Insights into egg coat assembly and egg-sperm interaction from the X-ray structure of full-length ZP3.

ZP3, a major component of the zona pellucida (ZP) matrix coating mammalian eggs, is essential for fertilization by acting as sperm receptor. By retaining a propeptide that contains a polymerization-blocking external hydrophobic patch (EHP), we determined the crystal structure of an avian homolog of ZP3 at 2.0 Å resolution. The structure unveils the fold of a complete ZP domain module in a homodimeric arrangement required for secretion and reveals how EHP prevents premature incorporation of ZP3 into the ZP. This suggests mechanisms underlying polymerization and how local structural differences, reflected by alternative disulfide patterns, control the specificity of ZP subunit interaction. Close relative positioning of a conserved O-glycan important for sperm binding and the hypervariable, positively selected C-terminal region of ZP3 suggests a concerted role in the regulation of species-restricted gamete recognition. Alternative conformations of the area around the O-glycan indicate how sperm binding could trigger downstream events via intramolecular signaling.

Effect of single-administration of D-sorbitol pretreatment on the bitterness and continued willingness to take asenapine: a randomized, single-blind, placebo-controlled, crossover trial.

BACKGROUND: Asenapine has unique orally-related side effects, such as a bitter taste induced by sublingual administration, which often results in discontinuation of the medication. While the FDA has approved black-cherry-flavored asenapine, several countries have prescribed only unflavored versions. Specifically, Asians commonly report experiencing the bitterness of asenapine because they are more sensitive to bitter tastes than other ethnic groups. In this study, with the aim of improving adherence by reducing the bitterness of asenapine, we investigated the effects of D-sorbitol, which reduced the bitterness parameters of taste sensors in our previous basic study on the bitterness and continuity of asenapine among patients with schizophrenia. METHODS: Twenty adult patients with schizophrenia were included in this single-blind, placebo-controlled, crossover trial. Participants rinsed their mouths with single-administration of D-sorbitol or a placebo prior to each administration of asenapine. We then conducted the questionnaires and assessed changes in the bitterness of asenapine (primary end point) and willingness to continue its use (secondary end point). RESULTS: D-sorbitol significantly improved the bitterness of asenapine (p = 0.038). Although it did not significantly increase the willingness to continue asenapine (p = 0.180), it did show improvement over the placebo in enhancing willingness to continue, especially in patients who were not accustomed to its taste. CONCLUSION: Our findings indicate that single-administration of D-sorbitol significantly reduces the bitterness of asenapine. In countries where flavored asenapine is not available, this finding could benefit patients who were not accustomed to its bitter taste. TRIAL REGISTRATION: This study was registered in the Japan Registry of Clinical Trials (jRCTs041210019) on May 14, 2021.

Simultaneous Tumor Shrinkage and Bronchial Perforation Induced by Nivolumab plus Cabozantinib Combination Therapy in a Patient with Collecting Duct Carcinoma.

Vascular endothelial growth factor receptor tyrosine kinase inhibitors are known to cause perforation as one of their severe side effects, and postoperative and postradiation therapy are known risk factors. However, there are few studies on perforation following tumor shrinkage. A 78-year-old woman with postoperative recurring left collecting duct carcinoma of the right hilar lymph nodes and mediastinum underwent eight courses of nivolumab plus cabozantinib, resulting in tumor shrinkage. Three days after the last administration, she developed fever and cough and was hospitalized for right lobar pneumonia. The patient received long-term antibiotics for bronchial fistula with the destruction of the bronchial wall and secondary lung abscess. When using nivolumab plus cabozantinib combination therapy for a tumor with bronchial invasion, physicians should be aware of bronchial perforation as the tumor shrinks.

Sensory evaluation of the bitterness of asenapine using D-sorbitol pretreatment: single-blind, placebo-controlled, crossover trial.

BACKGROUND: Antipsychotics are essential in the acute treatment of and maintenance therapy for schizophrenia, but medication adherence and long-term treatment continuity are needed to maximize their effectiveness. Each antipsychotic has various side effects, which may affect adherence. Some patients with schizophrenia are reluctant to take asenapine because of its unique oral-related side effects, such as the bitter taste caused by sublingual administration. Our previous basic research found that D-sorbitol lowered the bitterness parameters of the taste sensors. However, whether D-sorbitol has the same effect in patients remains unclear. Therefore, using a D-sorbitol solution, we aim to evaluate changes in the bitterness of asenapine among patients with schizophrenia. METHODS: In this single-blind, placebo-controlled, crossover trial, we plan to recruit 20 adult patients with schizophrenia spectrum disorder who take sublingual asenapine tablets. The participants will be divided into two groups (n = 10 each). Each group will be given a D-sorbitol or placebo solution on the first day for rinsing before taking the sublingual asenapine tablets. After a 1-day interval, the participants will rinse their mouths again with a different liquid. Questionnaires regarding changes in taste and the willingness to continue asenapine will be conducted before the start of the study and after each rinse. The primary and secondary end points will be a taste evaluation of bitterness, and the willingness to continue asenapine, respectively. Differences in questionnaire scores between the D-sorbitol and placebo solutions will be calculated and analyzed using a McNemar test. DISCUSSION: This study aims to determine the efficacy of D-sorbitol in masking the bitter taste of asenapine. To our knowledge, it is the first intervention study using D-sorbitol for bitter taste of asenapine in patients with schizophrenia. Evidence of the efficacy of D-sorbitol could result in D-sorbitol pretreatment being an easy and inexpensive means of improving adherence to asenapine. TRIAL REGISTRATION: This study was registered in the Japan Registry of Clinical Trials jRCTs041210019, on May 14, 2021. Ethics approval was obtained from the Nagoya University Clinical Research Review Board.

Acute Hepatitis E Infection during Chemotherapy for Lung Cancer: A Case Report.

Acute hepatitis E, one of the causes of acute liver injury, has been increasingly diagnosed in developed countries in recent years. Misdiagnosis of acute hepatitis E virus (HEV) infection as drug-induced liver injury (DILI) may lead to discontinuation of effective chemotherapy. Thus, viral hepatitis, including hepatitis E, must be ruled out in the diagnosis of DILI. A 78-year-old woman with lung adenocarcinoma and multiple bone metastases received maintenance therapy with pemetrexed + pembrolizumab for a year. Increased aspartate aminotransferase and alanine aminotransferase levels, indicating acute liver injury, were observed. Initially, DILI was suspected, and she was given medications to lower the levels of hepatic enzymes. She was later admitted to the hospital with the chief complaint of general malaise and anorexia. Serum aspartate aminotransferase and alanine aminotransferase levels were markedly elevated (381 and 854 U/L, respectively). Acute HEV infection was diagnosed based on the detection of serum HEV immunoglobulin A antibodies. The patient received liver support therapy, and the serum hepatic enzymes recovered to normal levels. Chemotherapy was resumed without any subsequent relapse of hepatic enzyme elevation. When DILI is suspected during chemotherapy, exclusion of viral hepatitis is mandatory, which can be achieved by measuring markers of hepatitis viruses, including HEV, and examining the patient's detailed medical history.

Shedding light on latent pathogenesis and pathophysiology of mental disorders: The potential of iPS cell technology.

Mental disorders are considered as one of the major healthcare issues worldwide owing to their significant impact on the quality of life of patients, causing serious social burdens. However, it is hard to examine the living brain-a source of psychiatric symptoms-at the cellular, subcellular, and molecular levels, which poses difficulty in determining the pathogenesis and pathophysiology of mental disorders. Recently, induced pluripotent stem cell (iPSC) technology has been used as a novel tool for research on mental disorders. We believe that the iPSC-based studies will address the limitations of other research approaches, such as human genome, postmortem brain study, brain imaging, and animal model analysis. Notably, studies using integrated iPSC technology with genetic information have provided significant novel findings to date. This review aimed to discuss the history, current trends, potential, and future of iPSC technology in the field of mental disorders. Although iPSC technology has several limitations, this technology can be used in combination with the other approaches to facilitate studies on mental disorders.

[Prognostic factors in light-chain cardiac amyloidosis: a retrospective analysis].

Among cases of amyloid light-chain (AL) amyloidosis, cardiac amyloidosis is particularly known to be associated with a poor prognosis. However, a few established prognostic indicators exist that consider other organ involvements and a patient's general condition. Between 2012 and 2019, we retrospectively reviewed 27 patients, who were diagnosed with AL amyloidosis at our hospital. The 3-year overall survival rate of patients with cardiac involvement was 20% (95% confidence interval [CI], 0.035-0.461) and that of patients without cardiac involvement was 85.7% (95%CI, 0.334-0.979) (p=0.021). Poor prognostic factors of AL amyloidosis included left ventricular ejection fraction <60%, hemoglobin < 10 g/dl, NT pro-BNP>1,800 pg/ml, BNP>400 pg/ml, difference free light chains>180 mg/l, New York Heart Association classification ≥3, Mayo stage IV disease, and cardiac amyloidosis. A study on four patients who died within 6 months of diagnosis revealed that all the patients had cardiac amyloidosis and Mayo stage IV disease, and they all did not receive sufficient chemotherapy. Although the number of treatment options for AL amyloidosis is expected to increase in the future, patients with poor prognostic factors have a poor prognosis and careful treatment decisions, including palliative care, are required.

Contribution of rat embryonic stem cells to xenogeneic chimeras in blastocyst or 8-cell embryo injection and aggregation.

The ultimate goal of regenerative medicine is the transplantation of a target organ generated by the patient's own cells. Recently, a method of organ generation using pluripotent stem cells (PSCs) and blastocyst complementation was reported. This approach is based on chimeric animal generation using an early embryo and PSCs, and the contribution of PSCs to the target organ is key to the method's success. However, the contribution rate of PSCs in target organs generated by different chimeric animal generation methods remains unknown. In this study, we used 8-cell embryo aggregation, 8-cell embryo injection, and blastocyst injection to generate interspecies chimeric mice using rat embryonic stem (ES) cells and then investigated the differences in the contribution rate of the rat ES cells. The rate of chimeric mouse generation was the highest using blastocyst injection, followed in order by 8-cell embryo injection and 8-cell embryo aggregation. However, the contribution rate of rat ES cells was the highest in chimeric neonates generated by 8-cell embryo injection, and the difference was statistically significant in the liver. Live functionality was confirmed by analyzing the expression of rat hepatocyte-derived drug-metabolizing enzyme. Collectively, these findings indicate that the 8-cell embryo injection method is the most suitable for generation of PSC-derived organs via chimeric animal generation, particularly for the liver.

Effect of Celecoxib on Differentiation of Human Induced Pluripotent Stem Cells into Hepatocytes Involves STAT5 Activation.

The liver abundantly expresses various drug-metabolizing enzymes and, thus, plays a central role in drug metabolism. In this regard, cytochrome P450 (CYP) is responsible for drug metabolism in the liver. Therefore, since CYP3A4 accounts for approximately 30% of the CYPs, the prediction of hepatic CYP3A4-mediated pharmacokinetics is essential for drug development. Human induced pluripotent stem cell-derived hepatocytes (hiHep) have become a major model of drug metabolism in drug development studies. However, drug metabolizing activities, such as those involving CYP3A4, are lower in hiHep than in human primary hepatocytes (HPHs). Recently, it was revealed that celecoxib upregulates the expression of CYPs to normal levels through the activation of signal transducer and transcriptional activation factor 5 (STAT5). Therefore, we investigated whether celecoxib treatment could normalize the low drug metabolism activities in hiHep. The mRNA expression levels of hepatic markers [asialoglycoprotein receptor 1 (ASGR1) and tyrosine aminotransferase (TAT)] and metabolic enzymes (UDP-glucuronosyltransferase 1A1 and CYP3A4) in hiHep significantly increased after celecoxib treatment. These mRNA expression levels were 7-, 1/3-, 1/2-, and 1/10-fold of the HPHs cultured for 48 hours, respectively. Furthermore, CYP3A4 activity significantly increased. To investigate the mechanism of CYP3A4 mRNA upregulation, we analyzed the phosphorylation of STAT5 after celecoxib treatment and found it to be significantly increased. Moreover, the increase in CYP3A4 mRNA expression was attenuated by cotreatment with STAT5 inhibitor. These results suggest that celecoxib promotes hepatocyte differentiation of hiHep by activating STAT5 and is useful for the generation of functional hiHep.

Avian Egg and Egg Coat.

An ovulated egg of vertebrates is surrounded by unique extracellular matrix, the egg coat or zona pellucida, playing important roles in fertilization and early development. The vertebrate egg coat is composed of two to six zona pellucida (ZP) glycoproteins that are characterized by the evolutionarily conserved ZP-domain module and classified into six subfamilies based on phylogenetic analyses. Interestingly, investigations of biochemical and functional features of the ZP glycoproteins show that the roles of each ZP-glycoprotein family member in the egg-coat formation and the egg-sperm interactions seemingly vary across vertebrates. This might be one reason why comprehensive understandings of the molecular basis of either architecture or physiological functions of egg coat still remain elusive despite more than 3 decades of intensive investigations. In this chapter, an overview of avian egg focusing on the oogenesis are provided in the first section, and unique features of avian egg coat, i.e., perivitelline layer, including the morphology, biogenesis pathway, and physiological functions are discussed mainly on chicken and quail in terms of the characteristics of ZP glycoproteins in the following sections. In addition, these features of avian egg coat are compared to mammalian zona pellucida, from the viewpoint that the structural and functional varieties of ZP glycoproteins might be associated with the evolutionary adaptation to their reproductive strategies. By comparing the egg coat of birds and mammals whose reproductive strategies are largely different, new insights into the molecular mechanisms of vertebrate egg-sperm interactions might be provided.

Effect of Oxidative Stress on Cardiovascular System in Response to Gravity.

Long-term habitation in space leads to physiological alterations such as bone loss, muscle atrophy, and cardiovascular deconditioning. Two predominant factors-namely space radiation and microgravity-have a crucial impact on oxidative stress in living organisms. Oxidative stress is also involved in the aging process, and plays important roles in the development of cardiovascular diseases including hypertension, left ventricular hypertrophy, and myocardial infarction. Here, we discuss the effects of space radiation, microgravity, and a combination of these two factors on oxidative stress. Future research may facilitate safer living in space by reducing the adverse effects of oxidative stress.

Glycosylated chicken ZP2 accumulates in the egg coat of immature oocytes and remains localized to the germinal disc region of mature eggs.

Vertebrate eggs are surrounded by an egg coat, which is a specific extracellular egg matrix consisting of several glycoproteins with a conserved zona pellucida (ZP) domain. Two mammalian egg coat subunits, ZP2 and ZP3, have been suggested to act as sperm receptors. In bird eggs, however, ZP2 has never been identified in the egg coat of mature oocytes and ovulated eggs. Here we report that chicken ZP2 is expressed in immature small follicles and remains as an egg-coat component locally in the germinal disc region of mature eggs. RT-PCR analysis indicated marked expression of the ZP2 and ZP4 genes in the granulosa cells of immature white follicles, whereas the ZP3 and ZPD genes showed marked expression in the cells of maturing yellow follicles. ZP2 was identified in the egg coat isolated from immature follicles as a heavily N-glycosylated glycoprotein of ∼200 kDa, which was enzymatically converted to a 70-kDa deglycosylated form. Immunoblotting and immunohistological analyses showed that ZP2 was localized around the germinal disc region of mature follicles. ZP2 was accumulated in the egg coat of immature white follicles at the earlier stages of oocyte development and became a minor component in the egg coat of maturing yellow follicles, except for the germinal disc region. Localization of ZP2 in the germinal disc region of mature eggs, where sperm bind to the egg coat at high density, suggests some role for ZP2 in the preferential binding and penetration of sperm in the germinal disc region of bird eggs.

Functional expression of recombinant human macrophage ß-glucan receptor dectin-1 using baculovirus-silkworm expression system.

Human macrophage dectin-1, a type II transmembrane ß-glucan receptor, was expressed as a fusion protein with an N-terminal hexahistidine tag in a baculovirus-silkworm expression system and assayed for binding activity. Recombinant dectin-1 specifically bound to some ß-glucans, and the neck domain and N-linked oligosaccharide chains of human dectin-1 did not affect the ligand binding activity and specificity of the receptor.

Analysis of human neuronal cells carrying ASTN2 deletion associated with psychiatric disorders.

Recent genetic studies have found common genomic risk variants among psychiatric disorders, strongly suggesting the overlaps in their molecular and cellular mechanism. Our research group identified the variant in ASTN2 as one of the candidate risk factors across these psychiatric disorders by whole-genome copy number variation analysis. However, the alterations in the human neuronal cells resulting from ASTN2 variants identified in patients remain unknown. To address this, we used patient-derived and genome-edited iPS cells with ASTN2 deletion; cells were further differentiated into neuronal cells. A comprehensive gene expression analysis using genome-edited iPS cells with variants on both alleles revealed that the expression level of ZNF558, a gene specifically expressed in human forebrain neural progenitor cells, was greatly reduced in ASTN2-deleted neuronal cells. Furthermore, the expression of the mitophagy-related gene SPATA18, which is repressed by ZNF558, and mitophagy activity were increased in ASTN2-deleted neuronal cells. These phenotypes were also detected in neuronal cells differentiated from patient-derived iPS cells with heterozygous ASTN2 deletion. Our results suggest that ASTN2 deletion is related to the common pathogenic mechanism of psychiatric disorders by regulating mitophagy via ZNF558.

Phenotypes for general behavior, activity, and body temperature in 3q29 deletion model mice.

Whole genome analysis has identified rare copy number variations (CNV) that are strongly involved in the pathogenesis of psychiatric disorders, and 3q29 deletion has been found to have the largest effect size. The 3q29 deletion mice model (3q29-del mice) has been established as a good pathological model for schizophrenia based on phenotypic analysis; however, circadian rhythm and sleep, which are also closely related to neuropsychiatric disorders, have not been investigated. In this study, our aims were to reevaluate the pathogenesis of 3q29-del by recreating model mice and analyzing their behavior and to identify novel new insights into the temporal activity and temperature fluctuations of the mouse model using a recently developed small implantable accelerometer chip, Nano-tag. We generated 3q29-del mice using genome editing technology and reevaluated common behavioral phenotypes. We next implanted Nano-tag in the abdominal cavity of mice for continuous measurements of long-time activity and body temperature. Our model mice exhibited weight loss similar to that of other mice reported previously. A general behavioral battery test in the model mice revealed phenotypes similar to those observed in mouse models of schizophrenia, including increased rearing frequency. Intraperitoneal implantation of Nano-tag, a miniature acceleration sensor, resulted in hypersensitive and rapid increases in the activity and body temperature of 3q29-del mice upon switching to lights-off condition. Similar to the 3q29-del mice reported previously, these mice are a promising model animals for schizophrenia. Successive quantitative analysis may provide results that could help in treating sleep disorders closely associated with neuropsychiatric disorders.

Mice with deficiency in Pcdh15, a gene associated with bipolar disorders, exhibit significantly elevated diurnal amplitudes of locomotion and body temperature.

Genetic factors significantly affect the pathogenesis of psychiatric disorders. However, the specific pathogenic mechanisms underlying these effects are not fully understood. Recent extensive genomic studies have implicated the protocadherin-related 15 (PCDH15) gene in the onset of psychiatric disorders, such as bipolar disorder (BD). To further investigate the pathogenesis of these psychiatric disorders, we developed a mouse model lacking Pcdh15. Notably, although PCDH15 is primarily identified as the causative gene of Usher syndrome, which presents with visual and auditory impairments, our mice with Pcdh15 homozygous deletion (Pcdh15-null) did not exhibit observable structural abnormalities in either the retina or the inner ear. The Pcdh15-null mice showed very high levels of spontaneous motor activity which was too disturbed to perform standard behavioral testing. However, the Pcdh15 heterozygous deletion mice (Pcdh15-het) exhibited enhanced spontaneous locomotor activity, reduced prepulse inhibition, and diminished cliff avoidance behavior. These observations agreed with the symptoms observed in patients with various psychiatric disorders and several mouse models of psychiatric diseases. Specifically, the hyperactivity may mirror the manic episodes in BD. To obtain a more physiological, long-term quantification of the hyperactive phenotype, we implanted nano tag® sensor chips in the animals, to enable the continuous monitoring of both activity and body temperature. During the light-off period, Pcdh15-null exhibited elevated activity and body temperature compared with wild-type (WT) mice. However, we observed a decreased body temperature during the light-on period. Comprehensive brain activity was visualized using c-Fos mapping, which was assessed during the activity and temperature peak and trough. There was a stark contrast between the distribution of c-Fos expression in Pcdh15-null and WT brains during both the light-on and light-off periods. These results provide valuable insights into the neural basis of the behavioral and thermal characteristics of Pcdh15-deletion mice. Therefore, Pcdh15-deletion mice can be a novel model for BD with mania and other psychiatric disorders, with a strong genetic component that satisfies both construct and surface validity.

New insights into the role of microheterogeneity of ZP3 during structural maturation of the avian equivalent of mammalian zona pellucida.

The egg coat including mammalian zona pellucida (ZP) and the avian equivalent, i.e., inner-perivitelline layer (IPVL), is a specialized extracellular matrix being composed of the ZP glycoproteins and surrounds both pre-ovulatory oocytes and ovulated egg cells in vertebrates. The egg coat is well known for its potential importance in both the reproduction and early development, although the underlying molecular mechanisms remain to be fully elucidated. Interestingly, ZP3, one of the ZP-glycoprotein family members forming scaffolds of the egg-coat matrices with other ZP glycoproteins, exhibits extreme but distinctive microheterogeneity to form a large number of isoelectric-point isoforms at least in the chicken IPVL. In the present study, we performed three-dimensional confocal imaging and two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE) of chicken IPVLs that were isolated from the ovarian follicles at different growth stages before ovulation. The results suggest that the relative proportions of the ZP3 isoforms are differentially altered during the structural maturation of the egg-coat matrices. Furthermore, tandem mass spectrometry (MS/MS) analyses and ZP1 binding assays against separated ZP3 isoforms demonstrated that each ZP3 isoform contains characteristic modifications, and there are large differences among ZP3 isoforms in the ZP1 binding affinities. These results suggest that the microheterogeneity of chicken ZP3 might be regulated to be associated with the formation of egg-coat matrices during the structural maturation of chicken IPVL. Our findings may provide new insights into molecular mechanisms of egg-coat assembly processes.

Diverse lectin-binding specificity of four ZP3 glycoprotein isoforms with a discrete isoelectric point in chicken egg coat.

The vertebrate egg coat corresponding to mammalian zona pellucida is a filamentous matrix composed of highly and heterogeneously glycosylated proteins designated ZP glycoproteins including ZP1 to 4, ZPD and ZPAX, and play important roles in species-specific egg-sperm interactions. Recent advance in structural biology of chicken ZP3 provided new insights into molecular mechanisms of the egg-coat function involving its carbohydrate moieties. In this study, chicken ZP3 was separated into four major and distinct isoforms with different pI in 2D-PAGE. To investigate the meanings of the ZP3 heterogeneity in egg-sperm interactions, we preliminary analyzed glycan diversity on the molecules by using lectin-staining assays. The four major ZP3 isoforms 4-7 (from acidic to basic) were recognized equally with PNA (Galß1-3GalNAc), but the isoforms 5-7 were recognized dominantly with WGA ((ß-GlcNAc)n, clustered Sia), PHA-E (bi- and triantennary N-glycan containing Galß1-4GlcNAcß1-2Manα1-6) and RCA I (terminal Galß1-4GlcNAc), respectively. Despite such sugar chain diversity among the ZP3 isoforms, a partner in the egg coat, ZP1, showed specific binding to each isoform equally. Localization of ZP1 and ZP3 in the egg-coat matrix were also analyzed.

Establishment of induced pluripotent stem cells from a patient with 16p13.11 duplication and VPS13B deletion.

VPS13B deletion and 16p13.11 duplication are related to mental disorders, such as schizophrenia. However, how these variants affect human neurons and contribute to the development of mental disorders is yet to be elucidated. In this study, we generated induced pluripotent stem cells (iPSCs) from a patient with 16p13.11 duplication and VPS13B deletion. The iPSCs indicated pluripotency marker expression and the differentiation capacity into three germ layers in vitro. Therefore, these iPSC lines will be useful tools to further understand the pathophysiology of mental disorders.