CCHCR1-astrin interaction promotes centriole duplication through recruitment of CEP72.

BACKGROUND: The centrosome is one of the most important non-membranous organelles regulating microtubule organization and progression of cell mitosis. The coiled-coil alpha-helical rod protein 1 (CCHCR1, also known as HCR) gene is considered to be a psoriasis susceptibility gene, and the protein is suggested to be localized to the P-bodies and centrosomes in mammalian cells. However, the exact cellular function of HCR and its potential regulatory role in the centrosomes remain unexplored. RESULTS: We found that HCR interacts directly with astrin, a key factor in centrosome maturation and mitosis. Immunoprecipitation assays showed that the coiled-coil region present in the C-terminus of HCR and astrin respectively mediated the interaction between them. Astrin not only recruits HCR to the centrosome, but also protects HCR from ubiquitin-proteasome-mediated degradation. In addition, depletion of either HCR or astrin significantly reduced centrosome localization of CEP72 and subsequent MCPH proteins, including CEP152, CDK5RAP2, and CEP63. The absence of HCR also caused centriole duplication defects and mitotic errors, resulting in multipolar spindle formation, genomic instability, and DNA damage. CONCLUSION: We conclude that HCR is localized and stabilized at the centrosome by directly binding to astrin. HCR are required for the centrosomal recruitment of MCPH proteins and centriolar duplication. Both HCR and astrin play key roles in keeping normal microtubule assembly and maintaining genomic stability.

Trichosanthin Promotes Anti-Tumor Immunity through Mediating Chemokines and Granzyme B Secretion in Hepatocellular Carcinoma.

Trichosanthin (TCS) is a type I ribosome-inactivating protein extracted from the tuberous root of the plant Trichosanthes. TCS shows promising potential in clinical drug abortion, anti-tumor and immunological regulation. However, the molecular mechanisms of its anti-tumor and immune regulation properties are still not well discovered. In the present study, we investigated the anti-tumor activity of TCS in hepatocellular carcinoma (HCC), both in vitro and in vivo. Both HCC cell lines and xenograft tumor tissues showed considerable growth inhibition after they were treated with TCS. TCS provoked caspase-mediated apoptosis in HCC cells and xenograft tumor tissues. The recruitment of CD8+ T cells to HCC tissues and the expression of chemokines, CCL2 and CCL22, were promoted upon TCS treatment. In addition, TCS induced an upregulation of Granzyme B (GrzB), TNF-α and IFN-γ in HCC tissues, which are the major cytotoxic mediators produced by T cells. Furthermore, TCS also resulted in an increase of mannose-6-phosphate receptor (M6PR), the major receptor of GrzB, in HCC tissues. In summary, these results suggest that TCS perhaps increases T-cell immunity via promoting the secretion of chemokines and accelerating the entry of GrzB to HCC cells, which highlights the potential role of TCS in anti-tumor immunotherapy.

COPI-TRAPPII activates Rab18 and regulates its lipid droplet association.

The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII-specific subunits by various methods including siRNA depletion and CRISPR-Cas9-mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII-deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis.

Acute kinematics and kinetics changes to wearable resistance during change of direction among soccer players.

This study determined the acute changes in kinematics and kinetics when an additional load equivalent to 5% body mass was attached to the torso during change of direction (COD). In this within-subject repeated measures study, 14 male soccer players (age: 18.29 ± 0.32 years) volunteered to participate. Subjects performed COD under two conditions in randomized order: (1) no WR, and (2) with WR. No significant differences between the loaded and unloaded conditions in actual COD angle, approach speed, braking time, propulsive time, contact time, COD completion time (all p > 0.05, ES = 0.05-0.11), and all measured kinematic parameters (all p > 0.05, ES = 0-0.18). Nonetheless, ankle plantar/dorsi flexion ROM had possibly small increase in the loaded condition (ES = 0.24). Kinetics analysis has shown that the loaded condition was likely to have small increase in relative peak vertical propulsive ground reaction force (GRF, p = 0.11, ES = 0.41), and possible small increases in relative peak braking GRF (vertical: p = 0.21, ES = 0.42; total: p = 0.22, ES = 0.38), relative peak total propulsive GRF (p = 0.24, ES = 0.26), and relative braking impulse (horizontal, vertical, and total; p = 0.27-0.41, ES = 0.26-0.28). WR did not significantly change the acute movement techniques, meanwhile induced small increases in important kinetic stimuli for potential adaptation in COD.

Mushroom extracts and compounds with suppressive action on breast cancer: evidence from studies using cultured cancer cells, tumor-bearing animals, and clinical trials.

This article reviews mushrooms with anti-breast cancer activity. The mushrooms covered which are better known include the following: button mushroom Agaricus bisporus, Brazilian mushroom Agaricus blazei, Amauroderma rugosum, stout camphor fungus Antrodia camphorata, Jew's ear (black) fungus or black wood ear fungus Auricularia auricula-judae, reishi mushroom or Lingzhi Ganoderma lucidum, Ganoderma sinense, maitake mushroom or sheep's head mushroom Grifola frondosa, lion's mane mushroom or monkey head mushroom Hericium erinaceum, brown beech mushroom Hypsizigus marmoreus, sulfur polypore mushroom Laetiporus sulphureus, Lentinula edodes (shiitake mushroom), Phellinus linteus (Japanese "meshimakobu," Chinese "song gen," Korean "sanghwang," American "black hoof mushroom"), abalone mushroom Pleurotus abalonus, king oyster mushroom Pleurotus eryngii, oyster mushroom Pleurotus ostreatus, tuckahoe or Fu Ling Poria cocos, and split gill mushroom Schizophyllum commune. Antineoplastic effectiveness in human clinical trials and mechanism of anticancer action have been reported for Antrodia camphorata, Cordyceps sinensis, Coriolus versicolor, Ganoderma lucidum, Grifola frondosa, and Lentinula edodes.

New ribosome-inactivating proteins and other proteins with protein synthesis-inhibiting activities.

Ribosome-inactivating proteins (RIPs) consist of three varieties. Type 1 RIPs are single-chained and approximately 30-kDa in molecular weight. Type 2 RIPs are double-chained and composed of a type 1 RIP chain and a lectin chain. Type III RIPs, such as maize b-32 barley and JIP60 which are produced as single-domain proenzymes, possess an N-terminal domain corresponding to the A domain of RIPs and fused to a C-terminal domain. In addition to the aforementioned three types of RIPs originating from flowering plants, there are recently discovered proteins and peptides with ribosome-inactivating and protein synthesis inhibitory activities but which are endowed with characteristics such as molecular weights distinctive from those of the regular RIPs. These new/unusual RIPs discussed in the present review encompass metazoan RIPs from Anopheles and Culex mosquitos, antimicrobial peptides derived from RIP of the pokeweed Phytolacca dioica, maize RIP (a type III RIP derived from a precursor form), RIPs from the garden pea and the kelp. In addition, RIPs with a molecular weight smaller than those of regular type 1 RIPs are produced by plants in the Cucurbitaceae family including the bitter gourd, bottle gourd, sponge gourd, ridge gourd, wax gourd, hairy gourd, pumpkin, and Chinese cucumber. A small type II RIP from camphor tree (Cinnamomum camphora) seeds and a snake gourd type II RIP with its catalytic chain cleaved into two have been reported. RIPs produced from mushrooms including the golden needle mushroom, king tuber mushroom, straw mushroom, and puffball mushroom are also discussed in addition to a type II RIP from the mushroom Polyporus umbellatus. Bacterial (Spiroplasma) RIPs associated with the fruitfly, Shiga toxin, and Streptomyces coelicolor RIP are also dealt with. The aforementioned proteins display a diversity of molecular weights, amino acid sequences, and mechanisms of action. Some of them are endowed with exploitable antipathogenic activities.

Rab proteins as regulators of lipid droplet formation and lipolysis.

Lipid droplets (LDs) are highly dynamic organelles that not only store neutral lipids but also are involved in multiple cellular processes. Dysregulation of lipogenesis or lipolysis greatly contributes to the pathogenesis of several human diseases, including obesity, diabetes, and fatty liver disease. Rab proteins have been found to be associated with LDs in proteomic studies and are also known to extensively regulate intracellular membrane traffic, suggesting that LDs actively communicate with other membrane compartments to maintain energy homeostasis. This review discusses recent studies that provide mechanistic insights into the regulation of LD formation and catabolism by Rab proteins in mammalian cells.

Sensitive and high resolution localization and tracking of membrane proteins in live cells with BRET.

Peripheral and integral membrane proteins can be located in several different subcellular compartments, and it is often necessary to determine the location of such proteins or to track their movement in living cells. Image-based colocalization of labeled membrane proteins and compartment markers is frequently used for this purpose, but this method is limited in terms of throughput and resolution. Here we show that bioluminescence resonance energy transfer (BRET) between membrane proteins of interest and compartment-targeted BRET partners can report subcellular location and movement of membrane proteins in live cells. The sensitivity of the method is sufficient to localize a few hundred protein copies per cell. The spatial resolution can be sufficient to determine membrane topology, and the temporal resolution is sufficient to track changes that occur in less than 1 second. BRET requires little user intervention, and is thus amenable to large-scale experimental designs with standard instruments.

A perspective from transport protein particle: vesicle tether and human diseases.

Vesicle-mediated transport of proteins is a highly regulated, multi-step process. When the vesicle is approaching its target membrane compartment, many factors are required to provide specificity and tethering between the incoming vesicle and the target membrane, before vesicle fusion can occur. Tethering factors, which include multisubunit complexes, coiled-coil proteins, with the help of small GTPases, provide the initial interaction between the vesicle and its target membrane. Of the multisubunit tethering factors, the transport protein particle (TRAPP) complexes function in a number of trafficking steps, including endoplasmic reticulum (ER)-to-Golgi transport, intra- and post-Golgi traffic and autophagosome formation. In this review, we summarize the updated progress in structure and function of TRAPP complexes as well as human diseases caused by genetic mutations in TRAPP.

Rab26 modulates the cell surface transport of α2-adrenergic receptors from the Golgi.

The molecular mechanisms underlying the transport from the Golgi to the cell surface of G protein-coupled receptors remain poorly elucidated. Here we determined the role of Rab26, a Ras-like small GTPase involved in vesicle-mediated secretion, in the cell surface export of α(2)-adrenergic receptors. We found that transient expression of Rab26 mutants and siRNA-mediated depletion of Rab26 significantly attenuated the cell surface numbers of α(2A)-AR and α(2B)-AR, as well as ERK1/2 activation by α(2B)-AR. Furthermore, the receptors were extensively arrested in the Golgi by Rab26 mutants and siRNA. Moreover, Rab26 directly and activation-dependently interacted with α(2B)-AR, specifically the third intracellular loop. These data demonstrate that the small GTPase Rab26 regulates the Golgi to cell surface traffic of α(2)-adrenergic receptors, likely through a physical interaction. These data also provide the first evidence implicating an important function of Rab26 in coordinating plasma membrane protein transport.

The associations of early specialization, sports volume, and maturity status with musculoskeletal injury in elite youth football players.

Background: Youth football in schools has experienced rapid growth in China. Despite the increase of players engaging in more frequent, intensive, and organized sports training at their early ages, the controversy over early specialization (ES) still exists. This study aims to: a) investigate the training situation of players in the Chinese School Football Programme and b) examine the associations of early specialization, sports volume, and maturity status with musculoskeletal injury. Methods: A cross-sectional survey was used. Players who participated in the National School Football Winter Camp were invited to fill out a questionnaire that included the data of maturity, ES, sports volume, and injury history (n = 88 boys and n = 90 girls). Results: The results have shown that 80.3% of the athletes were classified as ES, while 19.7% of them were classified as non-ES. Almost all athletes (96%) participated in a sport for more than 8 months in a year. Most athletes (75.8%) spent more than twice of the time on organized sports than leisure activities. 30.3% of the athletes trained on average more hours per week than the number of their ages. Binomial logistic regression models reflected the significant differences in the odds ratios (OR) of reporting a history of injury among athletes with different levels of specialization (p = 0.024) and the OR of reporting a history of leg injury among players with different weekly sports volumes (p = 0.038). Significant differences were also shown in the OR of players reporting foot injuries between players with different maturity states (p = 0.046), and the Chi-squared test showed significant differences in the OR of reporting acute injuries between players with different levels of specialization (p = 0.048) and weekly activity (p = 0.022). No significant differences were found between the remaining variables. Conclusion: Most school football elite players follow the ES pathway even though ES increases the risk of injury, especially acute injury. Pre-pubertal and early pubertal players have a higher incidence of foot injuries. Players who train more hours per week than their ages have more leg injuries and acute injuries. Therefore, priority protection and intervention should be carried out for populations with a high risk of injury.

Effects of plyometric training on kicking performance in soccer players: A systematic review and meta-analysis.

This systematic review and meta-analysis aimed to determine the pooled effect size (ES) of plyometric training (PT) on kicking performance (kicking speed and distance) in soccer players depending upon some related factors (i.e., age, gender, skill level, and intervention duration). This study was carried out according to the PRISMA guidelines. Four electronic databases-EBSCO, PubMed, Scopus, and Web of Science-were searched for relevant studies. A total of n = 16 studies yielding 17 ES with n = 553 participants were finally included in the meta-analysis. A random-effects model was used to calculate Hedge's g with a 95% confidence interval (CI), which showed that plyometric training had a large-sized positive effect on soccer kicking performance (g = 0.979, 95% CI [0.606, 1.353], p < 0.001). Subgroup analyses were performed according to participants' characteristics (i.e., age, gender, skill level) and intervention duration, demonstrating no significant differences between these subgroups. The study pointed out that plyometric training is a generally effective method to improve soccer players' kicking performance, which plays a crucial role in passing and shooting actions during games. As for soccer players and strength and conditioning coaches, the plyometric training aiming to enhance kicking performance has valuable implications in practice. Therefore, besides well-known training methods like power training in the weight room, plyometric training could be incorporated into the overall strength and conditioning programs for soccer players to reach high standards of kicking performance.

Regulation of α(2B)-adrenergic receptor-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation by ADP-ribosylation factor 1.

A number of signaling molecules are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway by G protein-coupled receptors. In this study, we have demonstrated that α(2B)-adrenergic receptor (α(2B)-AR) interacts with ADP-ribosylation factor 1 (ARF1), a small GTPase involved in vesicle-mediated trafficking, in an agonist activation-dependent manner and that the interaction is mediated through a unique double Trp motif in the third intracellular loop of the receptor. Interestingly, mutation of the double Trp motif and siRNA-mediated depletion of ARF1 attenuate α(2B)-AR-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) without altering receptor intracellular trafficking, whereas expression of the constitutively active mutant ARF1Q71L and ARNO, a GDP-GTP exchange factor of ARF1, markedly enhances the activation of Raf1, MEK1, and ERK1/2. These data strongly demonstrate that the small GTPase ARF1 modulates ERK1/2 activation by α(2B)-AR and provide the first evidence indicating a novel function for ARF1 in regulating the MAPK signaling pathway.

The effect of the video assistant referee (VAR) on referees' decisions at FIFA Women's World Cups.

Video assistant referee (VAR) has been implemented in women's football, aiming to improve referees' decision-making, but its impact has not yet been analyzed. This study intended to explore how the VAR affects refereeing decisions at Fédération Internationale de Football Association (FIFA) Women's World Cup competitions. The sample includes all 52 matches played in the 2015 tournament before VAR was introduced and all 52 matches played in the 2019 competition where VAR was deployed. For each match, data on ten variables were collected: first half playing time, second half playing time, total playing time, penalties, offsides, fouls, goals, corner kicks, yellow cards, and red cards. The match variables were compared before and after VAR implementation using a Mann-Whitney U test, a Bayesian analysis, a generalized linear model, and a non-clinical magnitude-based inference. The results demonstrated that after VAR was introduced, playing time during the first half [p < 0.001, BF 10 = 547.05, Cohen's d = 1.06, 90%CI (0.71, 1.40)], the second half [p < 0.001, BF 10 = 57.09, Cohen's d = 0.91, 90%CI (0.57, 1.25)], and the entire match [p < 0.001, BF 10 = 1,120.39, Cohen's d = 1.33, 90%CI (0.97, 1.69)] increased significantly with moderate to large effect sizes, while the number of penalties, offsides, and fouls did not vary significantly neither did the number of goals, corner kicks, yellow cards, and red cards. This study has practical implications for professionals in terms of a better understanding of VAR's impact on elite women's football.

Trichosanthin cooperates with Granzyme B to restrain tumor formation in tongue squamous cell carcinoma.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is a common type of oral cancer, with a relatively poor prognosis and low post-treatment survival rate. Various strategies and novel drugs to treat TSCC are emerging and under investigation. Trichosanthin (TCS), extracted from the root tubers of Tian-Hua-Fen, has been found to have multiple biological and pharmacological functions, including inhibiting the growth of cancer cells. Granzyme B (GrzB) is a common toxic protein secreted by natural killer cells and cytotoxic T cells. Our group has reported that TCS combined with GrzB might be a superior approach to inhibit liver tumor progression, but data relating to the use of this combination to treat TSCC remain limited. The aim of this study was to examine the effectiveness of TCS on TSCC processes and underlying mechanisms. METHODS: First, we screened the potential antitumor activity of TCS using two types of SCC cell lines. Subsequently, a subcutaneous squamous cell carcinoma xenograft model in nude mice was established. These model mice were randomly divided into four groups and treated as follows: control group, TCS treatment group, GrzB treatment group, and TCS/GrzB combination treatment group. Various tumorigenesis parameters, such as Ki67, PCNA, caspase-3, Bcl-2 and VEGFA, et al., were performed to determine the effects of these treatments on tumor development. RESULTS: Screening confirmed that the SCC25 line exhibited greater sensitivity than the SCC15 line to TCS in vitro studies. TCS or GrzB treatment significantly inhibited tumor growth compared with the inhibition seen in the control group. The TCS/GrzB combination inhibited tumor growth more than either drug alone. TCS treatment inhibited tumor proliferation by downregulating Ki67 and Bcl2 protein expression while accelerating tumor apoptosis. In the TCS/GrzB-treated group, expression of Ki67 was further downregulated, while the level of activated caspase-3 was increased, compared with their expression in either of the single drug treatment groups. CONCLUSION: These results suggest that the TCS/GrzB combination could represent an effective immunotherapy for TSCC.

Geoditin A induces oxidative stress and apoptosis on human colon HT29 cells.

Geoditin A, an isomalabaricane triterpene isolated from the marine sponge Geodia japonica, has been demonstrated to dissipate mitochondrial membrane potential, activate caspase 3, decrease cytoplasmic proliferating cell nuclear antigen (PCNA), and induce apoptosis of leukemia cells, but the underlying mechanism remains unclear [1]. In this study, we found fragmentation of Golgi structure, suppression of transferrin receptor expression, production of oxidants, and DNA fragmentation in human colon cancer HT29 cells after treatment with geoditin A for 24 h. This apoptosis was not abrogated by chelation of intracellular iron with salicylaldehyde isonicotinoyl hydrazone (SIH), but suppressed by N-acetylcysteine (NAC), a thiol antioxidant and GSH precursor, indicating that the cytotoxic effect of geoditin A is likely mediated by a NAC-inhibitable oxidative stress. Our results provide a better understanding of the apoptotic properties and chemotherapeutical potential of this marine triterpene.

Extracellular Matrix Remodeling During Palate Development.

The morphogenesis of the mammalian secondary plate is a series of highly dynamic developmental process, including the palate shelves vertical outgrowth, elevation to the horizontal plane and complete fusion in the midline. Extracellular matrix (ECM) proteins not only form the basic infrastructure for palatal mesenchymal cells to adhere via integrins but also interact with cells to regulate their functions such as proliferation and differentiation. ECM remodeling is essential for palatal outgrowth, expansion, elevation, and fusion. Multiple signaling pathways important for palatogenesis such as FGF, TGF ß, BMP, and SHH remodels ECM dynamics. Dysregulation of ECM such as HA synthesis or ECM breakdown enzymes MMPs or ADAMTS causes cleft palate in mouse models. A better understanding of ECM remodeling will contribute to revealing the pathogenesis of cleft palate.

Distinct Roles of TRAPPC8 and TRAPPC12 in Ciliogenesis via Their Interactions With OFD1.

The transport protein particle (TRAPP) complex was initially identified as a tethering factor for COPII vesicle. Subsequently, three forms (TRAPPI, II, and III) have been found and TRAPPIII has been reported to serve as a regulator in autophagy. This study investigates a new role of mammalian TRAPPIII in ciliogenesis. We found a ciliopathy protein, oral-facial-digital syndrome 1 (OFD1), interacting with the TRAPPIII-specific subunits TRAPPC8 and TRAPPC12. TRAPPC8 is necessary for the association of OFD1 with pericentriolar material 1 (PCM1). Its depletion reduces the extent of colocalized signals between OFD1 and PCM1, but does not compromise the structural integrity of centriolar satellites. The interaction between TRAPPC8 and OFD1 inhibits that between OFD1 and TRAPPC12, suggesting different roles of TRAPPIII-specific subunits in ciliogenesis and explaining the differences in cilium lengths in TRAPPC8-depleted and TRAPPC12-depleted hTERT-RPE1 cells. On the other hand, TRAPPC12 depletion causes increased ciliary length because TRAPPC12 is required for the disassembly of primary cilia. Overall, this study has revealed different roles of TRAPPC8 and TRAPPC12 in the assembly of centriolar satellites and demonstrated a possible tethering role of TRAPPIII during ciliogenesis.

Astrin: A Key Player in Mitosis and Cancer.

Astrin, which is a spindle-associated protein, was found to be closely related to mitotic spindle formation and maintenance. It interacts with other spindle-related proteins to play a key role in maintaining the attachment of the kinetochore-microtubule and integrity of centrosomes and promoting the centriole duplication. In addition, Astrin was quite recently found to be abnormally highly expressed in a variety of cancers. Astrin promotes the development of cancer by participating in various molecular pathways and is considered as a potential prognostic and survival predictor.

A Naturally Occurring Splice Variant of GGA1 Inhibits the Anterograde Post-Golgi Traffic of α2B-Adrenergic Receptor.

The regulatory mechanisms of cell surface targeting of nascent G protein-coupled receptors (GPCRs) en route from the endoplasmic reticulum through the Golgi remain poorly understood. We have recently demonstrated that three Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding proteins (GGAs) mediate the post-Golgi export of α2B-adrenergic receptor (α2B-AR), a prototypic GPCR, and directly interact with the receptor. In particular, GGA1 interaction with α2B-AR is mediated via its hinge domain. Here we determined the role of a naturally occurring truncated form of GGA1 (GGA1t) which lacks the N-terminal portion of the hinge domain in α2B-AR trafficking and elucidated the underlying mechanisms. We demonstrated that both GGA1 and GGA1t were colocalized and mainly expressed at the Golgi. In marked contrast to GGA1, the expression of GGA1t significantly attenuated the cell surface export of newly synthesized α2B-AR from the Golgi and in parallel receptor-mediated signaling. Furthermore, we found that GGA1t formed homodimers and heterodimers with GGA1. More interestingly, GGA1t was unable to bind the cargo α2B-AR and to recruit clathrin onto the trans-Golgi network. These data provide evidence implicating that the truncated form of GGA1 behaviors as a dominant-negative regulator for the cell surface export of α2B-AR and this function of GGA1t is attributed to its abilities to dimerize with its wide type counterpart and to inhibit cargo interaction and clathrin recruitment to form specialized transport vesicles.