ß2-microglobulin functions as an endogenous NMDAR antagonist to impair synaptic function.

Down syndrome (DS) is a neurological disorder with multiple immune-related symptoms; however, crosstalk between the CNS and peripheral immune system remains unexplored. Using parabiosis and plasma infusion, we found that blood-borne factors drive synaptic deficits in DS. Proteomic analysis revealed elevation of ß2-microglobulin (B2M), a major histocompatibility complex class I (MHC-I) component, in human DS plasma. Systemic administration of B2M in wild-type mice led to synaptic and memory defects similar to those observed in DS mice. Moreover, genetic ablation of B2m or systemic administration of an anti-B2M antibody counteracts synaptic impairments in DS mice. Mechanistically, we demonstrate that B2M antagonizes NMDA receptor (NMDAR) function through interactions with the GluN1-S2 loop; blocking B2M-NMDAR interactions using competitive peptides restores NMDAR-dependent synaptic function. Our findings identify B2M as an endogenous NMDAR antagonist and reveal a pathophysiological role for circulating B2M in NMDAR dysfunction in DS and related cognitive disorders.

The deubiquitinase USP6 affects memory and synaptic plasticity through modulating NMDA receptor stability.

Ubiquitin-specific protease (USP) 6 is a hominoid deubiquitinating enzyme previously implicated in intellectual disability and autism spectrum disorder. Although these findings link USP6 to higher brain function, potential roles for USP6 in cognition have not been investigated. Here, we report that USP6 is highly expressed in induced human neurons and that neuron-specific expression of USP6 enhances learning and memory in a transgenic mouse model. Similarly, USP6 expression regulates N-methyl-D-aspartate-type glutamate receptor (NMDAR)-dependent long-term potentiation and long-term depression in USP6 transgenic mouse hippocampi. Proteomic characterization of transgenic USP6 mouse cortex reveals attenuated NMDAR ubiquitination, with concomitant elevation in NMDAR expression, stability, and cell surface distribution with USP6 overexpression. USP6 positively modulates GluN1 expression in transfected cells, and USP6 down-regulation impedes focal GluN1 distribution at postsynaptic densities and impairs synaptic function in neurons derived from human embryonic stem cells. Together, these results indicate that USP6 enhances NMDAR stability to promote synaptic function and cognition.

Biallelic mutations in PMFBP1 cause acephalic spermatozoa.

The majority of men with defects in spermatogenesis remain undiagnosed. Acephalic spermatozoa is one of the diseases causing primary infertility. However, the causes underlying over half of affected cases remain unclear. Here, we report by whole-exome sequencing the identification of homozygous and compound heterozygous truncating mutations in PMFBP1 of two unrelated individuals with acephalic spermatozoa. PMFBP1 was highly and specifically expressed in human and mouse testis. Furthermore, immunofluorescence staining in sperm from a normal control showed that PMFBP1 localizes to the head-flagella junction region, and the absence of PMFBP1 was confirmed in patients harboring PMFBP1 mutations. In addition, we generated Pmfbp1 knock-out (KO) mice, which we found recapitulate the acephalic sperm phenotype. Label-free quantitative proteomic analysis of testicular sperm from Pmfbp1 KO and control mice showed 124 and 35 proteins, respectively, increased or decreased in sperm from KO mice compared to that found in control mice. Gene ontology analysis indicates that the biological process of Golgi vesicle transport was the most highly enriched in differentially expressed proteins, indicating process defects related to Golgi complex function may disturb formation of the head-neck junction. Collectively, our data indicate that PMFBP1 is necessary for sperm morphology in both humans and mice, and that biallelic truncating mutations in PMFBP1 cause acephalic spermatozoa.

A novel TEX11 mutation induces azoospermia: a case report of infertile brothers and literature review.

BACKGROUND: Testis-expressed gene 11 (TEX11) is an X-linked gene and essential for meiotic recombination and chromosomal synapsis. TEX11 deficiency causes meiotic arrest and male infertility, and many TEX11 mutations have been found in azoospermic and infertile men. CASE PRESENTATION: This study reported one novel TEX11 mutation (2653G → T, in exon 29, GenBank accession number, NM_031276) in two brothers with azoospermia. This mutation was firstly screened out by whole-exome sequencing (WES) and further verified by amplifying and sequencing the specific exon 29. Surprisingly, the same exonic missense mutation (W856C) was observed in two brothers but not in their mother. Histological analysis of testicular biopsy from both brothers revealed meiotic arrest and no post-meiotic round spermatids and mature spermatozoa were observed in the seminiferous tubules. TEX11 expression was observed strongly in spermatogonia and weakly in spermatocytes, but not in Sertoli cells and interstitial cells. CONCLUSIONS: We identified one novel TEX11 mutation in two brothers and summarized the literature regarding TEX11 mutations and male infertility. This study and previous literature indicate that TEX11 mutations are closely associated with male infertility, especially azoospermia, although auxiliary clinical analyses are needed to figure out the causes of male infertility.

Novel In Situ Hybridization Assay for Chromogenic Single-Molecule Detection of Human Papillomavirus E6/E7 mRNA.

RNA plays a vital role in the physiological and pathological processes of cells and tissues. However, RNA in situ hybridization applications in clinical diagnostics are still limited to a few examples. In this study, we developed a novel in situ hybridization assay for human papillomavirus (HPV) E6/E7 mRNA by taking advantage of specific padlock probing and rolling circle amplification, combined with chromogenic readout. We designed padlock probes for 14 types of high-risk HPV and demonstrated that E6/E7 mRNA could be visualized in situ as discrete dot-like signals using bright-field microscopy. Overall, the results are consistent with the clinical diagnostics lab's hematoxylin and eosin (H&E) staining and p16 immunohistochemistry test results. Our work thus shows the potential applications of RNA in situ hybridization for clinical diagnostics using chromogenic single-molecule detection, offering an alternative technical option to the current commercially available kit based on branched DNA technology. IMPORTANCE In situ detection of viral mRNA expression in tissue samples is of great value for pathological diagnosis to access viral infection status. Unfortunately, conventional RNA in situ hybridization assays lack sensitivity and specificity for clinical diagnostic purposes. Currently, the commercially available branched DNA technology-based single-molecule RNA in situ detection method offers satisfactory results. Here, we present our padlock probe- and rolling circle amplification-based RNA in situ hybridization assay for detecting HPV E6/E7 mRNA expression in formalin-fixed paraffin-embedded tissue sections, providing an alternative yet robust method for viral RNA in situ visualization that is also applicable to different types of diseases.

TMEM25 inhibits monomeric EGFR-mediated STAT3 activation in basal state to suppress triple-negative breast cancer progression.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor outcome and lacks of approved targeted therapy. Overexpression of epidermal growth factor receptor (EGFR) is found in more than 50% TNBC and is suggested as a driving force in progression of TNBC; however, targeting EGFR using antibodies to prevent its dimerization and activation shows no significant benefits for TNBC patients. Here we report that EGFR monomer may activate signal transducer activator of transcription-3 (STAT3) in the absence of transmembrane protein TMEM25, whose expression is frequently decreased in human TNBC. Deficiency of TMEM25 allows EGFR monomer to phosphorylate STAT3 independent of ligand binding, and thus enhances basal STAT3 activation to promote TNBC progression in female mice. Moreover, supplying TMEM25 by adeno-associated virus strongly suppresses STAT3 activation and TNBC progression. Hence, our study reveals a role of monomeric-EGFR/STAT3 signaling pathway in TNBC progression and points out a potential targeted therapy for TNBC.

Trisomy 21-induced dysregulation of microglial homeostasis in Alzheimer's brains is mediated by USP25.

Down syndrome (DS), caused by trisomy of chromosome 21, is the most significant risk factor for early-onset Alzheimer's disease (AD); however, underlying mechanisms linking DS and AD remain unclear. Here, we show that triplication of homologous chromosome 21 genes aggravates neuroinflammation in combined murine DS-AD models. Overexpression of USP25, a deubiquitinating enzyme encoded by chromosome 21, results in microglial activation and induces synaptic and cognitive deficits, whereas genetic ablation of Usp25 reduces neuroinflammation and rescues synaptic and cognitive function in 5×FAD mice. Mechanistically, USP25 deficiency attenuates microglia-mediated proinflammatory cytokine overproduction and synapse elimination. Inhibition of USP25 reestablishes homeostatic microglial signatures and restores synaptic and cognitive function in 5×FAD mice. In summary, we demonstrate an unprecedented role for trisomy 21 and pathogenic effects associated with microgliosis as a result of the increased USP25 dosage, implicating USP25 as a therapeutic target for neuroinflammation in DS and AD.

Lead affects apoptosis and related gene XIAP and Smac expression in the hippocampus of developing rats.

Lead (Pb) exposure poses devastating effects on central nervous system development of children. To replicate aspects of this neurotoxicity, we examined the effect of lead on the expression of apoptosis and of apoptosis-related genes, XIAP (X chromosome-linked inhibitor of apoptosis protein) and Smac (second mitochondrial activator of caspase), in the hippocampus of developing rats. A total of 48 rats (30-day old) were randomly divided into four groups for intragastrical perfusion of lead acetate [Pb(Ac)2]: untreated, low (2 mg/kg/d), medium (20 mg/kg/d), and high (200 mg/kg/d) dose groups. Pb content was determined in blood, and the apoptosis indexes and XIAP and Smac gene expression were analyzed in the hippocampus. There was a significant difference in apoptosis indexes (AI) between the exposed and control groups (p < 0.01). AI was highest in the high exposure group. XIAP gene expression was reduced in the exposed groups and the expression was negatively correlated with blood lead levels (BLLs) (p < 0.05). But the four groups did not differ in the expression of Smac (p > 0.05). Our data indicate that exposure to Pb(Ac)2 caused a dose-dependent and significant increase of apoptosis in the hippocampus of developing rats through depressing the expression of the XIAP but not the Smac genes.

[Congenital adrenal hyperplasia complicated by testicular adrenal rest tumors: one-case clinical analysis].

OBJECTIVE: To investigate the early diagnosis and treatment of congenital adrenal hyperplasia (CAH) complicated by testicular adrenal rest tumors (TART). METHODS: We retrospectively analyzed the clinical data of 1 case of late-onset CAH complicated by TART diagnosed and treated in Xiamen Women and Children Health Care Hospital. RESULTS: The patient was a 15 years old boy, short statured and dark skinned, with skin pigmentation in the gum and external genital, secondary sex characteristics of the adult and irregular tubercles palpable in the bilateral testes. Laboratory examinations showed obviously increased levels of ACTH, 17-KS, DHEA-S and progesterone and evidently decreased levels of FSH, LH and CO. The low-dose dexamethasone suppression test reduced ACTH and DHEA-S to normal. Imaging examinations revealed soft tissue density in the bilateral adrenal glands, especially on the right, and irregularly increased volume of the bilateral testes, particularly on the left, with heterogeneous signals and septas and surrounded by the fluid signals. Histopathological examinations showed the eosinophilic cytoplasm to be polygon- or round-shaped, interstitium-like cells arranged in line, and lipopigment in the endochylema. Immunohistochemical results were negative for testicular interstitial cell tumor. The clinical signs of the patient were improved after 3 months of dexamethasone treatment, the hyperplastic nodules in the left testis decreased obviously and those in the right testis disappeared after 6 months, and the hyperplastic nodules in the adrenal glands vanished after 9 months. CONCLUSION: Based on the clinical manifestations and the results of auxiliary examinations, this case was diagnosed as late-onset CAH complicated by TART, which was attributed to the continued surge of ACTH induced by corticoadrenal insufficiency. Sufficient dexamethasone treatment could make the TART decrease or disappear and the CAH vanish; it could also improve the clinical symptoms and bring the laboratory results to normal.

Lead and cadmium synergistically enhance the expression of divalent metal transporter 1 protein in central nervous system of developing rats.

Divalent metal transporter 1 (DMT1) can transport a large range of ions, including toxic lead (Pb) and cadmium (Cd), across membranes. In this study, a total of 24 rats were divided into four groups for intragastrical perfusion treatment: control, Pb alone, Cd alone, and Pb + Cd. Pb and Cd contents in blood were detected, and the mRNA and protein levels of DMT1 were analyzed in the cerebellum, cortex, and hippocampus. Both Pb and Cd levels were elevated in all groups perfused with Pb and/or Cd, except for Pb level in the Cd-alone group (P < 0.05). The mRNA level of DMT1 did not differ among the four groups (P > 0.05). However, the DMT1 protein expression was significantly increased by 0.9-, 1.0-, and 1.1-fold in cerebellum, cortex, and hippocampus of the Pb + Cd group than in controls, respectively. Pb and Cd exposure can synergistically induce DMT1 protein synthesis and has implications for transportation of toxic ions in the developing rat's brain.

Blood lead and cadmium levels and relevant factors among children from an e-waste recycling town in China.

BACKGROUND: Primitive electronic waste (e-waste) recycling is ongoing in Guiyu, and thus toxic heavy metals may keep on threatening to the health of local children. Some related factors may contribute to the elevation of blood lead levels (BLLs) or blood cadmium levels (BCLs). OBJECTIVE: To investigate the children's BLLs and BCLs in Guiyu and Chendian as compare to discuss the effects of primitive e-waste recycling activities on children's health. METHODS: Two hundred and seventy-eight children less than 8 years who lived in Guiyu and Chendian were observed, and their BLLs and BCLs were determined by graphite atomizer absorption spectrophotometer. Questionnaire survey for risk factors was also performed and data were analyzed using spearman correlation analyses and logistic regression analyses. RESULTS: Children living in Guiyu had significantly higher BLLs and BCLs as compared with those living in Chendian (p<0.01). In Guiyu, 70.8% of children (109/154) had BLLs>10 microg/dL, and 20.1% of children (31/154) had BCLs>2 microg/L, compared with 38.7% of children (48/124) had BLLs>10 microg/dL and 7.3% of children (9/124) had BCLs>2 microg/L in Chendian (p<0.01, respectively). We also observed a significant increasing trend in BLLs with increasing age in Guiyu (p<0.01). Mean height of children in Guiyu was significantly lower than that in Chendian (p<0.01). The risk factors related to children's BLLs and BCLs mainly included father's engagement in the work related to e-waste, children's residence in Guiyu and the amount of time that children played outside near the road everyday. CONCLUSIONS: There are close relationships between the BLLs, BCLs in children and the primitive e-waste recycling activities in Guiyu. Environmental pollution, especially lead pollution, has threatened the health of children living around e-waste recycling site.

[Determination of meconium lead level of newborn by graphite furnace atomic absorption spectrometry].

UNLABELLED: To establish a method for the determination of lead in meconium from 144 samples of newborn through nitric acid digestion by means of graphite furnace atomic absorption spectrometry. METHODS: after being baked for at least 12 h at 60 degrees C, 0.3 g of meconium was digested by nitric acid and hydrogen peroxide in turn at 80 degrees C under water-bath condition for 1h and then metered to the whole volume of to 2 mL. After correcting the background with D2 lamp, specimen basal corpuscle matched standard curve was used to detect the lead content. RESULTS: The mean lead content of 93 experimental samples was 1.934 microg x g(-1) with the standard deviation (SD) of 1.551, and that of the 51 control samples was 1.012 microg x g(-1), with the SD of 1.084. There was a significant difference in lead levels of in meconnt between the experimental group and control group (p = 0.000). CONCLUSION: The lead content of the experimental was significantly higher than that of the control group detected by this method. This method was stable and efficient.

Elevated blood lead levels of children in Guiyu, an electronic waste recycling town in China.

BACKGROUND: Electronic waste (e-waste) recycling has remained primitive in Guiyu, China, and thus may contribute to the elevation of blood lead levels (BLLs) in children living in the local environment. OBJECTIVES: We compared the BLLs in children living in the e-waste recycling town of Guiyu with those living in the neighboring town of Chendian. METHODS: We observed the processing of e-waste recycling in Guiyu and studied BLLs in a cluster sample of 226 children < 6 years of age who lived in Guiyu and Chendian. BLLs were determined with atomic absorption spectrophotometry. Hemoglobin (Hgb) and physical indexes (height and weight, head and chest circumferences) were also measured. RESULTS: BLLs in 165 children of Guiyu ranged from 4.40 to 32.67 microg/dL with a mean of 15.3 microg/dL, whereas BLLs in 61 children of Chendian were from 4.09 to 23.10 microg/dL with a mean of 9.94 microg/dL. Statistical analyses showed that children living in Guiyu had significantly higher BLLs compared with those living in Chendian (p < 0.01). Of children in Guiyu, 81.8% (135 of 165) had BLLs > 10 microg/dL, compared with 37.7% of children (23 of 61) in Chendian (p < 0.01). In addition, we observed a significant increasing trend in BLLs with increasing age in Guiyu (p < 0.01). It appeared that there was correlation between the BLLs in children and numbers of e-waste workshops. However, no significant difference in Hgb level or physical indexes was found between the two towns. CONCLUSIONS: The primitive e-waste recycling activities may contribute to the elevated BLLs in children living in Guiyu.

ALAD genotypes and blood lead levels of neonates and children from e-waste exposure in Guiyu, China.

Extensive e-waste recycling activity in Guiyu, China, has been conducted using primitive techniques for the last 20 years, resulting in serious heavy metal environmental contamination. A polymorphic variant of the δ-aminolevulinic acid dehydratase (ALAD) gene has been found to influence lead uptake and, thus, may influence an individual's susceptibility to lead toxicity. We therefore explored whether the ALAD gene polymorphism affects blood lead levels of newborns and children in Guiyu. A total of 273 newborns and 504 children, and a combination of 2004/2005 and 2006 independent recruitments were used for this study. Umbilical cord blood from newborns (Guiyu/exposed group 189 vs. Chaonan/reference group 84) and venous blood from children (exposed group, 319 vs. Chendian/reference group 185) were collected. Blood lead levels (BLLs) were measured via graphite furnace atomic absorption spectrometry (GFAAS) for all samples, while ALAD genotyping was performed using PCR-RFLP for 273 neonate cord blood and 246 children's blood. The median BBLs of neonates in exposed group vs. the reference group were 10.50 (2.36-40.78) vs. 7.79 (0.8-19.51) for 2004/2005 and 9.41 (9.28-47.60) vs. 5.49 (0.35-18.68) for 2006, while child mean BLLs were 15.31 ± 5.79 vs. 9.94 ± 4.05 for 2004/2005 and 13.17 ± 5.98 vs. 10.04 ± 4.85 for 2006. The genotype frequencies in newborns were 98.90 % for the ALAD-1/ALAD-1 homozygote and 1.10% for the ALAD-1/ALAD-2 heterozygotes, while the values were 95.93 and 4.07%, respectively, in children. The allele frequencies of the ALAD-1 and ALAD-2 were 99.45 and 0.55% for newborns, but 97.97 and 2.03% for children, respectively. No significant differences in blood lead levels were found between ALAD-1/ALAD-1 and ALAD-1/ALAD-2 either in newborns or in children. The frequency distribution of the ALAD-2 allele in newborns from the exposed group was lower than that of the reference group. There were no significant differences, between the two different ALAD genotypes in the lead load of newborns and children. The frequency distribution of ALAD gene does not influence the blood lead levels of newborns and children in this case, which means that the higher lead burden in the exposed children was possibly influenced by e-waste recycling, but not ALAD genotypes.

Monitoring of lead load and its effect on neonatal behavioral neurological assessment scores in Guiyu, an electronic waste recycling town in China.

Guiyu is the major electronic waste (e-waste) recycling town in China. The primary purpose of this study was to measure the lead levels in neonates and examine the correlation between lead levels and neurobehavioral development. One hundred full-term neonates from Guiyu and fifty-two neonates from neighboring towns (control group) in the late summer of 2006 were selected for study. The lead levels in the umbilical cord blood (CBPb) and lead levels in meconium (MPb) of neonates were determined with atomic absorption spectrophotometry. The neonatal behavioral neurological assessment (NBNA) was conducted on all neonates. A questionnaire related to the exposure to lead of pregnant women was used as a survey of the neonates' mothers. Compared with the control group, neonates in Guiyu had significantly higher levels of lead (P < 0.01), and the mean CBPb and MPb were 113.28 microg L(-1) and 2.50 microg g(-1), respectively. The relatively high lead levels in the neonates of the Guiyu group were found to correlate with their maternal occupation in relation to e-waste recycling. Neonates with high levels of lead load have lower NBNA scores (P < 0.01). There was a statistically significant difference in NBNA scores between the Guiyu group and the control group by t test (P < 0.05). No correlation was found between CBPb and NBNA scores; however, a negative correlation was found between MPb and NBNA scores (P < 0.01). There is a correlation between relatively high lead levels in the umbilical cord blood and meconium in neonates and the local e-waste recycling activities related to lead contamination. This study suggests that environmental lead contamination due to e-waste recycling have an impact on neurobehavioral development of neonates in Guiyu.

[Recombinant prokaryotic plasmid construction and high expression of FUS1 gene].

OBJECTIVE: To construct the prokaryotic plasmid of FUS1 gene for efficient FUS1 expression in E.coli strain Rosetta(DE3)2plys. METHODS: The full-length FUS1 gene was amplified by PCR from the total RNA of umbilical mesenchymal stem cells and cloned into pET-32a(+) vector followed by identification with PCR and sequencing. The recombinant plasmid pET-32a(+)-FUS1 was transformed into the E.coli strain Rosetta(DE3)2plys and the target protein expression was induced by IPTG. RESULTS: The plasmid pET-32a(+)-FUS1 was obtained successfully as verified by PCR and sequence analysis. High expression of the fused FUS1 protein was achieved after induction by low-concentration IPTG (25 micromol/L) for 3 h, and the recombinant FUS1 protein accounted for 40% of the total bacterial protein of Rosetta(DE3)2plys. CONCLUSION: The recombinant FUS1 plasmid has been successfully cloned, which allows highly efficient FUS1 expression in Rosetta (DE3)2 plys.