Prolonged infective SARS-CoV-2 omicron variant shedding in a patient with diffuse large B cell lymphoma successfully cleared after three courses of remdesivir.

We report a case of prolonged shedding of the infective SARS-CoV-2 omicron variant BA.1.1.2 in a 79-year-old male patient with diffuse large B-cell lymphoma, after receiving chemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP). The patient was admitted to our hospital in late March 2022 for the sixth course of R-CHOP chemotherapy. Initially, the patient tested negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using an in-hospital loop-mediated amplification assay with a nasopharyngeal swab, both on the day of admission and three days later. However, the patient developed fever and was diagnosed with coronavirus disease (COVID-19) six days after admission and was suspected to have contracted the infection in the ward. Viral shedding continued for more than three months, with confirmed viral infectivity. As compared to the original Wuhan-Hu-1/2019 strain, amino acid substitutions including S36 N in non-structural protein (NSP)2, S148P, S1265del and L1266I in NSP3, G105D in NSP4, G496S, A831V, or V987F in spike protein, and I45T in open-reading frame (ORF)9b were randomly detected in isolated viruses. Although the patient had received two doses of the BNT162b2 vaccine approximately six months earlier and the third dose on day 127 after the infection, both serum anti-spike and anti-nuclear protein IgG and IgM tests were negative at day 92, 114, and 149 after the infection. The patient finally cleared the virus after the third course of remdesivir and did not have further recurrence.

Y chromosome gr/gr subdeletion is associated with lower semen quality in young men from the general Japanese population but not in fertile Japanese Men.

Several case-control studies have investigated whether Y chromosome haplogroups or deletions are associated with spermatogenic failure. However, the relationships between Y chromosome haplogroups or deletions and semen quality in general population have not been elucidated. In this study, we assessed relationships between Y chromosome haplogroups or deletions and semen parameters in 791 fertile Japanese men and 1221 young men from the general Japanese population. We found that the haplogroup D2 (M55 lineage) was significantly associated with lower semen parameters, especially total motile sperm count (P = 0.00051, beta = -0.097), in men from the general population but not in fertile men. In addition, we found that the gr/gr subdeletion was associated with semen quality and in particular, strongly associated with decreased sperm motility (P = 0.00041, beta = -3.14) and total motile sperm count (P = 0.00031, beta = -0.099) in men from the general population but not in fertile men. The combined analysis of fertile Japanese men and men from the general Japanese population showed that the haplogroup D2 (M55 lineage) and the gr/gr subdeletion were strongly associated with reduced sperm motility (P = 0.00056, beta = -2.71, and P = 7.7 × 10(-5), beta = -3.05, respectively) and that haplogroup O2b1 was strongly associated with elevated sperm motility (P = 0.00089, beta = 2.94). These observations add further support for the view that the gr/gr subdeletion diminishes sperm motility that consequently may result in male infertility.

Y chromosome haplogroup d2* lineage is associated with azoospermia in Japanese males.

Several studies have investigated whether particular Y chromosome haplogroups are associated with spermatogenic failure in Japanese males; however, they produced differing results. In this study, to investigate the association of Y chromosome haplogroup with spermatogenic failure, we recruited 451 infertile patients and 730 fertile men from a Japanese population and typed their Y chromosome haplogroups. The infertile patients were suffering from varicocele, azoospermia, oligozoospermia, asthenozoospermia, obstructive azoospermia, karyotype abnormalities, microdeletions of the long arm of the Y chromosome, or other conditions that affect fertility. The frequency of haplogroup D2* was significantly higher (odds ratio = 2.28, 95% confidence interval = 1.44-3.61, P = 0.00034 using chi-square test) among the men with azoospermia than among the fertile men. None of the other Y haplogroups displayed associations with particular types of infertility. In conclusion, Y chromosome haplogroup D2* is associated with spermatogenic failure in Japanese males, suggesting that the Y chromosome lineage can have significant effects on spermatogenesis.

Climatic influence on the reproductive characteristics of Japanese males.

We previously performed a survey of the sperm characteristics of the partners of pregnant women in four cities in Japan. In the present study, we analyzed the sperm characteristics of these subjects and the correlations between these sperm characteristics and climatic changes or Y chromosome haplogroups. Our results showed that more haplogroup D2a1 males than O2b1 males were born in the first half of the year (January to June), whereas more O2b1 males were born in the last half of the year (July to December) (P<0.05). This was agreed and correlated with the seasonal variations in their mean sperm concentrations. The haplogroup C, D* and D2a1 males displayed lower sperm concentrations from March to May, followed by an increase in their sperm concentrations starting in June or July, while the O2b1 males displayed higher sperm concentrations in the first half of the year followed by a sudden decrease from July to August (P<0.05). We hypothesize that the Japanese climate has different effects on the sperm characteristics and reproductive seasonality of males from different lineages; and therefore, has influenced the modern population of Japan.

The male-determining gene SRY is a hybrid of DGCR8 and SOX3, and is regulated by the transcription factor CP2.

In mammals, sex is determined by the presence or absence of the Y chromosome that bears a male-dominant sex-determining gene SRY, which switches the differentiation of gonads into male testes. The molecular signaling mechanism turning on the switch, however, has remained unclear for 18 years since the identification of the gene. Here, we describe how this gene emerged and started to work. From amino acid homology, we realized that SRY is a hybrid gene between a portion of the first exon of DiGeorge syndrome critical region gene 8 (DGCR8) and the high-mobility group (HMG) box of SRY box-3 (SOX3) gene. We identified the regulatory sequence in the SRY promotor region by searching for a common motif shared with DGCR8 mRNA. From the motif search between DGCR8 mRNA and the SRY upstream sequence, we found that the transcription factor CP2 (TFCP2) binding motif is present in both. TFCP2 overexpression did not show a significant increase of SRY mRNA expression, and TFCP2 suppression by RNA interference (RNAi) significantly reduced SRY mRNA expression. Furthermore, electrophoretic mobility shift assay (EMSA) demonstrated that TFCP2 acts as a regulator by directly binding to the SRY promoter. We conclude that SRY is a hybrid gene composed of two genes, DGCR8 and SOX3; and TFCP2 is an essential transcription factor for SRY expression regulation.

Proteomics and transcriptome approaches to investigate the mechanism of human sex determination.

The SRY gene (sex-determining region on the Y chromosome) was isolated in 1990 and is known as the testis-determining factor on the Y chromosome. The SRY has been considered as a transcription factor since it contains an HMG box, which functions as a DNA-binding domain. However, a direct target for SRY remains to be identified. We have investigated the function of SRY through proteomics and transcriptome approaches, and by using two stable SRY-overexpressing cell lines (SRY1 and SRY2) in NT2/D1 cells derived from human testicular embryonal cell carcinoma. The results of 2-dimensional gel electrophoresis show that SRY overexpression causes a considerable downregulation of many chaperone proteins. SRY also upregulates laminin, which is important for Sertoli cell differentiation. Additionally, transcriptome analysis shows that SRY overexpression upregulates many zinc finger proteins and downregulates cellular growth factors with S or G(2)/M arrest of the cell cycle and inhibition of cellular proliferation.

Model mice for mild-form glycine encephalopathy: behavioral and biochemical characterizations and efficacy of antagonists for the glycine binding site of N-methyl D-aspartate receptor.

Glycine encephalopathy (GE) is caused by an inherited deficiency of the glycine cleavage system (GCS) and characterized by accumulation of glycine in body fluids and various neurologic symptoms. Coma and convulsions develop in neonates in typical GE while psychomotor retardation and behavioral abnormalities in infancy and childhood are observed in mild GE. Recently, we have established a transgenic mouse line (low-GCS) with reduced GCS activity (29% of wild-type (WT) C57BL/6) and accumulation of glycine in the brain (Stroke, 2007; 38:2157). The purpose of the present study is to characterize behavioral features of the low-GCS mouse as a model of mild GE. Two other transgenic mouse lines were also analyzed: high-GCS mice with elevated GCS activity and low-GCS-2 mice with reduced GCS activity. As compared with controls, low-GCS mice manifested increased seizure susceptibility, aggressiveness and anxiety-like activity, which resembled abnormal behaviors reported in mild GE, whereas high-GCS mice were less sensitive to seizures, hypoactive and less anxious. Antagonists for the glycine-binding site of the N-methyl-D-aspartate receptor significantly ameliorated elevated locomotor activity and seizure susceptibility in the low-GCS mice. Our results suggest the usefulness of low-GCS mice as a mouse model for mild GE and a novel therapeutic strategy.

Direct correlation between ischemic injury and extracellular glycine concentration in mice with genetically altered activities of the glycine cleavage multienzyme system.

BACKGROUND AND PURPOSE: Ischemia elicits the rapid release of various amino acid neurotransmitters. A glutamate surge activates N-methyl-d-aspartate (NMDA) glutamate receptors, triggering deleterious processes in neurons. Although glycine is a coagonist of the NMDA receptor, the effect of extracellular glycine concentration on ischemic injury remains controversial. To approach this issue, we examined ischemic injury in mice with genetically altered activities of the glycine cleavage multienzyme system (GCS), which plays a fundamental role in maintaining extracellular glycine concentration. METHODS: A mouse line with increased GCS activity (340% of C57BL/6 control mice) was generated by transgenic expression of glycine decarboxylase, a key GCS component (high-GCS mice). Another mouse line with reduced GCS activity (29% of controls) was established by transgenic expression of a dominant-negative mutant of glycine decarboxylase (low-GCS mice). We examined neuronal injury after transient occlusion of the middle cerebral artery in these mice by measuring extracellular amino acid concentrations in microdialysates. RESULTS: High-GCS and low-GCS mice had significantly lower and higher basal concentrations of extracellular glycine than did controls, respectively. In low-GCS mice, the extracellular glycine concentration reached 2-fold of control levels during ischemia, and infarct volume was significantly increased by 69% with respect to controls. In contrast, high-GCS mice had a significantly smaller infarct volume (by 21%). No significant difference was observed in extracellular glutamate concentrations throughout the experiments. An antagonist for the NMDA glycine site, SM-31900, attenuated infarct size, suggesting that glycine operated via the NMDA receptor. CONCLUSIONS: There is a direct correlation between ischemic injury and extracellular glycine concentration maintained by the GCS.

Overexpression of SOX15 inhibits proliferation of NT2/D1 cells derived from a testicular embryonal cell carcinoma.

SOX (Sry-related HMG box) family proteins, which have an evolutionarily conserved DNA binding domain, have crucial roles in cell differentiation. However, their target genes remain enigmatic. Some members of the SOX family may have roles in regulation of cell proliferation. We established stable NT2/D1 cell lines overexpressing SOX15 (SOX15-NT2/D1), and a modified 3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the SOX15-NT2/D1 cells exhibited significantly slower growth than the controls. Flow cytometry analysis revealed that an increased fraction of the SOX15-NT2/D1 cells were in G1-G0. In addition, a microarray analysis identified 26 genes that were up-regulated in the SOX15-NT2/D1 cells, but none that were down-regulated genes. Among the up-regulated genes, IGFBP5, S100A4, ID2, FABP5, MTSS1, PDCD4 have been shown to be related to cell proliferation and/or the cell cycle.

Y chromosomal STRs haplotypes in two populations from Bolivia.

In the present study, we typed our previously reported two microsatellite markers, DXYS241 and DXYS266 together with a basic set of nine Y-STRs (DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DXYS156Y, DYS413) on Y chromosomes from two Bolivian populations. Unrelated males from communities living at high- (N=59) and low- (N=142) altitude, were studied. Combining the alleles into 11 Y-STRs haplotypes revealed that the high-altitude population is significantly less diverse than the low-altitude population. Haplotype diversities of 0.927+/-0.029 and 0.996+/-0.002 were found within the high-altitude, and the low-altitude populations, respectively. Within the high-altitude population 40 haplotypes were detected, whereas in the low-altitude population 113 haplotypes were found. Only three haplotypes were shared between both populations. Haplotyping-based discrimination using the 11 Y-STRs including our new two microsatellite markers DXYS241 and DXYS266 was shown to be powerful than using the conventional 9 Y-STRs, especially for the low-altitude Bolivian population. This 11 Y-STRs-based haplotyping system shows a very high potential for discrimination and could provide an ideal tool for forensic analysis and population studies. Moreover, this study includes data about two Bolivian populations which were not previously reported, this will help in building a world-wide database for future use in forensic and legal studies.

A rapid and simple system of detecting deletions on the Y chromosome related with male infertility using multiplex PCR.

Around 10% of males with idiopathic azoospermia or oligozoospermia, which are important causes of male infertility, have partial deletions on the long arm of the Y chromosome. To develop a rapid and accurate detection system for screening major Y deletions found in infertile men, we developed a multiplex PCR system that can simultaneously amplify five loci on the Y chromosome, SRY, AMELY, DBY, RBMY, DAZ and one locus on the X chromosome, AMELX. The size of the PCR products was designed to increase gradually from the distal Yp to the distal Yq. Our system could detect deletions of three major candidate regions for the azoospermic factor, AZFa, AZFb and AZFc on the Y chromosome together with sex. The gradual increase in the size of the PCR products was convenient for imaging the location of deletions on the Y chromosome. Moreover, the multiplex PCR system was combined with microchip-based electrophoresis, and the PCR products derived from each locus were separated within 4 min. Our system is useful for screening Y chromosomes bearing the structural anomalies including three major AZF deletions found among azoospermic or oligozoospermic males.

A/G heterozygote of the A-3826G polymorphism in the UCP-1 gene has higher BMI than A/A and G/G homozygote in young Japanese males.

UCP-1 is suggested to have important roles for thermogenesis and energy expenditure. To elucidate whether the A-3826G polymorphism that is located in the 5' flanking region of the UCP-1 gene has roles in healthy young people, the polymorphism was genotyped among 251 young Japanese men whose mean age is 22.7 years old. We analyzed relationship between the A-3826G polymorphism and body mass index (BMI) or six biochemical parameters, serum concentration of total cholesterol (TC), high density lipoprotein (HDL) cholesterol, triglyceride (TG), asparatate aminotransferase (AST), alanine aminotransferase (ALT), fasting plasma glucose. The genotype frequencies were observed at the frequencies of 24.3% for AA, 48.2% for AG and 27.5% for GG, respectively. When BMI and the biochemical parameters were compared by ANOVA among individuals with each genotype, the statistical difference was observed only for BMI (P=0.016). Bonferroni's test demonstrated that the men with the AG genotype have higher BMI than those with the AA genotype (22.4+/-2.8 vs. 21.4+/-2.2) (P=0.04). The individuals with the AG genotype also showed trend to have higher BMI than those with the GG, although the difference was not statistically apparent (22.4+/-2.8 vs. 21.5+/-2.3) (P=0.07). Our results indicated that the young healthy Japanese men with the AG heterozygote showed higher BMI than those with other genotypes.

Expression analysis of a mouse orthologue of HSFY, a candidate for the azoospermic factor on the human Y chromosome.

Heat shock transcription factor on Y (HSFY) is located in one of three candidate regions for azoospermic factor (AZF), AZFb on the Y chromosome. We and others have already revealed that some azoospermic males are missing the regions of the Y chromosome including HSFY. Previously, we showed that murine HSFY-like sequence [mHSFYL (Riken cDNA 4933413G11Rik)], which is the mouse orthologue of HSFY, is exclusively expressed in testis. The sequences encoding the presumed DNA-binding domain in HSFY and mHSFYL were found in other mammals such as dogs, cows and chickens. To elucidate mHSFYL expression in the testes in detail, we carried out in situ hybridization. mHSFYL was predominantly expressed in round spermatids. Furthermore, we clarified the intracellular distribution of mHSFYL in COS1 cells with HA- or GFP-tagged proteins. Both HA-mHSFYL and GFP-mHSFYL were located in the nucleus. Our results suggest that HSFY/mHSFYL may have evolutionarily conserved functions for spermatogenesis.

Ultrafast diagnosis of the genetic-related disorders using the combined technologies of multiplex PCR and multichannel microchip electrophoresis.

For the diagnosis of unexplained male infertility a multiplex PCR for 6 markers, which are well-known as candidate genes for studying male infertility and located on the human Y-chromosome, has been designed. The multiplex PCR products have been separated on a 12 channel microchip electrophoresis system, which can analyze different samples simultaneously. By combining the technologies of multiplex PCR with multichannel microchip electrophoresis, the number of the DNA markers that can be screened simultaneously is increased to be 72 marker (12 x 6) in a single run while the electrophoresis analysis time is reduced to be only 180 s.

Multiplex PCR with multichannel microchip electrophoresis: an ultrafast analysis for genetic diseases.

A Y chromosomal polymorphic markers screening strategy using a multiplex polymerase chain reaction (PCR) and DNA microchip electrophoresis technology has recently been developed. It is a part of the human Y chromosome haplotyping system for studying Japanese population genetics and its relationship with male spermatogenic failure. This strategy is based on optimizing and modifying the primer set concentrations while keeping all other components of the PCR mixtures and conditions similar to those of a singleplex PCR. Well-balanced PCR products are obtained without changing even the DNA oligomer melting temperatures. Here, a panel of primer sets are used to amplify two groups of Y chromosome markers. The first consists of five markers and the second consists of seven markers. Both are possibly deleted in infertile men. The microchip electrophoresis technology is fast and sensitive, enables direct molecular typing of several Y chromosomal markers, and is separated by a difference of as many as six base pairs.

Two Y-chromosome-specific polymorphisms 12f2 and DFFRY in the Japanese population and their relations to other Y-polymorphisms.

This study of male-specific genetic markers in the Japanese population was carried out in an attempt to refine the existing theories concerning its population genetics and migration events. We examined the relation between the constructed haplotypes of three biallelic Y-chromosome-specific markers (YAP, 47z and SRY) and the results of studying two other Y-specific polymorphisms of both 12f2 and DFFRY markers. The 12f2 marker was completely absent in 14.7% of Japanese males; all of them were haplotype II males. None of the Japanese males from other haplotypes or other East Asian populations showed any deletion of 12f2. In all haplotype II Japanese men, we found that DFFRY gene harbors a (C-->T) substitution polymorphism that was not found in any other population of this study. These results suggested that although haplotype II Japanese males share with the other haplotype II men from different geographical areas in having the YAP insertion on their Y-chromosomes, their Y-chromosomal structure is somewhat characteristically different. They are probably descendants of the ancestral Jomonese population who lived in Japan before the Yayoi immigrants entered Japan approximately 2300 years ago. These findings suggested that linkage studies between Y-specific markers are helpful in understanding the migratory patterns in East Asia. We also suggested that Japanese males have characteristically different Y-chromosomes compared with other populations.

Roles of estrogen receptor alpha (ER alpha) in the regulation of the human Müllerian inhibitory substance (MIS) promoter.

Sex differentiation consists of multi-step pathway that involves expression of many different genes. Müllerian duct inhibitory substance (MIS) has a key role for regression of the Müllerian duct during male sex differentiation. Recently, endocrine disruptors (EDs), which often have estrogen-like activities, have caused concern over worldwide. It has been reported that estrogen regulates the MIS expression. Therefore, we tested whether ER alpha and ER beta influence the MIS promoter activity in the NT2/D1 cell line which expresses many sex differentiation-related genes such as SRY, SOX9, and DAX-1. RT-PCR analysis revealed that the NT2/D1 cells express both ER alpha and ER beta in addition to MIS. Under the low concentration of 17beta-estradiol (E2), the over-expression of exogenous ER alpha increased the MIS promoter activity 3.3-fold compared with the control. However, as E2 concentration was increased, the MIS promoter activity was decreased. For ER beta, we could not observe alterations of the MIS promoter activity. Furthermore, the over-expression of the exogenous SF-1 inhibited the activation of the MIS promoter with ER alpha. Although it remains unclear whether the effects of ER alpha on the MIS promoter are mediated through the genomic or the no-genomic actions, the present results suggest that ER alpha up-regulates the MIS promoter activity in the NT2/D1 cells under low concentrations of E2, and that the two ERs may work in different manners for the MIS promoter activation. The present findings may be useful to understand the molecular mechanisms by which EDs or estrogens affect the MIS expression.

Range of separation of potential tool for bioseparation, microchip electrophoresis system, for DNA polymorphisms on the human Y-chromosome.

For the requirement of a high, fast and sufficient technology to suit the needs of 21st century biotechnology, the separation range of a microchip electrophoresis system was studied. Two DNA fragments on the human Y-chromosome, SY594 (82 bp) and 12f2 (88 bp), were successfully separated with a reproducibility of 1.9% and an accuracy of 2.8%. Then, a mixture of 10 DNA markers ranging from 61 bp to 189 bp was successfully separated with high resolution. All of these results demonstrate the superiority of microchip electrophoresis as a tool for 21st century bioseparation.

[Sex differentiation and sex chromosomes].

The mechanisms for sex differentiation and the genes on the sex chromosomes are varied among different species. For human, SRY is the only testis-determining factor on the Y chromosome and triggers the cascade for male sex-determination. However, even if normal SRY exists, the haploinsufficienty of SOX9 or KTS+ splicing form of WT-1 can cause male-to-female sex reversal. Furthermore, the duplication of the partial region on the X chromosome including DAX-1 gene can also cause male-to-female sex reversal. The sex-determining system seems to be sensitive for the gene dosage or the gene expression level.