In Silico Reconstruction of Sperm Chemotaxis.

In echinoderms, sperm swims in random circles and turns in response to a chemoattractant. The chemoattractant evokes transient Ca2+ influx in the sperm flagellum and induces turning behavior. Recently, the molecular mechanisms and biophysical properties of this sperm response have been clarified. Based on these experimental findings, in this study, we reconstructed a sperm model in silico to demonstrate an algorithm for sperm chemotaxis. We also focused on the importance of desensitizing the chemoattractant receptor in long-range chemotaxis because sperm approach distantly located eggs, and they must sense the chemoattractant concentration over a broad range. Using parameters of the sea urchin, simulations showed that a number of sperm could reach the egg from millimeter-order distances with desensitization, indicating that we could organize a functional sperm model, and that desensitization of the receptor is essential for sperm chemotaxis. Then, we compared the model with starfish sperm, which has a different desensitization scheme and analyzed the properties of the model against various disturbances. Our approach can be applied as a novel tool in chemotaxis research.

Usefulness of the quantitative measurement of urine protein at a community-based health checkup: a cross-sectional study.

BACKGROUND: The dipstick urinalysis for proteinuria has been used for chronic kidney disease (CKD) screening at community-based health checkups; however, it has major drawbacks in that the result is only semi-quantitative and is influenced by urine concentration. METHODS: We conducted urine protein/creatinine ratio (UPCR) measurements of 590 participants who showed a result of more than trace proteinuria on a dipstick analysis and evaluated the usefulness of UPCR measurements in community-based health checkups. RESULTS: The UPCR values increased in accordance with the severity of the dipstick test findings, but statistical significance was only obtained between (±) and (1+), between (±) and (2+), and between (±) and (3+) groups. When the participants with (±) proteinuria were subjected to CGA classification (a classification of CKD by cause, glomerular filtration rate category, and albuminuria category) according to their UPCR data, a significant proportion of subjects (277, 77.0%) moved from the A2 category into A1, which is a less severe category. Conversely, 21 subjects (5.8%) were reclassified into a more severe category (A3). Thus, a dipstick test may produce a non-negligible number of false negatives as well as a large number of false positives. Similarly, the classifications of more than half of the subjects with (1+) or more severe proteinuria were changed based on their UPCR results. CONCLUSION: The dipstick urinalysis for proteinuria appears less reliable than expected, suggesting that the quantitative measurement of urine protein should be performed even during mass health checkups to ensure the early detection and prevention of CKD.

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation.

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43(-/-) salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43(-/-) samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43(-/-) phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.

Protein kinase A activity leads to the extension of the acrosomal process in starfish sperm.

Acrosomal vesicles (AVs) of sperm undergo exocytosis during the acrosome reaction, which is immediately followed by the actin polymerization-dependent extension of an acrosomal process (AP) in echinoderm sperm. In the starfish Asterias amurensis, a large proteoglycan, acrosome reaction-inducing substance (ARIS), together with asteroidal sperm-activating peptide (asterosap) and/or cofactor for ARIS, induces the acrosome reaction. Asterosap induces a transient elevation of intracellular cGMP and Ca2+ levels, and, together with ARIS, causes a sustained increase in intracellular cAMP and Ca2+ . Yet, the contribution of signaling molecules downstream of cAMP and Ca2+ in inducing AV exocytosis and AP extension remain unknown. A modified acrosome reaction assay was used here to differentiate between AV exocytosis and AP extension in starfish sperm, leading to the discovery that Protein kinase A (PKA) inhibitors block AP extension but not AV exocytosis. Additionally, PKA-mediated phosphorylation of target proteins occurs, and these substrates localize at the base of the AP, demonstrating that PKA activation regulates an AP extension step during the acrosome reaction. The major PKA substrate was further identified, from A. amurensis and Asterias forbesi sperm, as a novel protein containing six PKA phosphorylation motifs. This protein, referred to as PKAS1, likely plays a key role in AP actin polymerization during the acrosome reaction.

The CatSper channel: a polymodal chemosensor in human sperm.

The sperm-specific CatSper channel controls the intracellular Ca(2+) concentration ([Ca(2+)](i)) and, thereby, the swimming behaviour of sperm. In humans, CatSper is directly activated by progesterone and prostaglandins-female factors that stimulate Ca(2+) influx. Other factors including neurotransmitters, chemokines, and odorants also affect sperm function by changing [Ca(2+)](i). Several ligands, notably odorants, have been proposed to control Ca(2+) entry and motility via G protein-coupled receptors (GPCRs) and cAMP-signalling pathways. Here, we show that odorants directly activate CatSper without involving GPCRs and cAMP. Moreover, membrane-permeable analogues of cyclic nucleotides that have been frequently used to study cAMP-mediated Ca(2+) signalling also activate CatSper directly via an extracellular site. Thus, CatSper or associated protein(s) harbour promiscuous binding sites that can host various ligands. These results contest current concepts of Ca(2+) signalling by GPCR and cAMP in mammalian sperm: ligands thought to activate metabotropic pathways, in fact, act via a common ionotropic mechanism. We propose that the CatSper channel complex serves as a polymodal sensor for multiple chemical cues that assist sperm during their voyage across the female genital tract.

Novel hedgehog agonists promote osteoblast differentiation in mesenchymal stem cells.

Hedgehog (Hh) family members are involved in multiple cellular processes including proliferation, migration, differentiation, and cell fate determination. Recently, the novel Hh agonists Hh-Ag 1.3 and 1.7 were identified in a high-throughput screening of small molecule compounds that activate the expression of Gli1, a target of Hh signaling. This study demonstrates that Hh-Ag 1.3 and 1.7 strongly activate the expression of endogenous Gli1 and promote osteoblast differentiation in the mesenchymal stem cell line C3H10T1/2. Both compounds stimulated alkaline phosphatase activity in a dose-dependent manner, and induced osteoblast marker gene expression in C3H10T1/2 cells, which indicated that they had acquired an osteoblast identity. Of the markers, the expression of osterix/Sp7, a downstream target of runt-related transcription factor (Runx)2, was induced by Hh-Ag 1.7, which also rescued the osteoblast differentiation defect of RD-127, a mesenchymal cell line from Runx2-deficient mice. Hh-Ags also activated canonical Wnt signaling and synergized with low doses of BMP-2 to enhance osteoblastic potential. Thus, Hh-Ag 1.7 could be useful for bone healing in individuals with abnormalities in osteogenesis, such as osteoporosis patients and the elderly, and can contribute to the development of novel therapeutics for the treatment of bone fractures and defects.

High mortality rate in hemodialysis patients who undergo invasive cardiovascular procedures related to peripheral artery disease - community-based observational study in Kumamoto Prefecture - .

BACKGROUND: Cardiovascular disease is a major cause of mortality in hemodialysis patients. The aim was to assess the relationship of various invasive cardiovascular procedures (ICP) to clinical outcome in hemodialysis patients. METHODS AND RESULTS: A total of 5,813 patients at 76 facilities were on maintenance hemodialysis in Kumamoto Prefecture. Of these, 4,807 patients at 58 institutions were enrolled. Of 4,807 patients, 212 ICP (4.4%) were performed for various cardiovascular diseases in 189 patients (3.9%). ICP included PCI (n=80), endovascular treatment (n=59), radiofrequency catheter ablation (n=8), implantation of permanent pacemaker (n=15) and ICD (n=5), thoracotomy for valvular diseases (n=16), CABG (n=14), bypass surgery for peripheral artery disease (PAD; n=8), and artificial vessel replacement for aneurysm or aortic dissection (n=7). The overall mortality rate was 10.1% (19/189 patients). The mortality rate was highest in patients who underwent ICP for PAD, compared with other ICP (PAD, 18.2%; non-PAD, 6.7%, P=0.017). Infection and PAD were significant predictors of mortality (infection: OR, 8.30; 95% CI: 1.29-65.13, P=0.027; PAD: OR, 3.76; 95% CI: 1.35-10.48, P=0.012). The presence of inflammation/malnutrition factors was associated with high mortality (OR, 15.49; 95% CI: 3.22-74.12, P=0.0006). CONCLUSIONS: In this community-based registry study of 4,807 hemodialysis patients, the mortality rate of PAD patients was high despite ICP.

Primary vomerine osteotomy with gingivoperiosteoplasty for bilateral cleft lip and palate patients with protrusion and/or torsion of the premaxilla.

In patients with bilateral cleft lip and palate (BCLP) with protrusion and/or torsion of the premaxilla, achieving a favorable outcome with adequate facial and maxillary development remains challenging. In the present study, we report a retrospective cohort of patients with complete BCLP who were treated between 2017 and 2020 at a single center in Japan. We investigated the effects and complications of primary vomerine osteotomy (PVO) with gingivoperiosteoplasty (GPP) following pre-surgical orthodontics (PSO) for premaxillary protrusion and/or torsion. For patients with residual premaxillary protrusion and/or torsion after PSO, PVO, and GPP were performed. The distances and angles of the premaxilla were measured on dental casts before PSO, on the day of PVO, after PVO, and on the day of palatoplasty after cheiloplasty. We further assessed postoperative complications. From a total of 36 patients with complete BCLP after PSO, seven patients underwent PVO with GPP. Proper positioning of the premaxilla was achieved in all seven patients. The distance between the anterior edge of the premaxilla and the anterior edge of the lateral segment and the length of the premaxillary-lateral segment on both sides continued to decrease over time. Loosening of GPP sutures occurred in two cases, although no major complications such as necrosis of the premaxilla or fistula formation occurred. Vomerine osteotomy with GPP before primary cheiloplasty is a potential treatment option in BCLP when the premaxilla still protrudes despite PSO or because PSO cannot be applied.

An investigation into nutritional methods at the fifth day after birth of infants in association with cleft type and laterality.

In patients with clefts, the affection of other congenital malformations on the feeding is unclear. We investigated the other congenital malformations and nutritional intake of neonates with cleft lip and/or palate and examined their relationships associated with cleft type and laterality. The participants included 126 infants under treatment with a presurgical naso-alveolar molding (PNAM) or a Hotz-type plate. The survey items were gender, cleft type and side, presence and nature of other congenital malformations, birth weight and nutritional method at age of the fifth day. The number of infants was 36 (28.6%) of cleft lip and alveolus, 82 (65.1%) of cleft lip and palate, and 8 (6.3%) of cleft palate only. Forty-three patients (34.1%) had other various congenital malformations. The nutritional method included oral intake in 78.6% (n = 99) of cases and tube feeding with/without oral intake in 21.4% (n = 27) of cases. The rate of tube feeding was higher for right-sided clefts than that for left-sided clefts. This observation was consistent with the fact that right-sided clefts were associated with more significant other congenital malformations than those on the left-side. The nutritional method for infants with cleft lip and/or palate was related to the presence of other congenital malformations, not to cleft laterality or oral cleft itself under early treatment with PNAM plate. These results proposed that screening the general condition is essential for neonates with right-sided cleft lip with/without cleft palate compared to left-sided clefts, which should be conducted immediately after birth for planning the appropriate nutritional method.

Acrosome reaction-related steroidal saponin, Co-ARIS, from the starfish induces structural changes in microdomains.

Cofactor for acrosome reaction-inducing substance (Co-ARIS) is a steroidal saponin from the starfish Asterias amurensis. Saponins exist in many plants and few animals as self-defensive chemicals, but Co-ARIS has been identified as a cofactor for inducing the acrosome reaction (AR). In A. amurensis, the AR is induced by the cooperative action of egg coat components (ARIS, Co-ARIS, and asterosap); however, the mechanism of action of Co-ARIS is obscure. In this study we elucidated the membrane dynamics involved in the action of Co-ARIS. We found that cholesterol specifically inhibited the Co-ARIS activity for AR induction and detected the binding of labeled compounds with sperm using radioisotope-labeled Co-ARIS. Co-ARIS treatment did not reduce the content of sperm sterols, however, the condition was changed and localization of GM1 ganglioside on the periacrosomal region disappeared. We then developed a caveola-breaking assay, a novel method to detect the effect of chemicals on microdomains of culture cell, and confirmed the disturbance of somatic cell caveolae in the presence of Co-ARIS. Finally, by atomic force microscopy observations and surface plasmon resonance measurements using an artificial membrane, we revealed that Co-ARIS colocalized with GM1 clusters on the microdomains. Through this study, we revealed a capacitation-like event for AR in starfish sperm.

Novel conserved structural domains of acrosome reaction-inducing substance are widespread in invertebrates.

In the starfish Asterias amurensis, acrosome reaction inducing substance (ARIS) is the main factor responsible for allowing sperm to recognize the egg jelly and begin the acrosome reaction (AR). ARIS is a large proteoglycan-like molecule, and its pentasaccharide repeat, Fragment 1 (Fr. 1), is responsible for inducing AR. Here, we investigated the primary structure of ARIS for the first time in order to improve our understanding of its functionality. Electrophoretic analysis revealed that ARIS is a complex of three proteins, all of which are modified by the Fr. 1 sugar chain. Sequencing indicated that there are two novel, conserved domains in all three ARIS proteins: ARIS N-terminus (AR-N) and ARIS C-terminus (AR-C) domains. We also found that other echinoderms possess ARIS proteins that are capable of inducing the AR for homologous sperm, indicating that ARIS proteins may be a ubiquitous component for echinoderm fertilization. Moreover, we identified ARIS-like genes from Ctenophora to Protochordata.

A case with acute renal failure and subsequent nephrotic syndrome.

Nephrotic syndrome due to secondary amyloidosis is not so common, and the prognosis depends on primary disease. We report a case of secondary amyloidosis caused by Takayasu's arteritis. Sustained high fever and acute renal failure proceeded to the occurrence of nephrotic syndrome. Secondary amyloidosis was diagnosed by renal biopsy before the diagnosis of primary disease. She was completely recovered from nephrotic syndrome after two years' treatment with prednisolone, aspirin, and dimethyl sulfoxide. This rare case provides meaningful suggestions for the diagnosis and treatment of acute renal failure and nephrotic syndrome caused by secondary amyloidosis.

Regulation of the starfish sperm acrosome reaction by cGMP, pH, cAMP and Ca2+.

In the starfish, Asterias amurensis, three components in the jelly coat of eggs, namely acrosome reaction-inducing substance (ARIS), Co-ARIS and asterosap, act in concert on homologous spermatozoa to induce the acrosome reaction (AR). Molecular recognition between the sperm surface molecules and the egg jelly molecules must underlie signal transduction events triggering the AR. Asterosap is a sperm-activating molecule, which stimulates rapid synthesis of intracellular cGMP, pH and Ca2+. This transient elevation of Ca2+ level is caused by a K+-dependent Na+/Ca2+ exchanger, and the increase of intracellular pH is sufficient for ARIS to induce the AR. The concerted action of ARIS and asterosap could induce elevate intracellular cAMP levels in starfish sperm and the sustained increase in [Ca2+], which is essential for the AR. The signaling pathway induced by these factors seems to be synergistically regulated to trigger the AR in starfish sperm.

Long-term plasma levels and dose modulation of alacepril in patients with chronic renal failure.

Because most angiotensin-converting enzyme inhibitors are excreted into urine, any decrease in renal function increases the plasma levels of these drugs. This study was designed to investigate the appropriate doses of alacepril in patients with chronic renal failure. The total plasma concentration of captopril, an active metabolite of alacepril, was measured in 47 patients with chronic renal failure or normal renal function. Fifteen patients on chronic hemodialysis were also enrolled in this study. In patients treated with 12.5, 25 and 50 mg alacepril, the plasma concentration of captopril was linearly correlated with serum creatinine and creatinine clearance (Ccr). There was an approximately 40% decrease of the plasma captopril concentration after 4 h of hemodialysis. Among patients treated with 25 or 50 mg alacepril for 4.5 years, the plasma concentration of captopril gradually increased along with an increase in serum creatinine (from 2.0 to 5.8, and from 1.9 to 7.1 mg/dL, respectively). Although the plasma concentration of captopril was higher in the 50 mg group, the increase in serum creatinine during this period was not different between the two groups. The plasma aldosterone concentration did not increase during this period. These data suggest that alacepril should be reduced from 50 to 25 and 12.5 mg/day in patients with a serum creatinine level of greater than 2-3 and 4-6 mg/dL, respectively, in order to maintain a plasma level equivalent to that in subjects with normal renal function receiving 50 mg/day alacepril. For patients on chronic hemodialysis, 12.5 mg alacepril is the appropriate dose.

Validation of the high mortality rate of Malnutrition-Inflammation-Atherosclerosis syndrome: -Community-based observational study.

BACKGROUND: Malnutrition-Inflammation-Atherosclerosis (MIA) factors significantly and independently affect life prognosis of hemodialysis (HD) patients. We re-evaluated Japanese data, which have progressed ahead from a community-based observational study. The present study was designed to assess the contribution of these MIA factors to the mortality rate of Japanese HD patients in a community of 1.8 million people over a 36-month follow-up period. METHODS AND RESULTS: A total of 5813 patients at 76 facilities were on maintenance HD in the Kumamoto Prefecture. Specifically, 4807 of these patients at 58 institutions were enrolled. Patients who exhibited lower serum albumin and higher serum C-reactive protein levels were defined as "malnourished" and "inflamed", respectively, compared with the median values. Patients who underwent invasive procedures for atherosclerotic diseases were defined as "atherosclerotic". The 36-month all-cause mortality rate in Japanese HD patients was 12.4%. This rate directly correlated with the number of MIA factors. The odds ratio of the all-cause mortality rate markedly and significantly increased as the number of factors increased. The presence of 3 MIA factors in HD patients was a significant predictor of mortality, as evidenced by a multivariate logistic regression analysis. CONCLUSIONS: This study clearly demonstrated the close association between MIA syndrome and high mortality in Japanese HD patients. Early detection and the adjustment of MIA factors are mandatory.

Globoside accelerates the differentiation of dental epithelial cells into ameloblasts.

Tooth crown morphogenesis is tightly regulated by the proliferation and differentiation of dental epithelial cells. Globoside (Gb4), a globo-series glycosphingolipid, is highly expressed during embryogenesis as well as organogenesis, including tooth development. We previously reported that Gb4 is dominantly expressed in the neutral lipid fraction of dental epithelial cells. However, because its functional role in tooth development remains unknown, we investigated the involvement of Gb4 in dental epithelial cell differentiation. The expression of Gb4 was detected in ameloblasts of postnatal mouse molars and incisors. A cell culture analysis using HAT-7 cells, a rat-derived dental epithelial cell line, revealed that Gb4 did not promote dental epithelial cell proliferation. Interestingly, exogenous administration of Gb4 enhanced the gene expression of enamel extracellular matrix proteins such as ameloblastin, amelogenin, and enamelin in dental epithelial cells as well as in developing tooth germs. Gb4 also induced the expression of TrkB, one of the key receptors required for ameloblast induction in dental epithelial cells. In contrast, Gb4 downregulated the expression of p75, a receptor for neurotrophins (including neurotrophin-4) and a marker of undifferentiated dental epithelial cells. In addition, we found that exogenous administration of Gb4 to dental epithelial cells stimulated the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase signalling pathways. Furthermore, Gb4 induced the expression of epiprofin and Runx2, the positive regulators for ameloblastin gene transcription. Thus, our results suggest that Gb4 contributes to promoting the differentiation of dental epithelial cells into ameloblasts.

[A case of secondary hyperparathyroidism with an ectopic intrathyroid gland successfully diagnosed and controlled by percutaneous ethanol injection therapy (PEIT)].

Secondary hyperparathyroidism (II HPT) is a major complication in chronic dialysis patients, and percutaneous ethanol injection therapy (PEIT) has become a useful alternative treatment for II HPT. However, the existence of ectopic parathyroid glands is a major problem when conducting PEIT. Ectopic parathyroid gland accepts 10-35% of II HPT, and the missing glands cannot be detected consistently by any imaging techniques, including scintigraphy. Intrathyroid parathyroid gland is as rare as about 1% and recurrence of missing glands after parathyroidectomy (PTx) has been reported in some cases. We report here a 52-year-old female in whom an ectopic parathyroid gland was defected successfully and intact-PTH controlled by tentative PEIT. At the first examination, a left parathyroid adenoma and a right thyroid goiter were pointed out by ultrasonography, CT and scintigraphy. PEIT was applied twice to the left parathyroid adenoma, but intact-PTH was not decreased. Ultrasonography, CT, 201Tl-99mTc subtraction scintigraphy and fine needle aspiration biopsy (FNAB) were performed again to search for the existence of ectopic glands. The results suggested that the right intrathyroid tumor was an ectopic parathyroid gland. Consequently, tentative PEIT was applied to the right intrathyroid tumor, and successful control of intact-PTH and serum Ca was eventually achieved. To our knowledge, this is the first reported case of secondary hyperparathyroidism with an ectopic intrathyroid gland that was successfully controlled by PEIT. In this case, it was suggested that tentative PEIT of intrathyroid tumor was a useful method for detecting an ectopic parathyroid gland.