Unveiling a novel function of CD9 in surface compartmentalization of oocytes.

Gamete fusion is an indispensable process for bearing offspring. In mammals, sperm IZUMO1-oocyte JUNO recognition essentially carries out the primary step of this process. In oocytes, CD9 is also known to play a crucial role in gamete fusion. In particular, microvilli biogenesis through CD9 involvement appears to be a key event for successful gamete fusion, because CD9-disrupted oocytes produce short and sparse microvillous structures, resulting in almost no fusion ability with spermatozoa. In order to determine how CD9 and JUNO cooperate in gamete fusion, we analyzed the molecular profiles of each molecule in CD9- and JUNO-disrupted oocytes. Consequently, we found that CD9 is crucial for the exclusion of GPI-anchored proteins, such as JUNO and CD55, from the cortical actin cap region, suggesting strict molecular organization of the unique surface of this region. Through distinct surface compartmentalization due to CD9 governing, GPI-anchored proteins are confined to the appropriate fusion site of the oocyte.

Involvement in Fertilization and Expression of Gamete Ubiquitin-Activating Enzymes UBA1 and UBA6 in the Ascidian Halocynthia roretzi.

The extracellular ubiquitin-proteasome system is involved in sperm binding to and/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is ubiquitinated and degraded by the sperm proteasome during the sperm penetration of the VC and that a 700-kDa ubiquitin-conjugating enzyme complex is released upon sperm activation on the VC, which is designated the "sperm reaction". However, the de novo function of ubiquitin-activating enzyme (UBA/E1) during fertilization is poorly understood. Here, we show that PYR-41, a UBA inhibitor, strongly inhibited the fertilization of H. roretzi. cDNA cloning of UBA1 and UBA6 from H. roretzi gonads was carried out, and their 3D protein structures were predicted to be very similar to those of human UBA1 and UBA6, respectively, based on AlphaFold2. These two genes were transcribed in the ovary and testis and other organs, among which the expression of both was highest in the ovary. Immunocytochemistry showed that these enzymes are localized on the sperm head around a mitochondrial region and the follicle cells surrounding the VC. These results led us to propose that HrUBA1, HrUBA6, or both in the sperm head mitochondrial region and follicle cells may be involved in the ubiquitination of HrVC70, which is responsible for the fertilization of H. roretzi.

Visualization of cross-resistance between antimicrobial agents by asymmetric multidimensional scaling.

WHAT IS KNOWN AND OBJECTIVE: In our previous studies, we developed a cross-resistance rate (CRR) correlation diagram (CRR diagram) that visually captures the magnitude of CRRs between antimicrobials using scatter plots. We used asymmetric multidimensional scaling (MDS) to transform cross-resistance similarities between antimicrobials into a 2-dimensional map and attempted to visually express them. We also explored the antibiograms of Pseudomonas aeruginosa before and after the transfer to newly built hospitals, and we determined by the CRR diagram that the CRRs among ß-lactam antimicrobials other than carbapenems decreased substantially with the facility transfer. The present study tests whether the analysis of CRRs by asymmetric MDS can be used as new visual information that is easy for healthcare professionals to understand. METHOD: We tested the impact of changes in the nosocomial environment due to institutional transfers on CRRs among antimicrobials in asymmetric MDS, as well as contrasted the asymmetric MDS map and CRR diagram. RESULTS AND DISCUSSION: In the asymmetric MDS map, antimicrobial groups with the same mechanism of action were displayed close together, and antimicrobial groups with different mechanisms of action were displayed separately. The asymmetric MDS map drawn solely for antimicrobials belonging to the group with the same mechanism of action showed similarities to the CRR diagram. Also, the distance of each antimicrobial to other antimicrobials shown in the asymmetric MDS map was negatively correlated with the CRRs for them against that antimicrobial. WHAT IS NEW AND CONCLUSION: The asymmetric MDS map expresses the dissimilarity as distances between agents, and there are no meanings or units on the ordinate and abscissa axes of the output map. In contrast, the CRR diagram expresses the antimicrobials' resistance status as values, such as resistance rate and CRR. By analysing the CRRs in the asymmetric MDS, it is feasible to visually recognize cross-resistance similarities between antimicrobial groups as distances. The use of the asymmetric MDS combined with the CRR diagram allows us to visually understand the resistance and cross-resistance status of each antimicrobial agent as a 2-dimensional map, as well as to understand the trends and characteristics of the data by means of quantitative values.

Removal of the block to self-fertilization by low-calcium artificial seawater in the ascidian Ciona intestinalis.

Ascidians (Urochordate) are hermaphroditic marine invertebrates that release sperm and eggs to the surrounding seawater. However, several ascidians, including Ciona intestinalis and Halocynthia roretzi, show strict self-sterility due to a self/nonself-recognition mechanism in the interaction between sperm and the vitelline coat (VC) of the eggs. We have previously reported that sperm intracellular Ca2+ level drastically increased immediately after sperm binding to the VC of self eggs but not nonself eggs in C. intestinalis type A, which was potently inhibited by lowering the external Ca2+ concentration, suggesting that sperm Ca2+ influx occurs after sperm self-recognition on the VC. Here, we investigated whether self-sterility was abolished by lowering the external Ca2+ concentration in C. intestinalis. The results showed that the block to self-fertilization was removed by low-Ca2+ (∼1 mM) seawater without decreasing the fertilization rate. Such an effect was not observed with Mg2+ or K+. These results led us to conclude that a low-Ca2+ environment is sufficient to block the self-recognition signal upon fertilization. As low-Ca2+ seawater showed no effect on H. roretzi self-sterility, we propose that the mechanism of self-sterility in Ciona must be distinctive from that in Halocynthia.

Monitoring antimicrobial cross-resistance with cross-resistance rate correlation diagrams: Changes in antibiotic susceptibility of Pseudomonas aeruginosa due to hospital relocation.

WHAT IS KNOWN AND OBJECTIVE: Though most medical institutions calculate antimicrobial susceptibility and resistance rates of microbes isolated at their own facility as part of their efforts to promote the proper use of antibiotics, very few, if any, regularly monitor cross-resistance rates between antimicrobial agents. The authors have devised a tool in the form of a cross-resistance rate correlation diagram (CRR diagram) that allows easy identification of increases or decreases in, or changes in the pattern of, antimicrobial cross-resistance. The objective was to perform an analysis by CRR diagrams of the effect of relocation to a newly built facility on antimicrobial resistance and cross-resistance rates at a medical facility. METHODS: The Sakai City Medical Center relocated in July 2015 to a newly built facility located in a different primary medical care zone 3.5 km away. Based on the drug susceptibility test data compiled at the Sakai City Medical Center, resistance and cross-resistance rates of Pseudomonas aeruginosa before and after the relocation of the hospital facility were calculated, and the rates were assessed using CRR diagrams. RESULTS AND DISCUSSION: It was possible to confirm the effect of hospital relocation on antibiotic susceptibility of P aeruginosa in terms of changes in resistance and cross-resistance rates. The effect of the facility's relocation on cross-resistance rates was particularly notable with respect to ß-lactam antibiotics: cross-resistance rates among ß-lactams decreased substantially, represented as a large wedge-shaped change towards the origin on the CRR diagram. Rates of cross-resistance between classes of antibiotics with a different mechanism of antibiotic action changed little. WHAT IS NEW AND CONCLUSION: Including cross-resistance rates in the routine monitoring of resistance and susceptibility rates practiced by a medical institution can provide a comprehensive insight into the dynamics of bacterial flora in the facility. CRR diagrams, which allow visualization of the status and changes in cross-resistance, not only provide a new perspective for clinicians, but they also contribute to the proper use of antibiotics and serve as a tool in the education of healthcare professionals and students about antibiotic resistance.

Emerging Role of TCA Cycle-Related Enzymes in Human Diseases.

The tricarboxylic acid (TCA) cycle is the main source of cellular energy and participates in many metabolic pathways in cells. Recent reports indicate that dysfunction of TCA cycle-related enzymes causes human diseases, such as neurometabolic disorders and tumors, have attracted increasing interest in their unexplained roles. The diseases which develop as a consequence of loss or dysfunction of TCA cycle-related enzymes are distinct, suggesting that each enzyme has a unique function. This review aims to provide a comprehensive overview of the relationship between each TCA cycle-related enzyme and human diseases. We also discuss their functions in the context of both mitochondrial and extra-mitochondrial (or cytoplasmic) enzymes.

Sperm IZUMO1-Dependent Gamete Fusion Influences Male Fertility in Mice.

Sperm-egg fusion is accomplished through the interaction of a specific set of membrane proteins in each gamete: sperm IZUMO1 and oocyte JUNO. Recently, we found that alternative splicing of the Izumo1 gene generates a novel IZUMO1 isoform (IZUMO1_v2). Here, we obtained four mouse lines, having graded different levels of IZUMO1 protein by combining an original IZUMO1 (IZUMO1_v1) knockout with IZUMO1-null (both IZUMO1_v1 and _v2 disrupted) genetic background, in order to determine how the quantity of IZUMO1 influences male fertility. Subsequently, we clarified that the signal intensity from two quantitative assays, western blot and immunostaining analyses with a monoclonal antibody against mouse IZUMO1, were strongly correlated with average litter size. These results suggest that evaluating IZUMO1 protein levels is useful for predicting fecundity, and is a suitable test for male fertility.

Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins.

We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization.

Self-incompatibility response induced by calcium increase in sperm of the ascidian Ciona intestinalis.

Many hermaphroditic organisms possess a self-incompatibility system to avoid self-fertilization. Recently, we identified the genes responsible for self-sterility in a hermaphroditic primitive chordate (ascidian), Ciona intestinalis: sperm-side polycystin 1-like receptors s-Themis-A/B and egg-side fibrinogen-like ligands on the vitelline coat (VC) v-Themis-A/B. Here, we investigated the sperm behavior and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to self/nonself-recognition. We found that sperm motility markedly decreased within 5 min after attachment to the VC of self-eggs but not after attachment to the VC of nonself-eggs and that the apparent decrease in sperm motility was suppressed in low Ca(2+) seawater. High-speed video analysis revealed that sperm detached from the self-VC or stopped motility within 5 min after binding to the self-VC. Because s-Themis-B contains a cation channel domain in its C terminus, we monitored sperm [Ca(2+)](i) by real-time [Ca(2+)](i) imaging using Fluo-8H-AM (AAT Bioquest, Inc.). Interestingly, we found that sperm [Ca(2+)](i) rapidly and dramatically increased and was maintained at a high level in the head and flagellar regions when sperm interacted with the self-VC but not when the sperm interacted with the nonself-VC. The increase in [Ca(2+)](i) was also suppressed by low-Ca(2+) seawater. These results indicate that the sperm self-recognition signal triggers [Ca(2+)](i) increase and/or Ca(2+) influx, which elicits a self-incompatibility response to reject self-fertilization in C. intestinalis.

Usefulness of assessment of the Clinical Frailty Scale and the Dementia Assessment Sheet for Community-based Integrated Care System 21-items at the time of initiation of maintenance hemodialysis in older patients with chronic kidney disease.

INTRODUCTION: We examined whether the Clinical Frailty Scale (CFS), a widely adopted tool for stratifying the degree of frailty, and the Dementia Assessment Sheet for Community-based Integrated Care System 21-items (DASC-21), a simple tool for simultaneous assessment of impaired cognition and impaired ADL, at the time of initiation of hemodialysis is useful tool of older patients for the outcome and prognosis. METHODS: Data for 101 patients aged 75 years or older (mean age, 84.3 years) with ESRD who were initiated on hemodialysis and could be followed up for a period of 6 months were reviewed. RESULTS: The 6-month survival curves showed a significantly higher number of deaths in the frailty (CFS≥5) group than in the normal to vulnerable (CFS<5) group (p<0.01). The CFS level was also significantly higher (6.5±1.5) in patients who died within 6 months of dialysis initiation as compared with that (4.6±1.7) in patients who survived (p<0.01). On the other hand, the total score of DASC-21 was related to need for inpatient maintenance dialysis (p<0.01). The total score on the DASC-21 were found as showing significant correlations with the CFS level. The IADL outside the home was identified in the DASC-21 sub-analyses as being correlated with CFS. CONCLUSIONS: The CFS and the DASC-21 appeared to be a useful predictive tool of outcome and prognosis for older patients being initiated on hemodialysis. Assessment by the CFS or the DASC-21 might be useful for selecting the renal replacement therapy by shared decision-making and for advance care planning.

Metabolic syndrome and risk of progression of chronic kidney disease: a single-center cohort study in Japan.

Metabolic syndrome (MetS) is a risk factor for the development of diabetes and cardiovascular disease, and recently was linked to incident chronic kidney disease (CKD). The purpose of this study is to examine whether MetS is associated with CKD progression in Japanese at a single center. Outcome variables were a decrease in estimated glomerular filtration rate (eGFR) of 50 % or 25 ml/min/1.73 m(2), end-stage renal disease (ESRD), death, or a composite outcome of all three. There were 213 subjects in the analysis, 40.4 % of whom met the criteria for MetS. The group of subjects with MetS had higher urinary albumin-to-creatinine (UACR) levels. Survival curves stratified by MetS status showed early separation of the curves and a significantly higher survival rate in the group without MetS (P = 0.0086). Comparisons with normoalbuminuria and microalbuminuria showed that macroalbuminuria was equally associated with predicted composite outcome (GFR, ESRD, or death) both in the presence and absence of MetS. Multivariate analyses for all covariates showed that eGFR (hazard ratio (HR) 8.286, 95 % confidence interval (CI) 2.360-28.044, P = 0.0012) and the UACR (HR 2.338, 95 % CI 1.442-3.861, P = 0.0005) at baseline were independently associated with the composite outcomes. The results show that MetS was associated with albuminuria in a cohort of Japanese CKD patients, and both MetS and albuminuria were independently associated with CKD progression.

Sodium Hexametaphosphate Serves as an Inducer of Calcium Signaling.

In bacteria, polymers of inorganic phosphates, particularly linear polyphosphate, are used as alternative phosphate donors for adenosine triphosphate production. A six-chain form of sodium metaphosphate, sodium hexametaphosphate (SHMP), is believed to have no physiological functions in mammalian cells. In this study, we explored the possible effects of SHMP on mammalian cells, using mouse oocytes, which are useful for observing various spatiotemporal intracellular changes. Fertilization-competent oocytes were isolated from the oviducts of superovulated mice and cultured in an SHMP-containing medium. In the absence of co-incubation with sperm, SHMP-treated oocytes frequently formed pronuclei and developed into two-cell embryos owing to the increase in calcium concentration in the cytoplasm. We discovered an intriguing role for SHMP as an initiator of calcium rise in mouse oocytes, presumably in a wide variety of mammalian cells.

Identification of a syncytin gene in a non-rodent laboratory mammal, Suncus murinus.

An endogenous retrovirus-derived membrane protein, syncytin (SYN), contributes to placental function via trophoblast fusion. Multinuclear trophoblasts (syncytiotrophoblasts) physically and functionally mediate the interaction between fetal and maternal vessels in various ways. Suncus murinus (suncus) is a small mammalian species with a pregnancy duration of approximately 30 days, 1.5 times longer than mice. However, the molecular basis for the longer pregnancy duration is unknown. In this study, we first isolated two genes that encoded putative SYN proteins expressed in the suncus placenta, which were named syncytin-1-like proteins 1 and 2 (SYN1L1 and SYN1L2). When their expression vectors were introduced into cultured cells, suncus SYN1L2 was found to be active in cell fusion. Moreover, the SYN1L2 protein was homologous to a SYN1-like protein identified in greater mouse-eared bats (bat SYN1L) and was structurally compared with bat SYN1L and other SYN proteins, implying the presence of structural features of the SYN1L2 protein.

Comparison between serum free triiodothyronine levels and body fluid distribution in hemodialysis patients.

AIM: Low free triiodothyronine (fT3) has been associated with the presence of malnutrition-inflammation syndrome in patients with end-stage renal disease (ESRD) and decreased overall survival in ESRD. Since thyroid hormone has a particular effect on body fluid status, we hypothesized that hemodialysis patients with low-T3 syndrome might have interstitial edema. In this study, we examined the relationship between levels of thyroid hormone and body composition parameters in Japanese hemodialysis patients. METHODS: The subjects were 52 patients on maintenance hemodialysis. Serum levels of thyroid hormone and atrial natriuretic peptide (hANP) were measured. Body composition parameters were measured using a bioimpedance body composition analyzer. RESULTS: Serum fT3 had positive correlations with body mass index (BMI), body fat mass (BFM), total body water (TBW) and intracellular water (ICW), and negative correlations with the ratio of extracellular water to total body water (ECW/TBW) and hANP. There were no correlations between serum fT4 and any body composition parameter. The 49 patients with data at baseline and after 1 year were divided into groups with increased (n = 33) and decreased (n = 16) fT3 after 1 year. ΔBMI and ΔBFM were significantly lower and ΔTBW, ΔICW, ΔECW and ΔECW/TBW (changes over 1 year from baseline) were significantly higher in patients with decreased fT3 compared to those with increased fT3. There was no significant difference in ΔhANP or Δcardiothoracic ratio between the two groups. CONCLUSION: These results show that a decrease in fT3 might be associated with emaciation and interstitial edema in Japanese hemodialysis patients.

Mizoribine requires individual dosing due to variation of bioavailability.

BACKGROUND: Mizoribine (MZR) is an immunosuppressant used for the treatment of glomerular diseases, but there are few reports on the pharmacokinetics of MZR in children. METHODS: First, we performed a pharmacokinetic study on nine childhood-onset glomerular disease patients. The MZR dosages ranged from 1.8 to 14.5 mg/kg/dose. Pharmacokinetic parameters were analyzed using 38 MZR concentration-time curves. Second, nine patients who were newly treated with MZR were enrolled to validate the findings obtained from prior investigation. RESULTS: In the prior study, peak serum MZR concentration (C(max) ) was dose-dependent in each patient. Although proportionality between dosage and C(max) was observed in each patient, the regression coefficient was in a wide range from 0.075 to 1.04 and was specific to each patient. This variability was likely caused by individual variation of bioavailability. When the optimal time-point to monitor C(max) was investigated, the time-to-reach peak serum MZR concentration (T(max)) was similar among all the patients, which was from 2.5 to 3.5 h after administration of MZR. T(max) was most frequently observed at 3 h and the serum MZR concentration ratio relative to C(max) at 3 h was also highest (0.93 ± 0.07). In the following study, it was validated that monitoring C(3) is reproducible and reliable after adjusting the dosage of MZR to obtain target serum concentration. CONCLUSION: Individual dosing is required to optimize C(max) in childhood-onset glomerular disease patients. The safe dosage of MZR for each patient could be predicted by evaluating the serum MZR concentration 3 h after administration.

Mechanisms of Sperm-Egg Interactions: What Ascidian Fertilization Research Has Taught Us.

Fertilization is an essential process in terrestrial organisms for creating a new organism with genetic diversity. Before gamete fusion, several steps are required to achieve successful fertilization. Animal spermatozoa are first activated and attracted to the eggs by egg-derived chemoattractants. During the sperm passage of the egg's extracellular matrix or upon the sperm binding to the proteinaceous egg coat, the sperm undergoes an acrosome reaction, an exocytosis of acrosome. In hermaphrodites such as ascidians, the self/nonself recognition process occurs when the sperm binds to the egg coat. The activated or acrosome-reacted spermatozoa penetrate through the proteinaceous egg coat. The extracellular ubiquitin-proteasome system, the astacin-like metalloproteases, and the trypsin-like proteases play key roles in this process in ascidians. In the present review, we summarize our current understanding and perspectives on gamete recognition and egg coat lysins in ascidians and consider the general mechanisms of fertilization in animals and plants.

Identification and localization of the sperm CRISP family protein CiUrabin involved in gamete interaction in the ascidian Ciona intestinalis.

Ascidians are hermaphrodites, and most release sperm and eggs nearly simultaneously. Many species, including Halocynthia roretzi and Ciona intestinalis, are self-sterile. We previously reported that the interaction between a 12 EGF-like repeat-containing vitelline-coat (VC) protein, HrVC70, and a sperm GPI-anchored CRISP, HrUrabin, in lipid rafts plays a key role in self-/nonself-recognizable gamete interaction in H. roretzi. On the other hand, we recently identified two pairs of polymorphic genes responsible for self-incompatibility in C. intestinalis by positional cloning: The sperm polycystin 1-like receptors s-Themis-A/B and its fibrinogen-like ligand v-Themis-A/B on the VC. However, it is not known if the orthologs of HrVC70 and HrUrabin also participate in gamete interaction in C. intestinalis since they are from different orders. Here, we tested for a C. intestinalis ortholog (CiUrabin) of HrUrabin by searching the genome database and proteomes of sperm lipid rafts. The identified CiUrabin belongs to the CRISP family, with a PR domain and a GPI-anchor-attachment site. CiUrabin appears to be specifically expressed in the testis and localized at the surface of the sperm head, as revealed by Northern blotting and immunocytochemistry, respectively. The specific interaction between CiVC57, a C. intestinalis ortholog of HrVC70, and CiUrabin was confirmed by Far Western analysis, similarly to the interaction between HrVC70 and HrUrabin. The molecular interaction between CiVC57 and CiUrabin may be involved in the primary binding of sperm to the VC prior to the allorecognition process, mediated by v-Themis-A/B and s-Themis-A/B, during fertilization of C. intestinalis.

The effects of raloxifene on bone turnover markers and bone mineral density in women on maintenance hemodialysis.

BACKGROUND: Bone disease is caused not only by increased bone turnover accompanying secondary hyperparathyroidism but also by factors such as bone metabolic disorder accompanying kidney disease and postmenopausal or age-related osteoporosis in hemodialysis patients. In this study, we investigated the effects of raloxifene on bone turnover markers and bone mineral density (BMD) in female hemodialysis patients to determine involvement of estrogen deficiency in bone disease. METHODS: The subjects were 47 female patients on maintenance hemodialysis. Raloxifene hydrochloride (60 mg) was administered to 21 patients for 1 year, and these patients were compared with a control group of 26 patients. Serum levels of N-terminal cross-linking telopeptide of type I collagen (NTx), bone alkaline phosphatase, and intact parathyroid hormone were measured, and BMD was determined by quantitative heel ultrasound as the speed of sound (SOS) in the calcaneus over this period. RESULTS: NTx decreased after treatment with raloxifene for 1 year, but significantly increased in the control group. SOS increased after treatment with raloxifene for 1 year, but significantly decreased in the control group. Treatment with raloxifene resulted in a significant decrease of NTx and a significant increase of SOS in subgroups of patients aged <60 and ≥ 60 years. CONCLUSIONS: Treatment with raloxifene can suppress a rise in NTx and increase bone mineral density in patients around the time of menopause and in postmenopausal patients of advanced age. A reduction in bone mineral density caused by estrogen deficiency may be involved in the development of bone disease in female hemodialysis patients.

Fertilization of Ascidians: Gamete Interaction, Self/Nonself Recognition and Sperm Penetration of Egg Coat.

Fertilization is one of the most important events in living organisms to generate a new life with a mixed genetic background. To achieve successful fertilization, sperm and eggs must undergo complex processes in a sequential order. Fertilization of marine invertebrate Ciona intestinalis type A (Ciona robusta) has been studied for more than a hundred years. Ascidian sperm are attracted by chemoattractants from eggs and bind to the vitelline coat. Subsequently, sperm penetrate through the vitelline coat proteolytically and finally fuse with the egg plasma membrane. Here, we summarize the fertilization mechanisms of ascidians, particularly from sperm-egg interactions to sperm penetration of the egg coat. Since ascidians are hermaphrodites, inbreeding depression is a serious problem. To avoid self-fertilization, ascidians possess a self-incompatibility system. In this review, we also describe the molecular mechanisms of the self-incompatibility system in C. intestinalis type A governed by three allelic gene pairs of s-Themis and v-Themis.