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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Influence of analysis conditions for antimicrobial susceptibility test data on susceptibility rates.

BACKGROUND: To support effective antibiotic selection in empirical treatments, infection control interventions, and antimicrobial resistance containment strategies, many medical institutions collect antimicrobial susceptibility test data conducted at their facilities to prepare cumulative antibiograms. AIM: To evaluate how the setpoints of duplicate isolate removal period and data collection period affect the calculated susceptibility rates in antibiograms. METHODS: The Sakai City Medical Center is a regional core hospital for tertiary emergency medical care with 480 beds for general clinical care. In this study, all the Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae isolates collected at the Sakai City Medical Center Clinical Laboratory between July 2013 and December 2018 were subjected to antimicrobial susceptibility tests and the resulting data was analyzed. FINDINGS: The longer the duplicate isolate removal period, the fewer the isolates are available for every bacterial species. Differences in the length of the duplicate isolate removal period affected P. aeruginosa susceptibility rates to ß-lactam antibiotics by up to 10.8%. The setpoint of the data collection period affected the antimicrobial susceptibility rates by up to 7.3%. We found that a significant change in susceptibility could be missed depending on the setting of the data collection period, in preparing antibiogram of ß-lactam antibiotics for P. aeruginosa. CONCLUSIONS: When referring to antibiograms, medical professionals involved in infectious disease treatment should be aware that the parameter values, such as the duplicate isolate removal period and the data collection period, affect P. aeruginosa susceptibility rates especially to ß-lactam antibiotics. And antibiogram should be updated within the shortest time period that is practically possible, taking into account restrictions such as numbers of specimen.

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.

Alternative splicing of the Izumo1 gene ensures triggering gamete fusion in mice.

IZUMO1 is a sperm acrosomal membrane protein that is essential for mammalian fertilization through recognition of JUNO on the oocyte surface and accompanying IZUMO1-JUNO complex formation. Here, we report a new Izumo1 gene splicing variant (IZUMO1_v2) with a unique 52-amino-acid-long signal sequence transcribed from Exon 1b. Although the mRNA amount of Izumo1_v2 is 76 times lower than that of the original Izumo1 (IZUMO1_v1) in the testis, the cell-oocyte assay indicates that IZUMO1_v2-expressing COS-7 cells have the ability to attach to the oocyte equivalent of IZUMO1_v1. To clarify the physiological function of IZUMO1_v2, we produced an IZUMO1_v1-specific knockout mouse line with a nine-base deletion adjacent to the initial methionine codon of IZUMO1_v1 by the CRISPR/Cas9 system. The IZUMO1_v1 knockout male mice carry 0.19-fold lower level of IZUMO1 protein in the spermatozoon; however, reduction in fertility was only minimally affected compared to the wild-type mice, suggesting that only a small fraction of IZUMO1 is sufficient for triggering sperm-egg fusion. We propose that the alternative splicing generating IZUMO1_v2 might function as a fail-safe in mouse for when splicing is disturbed.

Structural Basis of the Human Endoglin-BMP9 Interaction: Insights into BMP Signaling and HHT1.

Endoglin (ENG)/CD105 is an essential endothelial cell co-receptor of the transforming growth factor ß (TGF-ß) superfamily, mutated in hereditary hemorrhagic telangiectasia type 1 (HHT1) and involved in tumor angiogenesis and preeclampsia. Here, we present crystal structures of the ectodomain of human ENG and its complex with the ligand bone morphogenetic protein 9 (BMP9). BMP9 interacts with a hydrophobic surface of the N-terminal orphan domain of ENG, which adopts a new duplicated fold generated by circular permutation. The interface involves residues mutated in HHT1 and overlaps with the epitope of tumor-suppressing anti-ENG monoclonal TRC105. The structure of the C-terminal zona pellucida module suggests how two copies of ENG embrace homodimeric BMP9, whose binding is compatible with ligand recognition by type I but not type II receptors. These findings shed light on the molecular basis of the BMP signaling cascade, with implications for future therapeutic interventions in this fundamental pathway.

Dual Impact of Tolvaptan on Intracellular and Extracellular Water in Chronic Kidney Disease Patients with Fluid Retention.

Objective Tolvaptan, an oral selective V2-receptor antagonist, is a water diuretic that ameliorates fluid retention with a lower risk of a worsening renal function than conventional loop diuretics. Although loop diuretics predominantly decrease extracellular water (ECW) compared with intracellular water (ICW), the effect of tolvaptan on fluid distribution remains unclear. We therefore examined how tolvaptan changes ICW and ECW in accordance with the renal function. Methods Six advanced chronic kidney disease patients (stage 4 or 5) with fluid retention were enrolled in this study. Tolvaptan (7.5 mg/day) added to conventional diuretic treatment was administered to remove fluid retention. The fluid volume was measured using a bioimpedance analysis device before (day 0) and after (day 5 or 6) tolvaptan treatment. Results Body weight decreased by 2.6%±1.3% (64.4±6.5 vs. 62.8±6.3 kg, p=0.06), and urine volume increased by 54.8%±23.9% (1,215±169 vs. 1,709±137 mL/day, p=0.03) between before and after tolvaptan treatment. Tolvaptan significantly decreased ICW (6.5%±1.5%, p=0.01) and ECW (7.5%±1.4%, p=0.02), which had similar reduction rates (p=0.32). The estimated glomerular filtration rate remained unchanged during the treatment (14.6±2.8 vs. 14.9±2.7 mL/min/1.732 m, p=0.35). Conclusion Tolvaptan ameliorates body fluid retention, and induces an equivalent reduction rate of ICW and ECW without a worsening renal function. Tolvaptan is a novel water diuretic that has a different effect on fluid distribution compared with conventional loop diuretics.

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.

Hypothermia and hypokalemia in a patient with diabetic ketoacidosis.

We present the case of a 36-year-old man with type-1 diabetes who was hospitalized with diabetic ketoacidosis (DKA). On admission, he had hypothermia, hypokalemia and combined metabolic and respiratory alkalosis, in addition to hyperglycemia. Hypothermia, hypokalemia and metabolic alkalosis, with a concurrent respiratory alkalosis, are not commonly seen in DKA. After admission, intravenous infusion of 0.45% saline was administered, which resulted in the development of pure metabolic acidosis. After starting insulin infusion, hypokalemia and hypophosphatemia became evident and finally resulted in massive rhabdomyolysis. Hyperkalemia accompanying oliguric acute kidney injury (AKI) warranted initiation of hemodialysis (HD) on Day-five. On the 45th hospital day, his urine output started to increase and a total of 22 HD sessions were required. We believe that in this case severe dehydration, hypothermia and hypokalemia might have contributed to the initial symptoms of DKA as well as the prolongation of AKI.

Changes in glomerular filtration rate after donation in living kidney donors: a single-center cohort study.

PURPOSE: A number of studies have reported on decline in glomerular filtration rate (GFR) after donation in Japanese living kidney donors. The purpose of the present study was to examine the clinicopathological factors associated with changes in GFR after donation in living kidney donors. METHODS: We reviewed the charts of living kidney donors (n = 294) and monitored estimated GFR (eGFR) values from the time of 0-h kidney biopsy until 3 years after donation. We assessed donor age, gender, body mass index, blood pressure, urinalysis, and several other clinical parameters including the severity of glomerulosclerosis and arteriosclerosis. RESULTS: The grade of arteriosclerosis in 0-h biopsy specimens was higher in the older donor group (57-76 years) than in the younger donor group (30-56 years). Mean donor eGFR at the time of the donation was 80.1 ± 13.6 ml/min/1.73 m(2). Most of the living kidney donors in this study developed stage 3 chronic kidney disease (CKD). The mean changes in eGFR at 1-3 years after donation showed a steady state that was distinct from the generally accepted notion that GFR declines with age. Multivariate regression analyses showed that the changes in eGFR were negatively associated with age (r = -0.21, P < 0.001) and preoperative eGFR (r = -0.18, P < 0.001), but not associated with the grade of glomerulosclerosis and arteriosclerosis. CONCLUSION: Donor age and pre-GFR at the time of nephrectomy were associated with decline in kidney function in living kidney donors after donation. Most of the donors developed stage 3 CKD within 3 years after donation but without subsequent progression, at least for several years.