Complement C1q-mediated microglial synaptic elimination by enhancing desialylation underlies sevoflurane-induced developmental neurotoxicity.

BACKGROUND: Repeated neonatal sevoflurane exposures led to neurocognitive disorders in young mice. We aimed to assess the role of microglia and complement C1q in sevoflurane-induced neurotoxicity and explore the underlying mechanisms. METHODS: Neonatal mice were treated with sevoflurane on postnatal days 6, 8, and 10, and the Morris water maze was performed to assess cognitive functions. For mechanistic explorations, mice were treated with minocycline, C1q-antibody ANX005, and sialidase-inhibitor N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA) before sevoflurane exposures. Western blotting, RT-qPCR, Golgi staining, 3D reconstruction and engulfment analysis, immunofluorescence, and microglial morphology analysis were performed. In vitro experiments were conducted in microglial cell line BV2 cells. RESULTS: Repeated neonatal sevoflurane exposures resulted in deficiencies in learning and cognition of young mice, accompanied by microglial activation and synapse loss. Sevoflurane enhanced microglia-mediated synapse elimination through C1q binding to synapses. Inhibition of microglial activation and phagocytosis with minocycline significantly reduced the loss of synapses. We further revealed the involvement of neuronal sialic acids in this process. The enhanced activity of sialidase by sevoflurane led to the loss of sialic acids, which facilitated C1q binding to synapses. Inhibition of C1q with ANX005 or inhibition of sialidase with NADNA significantly rescued microglia-mediated synapse loss and improved neurocognitive function. Sevoflurane enhanced the engulfment of BV2 cells, which was reversed by ANX005. CONCLUSIONS: Our findings demonstrated that C1q-mediated microglial synaptic elimination by enhancing desialylation contributed to sevoflurane-induced developmental neurotoxicity. Inhibition of C1q or sialidase may be a potential therapeutic strategy for this neurotoxicity.

The effect of sevoflurane exposure on cell-type-specific changes in the prefrontal cortex in young mice.

Sevoflurane, the predominant pediatric anesthetic, has been linked to neurotoxicity in young mice, although the underlying mechanisms remain unclear. This study focuses on investigating the impact of neonatal sevoflurane exposure on cell-type-specific alterations in the prefrontal cortex (PFC) of young mice. Neonatal mice were subjected to either control treatment (60% oxygen balanced with nitrogen) or sevoflurane anesthesia (3% sevoflurane in 60% oxygen balanced with nitrogen) for 2 hours on postnatal days (PNDs) 6, 8, and 10. Behavioral tests and single-nucleus RNA sequencing (snRNA-seq) of the PFC were conducted from PNDs 31 to 37. Mechanistic exploration included clustering analysis, identification of differentially expressed genes (DEGs), enrichment analyses, single-cell trajectory analysis, and genome-wide association studies (GWAS). Sevoflurane anesthesia resulted in sociability and cognition impairments in mice. Novel specific marker genes identified 8 distinct cell types in the PFC. Most DEGs between the control and sevoflurane groups were unique to specific cell types. Re-defining 15 glutamatergic neuron subclusters based on layer identity revealed their altered expression profiles. Notably, sevoflurane disrupted the trajectory from oligodendrocyte precursor cells (OPCs) to oligodendrocytes (OLs). Validation of disease-relevant candidate genes across the main cell types demonstrated their association with social dysfunction and working memory impairment. Behavioral results and snRNA-seq collectively elucidated the cellular atlas in the PFC of young male mice, providing a foundation for further mechanistic studies on developmental neurotoxicity induced by anesthesia.

Dexmedetomidine Infusion Versus Placebo During Light or Deep Anesthesia on Postoperative Delirium in Older Patients Undergoing Major Noncardiac Surgery: A Pilot Randomized Factorial Trial.

BACKGROUND: Postoperative delirium (POD) is common among older surgical patients and may be affected by dexmedetomidine and depth of anesthesia. We designed this pilot study to assess the feasibility of comparing dexmedetomidine with normal saline during light versus deep anesthesia on POD in older patients undergoing major noncardiac surgery. METHODS: In this pilot randomized factorial study, 80 patients aged 60 years or older undergoing major noncardiac surgery were randomized (1:1:1:1) to receive dexmedetomidine infusion 0.5 µg/kg/h or normal saline placebo during light (bispectral index [BIS] target 55) or deep (BIS target 40) anesthesia. Feasibility end points included consent rate and dropout rate, timely enrollment, blinded study drug administration throughout surgery, no inadvertent unmasking, achieving BIS target throughout >70% of surgery duration, and the process of twice-daily POD screening. In addition, we estimated the POD incidences in the 2 control groups (placebo and deep anesthesia) and treatment effects of dexmedetomidine and light anesthesia. RESULTS: Between November 1, 2021, and June 30, 2022, 78 patients completed the trial (mean [standard deviation, SD] age, 69.6 [4.6] years; 48 male patients [62%]; dexmedetomidine-deep, n = 19; dexmedetomidine-light, n = 20; placebo-deep, n = 19; placebo-light, n = 20). This study had a high consent rate (86%) and a low dropout rate (2.5%). Average recruitment was 5 patients at each center per month. Dexmedetomidine and normal saline were administered in a blinded fashion in all patients. Unmasking did not occur in either group. Approximately 99% of patients received the scheduled study drug infusion throughout the surgery. Approximately 81% of patients achieved the BIS targets throughout >70% of the surgery duration. The scheduled twice-daily POD screening was completed without exception. Overall, 10 of the 78 patients (13%; 95% confidence interval [CI], 7%-22%) developed POD. For the 2 reference groups, POD was observed in 7 of the 39 patients (17.9%; 95% CI, 9%-32.7%) in the placebo group and 7 of the 38 patients (18.4%; 95% CI, 9.2%-33.4%) in the deep anesthesia group. Regarding the treatment effects on POD, the estimated between-group difference was -10% (95% CI, -28% to 7%) for dexmedetomidine versus placebo, and -11% (95% CI, -28% to 6%) for light versus deep anesthesia. CONCLUSIONS: The findings of this pilot study demonstrate the feasibility of assessing dexmedetomidine versus placebo during light versus deep anesthesia on POD among older patients undergoing major noncardiac surgery, and justify a multicenter randomized factorial trial.

Single-nucleus Atlas of Sevoflurane-induced Hippocampal Cell Type- and Sex-specific Effects during Development in Mice.

BACKGROUND: Multiple neonatal exposures to sevoflurane induce neurocognitive dysfunctions in rodents. The lack of cell type-specific information after sevoflurane exposure limits the mechanistic understanding of these effects. In this study, the authors tested the hypothesis that sevoflurane exposures alter the atlas of hippocampal cell clusters and have neuronal and nonneuronal cell type-specific effects in mice of both sexes. METHODS: Neonatal mice were exposed to 3% sevoflurane for 2 h at postnatal days 6, 8, and 10 and analyzed for the exposure effects at postnatal day 37. Single-nucleus RNA sequencing was performed in the hippocampus followed by in situ hybridization to validate the results of RNA sequencing. The Morris Water Maze test was performed to test neurocognitive function. RESULTS: The authors found sex-specific distribution of hippocampal cell types in control mice alongside cell type- and sex-specific effects of sevoflurane exposure on distinct hippocampal cell populations. There were important changes in male but not in female mice after sevoflurane exposure regarding the proportions of cornu ammonis 1 neurons (control vs. sevoflurane, males: 79.9% vs. 32.3%; females: 27.3% vs. 24.3%), dentate gyrus (males: 4.2% vs. 23.4%; females: 36.2% vs. 35.8%), and oligodendrocytes (males: 0.6% vs. 6.9%; females: 5.9% vs. 7.8%). In male but not in female mice, sevoflurane altered the number of significantly enriched ligand-receptor pairs in the cornu ammonis 1, cornu ammonis 3, and dente gyrus trisynaptic circuit (control vs. sevoflurane, cornu ammonis 1-cornu ammonis 3: 18 vs. 42 in males and 15 vs. 21 in females; cornu ammonis 1-dentate gyrus: 21 vs. 35 in males and 12 vs. 20 in females; cornu ammonis 3-dentate gyrus: 25 vs. 45 in males and 17 vs. 20 in females), interfered with dentate gyrus granule cell neurogenesis, hampered microglia differentiation, and decreased cornu ammonis 1 pyramidal cell diversity. Oligodendrocyte differentiation was specifically altered in females with increased expressions of Mbp and Mag. In situ hybridization validated the increased expression of common differentially expressed genes. CONCLUSIONS: This single-nucleus RNA sequencing study reveals the hippocampal atlas of mice, providing a comprehensive resource for the neuronal and nonneuronal cell type- and sex-specific effects of sevoflurane during development.

Organocatalytic cycloaddition-elimination cascade for atroposelective construction of heterobiaryls.

The first chiral phosphoric acid (CPA) catalyzed cycloaddition-elimination cascade reaction of 2-naphthol- and phenol-derived enecarbamates with azonaphthalenes has been established, providing a highly atroposelective route to an array of axially chiral aryl-C3-benzoindoles in excellent yields with excellent enantioselectivities. The success of this strategy derives from the stepwise process involving CPA-catalyzed asymmetric formal [3 + 2] cycloaddition and subsequent central-to-axial chirality conversion by elimination of a carbamate. In addition, the practicality of this reaction had been verified by varieties of transformations towards functionalized atropisomers.

Copper(I)/DDQ-Mediated Double-Dehydrogenative Diels-Alder Reaction of Aryl Butenes with 1,4-Diketones and Indolones.

A copper(I)/DDQ-mediated double-dehydrogenative Diels-Alder (DDDA) reaction of simple butenes with 1,4-diketones and indolones has been established for the first time. This strategy is based on a tandem double-dehydrogenation/Diels-Alder reaction from nonprefunctionalized starting materials, in which both a diene and dienophile were in situ generated via activation of fourfold inert C(sp3)-H bonds in one catalytic system.

[Spatial Variation of Heavy Metals in Soils and Its Ecological Risk Evaluation in a Typical Carya cathayensis Production Area].

In recent years, the problem of soil pollution has become more and more serious. The problem of soil heavy metal pollution and its related human health risks has become a hot spot at home and abroad. Carya cathayensis is a unique high-grade woody nut and oil tree from China, and there are few reports on heavy metal pollution in Carya cathayensis plantation soils. Therefore, in order to study the spatial variability of heavy metals and the risk of pollution in Carya cathayensis soil and to promote the sustainable development of the Carya cathayensis industry, Lin'an, a typical Carya cathayensis plantation area, was selected for this study. A total of 188 soil samples were collected from the study area. We systematically studied the spatial heterogeneity of soil heavy metal content in the study area based on GIS technology, geostatistics, Moran's I, and other spatial analysis methods. The single factor pollution index method, the Nemerow comprehensive pollution index method, and the potential ecological risk assessment method were used to evaluate the heavy metal pollution in the study area. The results indicated that the mean content of soil cadmium (Cd), copper (Cu), zinc (Zn), lead (Pb), nickel (Ni), and chromium (Cr) were 0.37, 40.76, 87.61, 30.10, 28.33, 56.57 mg·kg-1, respectively. The average values of Cd and Cu were 1.33 and 2.87 times of the background values, respectively, and the average content of other heavy metals did not exceed the background values. The results of the single factor Nemerow pollution index and potential ecological risk assessment methods showed that heavy metals in the study area exceeded the soil background values in some samples, and the second grade standard of soil environmental quality was exceed for Cd, Cu, Zn, Pb, and Ni in 31.38%, 31.38%, 2.65%, 0.53%, and 17.02% of the samples, respectively. This indicated that the soils in the study area had different accumulation characteristics for Cd, Cu, Zn, Pb, Ni, and Cr, and the local soil had reached pollution levels for Cd, Cu, Zn, Pb, and Ni. Among them, Cd was the most serious, reaching the degree of strong ecological damage, followed by Cu. In general, the heavy metal contents indicated a moderate degree of ecological damage. Based on the analysis of the semi-variance function, the Cd, Cu, and Ni in the soil were best fit with exponential models, the Zn and Pb were better fit with the Gaussian model, and Cr was consistent with the spherical model. Cd, Cu, Pb, Ni, and Cr had the strong spatial autocorrelation, with Nugget/Sill ratios of 12.1%, 4.6%, 14.9%, 2.6%, and 11.2%, respectively, while the Nugget/Sill ratio of Zn was 48.8%, indicating a medium spatial autocorrelation. Moran's I and Kriging interpolation results found that the heavy metals Cd, Cu, Zn, Pb, Ni, and Cr all had obvious spatial distribution patterns and local spatial aggregation phenomena. The high values of heavy metals in soils were mainly found in Taiyang, Daoshi, Qingliangfeng, Heqiao, and Tuankou, and the probability of the risk for contamination by Cd and Cu was higher in the study area. The high values of Cd, Cu, Zn, Ni, and Cr were mainly related to mining, while Pb was closely related to the application of potassium.

MicroRNA-107: a novel promoter of tumor progression that targets the CPEB3/EGFR axis in human hepatocellular carcinoma.

MicroRNAs (miRNAs) are dysregulated in many types of malignancies, including human hepatocellular carcinoma (HCC). MiR-107 has been implicated in several types of cancer regulation; however, relatively little is known about miR-107 in human HCC. In the present study, we showed that the overexpression of miR-107 accelerates the tumor progression of HCC in vitro and in vivo through its new target gene, CPEB3. Furthermore, our results demonstrated that CPEB3 is a newly discovered tumor suppressor that acts via the EGFR pathway. Therefore, our study demonstrates that the newly discovered miR-107/CPEB3/EGFR axis plays an important role in HCC progression and might represent a new potential therapeutic target for HCC treatment.

Silver catalyzed decarboxylative direct C2-alkylation of benzothiazoles with carboxylic acids.

A novel and efficient silver catalyzed decarboxylative direct C2-alkylation of benzothiazoles with carboxylic acids for the synthesis of 2-alkyl benzothiazoles was developed.

Antitumor effects of mutant endostatin are enhanced by Bcl-2 antisense oligonucleotides in UM-UC-3 bladder cancer cell line.

BACKGROUND: Endostatin is a potent inhibitor of tumor angiogenesis. In the preliminary studies, we developed a mutant endostatin containing Arg-Gly-Asp-Arg-Gly-Asp (RGDRGD) sequences. In this study, we compared the antitumor effects of mutant endostatin and Bcl-2 antisense oligonucleotides both in combination and individually. METHODS: The artificially synthesized Bcl-2 ASODN (antisense oligonucleotides) included a translation-initiation site and was transfected into the bladder cancer cells by Lipofectamine. Cell growth was investigated by the tumor cell growth chart, MTT assay, caspase-3 activity detection assay, AO/EB fluorescein stain, and the annexin V-FITC apoptosis detection assay. In the in vivo study, UM-UC-3 bladder cancer cells were subcutaneously implanted into nude mice and the growth of tumor was examined. The ultrastructure of the tumor tissues in the treated and control groups were observed. RESULTS: The cell growth chart showed that the cell population of the treated combination group decreased by 52.04% compared to the control group. The inhibition rate of the treated combination group was (79.66 ± 6.79)%, whereas those of the individual ASODN and ES groups were (53.39 ± 3.22)% and (50.22 ± 5.46)% respectively. In the caspase-3 activity detection using AO/EB fluorescein stain and annexin V-FITC apoptosis detection assay, the co-inhibitory effect was higher than the individual inhibitory effects (P < 0.05). There were significant differences in the inhibition of the solid tumor growth in the in vivo study. CONCLUSIONS: Our findings indicated that Bcl-2 antisense oligonucleotides enhance the antitumor effects of mutant endostatin both in vitro and in vivo. We noted the synergistic effects of Bcl-2 antisense oligonucleotides combined with mutant endostatin.

Novel cathode interlayers based on neutral alcohol-soluble small molecules with a triphenylamine core featuring polar phosphonate side chains for high-performance polymer light-emitting and photovoltaic devices.

A new family of neutral alcohol-soluble small molecular materials comprised of electron-rich triphenylamine (TPA) and fluorene featuring phosphonate side chains (FEP) is reported, namely 3TPA-FEP, 2TPA-2FEP and TPA-3FEP, which have different TPA and FEP contents. Due to their good solubility in polar solvents like alcohol, multilayer devices can be fabricated by a wet process from orthogonal solvents. Polymer light-emitting devices with these materials as a cathode interlayer and Al as the cathode show greatly enhanced efficiencies in contrast to control devices without such a cathode interlayer, and their efficiencies are comparable with or even higher than devices with the low work-function metal Ba/Al as the cathode. In addition, high-performance polymer solar cells based on the poly[N-9''-hepta-decanyl-2,7-carbazole-alt-5,5-(4',7'-di-2-thienyl-2',1',3'-benzothiadiazole)] (PCDTBT):[6,6]-phenyl C71 -butyric acid methyl ester (PC71 BM) system are also achieved with power conversion efficiencies of 7.21%, 6.90% and 6.89%, by utilizing 3TPA-FEP, 2TPA-2FEP and TPA-3FEP as the cathode interlayer, respectively. These efficiencies are also much higher than those for control devices without the cathode interlayer. Although TPA is well-known as a hole-transport unit, the current findings indicate that alcohol-soluble TPA-based small molecules are also a promising cathode interlayer for both electron injection and extraction.

Novel bipolar host materials based on 1,3,5-triazine derivatives for highly efficient phosphorescent OLEDs with extremely low efficiency roll-off.

Recently, bipolar host materials have attracted considerable attention because they can achieve balanced charge injection/transport in phosphorescent organic light emitting diodes (PhOLEDs) and consequently obtain excellent device performance. In this work, two bipolar host materials, namely, 3-(4,6-diphenyl-1,3,5-triazin-2-yl)-9-phenyl-9H-carbazole (DPTPCz) and 3-(4,6-diphenoxy-1,3,5-triazin-2-yl)-9-phenyl-9H-carbazole (DPOTPCz), have been designed, synthesized and characterized. With high triplet energy levels of 2.78 and 2.86 eV for DPTPCz and DPOTPCz, respectively, two compounds are considered promising bipolar host materials for PhOLEDs. Blue and green PhOLEDs based on these two new compounds show excellent performances. The phosphorescent devices based on DPTPCz exhibit maximum external quantum efficiencies of 14.4% (for blue device) and 21.2% (for green device), and maintain high efficiencies of 11.9% and 20.0% even at a high luminance of 10,000 cd m(-2).

[The genetic stability of recombinant adenovirus expressing human rotavirus VP6 gene which used Ad41 as vector].

OBJECTIVE: To investigate the genetic stability of non-replicating recombinant adenovirus which used Ad41 as vector and could express VP6 gene of group A rotavirus during continous passage, in order to develop the vaccine of rotavirus. METHODS: The recombinant adenovirus rvAd41-VP6 (o) was prepared by our laboratory early, it then was continuously propagated on 293TE7 cells for 14 passages. After that samples of the infected cells were collected at every 2 passages for the detection of the integration of the VP6 gene by PCR, and the expression of the target protein was detected by Western Blot analysis. RESULTS: Analysis by PCR revealed that, there was stable integration of specific VP6 gene in the rvAd41-VP6 (o), Western Blot analysis confirmed that rvAd41-VP6 (o) could stably expressed the group-specific antigen structural protein VP6 (o), and it had preferable genetic stability. CONCLUSION: The recombinant adenovirus rvAd41-VP6 (o) which could stably express the VP6 (o) gene had favorable biological property in vitro, and it has provided a basis for further research of animal immunization.

Replication and transcription of human papillomavirus type 58 genome in Saccharomyces cerevisiae.

BACKGROUND: To establish a convenient system for the study of human papillomavirus (HPV), we inserted a Saccharomyces cerevisiae selectable marker, Ura, into HPV58 genome and transformed it into yeast. RESULTS: HPV58 genome could replicate extrachromosomally in yeast, with transcription of its early and late genes. However, with mutation of the viral E2 gene, HPV58 genome lost its mitotic stability, and the transcription levels of E6 and E7 genes were upregulated. CONCLUSIONS: E2 protein could participate in viral genome maintenance, replication and transcription regulation. This yeast model could be used for the study of certain aspects of HPV life cycle.

Dendritic cells (DCs) transfected with a recombinant adenovirus carrying chlamydial major outer membrane protein antigen elicit protective immune responses against genital tract challenge infection.

Chlamydia trachomatis, an obligate intracellular bacterial pathogen, is the major cause of sexually transmitted diseases worldwide. Although a variety of strategies have been taken to promote the development of a protective vaccine, no ideal vaccine has been generated so far. In this study, we transfected dendritic cells (DCs) with recombinant adenovirus carrying C. trachomatis serovar E major outer membrane protein gene (Ad-MOMP), and investigated their ability to induce specific protection against genital tract chlamydial challenge infection. The results showed that when DCs were transfected with Ad-MOMP in vitro, the DCs exhibited increased expression of CD80 and MHC-II molecules as well as enhanced IL-12 secretion and were able to stimulate T-cell proliferation. The level of IFN-gamma secreted by stimulated T cells was also up-regulated significantly. When the Ad-MOMP transfected DCs were adoptively transferred intravenously to naive mice, they generated Th1-biased cytokine production and mucosal IgA responses specific for C. trachomatis. More importantly, the mice immunized with Ad-MOMP-DC mounted protection against genital tract challenge infection, shown by lower body mass loss, lower chlamydial loads, and less severe pathological changes. In conclusion, Ad-MOMP transfected DCs are capable of inducing effective protective immune responses against C. trachomatis genital infection.

Translational comparison of HPV58 long and short L1 mRNAs in yeast (Saccharomyces cerevisiae) cell-free system.

Recently, we developed a yeast cell-free system for translation of human papillomavirus (HPV) 58 short L1 mRNA and VLP assembly in vitro. In the present study, we examined the translation efficiency of HPV58 long and short L1 mRNAs in our established system. Production of HPV 58 long and short L1 proteins was significantly dependent on the amounts of the L1 mRNAs used and the time length of translation reaction. Compared with the long L1 mRNA, the short L1 mRNA consistently exhibited higher translation efficiency in all the experiments conducted. Supplementation of exogenous amino acids significantly enhanced translation of the long L1 mRNA in this system. Furthermore, a single amino acid leucine can be rate-limiting for translation of the long L1 mRNA in this system. Electron microscopy demonstrated that HPV 58 virus-like particles (VLPs) were assembled from both long and short L1 capsid proteins in the yeast cell-free system.

Constitutive activation of signal transducer and activator of transcription 3 regulates expression of vascular endothelial growth factor in human meningioma differentiation.

PURPOSE: Janus tyrosine kinases (JAKs) and signal transducer and activator of transcription factors (STATs), especially STAT3, are constitutively activated in human cancers. The function of STAT3 in the pathogenesis of meningioma remains unknown. In this study, we investigated the role of JAK1/STAT3 regulating vascular endothelial growth factor (VEGF) expression in the occurrence and progression of human meningioma. METHODS: We detected the expression of JAK1, p-JAK1, STAT3, p-STAT3, and VEGF in human meningioma and normal dura tissues by RT-PCR, Western blot analysis, and immunohistochemistry. RESULTS: JAK1, p-JAK1, STAT3, p-STAT3, and VEGF showed high expression in grade I and grade II meningioma. The level of STAT3 activation was associated with VEGF expression; all meningioma tumors that expressed p-STAT3 also expressed VEGF. Both frequency of positivity and expression were enhanced with increasing tumor grade; high frequencies and levels were found in grade II tumors, with no expression detected in normal dura tissues (P < 0.05). CONCLUSIONS: VEGF is directly regulated by constitutive STAT3 activity and associated with meningioma differentiation. STAT3 has an important role in the occurrence and development of human meningioma by regulating VEGF expression.

The antiarrhythmic effect and possible ionic mechanisms of pilocarpine on animal models.

This study was designed to evaluate the effects of pilocarpine and explore the underlying ionic mechanism, using both aconitine-induced rat and ouabain-induced guinea pig arrhythmia models. Confocal microscopy was used to measure intracellular free-calcium concentrations ([Ca(2+)](i)) in isolated myocytes. The current data showed that pilocarpine significantly delayed onset of arrhythmias, decreased the time course of ventricular tachycardia and fibrillation, reduced arrhythmia score, and increased the survival time of arrhythmic rats and guinea pigs. [Ca(2+)](i) overload induced by aconitine or ouabain was reduced in isolated myocytes pretreated with pilocarpine. Moreover, M(3)-muscarinic acetylcholine receptor (mAChR) antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine-methiodide) partially abolished the beneficial effects of pilocarpine. These data suggest that pilocarpine produced antiarrhythmic actions on arrhythmic rat and guinea pig models induced by aconitine or ouabain via stimulating the cardiac M(3)-mAChR. The mechanism may be related to the improvement of Ca(2+) handling.

Mutational analysis of proto-oncogene Dbl on Xq27 in testicular germ cell tumors reveals a rare SNP in a patient with bilateral undescended testis.

OBJECTIVES: An abundance of X chromosomes in testicular germ cell tumors (TGCTs), and a candidate TGCTs susceptibility gene (TGCT1) on Xq27 highlight the potential involvement of X chromosomes in TGCT pathogenesis. However, the TGCT1 on Xq27 has so far not been identified. We hypothesized that a somatic mutation of dbl oncogene on Xq27 may play a role for the development of TGCTs. METHODS: We have screened 41 TGCT tissues for dbl mutations using single-strand conformation polymorphism (SSCP) analysis. These tissues are composed of 25 seminomatous TGCTs tissues and 16 non-seminomatous TGCTs tissues, including two cases with a rhabdomyosarcoma component. RESULTS: Somatic mutations were not detected in the 25 exons of dbl in these TGCTs. However, we found a rare single nucleotide polymorphism (SNP) (T to C nucleotide change) within intron 22 in one out of the 41 TGCTs cases (2%). Furthermore, the sample with the rare SNP was identified as the sole TGCTs case associated with bilateral undescended testis in our series. CONCLUSIONS: Our results indicate that proto-oncogene dbl is not a major target for sporadic TGCTs. However, the rare SNP in dbl may affect the susceptibility to undescended testis. Determining the frequency of this SNP in patients with various types of undescended testis in different ethnic groups is a warranted study.

Expression of HPV 58 long and short L1 capsid proteins in primary mouse keratinocyte cultures.

We studied expression of HPV 58 long and short L1 proteins in primary mouse keratinocyte (KC) cultures by transient transfection of the L1 expression constructs. Following transient transfection, long and short L1 open reading frames (ORFs) were transcribed continuously for 9 days; however, no significant difference was detected between the long and short L1 mRNA levels measured by quantitative RT-PCR. Western blot analysis showed that both long and short L1 proteins were continuously detected in L1-transfected KCs for 9 days post-transfection and the significantly increased signals of the L1 proteins over time were associated with KC differentiation. Moreover, L1 protein was more abundant in KCs transfected with the short L1 ORF than the long L1 ORF. In vitro translation of the L1 mRNAs indicated further that the short L1 mRNA had significantly higher translation efficiency than the long L1 mRNA in cell-free lysate system. The L1 proteins expressed from the two L1 mRNAs in KCs were similarly stable. Thus, approximate 40% lower level of expression of the L1 protein in KCs transfected with the long L1 ORF was probably due to a stem-loop structure with high DeltaG value downstream the first AUG codon in its mRNA secondary structure. This stem-loop structure might prevent efficient binding of the ribosome to mRNA and therefore reduced translation.