Vacuolar Structures Research Articles

Potential engineering applications of volcanic scoria from Mount Cameroon area, Central Africa: evidence from petrography and geochemistry

This study was initiated to explore the suitability of volcanic scoria from Mount Cameroon along the Cameroon Volcanic Line (CVL) in engineering applications. The mineral composition has been obtained through thin sections observation and XRD method. The XRF analytical methods have been used to determine major element composition. The studied rocks are dark gray to reddish in color with a vacuolar structure and porphyritic microlithic texture recalling the Strombolian dynamism. They are mainly made up of pyroxene, olivine, plagioclase, opaque minerals, quartz, magnetite and iddingsite. The presence of phenocrysts of olivine and clinopyroxene is due to mantle contamination. The main chemical components of scoria of Mount Cameroon are silica, alumina, iron and calcium oxides. The abundant vitreous phases are responsible for the acidic character revealed by high SiO2 + Al2O3 contents. Fe2O3, MnO and MgO are hosted by ferromagnesian minerals while SiO2, Al2O3, K2O, NaO and P2O5 are found in acidic vitreous phases. The mineral and chemical composition of the scoria of Mount Cameroun is similar to those of scoria previously studied around the world. They can be used as absorbents for metals because of their high adsorption capacity and affinity for metals. The high SiO2 + Al2O3 + Fe2O3 contents and low LOI values show that the scoria from Mount Cameroon are suitable to the use in cement and concretes. They can improve the resistance to sulfate, and chlorite and alkali silica reaction. According to the chemical composition, the rocks are conforming to French standard NFP18310. Their high CaO contents are favorable to improve the quality of scoria blended cements.

New promising antiseptics for individual antiviral protection against seasonal infections including COVID-19

Seasonal viral infections of the upper respiratory tract are widespread throughout the world. The SARS-CoV-2 pandemic has revealed the importance of timely personalized prevention of acute viral diseases and related bacterial complications. One of the main directions in the preventing viral infections relies on the development of effective antiseptics to inactivate viruses on the hands and mucous membranes. To effectively destroy SARS-CoV-2, the WHO recommends two antiseptics: a 70% ethanol solution and sodium hypochlorite. The proposed antiseptics are outdated and have an irritating effect on the skin and mucous membranes. In connection with this, a promising research direction includes development of new antiseptics with selective toxicity, low volatility, low coefficients in the oil-water system, weak lipophilic properties, low absorption into the vacuolar skin structures and low content of toxic impurities. One of these antiseptics is anavidin. The development and selection of molecules has been carried out, the next planned stage will be assessment of the effectiveness and safety of new molecules, including preclinical and early phase clinical trials, also analyzing prevention of viral infections, e.g. COVID-19.

Cataloguing the postnatal small intestinal transcriptome during the period of susceptibility to necrotizing enterocolitis.

In the first postnatal month, the developing mouse intestine shifts from an immature to a mature intestine that will sustain the organism throughout the lifespan. Here, we surveyed the mouse intestine in C57Bl/6 mice by RNA-Seq to evaluate the changes in gene expression over time from the day of birth through 1 month of age in both the duodenum and ileum. We analyzed gene expression for changes in gene families that correlated with the periods of NEC susceptibility or resistance. We highlight that increased expression of DNA processing genes and vacuolar structure genes, tissue development and morphogenesis genes, and cell migration genes all correlated with NEC susceptibility, while increases in immunity gene sets, intracellular transport genes, ATP production, and intracellular metabolism genes correlated with NEC resistance. Using trends identified in the RNA analyses, we further evaluated expression of cellular markers and epithelial regulators, immune cell markers, and adenosine metabolism components. We confirmed key changes with qRT-PCR and immunofluorescence. In addition, we compared some findings to humans using human intestinal biopsies and organoids. This dataset can serve as a reference for other groups considering the role of single molecules or molecular families in early intestinal and postnatal development.

Light at the end of the tunnel: FRAP assays combined with super resolution microscopy confirm the presence of a tubular vacuole network in meristematic plant cells.

Plant vacuoles play key roles in cellular homeostasis, performing catabolic and storage functions, and regulating pH and ion balance. Despite their essential role, there is still no consensus on how vacuoles are established. A model proposing that the endoplasmic reticulum is the main contributor of membrane for growing vacuoles in meristematic cells has been challenged by a study proposing that plant vacuoles are formed de novo by homotypic fusion of multivesicular bodies (MVBs). Here, we use the Arabidopsis thaliana root as a model system to provide a systematic overview of successive vacuole biogenesis stages, starting from the youngest cells proximate to the quiescent center. We combine in vivo high- and super-resolution (STED) microscopy to demonstrate the presence of tubular and connected vacuolar structures in all meristematic cells. Using customized fluorescence recovery after photobleaching (FRAP) assays, we establish different modes of connectivity and demonstrate that thin, tubular vacuoles, as observed in cells near the quiescent center, form an interconnected network. Finally, we argue that a growing body of evidence indicates that vacuolar structures cannot originate from MVBs alone but receive membrane material from different sources simultaneously.

Butylated hydroxyanisole controls anthracnose on postharvest citrus fruit by inducing autophagosome formation in Colletotrichum gloeosporioides

Citrus anthracnose, caused by Colletotrichum gloeosporioides, is a preharvest and postharvest disease that results in economic losses by affecting citrus fruit quality, shelf life, and market value. To date, research on environmental safety, high efficiency and viable alternatives has not been reported. In this study, butylated hydroxyanisole (BHA) was screened and verified to inhibit C. gloeosporioides of citrus. The results showed that BHA could completely inhibit mycelial growth in vitro (0.25 g/L) and absolutely lose its virulence in vivo (20 × Minimum fungicidal concentration (MFC) = 5 g/L). Furthermore, some transparent irregularly shaped vacuolar structures in the cytosol of the BHA-treated conidia were detected by Transmission Electron Microscope (TEM), and autophagosome formation was verified by monodansylcadaverine(MDC) staining. In addition, staining experiments indicated that BHA induced the accumulation of excessive reactive oxygen species (ROS, the DCFH-DA staining rates of C. gloeosporioides conidia treated with 1/2 × MIC and MIC were 26.4% and 83%, respectively), which implied that BHA destroyed spore organelles and damaged cell membrane integrity (the electrical conductivity of conidia treated with 1/2 × Minimum inhibitory concentration (MIC), MIC and MFC was 48.37%, 64.79% and 97.95%, respectively), further resulting in reduced pathogenicity and virulence. In summary, this study provides evidence that BHA has the potential to be a valuable alternative for controlling citrus anthracnose in the future.

Early-Life Exposure to the Mycotoxin Fumonisin B1 and Developmental Programming of the Ovary of the Offspring: The Possible Role of Autophagy in Fertility Recovery.

Mycotoxins are produced by more than one hundred fungi and produce secondary metabolites that contaminate various agricultural commodities, especially rice and corn. Their presence in the food chain is considered a serious problem worldwide. In recent years, a link between exposure to mycotoxins and impaired fertility has been suggested. Consequently, it has become vital to investigate the interactive effects of these mycotoxins on ovarian function. In this study, we investigated the intergenerational effects of the mycotoxin fumonisin B1 (FB1) on ovarian structure and function. Virgin Wistar albino female rats were separated into control and FB1 treatment groups and examined from day 6 of pregnancy until delivery (20 and 50 mg/kg b.w./day). The obtained female rats of the first (F1) and second generations (F2) were euthanized at 4 weeks of age, and ovary samples were collected. We found that the ovary weight index increased with the high dose of the treatment (50 mg/kg b.w./day) among both F1 and F2, in a manner similar to that observed in polycystic ovary syndrome. As expected, FB1 at a high dose (50 mg/kg b.w.) reduced the number of primordial follicles in F1 and F2, leading to an accelerated age-related decline in reproductive capacity. Moreover, it reduced the fertility rate among the F1 female rats by affecting follicle growth and development, as the number of secondary and tertiary follicles decreased. Histopathological changes were evidenced by the altered structures of most of the growing follicle oocytes, as revealed by a thinning irregular zona pellucida and pyknosis in granulosa cells. These findings are concomitant with steroidogenesis- and folliculogenesis-related gene expression, as evidenced by the decrease in CYP19 activity and estrogen receptor beta (ESR2) gene expression. Additionally, GDF-9 mRNA levels were significantly decreased, and IGF-1 mRNA levels were significantly increased. However, the results from the ovaries of the F2 treatment groups were different and unexpected. While there was no significant variation in CYP19 activity compared to the control, the ESR2 significantly increased, leading to stereological and histopathological changes similar to those of the control, except for some altered follicles. The hallmark histological feature was the appearance of vacuolar structures within the oocyte and between granulosa cell layers. Interestingly, the autophagic marker LC3 was significantly increased in the F2 offspring, whereas this protein was significantly decreased in the F1 offspring. Therefore, we suggest that the promotion of autophagy in the ovaries of the F2 offspring may be considered a recovery mechanism from the effect of prenatal FB1 exposure. Thus, autophagy corrected the effect of FB1 during the early life of the F1 female rats, leading to F2 offspring with ovarian structure and function similar to those of the control. However, the offspring, treated female rats may experience early ovarian aging because their ovarian pool was affected.

Optical Diffraction Tomography and Raman Confocal Microscopy for the Investigation of Vacuoles Associated with Cancer Senescent Engulfing Cells.

Wild-type p53 cancer therapy-induced senescent cells frequently engulf and degrade neighboring ones inside a massive vacuole in their cytoplasm. After clearance of the internalized cell, the vacuole persists, seemingly empty, for several hours. Despite large vacuoles being associated with cell death, this process is known to confer a survival advantage to cancer engulfing cells, leading to therapy resistance and tumor relapse. Previous attempts to resolve the vacuolar structure and visualize their content using dyes were unsatisfying for lack of known targets and ineffective dye penetration and/or retention. Here, we overcame this problem by applying optical diffraction tomography and Raman spectroscopy to MCF7 doxorubicin-induced engulfing cells. We demonstrated a real ability of cell tomography and Raman to phenotype complex microstructures, such as cell-in-cells and vacuoles, and detect chemical species in extremely low concentrations within live cells in a completely label-free fashion. We show that vacuoles had a density indistinguishable to the medium, but were not empty, instead contained diluted cell-derived macromolecules, and we could discern vacuoles from medium and cells using their Raman fingerprint. Our approach is useful for the noninvasive investigation of senescent engulfing (and other peculiar) cells in unperturbed conditions, crucial for a better understanding of complex biological processes.

ClpP/ClpX deficiency impairs mitochondrial functions and mTORC1 signaling during spermatogenesis

Caseinolytic protease proteolytic subunit (ClpP) and caseinolytic protease X (ClpX) are mitochondrial matrix peptidases that activate mitochondrial unfolded protein response to maintain protein homeostasis in the mitochondria. However, the role of ClpP and ClpX in spermatogenesis remains largely unknown. In this study, we demonstrated the importance of ClpP/ClpX for meiosis and spermatogenesis with two conditional knockout (cKO) mouse models. We found that ClpP/ClpX deficiency reduced mitochondrial functions and quantity in spermatocytes, affected energy supply during meiosis and attenuated zygotene-pachytene transformation of the male germ cells. The dysregulated spermatocytes finally underwent apoptosis resulting in decreased testicular size and vacuolar structures within the seminiferous tubules. We found mTORC1 pathway was over-activated after deletion of ClpP/ClpX in spermatocytes. Long-term inhibition of the mTORC1 signaling via rapamycin treatment in vivo partially rescue spermatogenesis. The data reveal the critical roles of ClpP and ClpX in regulating meiosis and spermatogenesis.

Dietary coriander (Coriandrum sativum L) oil improves growth, nutrient utilization, antioxidant status, tissue histomorphology and reduces omega-3 fatty acid production in Nile tilapia (Oreochromis niloticus)

Coriander (Coriandrum sativum L.) is recently emerging as an antimicrobial growth promoter in aquafeed formulations. To assess the effect of coriander seed oil in the feed of Nile tilapia five iso-nitrogenous (crude protein 40%) and iso-lipidic (crude lipid 7%) diets were prepared with graded levels of coriander oil i. e., 0, 5, 10, 15 and 20 g/Kg referred as C0, C5, C10, C15 and C20, respectively. The fish were fed with the experimental diets twice daily for a period of 60 days in triplicate. After the feeding experiment, there observed a linear pattern in weight gain with the dietary coriander oil levels. The expression of hepatic insulin like growth factor 1 gene increased significantly in coriander oil supplemented groups in dose dependent manner in hepatic tissues of tilapia. The viscerosomatic and hepatosomatic index decreased significantly in the coriander oil treated groups. The crude protein contents increased significantly in the experimental groups; however, it did not vary significantly in between the groups. The crude lipid contents increased significantly in the last three groups (C10, C15 and C20) compared to C0 and C5. The serum glucose content decreased significantly in the last three groups i.e., C10, C15 and C20 coriander fed groups compared to C0. Whereas, the HDL level increased significantly in the 20 g/Kg treatment group compared to rest of the groups. Catalase activity increased significantly in the liver tissue of coriander oil treated groups compared to control. The liver histomorphology showed signs of storage of carbohydrate or lipid reserves in the form of vacuoles in the control group. However, in the coriander supplemented groups there was a reduction in hepatic vacuolar structures. The appearance of intestinal villi was enriched in coriander oil supplemented groups via branching. The Δ6/Δ5 desaturase increased significantly in the experimental groups compared to control. The Δ4 desaturase and elongase decreased significantly beyond 6.3% and 11.7% of coriander oil in the diet, respectively. There observed a significant increase in the percentage of saturated fatty acids in dose dependent manner. However, the unsaturated fatty acids including omega-3 fatty acids decreased significantly with an increase dose of coriander oil in feed of Nile tilapia. It can be concluded from the above study that coriander essential oil can be used for better growth performance, intestinal nutrient absorption, antioxidant activity and decreasing hyperglycemia in Nile tilapia. However, higher inclusion level (above 5 g/Kg) led to decrease in the wholebody omega-3 fatty acid production.

Qirong Tablets inhibits apoptosis of ovarian granulosa cells via PI3K/Akt/ HIF-1 signaling pathway

This study aims to observe the effect and explore the mechanism of Qirong Tablets in the treatment of premature ovarian insufficiency(POI) in mice via the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/hypoxia inducible factor 1(HIF-1) signaling pathway. Sixty SPF female BALB/c mice were randomly divided into normal group, model group, positive control group, Qirong Tablets low-, medium-and high-dose group. The normal group was intraperitoneally injected with the same amount of normal saline, and the other groups were intraperitoneally injected with cyclophosphamide 120 mg·kg~(-1)·d~(-1) once to establish a POI animal model. After the model was successfully established, the low-, medium-and high-dose groups of Qirong Tablets were administered orally with 0.6, 1.2, 2.4 mg·kg~(-1)·d~(-1) respectively. The positive control group was given 0.22 mg·kg~(-1)·d~(-1) Clementine Tablets by intragastric administration, and the normal group and model group were given intragastric administration with the same amount of normal saline, and the treatment was 28 d as a course of treatment. After drug intervention, enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of estradiol(E_2), follicle-stimulating hormone(FSH), luteinizing hormone(LH), and anti-mullerian hormone(AMH) in peripheral blood, and hematoxylin-eosin(HE) staining to observe the ovarian tissue. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay was used to detect the apoptosis of granulosa cells, and Western blot to determine the expression levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, PI3K, Akt, and HIF-1. Compared with the normal group, the modeling of POI caused loose or destroyed ovarian tissue with vacuolar structures, edema and fibrosis in the ovarian interstitium, disordered or loose arrangement of granulosa cells, and reduced normal follicles. Compared with the model group, drug interventions restored the ovarian tissue and follicles at all the development stages and reduced atretic follicles. Compared with the normal group, the modeling of POI lowered the serum level of E_2 and AMH(P<0.01), and elevated the level of FSH and LH(P<0.01). Compared with the model group, high-dose Qirong Tablets elevated the levels of E_2 and AMH(P<0.05), and lowered the levels of FSH and LH(P<0.05). Compared with the normal group, the modeling of POI up-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 and down-regulated the protein level of Bcl-2 in the ovarian tissue(P<0.01). Compared with the model group, low-, medium-, and high-dose Qirong Tablets down-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 proteins and up-regulated the protein level of Bcl-2 in the ovarian tissue(P<0.05). In conclusion, Qirong Tablets can up-regulate the expression Bcl-2, down-regulate the expression of Bax and caspase-3 in POI mice. Qirong Tablets may inhibit the apoptosis of follicular granulosa cells in mice, thereby delaying ovarian aging, improving reproductive axis function, and strengthening ovarian reserve capacity, which may be associated with the inhibition of PI3K/Akt/HIF-1 pathway.

Machine perfusion preservation with hemoglobin based oxygen vesicles alleviate ultrastructural damages in porcine liver donated after cardiac death.

Midthermic machine perfusion (MMP) of post-circulatory arrest donor liver grafts has the advantage of preserving the functional ultrastructure of hepatocytes in donor grafts. It was reported that oxygenation during MMP reduces portal venous resistance and increases bile production. The MMP with hemoglobin-based oxygen vesicles (HbV) keeps the lower aspartate aminotransferase level (an indicator of liver injury) and maintains the functional ultrastructure of mitochondria in the hepatocytes. To evaluated differences of ultrastructural damages in donor livers between the MMP with and without HbV, porcine liver grafts after 60 min of warm ischemia were perfused at 22°C for 4 h with or without HbV, and a part of liver grafts were analyzed by transmission electron microscopy (TEM) and osmium-maceration scanning electron microscopy (OM-SEM). The remaining grafts were perfused with autologous blood at 38°C for 2 h in an isolated liver reperfusion model (IRM) that mimics the inside of the body after transplantation, and then analyzed by TEM and OM-SEM. Hepatocytes after MMP had small round mitochondria with rod-shaped cristae and reticulovesicular rough endoplasmic reticulum (rER) in both HbV(+) and HbV(-) livers. After IRM of HbV(+) livers, the well-developed lamellar rER was often found in hepatocytes. Liver sinusoidal endothelial cells (LSECs) after MMP contained some large vacuolar structures containing amorphous garbage in the cytoplasm, and their size along with appearance frequency were smaller and lower, respectively, in HbV(+) livers than HbV(-). Oxygenation during the MMP by using HbV suppressed the ultrastructural damages in donor livers, in particular for the LSECs. RESEARCH HIGHLIGHTS: Liver sinusoidal endothelial cells after midthermic machine perfusion had large vacuolar organelles with amorphous garbage. Oxygenation during the perfusion made them less and smaller, ultrastructurally supporting its utility.

Ferroptosis is involved in testicular injury induced by TDCIPP in adolescent male mice

Objective: To investigate the role of ferroptosis in testicular injury in adolescent male mice induced by TDCIPP. Methods: In December 2021, 30 healthy 3-week-old male C57BL/6 mice, with a body weight of (13±2) g, were selected and fed adaptive for one week. They were divided into control group, low-dose group, medium-dose group, high-dose group and iron death inhibitor group according to a random number table, with 6 mice in each group. Mice in low, medium and high dose groups were treated with 5, 25 and 125 mg/ (kg·d) TDCIPP for 28 days, respectively, while the control group was treated with the same amount of corn oil for 28 days. The iron death inhibitor group was given 125 mg/ (kg·d) TDCIPP intragastric administration for 28 days, and 30 mg/kg DFO saline solution was intraperitoneally injected three times a week. After the treatment, the mice were killed, the epididymis was separated, and sperm count was performed. HE staining was used to observe the morphological changes of mouse testis, and iron content in testis was detected by tissue iron detection kit. The level of reactive cxygen species, MDA content, and the mitochondrial membrane potential level of mice were detected. Western blot analysis of testicular glutathione peroxidase (GPX4) and internal cyclooxygenase-2 (COX2) protein expression. Results: Compared with the control group, the spermatogenic cells in the testes of mice treated with medium-and high-dose of TDCIPP were disorderly arranged, showing a vacuolar structure. the number of sperm in the epididymis was significantly reduced (P=0.009, 0.004), while the sperm deformity rate was significantly increased (P=0.010, 0.000). Moreover, the content of ROS, iron ion and MDA in the testes increased significantly (P<0.05), and the mitochondrial membrane potential of mouse testicular cells decreased significantly (P<0.05). The expression of GPX4 proteins decreased (P<0.05). while the expression of COX2 increased significantly (P<0.01). Compared with high-dose group group, spermatogenic cells in ferroptosis inhibitor group were closely arranged and normal, and ROS and Fe contents in testicular tissue were significantly decreased (P<0.01) ; GPX4 protein expression was significantly increased while COX2 protein expression was significantly decreased (P<0.05) . Conclusion: Ferroptosis is involved in TDCIPP-induced testicular damage in male pubertal mice.

P-001 ClpX is required in maintaining mitochondrial functions during meiosis and spermatogenesis

Abstract Study question What is the role of ClpX in spermatogonia differentiation and spermatogenesis? Summary answer ClpX is required to maintain normal mitochondrial functions in spermatocytes, deficiency of ClpX resulted in meiosisarrest and diruption of spermatogenesis in mice. What is known already ClpX is mitochondria-specific quality control protease, it maintains proteostasis via degrading misfolded or damagedproteins in mitochondrial matrix. ClpX is a AAA+ protease that uses the energy of ATP binding and hydrolysis to degradeunfolded or misfolded proteins. ClpX is reported working as a complex with ClpP, the complex consists of a hexamer ofClpX and a tetradecamer of ClpP. ClpX recognizes the protein substrates via binding with their unstructured peptidesequences, termed as degradation tags or recognition signals. The fragments of the cleaved polypeptides can then exitthe chamber and be further degraded. Study design, size, duration Study design: we generated a germ cell specific ClpX knockout mice line and evaluated size, weight, inner structure oftestis tissue. We also applied biochemical techniques and high-throughput sequencing techniques to investigate themechanism of ClpX in regulating spermatogenesis. Size: more than 50 mice were used in this study, including the control mice. Duration: 2 years Participants/materials, setting, methods Participants/materials: we generated a ClpX conditional knockout (cKO) mice line by CRISPR-gene editing and Cre-LoxP system, which specifically knockout ClpX in the germ cells of male mice. Setting: the siblings of ClpX WT mice and ClpX cKO mice were compared in various of experiments. Mehods: we performed morphology comparement to check the weight and size of tetes in the control and ClpX cKOmice, and applied immunofluorescence, western blot, histological study, high-throughput sequencing andpharmocological treatment. Main results and the role of chance We found ClpX deficiency reduced mitochondrial functions and quantity in spermatocytes, affected energy supply duringmeiosis and attenuated zygotene-pachytene transformation of the male germ cells. The affected spermatocytesexhibited disorder of chromosome synapsis and cross-over events. Their telomeres failed to attach to the nuclearenvelop, which was associated with failure of α-tubulin formation in the ClpX deficient spermatocytes. The dysregulatedspermatocytes finally underwent apoptosis resulting in decreased testicular size and vacuolar structures within theseminiferous tubules. Both transcriptome analysis and m6A-methylation sequencing analysis highlighted dysregulationof metabolic pathways including the mTOR signaling in the isolated spermatocytes. We confirmed over-activation of themTORC1 pathway with increased expression of phosphorylated S6 and 4EBP after deletion of ClpX in spermatocytes.Long-term inhibition of the mTORC1 signaling via rapamycin treatment in vivo partially rescue spermatogenesis in theseminiferous tubules with much less apoptotic germ cells and formation of late stage of meiotic germ cells, such asround spermatids and elongated spermatozoon. The data reveal the novel roles of ClpX in regulating meiosis andspermatogenesis. Limitations, reasons for caution This study is based on animal experiments, thus, these results only indicated the important roles of ClpX in micespermatogenesis rather than human beings. Clinical data has not yet found a Clpx gene mutation in reproductivedisease. Wider implications of the findings Clinical report has already found the mutation in ClpP can induce autosomal recessive Perrault syndrome, while themutation of ClpX has not been reported yet from clinical data. Our resutls demonstrated severe effect onspermatogenesis after knockingout of ClpX, this might give some insights to the clinic. Trial registration number not applicable

HSP90α is needed for the survival of rod photoreceptors and regulates the expression of rod PDE6 subunits

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the stability of a small set of proteins essential in various cellular pathways. Cytosolic HSP90 has two closely related paralogs: HSP90α and HSP90β. Due to the structural and sequence similarities of cytosolic HSP90 paralogs, identifying the unique functions and substrates in the cell remains challenging. In this article, we assessed the role of HSP90α in the retina using a novel HSP90α murine knockout model. Our findings show that HSP90α is essential for rod photoreceptor function but was dispensable in cone photoreceptors. In the absence of HSP90α, photoreceptors developed normally. We observed rod dysfunction in HSP90α knockout at 2months with the accumulation of vacuolar structures, apoptotic nuclei, and abnormalities in the outer segments. The decline in rod function was accompanied by progressive degeneration of rod photoreceptors that was complete at 6months. The deterioration in cone function and health was a "bystander effect" that followed the degeneration of rods. Tandem mass tag proteomics showed that HSP90α regulates the expression levels of <1% of the retinal proteome. More importantly, HSP90α was vital in maintaining rod PDE6 and AIPL1 cochaperone levels in rod photoreceptor cells. Interestingly, cone PDE6 levels were unaffected. The robust expression of HSP90β paralog in cones likely compensates for the loss of HSP90α. Overall, our study demonstrated the critical need for HSP90α chaperone in the maintenance of rod photoreceptors and showed potential substrates regulated by HSP90α in the retina.

Effects of low dose radiation on behavior rhythm of zebrafish (Danio rerio)

Biological rhythm refers to the internal regulation of various life activities of an organism, which are determined by the specific time structure sequences of each individual. Behavior rhythm is the most intuitive embodiment of biological rhythm. To study the effect of low dose radiation on behavioral rhythm, zebrafish (Danio rerio) was used as a model organism in this study. The early embryos of zebrafish were irradiated at doses of 0.01, 0.1, and 1 Gy to observe the changes in zebrafish development, circadian rhythm, key clock genes, related RNA and protein expression, and melatonin. The results revealed that 0.1 and 1 Gy radiation could lead to different degrees of telencephalic nerve cell apoptosis and the formation of vacuolar structures. 0.1 and 1 Gy radiation could reduce the hatching rate of zebrafish embryos at 72 hpf and delay embryo hatching. The analysis of circadian behavior at 120 hpf demonstrated that 1 Gy dose of radiation altered the circadian rhythm of zebrafish, as well as decreased the distance, amplitude, and phase of movement. RT-PCR analysis of the key clock genes (bmal1b, clock1a, per1b, per2, cry2, and nr1d1) involved in regulating circadian rhythm was performed. The results showed that 1 Gy radiation could interfere with the expression of clock genes in zebrafish embryos and upregulate bmal1b, clock1a, and per1b. Western blot experiments further verified the protein expression of key clock genes, bmal1b and clock. Detection of melatonin secretion at different time points over 24 h showed that radiation doses of 0.1 and 1 Gy could increase melatonin secretion. Based on these findings, it is speculated that a certain dose of radiation may affect melatonin secretion, which impacts the telencephalon structure and ontogeny of zebrafish, delays hatching, and changes the circadian rhythm. This effect is thought to be achieved through upregulating the expression of circadian rhythm genes, clock1a and per1b and related proteins, which may be responsible for the abnormal circadian rhythm caused by radiation.

Fatty Acyl Coenzyme A Synthetase Fat1p Regulates Vacuolar Structure and Stationary-Phase Lipophagy in Saccharomyces cerevisiae.

During yeast stationary phase, a single spherical vacuole (lysosome) is created by the fusion of several small ones. Moreover, the vacuolar membrane is reconstructed into two distinct microdomains. Little is known, however, about how cells maintain vacuolar shape or regulate their microdomains. Here, we show that Fat1p, a fatty acyl coenzyme A (acyl-CoA) synthetase and fatty acid transporter, and not the synthetases Faa1p and Faa4p, is essential for vacuolar shape preservation, the development of vacuolar microdomains, and cell survival in stationary phase of the yeast Saccharomyces cerevisiae. Furthermore, Fat1p negatively regulates general autophagy in both log- and stationary-phase cells. In contrast, Fat1p promotes lipophagy, as the absence of FAT1 limits the entry of lipid droplets into the vacuole and reduces the degradation of liquid droplet (LD) surface proteins. Notably, supplementing with unsaturated fatty acids or overexpressing the desaturase Ole1p can reverse all aberrant phenotypes caused by FAT1 deficiency. We propose that Fat1p regulates stationary phase vacuolar morphology, microdomain differentiation, general autophagy, and lipophagy by controlling the degree of fatty acid saturation in membrane lipids. IMPORTANCE The ability to sense environmental changes and adjust the levels of cellular metabolism is critical for cell viability. Autophagy is a recycling process that makes the most of already-existing energy resources, and the vacuole/lysosome is the ultimate autophagic processing site in cells. Lipophagy is an autophagic process to select degrading lipid droplets. In yeast cells in stationary phase, vacuoles fuse and remodel their membranes to create a single spherical vacuole with two distinct membrane microdomains, which are required for yeast lipophagy. In this study, we discovered that Fat1p was capable of rapidly responding to changes in nutritional status and preserving cell survival by regulating membrane lipid saturation to maintain proper vacuolar morphology and the level of lipophagy in the yeast S. cerevisiae. Our findings shed light on how cells maintain vacuolar structure and promote the differentiation of vacuole surface microdomains for stationary-phase lipophagy.

Preparation of a biobased polyelectrolyte complex from chitosan and sodium carboxymethyl cellulose and its antibacterial characteristics

Using chitosan (CTS) and sodium carboxymethyl cellulose (CMCNa) as raw biobased materials, polyelectrolyte complex (PEC), which is the product of strong electrostatic interaction between two bio-based polyelectrolytes with opposite charges, was attempted to prepare. To enlarge the reactive contact area between CTS and CMCNa, the crosslinked vacuolar structure of PEC was prepared without addition of cross-linked agent. The preparation conditions had a significant impact on the yield of PEC and the bibulous rate of PEC. When pH, mass ratio of CMC-Na-to-CTS, stirring speed and reaction system temperature were 5, 1:2 [(1 wt% CMCNa, 2 wt% CTS), CMC-Na:CTS = 1:1 (v/v)], 800 rpm, 2 min and 25 °C, the yield of PEC reached 71.2 %. The prepared PEC was characterized by XRD and FT-IR. Afterwards, the antibacterial performance of PEC was examined. The prepared PEC had certain bacteriostatic effect on gram-positive and gram-negative bacteria. The bacteriostasis ratios of PEC against Escherichia coli and Staphylococcus aureus were 18.7 % and 31.3 %, respectively. By controlling the combination parameters of the preparation system, an effective strategy was successfully developed for preparation of biobased PEC with bacteriostatic and crosslinked vacuolar structure through simple physical blending without the application of additional crosslinker.

Lack of Low-affinity Phosphate Transporter Pho91 Alters Lipid Metabolism in Yeast Saccharomyces cerevisiae

Objective: The current study tries to elucidate the impact of low-affinity Pi transporter Pho91 (using pho91Δ cells) on lipid metabolism and cellular organelles morphology (mitochondria and vacuole) in yeast Saccharomyces cerevisiae. Methods: The lipid profile was performed using thin layer chromatography (TLC), and the membrane defect was determined using DiOC6 staining, lipid droplets (LDs) were observed by Nile red staining, mitochondrial morphology was observed using aconitase 1 GFP, vacuolar morphology was studied by FM4-64 staining and were performed with the aid of laser scanning fluorescent microscope. Findings: Pho91 is a low-affinity phosphate (Pi) transporter in the vacuoles that functions under Pi-rich conditions, but its role in lipid metabolism is largely unknown. In this study, we used defined synthetic complete media (SC). The deletion of Pho91 depicted a moderate growth defect but increased the major phospholipids PC, PE, and PI. Alterations in the membrane phospholipids resulted in defective membrane morphology. In pho91 mutant (pho91Δ), the neutral lipids TAG and SE were increased and stored as LDs. The LD numbers were increased in pho91Δ cells than in WT cells. Altered phospholipids also defective mitochondrial morphology and enlarged vacuoles in pho91 deletion. Novelty: In the absence of low-affinity Pi transporter pho91, it increases phospholipid, neutral lipid levels, LD numbers, and impacted mitochondria along with vacuolar structures. Keywords: Phosphate transporter; Low affinity; Phospholipids; Neutral lipids; Lipid droplets

Honokiol reduces cadmium-induced oxidative injury and endosomal/lysosomal vacuolation via protecting mitochondrial function in quail (Coturnix japonica) liver tissues

Cadmium (Cd) pollution in environment is toxic to birds. This study aimed to assess antagonistic effect of honokiol (HNK) on Cd-induced quail (Coturnix japonica) liver tissue damage and Cd-induced vacuolation in hepatocytes. We found that HNK alleviated Cd-induced liver pathological damage marked by elevated serum liver biochemical indicators, disordered antioxidant levels and trace elements in quails. HNK reduced Cd-induced liver cell apoptosis as assessed by western blotting and TUNEL staining. The ultrastructure of hepatocytes under transmission electron microscope revealed that Cd induced mitochondrial damage in addition to abnormal enlargement and increased vacuolar structure of cells. Mitochondrial damage and vacuolization were reduced in the HNK + Cd group. Cd induced an increase in the levels of endosomal/lysosomal-related genes, while HNK treatment reversed this effect. Finally, we demonstrated that vacuolation in buffalo rat liver 3A (BRL 3A) cells occurred primarily due to Cd-induced oxidative stress damage that reduces mitochondrial ATP content and indirectly led to dysfunction of ATP-dependent lipid kinase PIKfyve complex. In summary, we are the first to report that Cd induces abnormal enlargement of endosome/lysosomes in quail liver cells and HNK alleviated this phenomenon by reducing mitochondrial damage and increasing intracellular ATP level. This study demonstrated the toxic effect of Cd pollution on birds and how HNK mitigated these effect at the cellular level. Overall, more research on Cd pollution and HNK use in animal husbandry is warranted.

Exploring the synergic mechanism of Ligusticum striatum DC. and borneol in attenuating BMECs injury and maintaining tight junctions against cerebral ischaemia based on the HIF-1α/VEGF signalling pathway

Ethnopharmacological relevanceLigusticum striatum DC., also known as Ligusticum chuanxiong Hort. (LCH), is widely used in China for its excellent effect in ischaemic stroke (IS) patients, and borneol (BO) has been confirmed to maintain the blood‒brain barrier (BBB) after stroke. They are often used as a combination in the prescriptions of IS patients. Although the advantage of their combined treatment in improving brain ischaemia has been verified, their synergistic mechanism on BBB maintenance is still unclear. Aim of the studyThis study was designed to evaluate the synergistic effect of maintaining the BBB between LCH and BO against IS and to further explore the potential mechanism. Materials and methodsAfter primary mouse brain microvascular endothelial cells (BMECs) were extracted and identified, the duration of oxygen-glucose deprivation (OGD) and the doses of LCH and BO were optimized. Then, the cells were divided into five groups: control, model, LCH, BO, and LCH + BO. Cell viability, injury degree, proliferation and migration were detected by CCK-8, LDH, EdU and wound-healing assays, respectively. Hoechst 33342 staining was adopted to detect the apoptosis rate, and western blotting was employed to observe the expressions of Bax, Bcl-2, caspase-3 and cleaved caspase-3. The TEER value and NaF permeability were measured to assess tight junction (TJ) function, while ZO-1, occludin and claudin-5 were also probed by western blotting. Moreover, the HIF-1α/VEGF pathway was observed to explore the underlying mechanism of BBB maintenance. In vivo, global cerebral ischaemia/reperfusion (GCIR) surgery was performed to establish an IS model. After treatment with LCH (200 mg/kg) and/or BO (160 mg/kg), histopathological structure and BMECs repair were observed by HE staining and immunohistochemistry of vWF. Meanwhile, TJ-associated proteins in vivo were also detected by western blotting. ResultsBasically, LCH and BO had different emphases. LCH significantly attenuated the vacuolar structure, nuclear pyknosis and neuronal loss of GCIR mice, while BO focused on promoting BMECs proliferation and angiogenesis and inhibiting the degradation of TJ-associated proteins in vivo after IS. Interestingly, their combination further enhanced these effects. OGD injury markedly reduced the viability, proliferation and migration of primary BMECs; decreased the ratio of Bcl-2/Bax, TEER value, and the expressions of ZO-1, occludin and claudin-5; induced LDH release and apoptosis; and increased the cleaved caspase-3/caspase-3 ratio and NaF permeability. Meanwhile, BO might be the main contributor to the combinative treatment in ameliorating OGD-induced damage of BMECs and degradation of TJ-related proteins, and the potential mechanism might be involved in upregulating the HIF-1α/VEGF signalling pathway. Although LCH showed no obvious improvement, it could enhance the therapeutic effect of BO. Interestingly, their combination even produced some new improvements, including the reduction of cleaved caspase-3 and increase in TEER value, none of which were exhibited in their monotherapies. ConclusionsLCH and BO exhibited complementary therapeutic features in alleviating cerebral ischaemic injury by inhibiting BMECs apoptosis, maintaining the BBB and attenuating the loss of neurons. LCH preferred to protect ischaemic neurons, while BO played a key role in protecting BMECs, maintaining the BBB and TJs by activating the HIF-1α/VEGF signalling pathway.