilirubin (T. Bil.) were estimated biochemically; liver tissues were collected and divided into two parts: the first part were fixed for histopathological examination by H&E staining whereas the other part was processed for RNA extraction and further for real time PCR examination of the mRNA expression of IL1β and TNFα. Cefepime could significantly increase the concentration of ALT, AST, T. Bil. as well as the mRNA expression of proinflammatory cytokines IL1β and TNFα; histopathological changes in the form of hepatic lobules distortion, marked degeneration, hepatocellular vacuolation and necrosis, nuclear pyknosis, scattered apoptosis, distortion of portal areas and marked fibrosis were observed after two weeks of cefepime administration; co- administration of vitamin C with cefepime or administration administering it for 7 days after cefepime could successfully improve the levels of ALT, AST, T. Bil., histopathological and mRNA expression of IL1β and TNFα. The current study suggested that cefepime has hepatotoxic effects shown as alternation of the liver enzymes, changing in the histopathological features as well as stimulating the inflammation in the liver tissue. Vitamin C can be used as a protective or recovering drug against cefepime-induced hepatotoxicity.

Photothermal effect of albumin-modified gold nanorods diminished neuroblastoma cancer stem cells dynamic growth by modulating autophagy

Here, we investigated the photothermal effect of gold nanorods (GNRs) on human neuroblastoma CD133+ cancer stem cells (CSCs) via autophagic cell death. GNRs were synthesized using Cetyltrimethylammonium bromide (CTAB), covered with bovine serum albumin (BSA). CD133+ CSCs were enriched from human neuroblastoma using the magnetic-activated cell sorting (MACS) technique. Cells were incubated with GNRs coated with BSA and exposed to 808-nm near-infrared laser irradiation for 8 min to yield low (43 °C), medium (46 °C), and high (49 °C) temperatures. After 24 h, the survival rate and the percent of apoptotic and necrotic CSCs were measured using MTT assay and flow cytometry. The expression of different autophagy-related genes was measured using polymerase chain reaction (PCR) array analysis. Protein levels of P62 and LC3 were detected using an enzyme-linked immunosorbent assay (ELISA). The viability of CSC was reduced in GNR-exposed cells compared to the control group (p < 0.05). At higher temperatures (49 °C), the percent of apoptotic CSCs, but not necrotic cells, increased compared to the lower temperatures. Levels of intracellular LC3 and P62 were reduced and increased respectively when the temperature increased to 49 °C (p < 0.05). These effects were non-significant at low and medium temperatures (43 and 46 °C) related to the control CSCs (p > 0.05). The clonogenic capacity of CSC was also inhibited after photothermal therapy (p < 0.05). Despite these changes, no statistically significant differences were found in terms of CSC colony number at different temperatures regardless of the presence or absence of HCQ. Based on the data, the combination of photothermal therapy with HCQ at 49 °C can significantly abort the CSC clonogenic capacity compared to the control-matched group without HCQ (p < 0.0001). PCR array showed photothermal modulation of CSCs led to alteration of autophagy-related genes and promotion of co-regulator of apoptosis and autophagy signaling pathways. Factors related to autophagic vacuole formation and intracellular transport were significantly induced at a temperature of 49 °C (p < 0.05). We also note the expression of common genes belonging to autophagy and apoptosis signaling pathways at higher temperatures. Data showed tumoricidal effects of laser-irradiated GNRs by the alteration of autophagic response and apoptosis.

Ultrastructure change and transcriptome analysis of GA3 treatment on seed germination of moso bamboo(Phyllostachys edulis)

ABSTRACT Exploring the mechanism of Gibberellic acid (GA3) treatment on seed germination of moso bamboo can lay a foundation for its future breeding and research. In this study, the germination-related indicators (germination rate, germination potential, vigor index, respiration rate) with different content of GA3 treatment were measured, and the ultrastructure of moso bamboo seeds treated with low and high GA3 concentrations was observed during the germination process. In addition, the transcriptome data of the germination seeds, with and without GA3 treatment were analyzed. The results showed that the low GA3 concentration (10 mol/L) increased the germination rate, germination potential, vigor index and respiration rate, thus promoting the germination of moso bamboo seeds, but a high concentration of GA3 (50 mol/L) inhibited the seed germination. The low GA3 concentration accelerated the decomposition of starch and fat and promoted the vacuole formation of cells, but the high GA3 concentration damaged organelles and increased the endocytosis of cells. Compared with untreated moso bamboo seeds, the seeds had fewer genes expressed after GA3 treatment. Starch and carbon metabolism play a very important role in seed development and embryo viability, whether the seed is treated with GA3 or not. After hormone treatment, GID1 and DELLA-related genes homologous to rice genes is not expressed, but the expression of PIF4, PIF5, GA3 ox2, GA2 oxs, etc., were up-regulated.

Strain-Specific Behavior of Mycobacterium tuberculosis in Interruption of Autophagy Pathway in Human Alveolar Type II Epithelial A549 Cells

Background:Autophagy induction has been shown to differ in magnitude depending on the mycobacterial species. However, few studies have investigated the specific autophagic capacity of different Mtb strains in ATs. This study aimed to elucidate the host autophagic response to different Mtb strains in ATs responsible for TB in the capital of Iran, Tehran. Methods:A549 cells were infected with three different Mtb clinical isolates (Beijing, NEW1, and CAS1/Delhi) and the reference strain H37Rv. Following RNA extraction, the expression of eight ATG genes, four mycobacterial genes, and three miRNAs was evaluated using quantitative RT-PCR. Results:The results revealed that all four strains influenced the autophagy pathway in various ways at different magnitudes. The Beijing and H37Rv strains could inhibit autophagosome formation, whereas the CAS and NEW1 strains induced autophagosome formation. The expression of genes involved in the fusion of autophagosomes to lysosomes (LAMP1) indicated that all the studied strains impaired the autophagolysosomal fusion; this result is not unexpected as Mtb can block the autophagolysomal fusion. In addition, the Beijing and H37RV strains prevented the formation of autophagic vacuoles, besides mycobacterial targeting of lysosomes and protease activity.Conclusion:This preliminary study improved our understanding of how Mtb manages to overcome the host immune system, such as autophagy, and evaluated the genes used by specific strains during this process. Further studies with a large number of Mtb strains, encompassing the other main Mtb lineages, are inevitable.

Investigation of the cell structure and organelles during autolytic PCD of Antirrhinum majus "Legend White" petals.

One of the classes of the plant developmental programmed cell death (PCD) is vacuolar cell death or autolysis. The results of the transmission electron microscope (TEM) studies indicated that this type of PCD occurs during the petal senescence of Antirrhinum majus "Legend White" flowers. The major hallmarks of the process related to the ultrastructure of the cells involved chloroplast degradation, vacuolation, chromatin condensation, cell wall swelling, degradation of Golgi apparatus, protoplasmic shrinkage, degradation of the endoplasmic reticulum, nuclear fragmentation, rupture of tonoplast, and plasma membrane. Macroautophagy and microautophagy processes were also clearly observed during vacuole formation. As in yeasts, in the present study, Golgi apparatus became autophagosome-like structures during degradation that had autophagy activity and then disappeared. Our results revealed a type of selective microautophagy, piecemeal microautophagy of the nucleus (PMN), in nuclear degradation during PCD of petals that has not previously been reported in plants. Moreover, vesicular structures, such as paramural and multilamellar bodies, were observed in some stages.

Cixutumumab reveals a critical role for IGF-1 in adipose and hepatic tissue remodelling during the development of diet-induced obesity

ABSTRACT High fat diet (HFD)-induced obesity leads to perturbation in the storage function of white adipose tissue (WAT) resulting in deposition of lipids in tissues ill-equipped to deal with this challenge. The role of insulin like growth factor-1 (IGF-1) in the systemic and organ-specific responses to HFD is unclear. Using cixutumumab, a monoclonal antibody that internalizes and degrades cell surface IGF-1 receptors (IGF-1 R), leaving insulin receptor expression unchanged we aimed to establish the role of IGF-1 R in the response to a HFD. Mice treated with cixutumumab fed standard chow developed mild hyperinsulinemia with no change in WAT. When challenged by HFD mice treated with cixutumumab had reduced weight gain, reduced WAT expansion, and reduced hepatic lipid vacuole formation. In HFD-fed mice, cixutumumab led to reduced levels of genes encoding proteins important in fatty acid metabolism in WAT and liver. Cixutumumab protected against blunting of insulin-stimulated phosphorylation of Akt in liver of HFD fed mice. These data reveal an important role for IGF-1 R in the WAT and hepatic response to short-term nutrient excess. IGF-1 R inhibition during HFD leads to a lipodystrophic phenotype with a failure of WAT lipid storage and protection from HFD-induced hepatic insulin resistance.

Antibiotic-Induced Changes in Pigment Accumulation, Photosystem II, and Membrane Permeability in a Model Cyanobacterium.

Fremyella diplosiphon is a well-studied a model cyanobacterium for photosynthesis due to its efficient light absorption potential and pigment accumulation. In the present study, the impact of ampicillin, tetracycline, kanamycin, and cefotaxime on pigment fluorescence and photosynthetic capacity in Fremyella diplosiphon strains B481-WT and B481-SD was investigated. Our results indicated that both strains exposed to kanamycin from 0.2 to 3.2 mg/L and tetracycline from 0.8 to 12.8 mg/L enhanced growth and pigment accumulation. Additionally, B481-SD treated with 0.2–51.2 mg/L ampicillin resulted in a significant enhancement of pigment fluorescence. A detrimental effect on growth and pigmentation in both the strains exposed to 6.4–102.5 mg/L kanamycin and 0.8–102.5 mg/L cefotaxime was observed. Detection of reactive oxygen species revealed highest levels of oxidative stress at 51.2 and 102.5 mg/L kanamycin for B481-SD and 102.5 mg/L for B481-WT. Membrane permeability detected by lactate dehydrogenase assay indicated maximal activity at 0.8 mg/L ampicillin, kanamycin, and tetracycline treatments on day 6. Abundant vacuolation, pyrophosphate, and cyanophycin granule formation were observed in treated cells as a response to antibiotic stress. These findings on the hormetic effect of antibiotics on F. diplosiphon indicate that optimal antibiotic concentrations induce cellular growth while high concentrations severely impact cellular functionality. Future studies will be aimed to enhance cellular lipid productivity at optimal antibiotic concentrations to disintegrate the cell wall, thus paving the way for clean bioenergy applications.

Parkin, as a Regulator, Participates in Arsenic Trioxide-Triggered Mitophagy in HeLa Cells.

Parkin is a well-established synergistic mediator of mitophagy in dysfunctional mitochondria. Mitochondria are the main target of arsenic trioxide (ATO) cytotoxicity, and the effect of mitophagy on ATO action remains unclear. In this study, we used stable Parkin-expressing (YFP-Parkin) and Parkin loss-of-function mutant (Parkin C431S) HeLa cell models to ascertain whether Parkin-mediated mitophagy participates in ATO-induced apoptosis/cell death. Our data showed that the overexpression of Parkin significantly sensitized HeLa cells to ATO-initiated proliferation inhibition and apoptosis; however, the mutation of Parkin C431S significantly weakened this Parkin-mediated responsiveness. Our further investigation found that ATO significantly downregulated two fusion proteins (Mfn1/2) and upregulated fission-related protein (Drp1). Autophagy was also activated as evidenced by the formation of autophagic vacuoles and mitophagosomes, increased expression of PINK1, and recruitment of Parkin to impaired mitochondria followed by their degradation, accompanied by the increased transformation of LC3-I to LC3-II, increased expression of Beclin1 and decreased expression of P62 in YFP-Parkin HeLa cells. Enhanced mitochondrial fragmentation and autophagy indicated that mitophagy was activated. Furthermore, during the process of mitophagy, the overproduction of ROS implied that ROS might represent a key factor that initiates mitophagy following Parkin recruitment to mitochondria. In conclusion, our findings indicate that Parkin is critically involved in ATO-triggered mitophagy and functions as a potential antiproliferative target in cancer cells.

IKKγ/NEMO Localization into Multivesicular Bodies

The NF-κB pathway is central pathway for inflammatory and immune responses, and IKKγ/NEMO is essential for NF-κB activation. In a previous report, we identified the role of glycogen synthase kinase-3β (GSK-3β) in NF-κB activation by regulating IKKγ/NEMO. Here, we show that NEMO phosphorylation by GSK-3β leads to NEMO localization into multivesicular bodies (MVBs). Using the endosome marker Rab5, we observed localization into endosomes. Using siRNA, we identified the AAA-ATPase Vps4A, which is involved in recycling the ESCRT machinery by facilitating its dissociation from endosomal membranes, which is necessary for NEMO stability and NF-κB activation. Co-immunoprecipitation studies of NEMO and mutated NEMO demonstrated its direct interaction with Vps4A, which requires NEMO phosphorylation. The transfection of cells by a mutated and constitutively active form of Vps4A, Vps4A-E233Q, resulted in the formation of large vacuoles and strong augmentation in NEMO expression compared to GFP-Vps4-WT. In addition, the overexpression of the mutated form of Vps4A led to increased NF-κB activation. The treatment of cells with the pharmacologic V-ATPase inhibitor bafilomycin A led to a dramatic downregulation of NEMO and, in this way, inhibited NF-κB signal transduction. These results reveal an unexpected role for GSK-3β and V-ATPase in NF-κB signaling activation.

Identification of Key Pathways Involved in White Strain of Hypsizygus marmoreus Extracts-Induced Cell Death of Human Hepatoma Hep3B Cells by Next Generation Sequencing.

White strain of Hypsizygus marmoreus is named as white genius mushroom (WGM) and is a popular food in Taiwan. We have confirmed the cytotoxicity of WGM extracts on human Hep3B liver cancer cells. A total of 8711 significantly differential genes were identified through large-scale transcriptome sequencing. According to the KEGG pathway enrichment analysis, autophagy, mitophagy and apoptosis pathways were identified as significant in WGM extracts-treated cells. WGM extracts induced a dose-dependent generation of reactive oxygen species (ROS) and membrane-enclosed vacuoles in Hep3B cells. The inhibition of ROS by the ROS scavengers blocked the induction of cell death and vacuoles formation. We suggested that the cell death and membrane-enclosed vacuoles induced by WGM extracts are dependent on ROS production in Hep3B cells. (2E,6E)-3,7,11,15,19,23,27,31,35-Nonamethylhexatriaconta-2,6,34-triene-1,11,15,19,23,27,31-heptol and (18:2) lysophosphatidylcholine were identified in WGM extracts. In addition to being a very popular edible mushrooms, WGM may be developed into a dietary supplement or dietary chemopreventive agent for the cancer treatment.

Pamidronate, a promising repositioning drug to treat leishmaniasis, displays antileishmanial and immunomodulatory potential

Visceral leishmaniasis (VL) is an infectious disease caused by Leishmania infantum (L. infantum). Currently, there are no vaccines and/or prophylactic therapies against VL, and the recentpharmacological approaches come from the drug repositioning strategy. Here, we evaluated the anticancer drug pamidronate (PAM) to identify a new therapeutic option for the treatment of human VL. We assessed its in vitro antileishmanial activity against the promastigote and amastigote forms of L. infantum by evaluating cell cytotoxicity. The antileishmanial and immunomodulatory activities were assessed using human peripheral blood leukocytes ex vivo. PAM induced the formation of vacuoles in the cytoplasm of the promastigotes and alterations in the morphology of the kinetoplast and mitochondria in vitro, which indicates anti-promastigote activity. PAM also reduced the number of infected macrophages and intracellular amastigotes in a concentration-dependent manner, with cell viability above 70%. In ex vivo, PAM reduced the internalized forms of L. infantum in the classical monocyte subpopulation. Furthermore, it enhanced IL-12 and decreased IL-10 and TGF-β by monocytes and neutrophils. Increased IFN-γ and TNF levels for CD8- and CD8+ T lymphocytes and B lymphocytes, respectively, were observed after the treatment with PAM, as well as a reduction in IL-10 by the lymphocyte subpopulations evaluated. Taken together, our results suggest that PAM may be eligible as a potential therapeutic alternative for drug repurposing to treat human visceral leishmaniasis.

Maturation study of vitrinite in carbonaceous shales and coals: Insights from hydrous pyrolysis

The presence of vitrinite in sedimentary rocks of post-Silurian age allows its reflectance to be used to estimate the thermal maturation of organic matter in petroleum systems. Increasing reflectance of vitrinite, which is primarily driven by aromaticity, depends primarily on the time and temperature attributes of its evolutionary pathway. This study evaluated carbonaceous shales proximal to coal measures and coal samples via isothermal hydrous pyrolysis (HP) to compare differences in the maturation pathways of vitrinite. Sample residues were analysed via vitrinite reflectance (VRo), geochemical screening tests (organic carbon and programmed temperature pyrolysis), and infrared spectroscopy. The study included samples from Indian and North American basins, to observe differences in vitrinite evolution with respect to enclosing mineral matrix, starting degree of aromaticity, organic matter types, stratigraphic age, and depositional environment. The organic content of HP residues shows an intuitive response to the thermal stress of HP, e.g., a general depletion of total organic carbon (TOC) content, pyrolyzate (S2), and hydrogen index with increasing HP temperature. Infrared proxies including C-factor and CH2/CH3 generally decrease with increasing thermal maturity indicating loss of O via CO2 generation and the thermal cracking of aliphatic organic matter. Tmax, production index (PI), and VRo show intuitive increasing values with respect to HP temperature. The least mature sample (0.48 ± 0.05% VRo) generally experienced the maximum change in these parameters during maturation, whereas the most mature sample (0.99 ± 0.06% VRo) generally showed the least change. This observation is consistent with higher kinetic barriers to reaction in more aromatic vitrinite which contains higher bond dissociation energies. Devolatilization of vitrinite during HP causes formation of gas evacuation vacuoles and contraction cracks in the vitrinite grains of both coal and carbonaceous shale. Similarities in vitrinite response to HP between coal and carbonaceous shale suggest that thermal evolution of the vitrinite maceral is principally controlled by inherent rate-limiting kinetic parameters related to its molecular structure. Whereas, the stratigraphic age, sedimentary environment, surrounding organic matter, lithology, and catalysis by mineral composition have less effect. To further improve our understanding of vitrinite aromatization and kinetic parameters, future studies of vitrinite reflectance thermal evolution with temperature should include coal and carbonaceous shale from the same stratigraphic section and extant woody tissue from modern vascular plants.

Vacuoles in Bryophytes: Properties, Biogenesis, and Evolution.

Vacuoles are the most conspicuous organelles in plants for their indispensable functions in cell expansion, solute storage, water balance, etc. Extensive studies on angiosperms have revealed that a set of conserved core molecular machineries orchestrate the formation of vacuoles from multiple pathways. Usually, vacuoles in seed plants are classified into protein storage vacuoles and lytic vacuoles for their distinctive morphology and physiology function. Bryophytes represent early diverged non-vascular land plants, and are of great value for a better understanding of plant science. However, knowledge about vacuole morphology and biogenesis is far less characterized in bryophytes. In this review, first we summarize known knowledge about the morphological and metabolic constitution properties of bryophytes' vacuoles. Then based on known genome information of representative bryophytes, we compared the conserved molecular machinery for vacuole biogenesis among different species including yeast, mammals, Arabidopsis and bryophytes and listed out significant changes in terms of the presence/absence of key machinery genes which participate in vacuole biogenesis. Finally, we propose the possible conserved and diverged mechanism for the biogenesis of vacuoles in bryophytes compared with seed plants.

Inhibition of USP14 enhances anti-tumor effect in vemurafenib-resistant melanoma by regulation of Skp2.

The mutation of BRAF V600E often occurred in melanoma and results in tumorigenesis. BRAF mutation drives hyperactivation of the RAF-MAPK-ERK pathway. The acquired drug resistance upon prolonged use of BRAF inhibitors (such as vemurafenib) still remains the main obstacle. Previously, we have found that E3 ligase Skp2 over-expresses vemurafenib-resistant melanoma cells, and knockdown of Skp2 enhances the anti-tumor effect of vemurafenib. Interestingly, the literature has reported that the selective USP14/UCHL5 inhibitor b-AP15 displays great potential in melanoma therapy; however, the molecular mechanism still remains unknown. In vitro, the effect of the combination regimen of vemurafenib (Vem, PLX4032) and b-AP15 on vem-sensitive and vem-resistant melanoma has been investigated by wound healing, colony formation, transwell invasion assay, flow cytometry, lysosome staining, and ROS detection. In vivo, the combination effect on vem-resistant melanoma has been evaluated with a nude mice xenograft tumor model. GST-pulldown and co-immunoprecipitation (co-IP) assays have been applied to investigate the interactions between USP14, UCHL5, and Skp2. Cycloheximide (CHX) assay and ubiquitination assays have been used to explore the effect of USP14 on Skp2 protein half-life and ubiquitination status. In the present study, we have revealed that repression of USP14 sensitizes vemurafenib resistance in melanoma through a previously unappreciated mechanism that USP14 but not UCHL5 stabilizes Skp2, blocking its ubiquitination. K119 on Skp2 is required for USP14-mediated deubiquitination and stabilization of Skp2. Furthermore, the mutated catalytic activity amino acid cysteine (C) 114 on USP14 abrogates stabilization of Skp2. Stabilization of Skp2 is required for USP14 to negatively regulate autophagy. The combination regimen of Skp2 inhibitor vemurafenib and USP14/UCHL5 inhibitor b-AP15 dramatically inhibits cell viability, migration, invasion, and colony formation in vemurafenib-sensitive and vemurafenib-resistant melanoma. Vemurafenib and b-AP15 hold cells in the S phase thus leading to apoptosis as well as the formation of the autophagic vacuole in vemurafenib-resistant SKMEL28 cells. The enhanced proliferation effect of USP14 and Skp2 is mainly due to a more effective reduction of cell apoptosis and autophagy. Further evaluation of various protein alterations has revealed that the increased expression of cleaved-PARP, LC3, and decreased Ki67 are more obvious in the combination of vemurafenib and b-AP15 treatment than those in single-drug treatment. Moreover, the co-treatment of vemurafenib and b-AP15 dramatically inhibits the growth of vemurafenib-resistant melanoma xenograft in vivo. Collectively, our findings have demonstrated that the combination of Skp2 inhibitor and USP14 inhibitor provides a new solution for the treatment of BRAF inhibitor resistance melanoma.

Disulfiram oxy-derivatives induce entosis or paraptosis-like death in breast cancer MCF-7 cells depending on the duration of treatment

BackgroundDithiocarbamates and derivatives (including disulfiram, DSF) are currently investigated as antineoplastic agents. We have revealed earlier the ability of hydroxocobalamin (vitamin В12b) combined with diethyldithiocarbamate (DDC) to catalyze the formation of highly cytotoxic oxidized derivatives of DSF (DSFoxy, sulfones and sulfoxides). MethodsElectron and fluorescent confocal microscopy, molecular biology and conventional biochemical techniques were used to study the morphological and functional responses of MCF-7 human breast cancer cells to treatment with DDC and B12b alone or in combination. ResultsDDC induces unfolded protein response in MCF-7 cells. The combined use of DDC and B12b causes MCF-7 cell death. Electron microscopy revealed the separation of ER and nuclear membranes, leading to the formation of both cytoplasmic and perinuclear vacuoles, with many fibers inside. The process of vacuolization coincided with the appearance of ER stress markers, a marked damage to mitochondria, a significant inhibition of 20S proteasome, and actin depolimerization at later stages. Specific inhibitors of apoptosis, necroptosis, autophagy, and ferroptosis did not prevent cell death. A short- time (6-h) exposure to DSFoxy caused a significant increase in the number of entotic cells. ConclusionsThese observations indicate that MCF-7 cells treated with a mixture of DDC and B12b die by the mechanism of paraptosis. A short- time exposure to DSFoxy caused, along with paraptosis, a significant activation of the entosis and its final stage, lysosomal cell death. General significanceThe results obtained open up opportunities for the development of new approaches to induce non-apoptotic death of cancer cells by dithiocarbamates.

Chaperone-Mediated Autophagy and Its Implications for Neurodegeneration and Cancer.

Proteostasis, also known as protein homeostasis, is critical for cell survival. Autophagy is a cellular process that degrades and recycles damaged or long-lived proteins, misfolded proteins, and damaged or abnormal organelles in order to preserve homeostasis. Among the three forms of autophagy, chaperone-mediated autophagy (CMA) is distinct from macroautophagy and microautophagy; it does not require the formation of vacuoles and only degrades selected individual proteins. CMA helps to maintain cellular homeostasis by regulating protein quality, bioenergetics, and substrate-associated cellular processes at the right moment. This pathway’s dysfunction has been linked to several diseases and disorders. Neurodegenerative diseases and cancer have received the most attention. In various neurodegenerative disorders, especially in their later stages, CMA activity declines. CMA has been shown to act as a tumor suppressor in cancer by destroying specific tumor promoters. Once a tumor has grown, it also helps tumor survival and the metastatic cascade. The presence of changes in CMA in these diseases disorders raises the idea of targeting CMA to restore cellular homeostasis as a potential therapeutic method. Manipulation of CMA activity may be effective therapeutic strategies for treating these diseases. Therefore, in this paper; we introduce the basic processes, regulatory mechanisms, and physiological functions of CMA; evidences supporting the role of impaired CMA function in neurodegeneration and cancer; and the potential of how targeting CMA could be a promising therapeutic method for the two diseases.

A Search for NovelLegionella pneumophilaEffector Proteins Reveals a Strain Specific Nucleotropic Effector.

Legionella pneumophila is an accidental human pathogen that causes the potentially fatal Legionnaires’ disease, a severe type of pneumonia. The main virulence mechanism of L. pneumophila is a Type 4B Secretion System (T4SS) named Icm/Dot that transports effector proteins into the host cell cytosol. The concerted action of effectors on several host cell processes leads to the formation of an intracellular Legionella-containing vacuole that is replication competent and avoids phagolysosomal degradation. To date over 300 Icm/Dot substrates have been identified. In this study, we searched the genome of a L. pneumophila strain (Pt/VFX2014) responsible for the second largest L. pneumophila outbreak worldwide (in Vila Franca de Xira, Portugal, in 2014) for genes encoding potential novel Icm/Dot substrates. This strain Pt/VFX2014 belongs to serogroup 1 but phylogenetically segregates from all other serogroup 1 strains previously sequenced, displaying a unique mosaic genetic backbone. The ability of the selected putative effectors to be delivered into host cells by the T4SS was confirmed using the TEM-1 β-lactamase reporter assay. Two previously unknown Icm/Dot effectors were identified, VFX05045 and VFX10045, whose homologs Lpp1450 and Lpp3070 in clinical strain L. pneumophila Paris were also confirmed as T4SS substrates. After delivery into the host cell cytosol, homologs VFX05045/Lpp1450 remained diffused in the cell, similarly to Lpp3070. In contrast, VFX10045 localized to the host cell nucleus. To understand how VFX10045 and Lpp3070 (94% of identity at amino acid level) are directed to distinct sites, we carried out a comprehensive site-directed mutagenesis followed by analyses of the subcellular localization of the mutant proteins. This led to the delineation of region in the C-terminal part (residues 380 to 534) of the 583 amino acid-long VFX10045 as necessary and sufficient for nuclear targeting and highlighted the fundamental function of the VFX10045-specific R440 and I441 residues in this process. These studies revealed a strain-specific nucleotropism for new effector VFX10045/Lpp3070, which anticipates distinct functions between these homologs.