Global profiling of protein lactylation in Caenorhabditis elegans.

Lactylation, as a novel posttranslational modification, is essential for studying the functions and regulation of proteins in physiological and pathological processes, as well as for gaining in-depth knowledge on the occurrence and development of many diseases, including tumors. However, few studies have examined the protein lactylation of one whole organism. Thus, we studied the lactylation of global proteins in Caenorhabditis elegans to obtain an in vivo lactylome. Using an MS-based platform, we identified 1836 Class I (localization probabilities > 0.75) lactylated sites in 487 proteins. Bioinformatics analysis showed that lactylated proteins were mainly located in the cytoplasm and involved in the tricarboxylic acid cycle (TCA cycle) and other metabolic pathways. Then, we evaluated the conservation of lactylation in different organisms. In total, 41 C. elegans proteins were lactylated and homologous to lactylated proteins in humans and rats. Moreover, lactylation on H4K80 was conserved in three species. An additional 238 lactylated proteins were identified in C. elegans for the first time. This study establishes the first lactylome database in C. elegans and provides a basis for studying the role of lactylation.

Global profiling of lysine lactylation in human lungs.

Lactate is closely related to various cellular processes, such as angiogenesis, responses to hypoxia, and macrophage polarization, while regulating natural immune signaling pathways and promoting neurogenesis and cognitive function. Lysine lactylation (Kla) is a novel posttranslational modification, the examination of which may lead to new understanding of the nonmetabolic functions of lactate and the various physiological and pathological processes in which lactate is involved, such as infection, tumorigenesis and tumor development. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), researchers have identified lactylation in human gastric cancer cells and some other species, but no research on lactylation in human lungs has been reported. In this study, we performed global profiling of lactylation in human lungs under normal physiological conditions, and 724 Kla sites in 451 proteins were identified. After comparing the identified proteins with those reported in human lactylation datasets, 141 proteins that undergo lactylation were identified for the first time in this study. Our work expands the database on human lactylation and helps advance the study on lactylation function and regulation under physiological and pathological conditions.

New insights into the pathogenesis of Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) is characterized by defects of multiple tissue-specific lysosome-related organelles (LROs), typically manifesting with oculocutaneous albinism or ocular albinism, bleeding tendency, and in some cases with pulmonary fibrosis, inflammatory bowel disease or immunodeficiency, neuropsychological disorders. Eleven HPS subtypes in humans and at least 15 subtypes in mice have been molecularly identified. Current understanding of the underlying mechanisms of HPS is focusing on the defective biogenesis of LROs. Compelling evidences have shown that HPS protein-associated complexes (HPACs) function in cargo transport, cargo recycling, and cargo removal to maintain LRO homeostasis. Further investigation on the molecular and cellular mechanism of LRO biogenesis and secretion will be helpful for better understanding of its pathogenesis and for the precise intervention of HPS.

Byproduct Surplus: Lighting the Depreciative Europium in China's Rare Earth Boom.

Europium (Eu) is often regarded as a critical mineral due to its byproduct nature, importance to lighting technologies, and global supply concentration. However, the existing indicator-based criticality assessments have limitations to capture Eu's supply chain information and thus fall short of reflecting its true criticality. This study quantified the flows and stocks of Eu in mainland China from 1990 to 2018. Results show that: (1) China's Eu demand decreased by 75% from 2011 to 2018, as a result of the lighting technology transition from fluorescent lamps to light-emitting diodes, which significantly reduced Eu's importance; (2) the supply of Eu mined as a byproduct kept increasing together with the growing rare earth production, which caused a substantial supply surplus being ≈1900 t by 2018; (3) despite the leading role of China in global Eu production, Eu mined in China was exported mainly in the form of intermediate and final products, and ≈90% Eu embedded in domestically produced final products was used for export recently. This study indicates that Eu's criticality is not as severe as previously assessed and highlights the necessity of material flow analysis for a holistic and dynamic view on the entire supply chain of critical minerals.

Inhibition of N-WASP affects actin-mediated cytokinesis during porcine oocyte maturation.

N-WASP is the mammalian ortholog of WASP which is an actin nucleation promoting factor and has been reported to regulate actin nucleation and polymerization for multiple cell activities. However, the expression and functions of N-WASP in porcine oocytes are still unclear. In this study, we showed that N-WASP expressed at all stages during porcine oocyte maturation, and immunofluorescence staining indicated that N-WASP mainly accumulated at the cortex in different stages of meiosis. Inhibition of N-WASP activity by Wiskostatin significantly decreased the rate of first polar body extrusion and disturbed the cell cycle progression of porcine oocytes. Further analysis indicated that cortical actin distribution was interfered by N-WASP inhibition, and this might be through its regulatory roles on the expression and localization of ARP2, a key component of actin nucleator Arp2/3 complex. Moreover, the expression of N-WASP decreased after ROCK activity inhibition, indicating a ROCK-N-WASP-ARP2/3 pathway for actin assembly in porcine oocytes. Taken together, these results suggest that N-WASP is critical for the regulation of actin filaments for cytokinesis during porcine oocyte maturation.

[Laboratory design and practice for undergraduates: Using RNAi to modulate gene expression].

RNA interference is a gene silencing phenomenon mediated by short double-stranded RNAs, which has become a widely used research technology for reverse genetics. In order to make students understand the technology better, the students were required to select target genes, to design small interfering RNAs (siRNAs) and primers, and then to test the effect of gene silencing mediated by siRNAs. Taking the fifth group in 2018 as an example, Mus musculus acyl-CoA synthetase long-chain family member 1 (Acsl1) was selected as the target gene, two pairs of siRNAs targeting Acsl1 mRNA were designed and transfected into 3T3-L1 by electroporation, then the total RNAs were extracted and synthesized to cDNA, and the expression levels of mRNAs were finally tested by relative quantitative PCR. The results showed that both pairs of siRNAs had more than 60% silencing effects. In the past three years, about 83% of the students completed all the experiments successfully and screened out at least a pair of effective siRNA. This teaching practice for undergraduates enhances students' understanding of RNA interference principle and technology, and exercises students' lab experience and scientific research ability.

ER stress mediated degradation of diacylglycerol acyltransferase impairs mitochondrial functions in TMCO1 deficient cells.

In eukaryotic cells, Endoplasmic Reticulum (ER) is an interconnected membranous organelle and plays important roles in protein synthesis and lipid metabolism. We have previously demonstrated that TMCO1 is an ER Ca2+ channel actively preventing ER Ca2+ overloading. Recently, we also found that TMCO1 deficiency in mouse granulosa cells (GCs) caused abnormal Ca2+ signaling, ER stress and enhanced reactive oxygen species (ROS). In this study, we further examined the roles of TMCO1 in lipid metabolism and mitochondrial functions. Intriguingly, we found that TMCO1 deletion reduced the number of lipid droplets (LDs) and the content of triglyceride (TG), which was due to ER stress associated degradation (ERAD) of the important enzyme in catalyzing TG synthesis, diacylglycerol acyltransferase 2 (DGAT2). Hypofunction in transforming non-esterification fatty acid (NEFA) to TG caused NEFA deposit, a potential risk of mitochondrial dysfunction. Furthermore, in TMCO1 deficient cells, mitochondria volume decreased and inefficient oxidative phosphorylation was detected, which underlined enhanced mitophagy and impaired mitochondrial functions. Taken these data together, we for the first time revealed the role of TMCO1 in regulating lipid-metabolism and mitochondrial function. This study may provide new insights into understanding TMCO1 defect syndrome.

EB1 traps STIM1 and regulates local store-operated Ca2+ entry.

STIM1 activates store-operated Ca2+ entry when Ca2+ in the endoplasmic reticulum (ER) is depleted. In this issue, Chang et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201711151) demonstrate that EB1 traps STIM1 at dynamic contacts between the ER and microtubule plus ends, delaying STIM1 translocation to ER-plasma membrane junctions and preventing Ca2+ overload.

[Establishment of A Serum-Free Culture System Based on Heparin and Anti-CD16 Antibody for Expansion of Human Cord Blood NK Cells].

OBJECTIVE: To establish a novel method for ex vivo expansion of natural killer cells from human umbilical blood, so as to provide the basis for NK cell therapy. METHODS: Mononucleated cells from human umbilical blood were harvested and suspended in a serum-free medium containing 5% autologous plasma, recombinant human IL-15 (50 ng/ml) and hydrocortisone sodium succinate (5×10-8 mol/L) at a concentration of 1.5×106/ml, then the cells were seeded into flasks pre-coated with heparin sodium (100 U/cm2) or/and anti-human CD16 antibody (1 µg/cm2). After culture for 2 weeks, the cells were harvested and counted. Ratios of CD3-/CD56+ of the cells were determined by flow cytometry. MTT test was performed to assess the cytotoxicity against K562 cells with graded ratios of effector/target cells. RESULTS: In contrast to the cells in flasks without pre-coating, the attached colonies appeared predominantly within 1 week of culture from heparin- and antibody-coated groups. The cell numbers from the pre-coated groups were significantly higher than that of uncoated one after culture for 2 weeks. Furthermore, the ratios of CD3-/CD56+ cells were much higher in pre-coated groups, and that of the cells from flasks pre-coated with heparin and antibody were the highest (all the P values <0.01). MTT test showed that the cytotoxic activity of the cells stimulated by precoating were much more potent than that of the cells without the stimulation. CONCLUSION: Advantageous expansion of NK cells can be achieved by precoating with heparin and anti-CD16 antibody, and also by supplement with IL-15 and hydrocortisone into the media, so the umbilical NK cells with high purity and potent cytotoxicity can be obtained.

TMCO1 is essential for ovarian follicle development by regulating ER Ca2+ store of granulosa cells.

TMCO1 (transmembrane and coiled-coil domains 1) is an endoplasmic reticulum (ER) transmembrane protein that actively prevents Ca2+ stores from overfilling. To characterize its physiological function(s), we generated Tmco1-/- knockout (KO) mice. In addition to the main clinical features of human cerebrofaciothoracic (CFT) dysplasia spectrum, Tmco1-/- females manifest gradual loss of ovarian follicles, impaired ovarian follicle development, and subfertility with a phenotype analogous to the premature ovarian failure (POF) in women. In line with the role of TMCO1 as a Ca2+ load-activated Ca2+ channel, we have detected a supernormal Ca2+ signaling in Tmco1-/- granulosa cells (GCs). Interestingly, although spontaneous Ca2+ oscillation pattern was altered, ER Ca2+ stores of germinal vesicle (GV) stage oocytes and metaphase II (MII) arrested eggs were normal upon Tmco1 ablation. Combined with RNA-sequencing analysis, we also detected increased ER stress-mediated apoptosis and enhanced reactive oxygen species (ROS) level in Tmco1-/- GCs, indicating the dysfunctions of GCs upon TMCO1 deficiency. Taken together, these results reveal that TMCO1 is essential for ovarian follicle development and female fertility by maintaining ER Ca2+ homeostasis of GCs, disruption of which causes ER stress-mediated apoptosis and increased cellular ROS level in GCs and thus leads to impaired ovarian follicle development.

Profilin 1 plays feedback role in actin-mediated polar body extrusion in mouse oocytes.

Mammalian oocytes undergo several crucial processes during meiosis maturation, including spindle formation and migration and polar body extrusion, which rely on the regulation of actin. As a small actin-binding protein, profilin 1 plays a central role in the regulation of actin assembly. However, the functions of profilin 1 in mammalian oocytes are uncertain. To investigate the function of profilin 1 in oocytes, immunofluorescent staining was first used to examine profilin 1 localisation. The results showed that profilin 1 was localised around the meiotic spindles and was colocalised with cytoplasmic actin. Knockdown (KD) of profilin 1 with specific morpholino microinjection resulted in failure of polar body extrusion. This failure resulted from an increase of actin polymerisation both at membranes and in the cytoplasm. Furthermore, western blot analysis revealed that the expression of Rho-associated kinase (ROCK) and phosphorylation levels of myosin light chain (MLC) were significantly altered after KD of profilin 1. Thus, the results indicate that a feedback mechanism between profilin, actin and ROCK-MLC2 regulates actin assembly during mouse oocyte maturation.

The small GTPase CDC42 regulates actin dynamics during porcine oocyte maturation.

The mammalian oocyte undergoes an asymmetric division during meiotic maturation, producing a small polar body and a haploid gamete. This process involves the dynamics of actin filaments, and the guanosine triphosphatase (GTPase) protein superfamily is a major regulator of actin assembly. In the present study, the small GTPase CDC42 was shown to participate in the meiotic maturation of porcine oocytes. Immunofluorescent staining showed that CDC42 was mainly localized at the periphery of the oocytes, and accumulated with microtubules. Deactivation of CDC42 protein activity with the effective inhibitor ML141 caused a decrease in actin distribution in the cortex, which resulted in a failure of polar body extrusion. Moreover, western blot analysis revealed that besides the Cdc42-N-WASP pathway previously reported in mouse oocytes, the expression of ROCK and p-cofilin, two molecules involved in actin dynamics, was also decreased after CDC42 inhibition during porcine oocyte maturation. Thus, our study demonstrates that CDC42 is an indispensable protein during porcine oocyte meiosis, and CDC42 may interact with N-WASP, ROCK, and cofilin in the assembly of actin filaments during porcine oocyte maturation.

Inhibition of Rac1 GTPase activity affects porcine oocyte maturation and early embryo development.

Mammalian oocyte asymmetric division relies on the eccentric positioning of the spindle, resulting in the polar body formation. Small signaling G protein Rac1 is a member of GTPases, which regulates a diverse array of cellular events, including the control of cell growth, cytoskeletal reorganization, and the activation of protein kinases. However, effects of Rac1 on the porcine oocyte maturation and early embryo development are not fully understood. In present study we investigated the role of Rac1 in oocyte maturation and embryo cleavage. We first found that Rac1 localized at the cortex of the porcine oocytes, and disrupting the Rac1 activities by treating with NSC 23766 led to the failure of polar body emission. In addition, a majority of treated oocytes exhibited abnormal spindle morphology, indicating that Rac1 may involve into porcine oocyte spindle formation. This might be due to the regulation of Rac1 on MAPK, since p-MAPK expression decreased after NSC 23766 treatments. Moreover, we found that the position of most meiotic spindles in treated oocytes were away from the cortex, indicating the roles of Rac1 on meiotic spindle positioning. Our results also showed that inhibition of Rac1 activity caused the failure of early embryo development. Therefore, our study showed the critical roles of Rac1 GTPase on porcine oocyte maturation and early embryo cleavage.

TMCO1 Is an ER Ca(2+) Load-Activated Ca(2+) Channel.

Maintaining homeostasis of Ca(2+) stores in the endoplasmic reticulum (ER) is crucial for proper Ca(2+) signaling and key cellular functions. The Ca(2+)-release-activated Ca(2+) (CRAC) channel is responsible for Ca(2+) influx and refilling after store depletion, but how cells cope with excess Ca(2+) when ER stores are overloaded is unclear. We show that TMCO1 is an ER transmembrane protein that actively prevents Ca(2+) stores from overfilling, acting as what we term a "Ca(2+) load-activated Ca(2+) channel" or "CLAC" channel. TMCO1 undergoes reversible homotetramerization in response to ER Ca(2+) overloading and disassembly upon Ca(2+) depletion and forms a Ca(2+)-selective ion channel on giant liposomes. TMCO1 knockout mice reproduce the main clinical features of human cerebrofaciothoracic (CFT) dysplasia spectrum, a developmental disorder linked to TMCO1 dysfunction, and exhibit severe mishandling of ER Ca(2+) in cells. Our findings indicate that TMCO1 provides a protective mechanism to prevent overfilling of ER stores with Ca(2+) ions.

Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation.

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3 and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications.

RhoA-mediated MLC2 regulates actin dynamics for cytokinesis in meiosis.

During oocyte meiosis, the bipolar spindle forms in the central cytoplasm and then migrates to the cortex. Subsequently, the oocyte extrudes the polar body through two successive asymmetric divisions, which are regulated primarily by actin filaments. Myosin light chain2 (MLC2) phosphorylation plays pivotal roles in smooth muscle contraction, stress fiber formation, cell motility and cytokinesis. However, whether MLC2 phosphorylation participates in the oocyte polarization and asymmetric division has not been clarified. The present study investigated the expression and functions of MLC2 during mouse oocyte meiosis. Our result showed that p-MLC2 was localized in the oocyte cortex, with a thickened cap above the chromosomes. Meanwhile, p-MLC2 was also localized in the poles of spindle. Disruption of MLC2 activity by MLC2 knock down (KD) caused the failure of polar body extrusion. Immunofluorescent staining showed that a large proportion of oocytes arrested in telophase stage and failed to undergo cytokinesis after culturing for 12 hours. In the meantime, actin filament staining at oocyte membrane and cytoplasm were reduced in MLC2 KD oocytes. Finally, we found that the phosphorylation of MLC2 protein levels was decreased after disruption of RhoA activity. Above all, our data indicated that the RhoA-mediated MLC2 regulates the actin organization for cytokinesis during mouse oocyte maturation.

Adult neural progenitor cells from Huntington's disease mouse brain exhibit increased proliferation and migration due to enhanced calcium and ROS signals.

OBJECTIVES: Huntington's disease (HD) is an inherited human neurodegenerative disorder characterized by uncontrollable movement, psychiatric disturbance and cognitive decline. Impaired proliferative/differentiational potentials of adult neural progenitor cells (ANPCs) have been thought to be a pathogenic mechanism involved in it. In this study, we aimed to elucidate intrinsic properties of ANPCs subjected to neurodegenerative condition in YAC128 HD mice. MATERIALS AND METHODS: ANPCs were isolated from the SVZ regions of 4-month-old WT and YAC128 mice. Cell proliferation, migration and neuronal differentiation in vitro were compared between these two genotypes with/without Ca(2+) inhibitors or ROS scavenger treatments. Differences in ANPC proliferation and differentiation capabilities in vivo between the two genotypes were evaluated using Ki-67 and Doublecortin (DCX) immunofluorescence respectively. RESULTS: Compared to WT ANPCs, YAC128 ANPCs had significantly enhanced cell proliferation, migration and neuronal differentiation in vitro, accompanied by increased Ca(2+) and ROS signals. Raised proliferation and migration in YAC128 ANPCs were abolished by Ca(2+) signalling antagonists and ROS scavenging. However, in vivo, HD ANPCs failed to show any elevated proliferation or differentiation. CONCLUSIONS: Increased Ca(2+) signalling and higher level of ROS conferred HD ANPC enhancement of proliferation and migration potentials. However, the in vivo micro-environment did not support endogenous ANPCs to respond appropriately to neuronal loss in these YAC128 mouse brains.

Acrylamide toxic effects on mouse oocyte quality and fertility in vivo.

Acrylamide is an industrial chemical that has attracted considerable attention due to its presumed carcinogenic, neurotoxic, and cytotoxic effects. In this study we investigated possible acrylamide reproductive toxic effects in female mice. Mice were fed an acrylamide-containing diet for 6 weeks. Our results showed the following effects of an acrylamide-containing diet. (1) Ovary weights were reduced in acrylamide-treated mice and oocyte developmental competence was also reduced, as shown by reduced GVBD and polar body extrusion rates. (2) Acrylamide feeding resulted in aberrant oocyte cytoskeletons, as shown by an increased abnormal spindle rate and confirmed by disrupted γ-tubulin and p-MAPK localization. (3) Acrylamide feeding resulted in oxidative stress and oocyte early stage apoptosis, as shown by increased ROS levels and p-MAPK expression. (4) Fluorescence intensity analysis showed that DNA methylation levels were reduced in acrylamide-treated oocytes and histone methylation levels were also altered, as H3K9me2, H3K9me3, H3K4me2, and H3K27me3 levels were reduced after acrylamide treatment. (5) After acrylamide feeding, the litter sizes of acrylamide-treated mice were significantly smaller compared to thus of control mice. Thus, our results indicated that acrylamide might affect oocyte quality through its effects on cytoskeletal integrity, ROS generation, apoptosis induction, and epigenetic modifications.

Aflatoxin B1 is toxic to porcine oocyte maturation.

As a toxic secondary metabolite of Aspergillus species, Aflatoxin B1 (AFB1) is a major food and feed contaminant in tropical and sub-tropical regions with high temperature and humidity. It has been reported to be toxic to the female reproductive system in laboratory and domestic animals. In the present study, the influence of acute exposure to AFB1 (10 and 50 µM, 44h) on porcine oocyte maturation and its possible mechanism were investigated. The maturation rates of oocytes decreased significantly in the presence of 50 µM of AFB1. Cell cycle analysis showed that most oocytes were arrested at germinal vesicle breakdown or meosis I stage. However, actin assembly, spindle structure and chromosome alignment were not disrupted after exposure to 50 µM AFB1. Further study showed that DNA methylation levels increased in treated oocytes (50 µM). Histone methylation levels were also analysed after treatment (50 µM): H3K27me3 and H3K4me2 levels decreased, whereas H3K9me3 level increased, indicating that epigenetic modification was affected. AFB1 treatment (50 µM) also induced oxidative stress and further led to autophagy, as shown by accumulation of reactive oxygen species, up-regulated LC3 protein expression and increased mRNA levels of ATG3, ATG5 and ATG7. Annexin V-FITC staining assay revealed that AFB1 treatment (50 µM) resulted in oocyte early apoptosis, which was confirmed by increased Bak, Bax, Bcl-xl mRNA levels. Collectively, our results suggest that AFB1 disrupts porcine oocyte maturation through changing epigenetic modifications as well as inducing oxidative stress, excessive autophagy and apoptosis.

The Dynamin 2 inhibitor Dynasore affects the actin filament distribution during mouse early embryo development.

Dynamin 2 is a large GTPase notably involved in clathrin-mediated endocytosis, cell migration and cytokinesis in mitosis. Our previous study identified that Dynamin 2 regulated polar body extrusion in mammalian oocytes, but its roles in early embryo development, remain elusive. Here, we report the critical roles of Dynamin 2 in mouse early embryo development. Dynamin 2 accumulated at the periphery of the blastomere during embryonic development. When Dynamin 2 activity was inhibited by Dynasore, embryos failed to cleave to the 2-cell or 4-cell stage. Moreover, the actin filament distribution and relative amount were aberrant in the treatment group. Similar results were observed when embryos were cultured with Dynasore at the 8-cell stage; the embryos failed to undergo compaction and develop to the morula stage, indicating a role of Dynamin 2 in embryo cytokinesis. Therefore, our data indicate that Dynamin 2 might participate in the early embryonic development through an actin-based cytokinesis.