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The impact of carbon/nitrogen (C/N) ratio on sequencing batch biofilm reactor (SBBR) initiated with different seed sludges for treating actual mariculture effluent was explored. Increasing the C/N ratio significantly enhanced the nitrogen removal efficiency, achieving average removal efficiency of 95% for ammonia nitrogen and 73% for total nitrogen at ratio of 30, while the impact of seed sludge was minimal. High C/N ratio promoted the secretion of tightly bound extracellular polymeric substances (TB-EPS), which showed significant correlation with nitrogen removal. Interactions between bacteria and archaea were enhanced and conditionally rare or abundant taxa were the keystone taxa. High C/N ratio inhibited the relative abundance of ammonia-oxidizing archaea (Candidatus_Nitrosopumilus) and bacteria (Nitrosomonas), but promoted the heterotrophic nitrification-aerobic denitrification bacteria (Halomonas). The expression of nitrogen removal functional genes significantly correlated with functional genera. This study emphasized the crucial role of high C/N ratios in biological nitrogen removal from actual mariculture effluent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ribosomal DNA (rDNA) repeats harbor ribosomal RNA (rRNA) genes and intergenic spacers (IGS). RNA polymerase (Pol) I transcribes rRNA genes yielding rRNA components of ribosomes. IGS-associated Pol II prevents Pol I from excessively synthesizing IGS non-coding RNAs (ncRNAs) that can disrupt nucleoli and rRNA production. Here, compartment-enriched proximity-dependent biotin identification (compBioID) revealed the TATA-less-promoter-binding TBPL1 and transcription-regulatory PAF1 with nucleolar Pol II. TBPL1 localizes to TCT motifs, driving Pol II and Pol I and maintaining its baseline ncRNA levels. PAF1 promotes Pol II elongation, preventing unscheduled R-loops that hyper-restrain IGS Pol I-associated ncRNAs. PAF1 or TBPL1 deficiency disrupts nucleolar organization and rRNA biogenesis. In PAF1-deficient cells, repressing unscheduled IGS R-loops rescues nucleolar organization and rRNA production. Depleting IGS Pol I-dependent ncRNAs is sufficient to compromise nucleoli. We present the nucleolar interactome of Pol II and show that its regulation by TBPL1 and PAF1 ensures IGS Pol I ncRNAs maintaining nucleolar structure and function., (© 2024. The Author(s).)

Background: NUP98 rearrangements (NUP98-r) are rare but overrepresented mutations in pediatric acute myeloid leukemia (AML) patients. NUP98-r is often associated with chemotherapy resistance and a particularly poor prognosis. Therefore, characterizing pediatric AML with NUP98-r to identify aberrations is critically important., Methods: Here, we retrospectively analyzed the clinicopathological features, genomic and transcriptomic landscapes, treatments, and outcomes of pediatric patients with AML., Results: Nine patients with NUP98-r mutations were identified in our cohort of 142 patients. Ten mutated genes were detected in patients with NUP98-r. The frequency of FLT3-ITD mutations differed significantly between the groups harboring NUP98-r and those without NUP98-r (P = 0.035). Unsupervised hierarchical clustering via RNA sequencing data from 21 AML patients revealed that NUP98-r samples clustered together, strongly suggesting a distinct subtype. Compared with that in the non-NUP98-r fusion and no fusion groups, CMAHP expression was significantly upregulated in the NUP98-r samples (P < 0.001 and P = 0.001, respectively). Multivariate Cox regression analyses demonstrated that patients harboring NUP98-r (P < 0.001) and WT1 mutations (P = 0.030) had worse relapse-free survival, and patients harboring NUP98-r (P < 0.008) presented lower overall survival., Conclusions: These investigations contribute to the understanding of the molecular characteristics, risk stratification, and prognostic evaluation of pediatric AML patients., (© 2024. The Author(s).)

The construction of aerobic denitrification (AD) systems in an antibiotic-stressed environment is a serious challenge. This study investigated strategy of cyclic stress with concentration gradient (5-30 mg/L) of sulfamethoxazole (SMX) in a sequencing batch reactor (SBR), to achieve operation of AD. Total nitrogen removal efficiency of system increased from about 10 % to 95 %. Original response of abundant-rare genera to antibiotics was changed by SMX stress, particularly conditionally rare or abundant taxa (CRAT). AD process depends on synergistic effect of heterotrophic nitrifying aerobic denitrification bacteria (Paracoccus, Thauera, Hypomicrobium, etc). AmoABC, napA, and nirK were functionally co-expressed with multiple antibiotic resistance genes (ARGs) (acrR, ereAB, and mdtO), facilitating AD process. ARGs and TCA cycling synergistically enhance the antioxidant and electron transport capacities of AD process. Antibiotic efflux pump mechanism played an important role in operation of AD. The study provides strong support for regulating activated sludge to achieve in situ AD function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Low temperatures limit the denitrification wastewater in activated sludge systems, but this can be mitigated by addition of redox mediators (RMs). Here, the effects of chlorophyll (Chl), 1,2-naphthoquinone-4-sulfonic acid (NQS), humic acid (HA), and riboflavin (RF), each tested at three concentrations, were compared for denitrification performance at low temperature, by monitoring the produced extracellular polymeric substances (EPS), and characterizing microbial communities and their metabolic potential. Chl increased the denitrification rate most, namely 4.12-fold compared to the control, followed by NQS (2.62-fold increase) and HA (1.35-fold increase), but RF had an inhibitory effect. Chl promoted the secretion of tryptophan-like and tyrosine-like proteins in the EPS and aided the conversion of protein from tightly bound EPS into loosely bound EPS, which improved the material transfer efficiency. NQS, HA, and RF also altered the EPS components. The four RMs affected the microbial community structure, whereby both conditionally abundant taxa (CAT) and conditionally rare or abundant taxa (CRAT) were key taxa. Among them, CRAT members interacted most with the other taxa. Chl promoted Flavobacterium enrichment in low-temperature activated sludge systems. In addition, Chl promoted the abundance of nitrate reduction genes narGHI and napAB and of nitrite reduction genes nirKS, norBC, and nosZ. Moreover, Chl increased abundance of genes involved in acetate metabolism and in the TCA cycle, thereby improving carbon source utilization. This study increases our understanding of the enhancement of low-temperature activated sludge by RMs, and demonstrates positive effects, in particular by Chl., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: West Nile virus is a severe zoonotic pathogen that can cause severe central nervous system symptoms in humans and horses, and is fatal for birds, chickens and other poultry. With no specific drugs or vaccines available, antibody-based therapy is a promising treatment. This study aims to develop neutralizing antibodies against West Nile virus and assess their cross-protective potential against Japanese encephalitis virus., Methods: Monoclonal antibodies against WNV and JEV were isolated by hybridoma technology. The therapeutic efficacy of these antibodies was evaluated using a mouse model, and a humanized version of the monoclonal antibody was generated for potential human application., Results: In this study, we generated eight monoclonal antibodies that exhibit neutralizing activity against WNV. Their therapeutic effects against WNV were validated both in vivo and in vitro. Among these antibodies, C9-G11-F3 also exhibited cross-protective activity against JEV. We also humanized the antibody to ensure that it could be used for WNV infection treatment in humans., Conclusion: This study highlights the importance of neutralizing antibodies as a promising approach for protection against West Nile virus infection and suggests their potential utility in the development of therapeutic interventions., (© 2023 The Author(s).)

Recent studies show that antiviral systems are remarkably conserved from bacteria to mammals, demonstrating that unique insights into these systems can be gained by studying microbial organisms. Unlike in bacteria, however, where phage infection can be lethal, no cytotoxic viral consequence is known in the budding yeast Saccharomyces cerevisiae even though it is chronically infected with a double-stranded RNA mycovirus called L-A. This remains the case despite the previous identification of conserved antiviral systems that limit L-A replication. Here, we show that these systems collaborate to prevent rampant L-A replication, which causes lethality in cells grown at high temperature. Exploiting this discovery, we use an overexpression screen to identify antiviral functions for the yeast homologs of polyA-binding protein (PABPC1) and the La-domain containing protein Larp1, which are both involved in viral innate immunity in humans. Using a complementary loss of function approach, we identify new antiviral functions for the conserved RNA exonucleases REX2 and MYG1 ; the SAGA and PAF1 chromatin regulatory complexes; and HSF1 , the master transcriptional regulator of the proteostatic stress response. Through investigation of these antiviral systems, we show that L-A pathogenesis is associated with an activated proteostatic stress response and the accumulation of cytotoxic protein aggregates. These findings identify proteotoxic stress as an underlying cause of L-A pathogenesis and further advance yeast as a powerful model system for the discovery and characterization of conserved antiviral systems.

Supports can widely affect or even dominate the catalytic activity and selectivity of nanoparticles because atomic geometry and electronic structures of active sites can be regulated, especially at the interface of nanoparticles and supports. However, the underlying mechanisms of most systems are still not fully understood yet. Herein, we construct the interface of Co 3 O 4 /TiO 2 to boost ammonium perchlorate (AP) catalytic decomposition. This catalyst shows enhanced catalytic performance. With the addition of 2 wt % Co 3 O 4 /TiO 2 catalysts, AP decomposition peak temperature decreases from 435.7 to 295.0 °C and activation energy decreases from 211.5 to 137.7 kJ mol -1 . By combining experimental and theoretical studies, we find that Co 3 O 4 nanoparticles can be strongly anchored onto TiO 2 supports accompanied by charge transfer. Moreover, at the interfaces in the Co 3 O 4 /TiO 2 nanostructure, NH 3 adsorption can be enhanced through hydrogen bonds. Our research studies provide new insights into the promotion effects of the nanoparticle/support system on the AP decomposition process and inspire the design of efficient catalysts.

Background: Approximately 30% of Chinese individuals with cytogenetically normal acute myeloid leukemia (CN-AML) have biallelic CEBPA (bi CEBPA ) mutations. The prognosis and optimal therapy for these patients are controversial in clinical practice., Methods: In this study, we performed targeted region sequencing of 236 genes in 158 individuals with this genotype and constructed a nomogram model based on leukemia-free survival (LFS). Patients were randomly assigned to a training cohort ( N =111) and a validation cohort ( N =47) at a ratio of 7:3. Risk stratification was performed by the prognostic factors to investigate the risk-adapted post-remission therapy by Kaplan-Meier method., Results: At least 1 mutated gene other than CEBPA was identified in patients and mutation number was associated with LFS (61.6% vs. 39.0%, P =0.033), survival (85.6% vs. 62.9%, P  =0.030) and cumulative incidence of relapse (CIR) (38.4% vs. 59.5%, P =0.0496). White blood cell count, mutations in CFS3R , KMT2A and DNA methylation related genes were weighted to construct a nomogram model and differentiate two risk subgroups. Regarding LFS, low-risk patients were superior to the high-risk (89.3% vs. 33.8%, P < 0.001 in training cohort; 87.5% vs. 18.2%, P =0.009 in validation cohort). Compared with chemotherapy, allogenic hematopoietic stem cell transplantation (allo-HSCT) improved 5-year LFS (89.6% vs. 32.6%, P < 0.001), survival (96.9% vs. 63.6%, P =0.001) and CIR (7.2% vs. 65.8%, P < 0.001) in high-risk patients but not low-risk patients (LFS, 77.4% vs. 88.9%, P =0.424; survival, 83.9% vs. 95.5%, P =0.173; CIR, 11.7% vs. 11.1%, P =0.901)., Conclusions: Our study indicated that bi CEBPA mutant-positive CN-AML patients could be further classified into two risk subgroups by four factors and allo-HSCT should be recommended for high-risk patients as post-remission therapy. These data will help physicians refine treatment decision-making in bi CEBPA mutant-positive CN-AML patients., Competing Interests: Authors S-BC, FL, TZ, L-XL, and C-CW are employed by Acornmed Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RL declared a past co-authorship with the authors to the handling editor., (Copyright © 2021 Wu, Jiang, Chang, Zhou, Wang, Wang, Cao, Li, Sun, Cao, Lou, Zhou, Liu, Wang, Wang, Jiang, Xu, Zhang, Liu, Huang and Ruan.)

FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most common somatic mutations in acute myeloid leukemia (AML). However, the molecular structure characteristics and widely accepted prognostic factors for FLT3-ITD are still not well described. This study aimed to retrospectively examine 81 patients with FLT3-ITD-positive AML diagnosed and treated at the First Affiliated Hospital of Zhejiang University from December 2013 to March 2018 using the next-generation sequencing 185-gene platform. High variant allele frequency (VAF) [> 0.48, P = 0.0089 for overall survival (OS), P = 0.13 for relapse-free survival (RFS)], multiple ITDs (> 1 ITDs, P = 0.011 for OS, P = 0.033 for RFS) and longer insertion length (> 69 bp, P = 0.14 for OS, P = 0.0078 for RFS) predicted poor survival. The study further proposed an easily applicable scoring model for OS using the Least Absolute Shrinkage and Selector Operation (LASSO) Cox regression model. Also, an independent cohort of 30 patients was used for external model validation. The mode was expressed as follows: 0.659 × FLT3-ITD VAF + 0.375 × FLT3-ITD number + 0.807 × Age + 0.688 × DNMT3A + 1.939 × U2AF1 (FLT3-ITD VAF > 0.48 scored 1; FLT3-ITD number scored 1 if carried 1 ITD, 2 if carried ≥ 2 ITDs; age > 44 years scored 1, the presence of DNMT3A or U2AF1 scored 1; 0 for other conditions). It categorized patients into low-risk (L-R, score < 1, n = 20) and high-risk (H-R, score ≥ 1, n = 61) groups based on the risk score with a significant difference in survival (3-year OS, P < 0.0001; 3-year RFS, P = 0.0005). A prognostic nomogram that integrated these five factors was developed with a concordance index calculation [OS: 0.68, 95% CI (0.64-0.72)]., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (AJCR Copyright © 2020.)

About 25% of patients with newly diagnosed acute myeloid leukaemia (AML) have normal cytogenetics and no nucleophosmin 1 (NPM1) mutation or Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD). The prognosis and best therapy for these patients is controversial. We evaluated 158 newly diagnosed adults with this genotype who achieved histological complete remission within two cycles of induction therapy and were assigned to two post-remission strategies with and without an allotransplant. Targeted regional sequencing at diagnosis was performed and data were used to estimate their prognosis, including relapse and survival. In multivariable analyses, having wild-type or mono-allelic mutated CCAAT/enhancer-binding protein alpha (CEBPA) [hazard ratio (HR) 2·39, 95% confidence interval (CI) 1·08-5·30; P = 0·032), mutated NRAS (HR 2·67, 95% CI 1·36-5·25; P = 0·004), mutated colony-stimulating factor 3 receptor (CSF3R) (HR 2·85, 95% CI 1·12-7·27; P = 0·028) and a positive measurable residual disease (MRD)-test after the second consolidation cycle (HR 2·88, 95% CI 1·32-6·30; P = 0·008) were independently correlated with higher cumulative incidence of relapse (CIR). These variables were also significantly associated with worse survival (HR 3·02, 95% CI 1·17-7·78, P = 0·022; HR 3·62, 95% CI 1·51-8·68, P = 0·004; HR 3·14, 95% CI 1·06-9·31, P = 0·039; HR 4·03, 95% CI 1·64-9·89, P = 0·002; respectively). Patients with ≥1 of these adverse-risk variables benefitted from a transplant, whereas the others did not. In conclusion, we identified variables associated with CIR and survival in patients with AML and normal cytogenetics without a NPM1 mutation or FLT3-ITD., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Hereditary spherocytosis (HS) is often misdiagnosed due to lack of specific diagnostic methods. Our study summarized clinical characteristics and described the diagnostic workflow for mild and moderate HS in Chinese individuals, using data from 20 adults, 8 of whom presented a familial history for HS. We used scanning electron microscopy (SEM) to diagnose HS. We observed reduced eosin maleimide fluorescence activity (5.50 mean channel fluorescence (MCF) units) in the 10 cases of HS, which differed significantly when compared with 10 normal adults (15.50 units), iron deficiency anemia (15.50 MCF units), and megaloblastic anemia (12.00 MCF units) values (P < .05). Next generation sequencing results revealed that 9 out of 10 patients were found to have mutations in the spectrin alpha chain (SPTB), anchor protein (ANK1), and SLC4A1 genes. These mutations were not reported in the Human Gene Mutation Database (HGMD), 1000 human genome, ExAC, and dbSNP147 databases. Splenectomy proved to be beneficial in alleviating HS symptoms in 10 cases. It was found that for the diagnosis of HS, SEM and next generation gene sequencing method proved to be more ideal than red blood cell membrane protein analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting., (© 2020 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Objective: Chromosome aberrations are generally considered as one of the most substantial causative factors contributing to spontaneous miscarriages. Cytogenetic analyses like G-banded karyotype and chromosomal microarray analyses are often performed to further investigate the chromosome status of a miscarried fetus., Study Design: Here, we describe a novel method, AnnoCNV, to detect DNA copy number variations (CNVs) using low coverage whole genome sequencing (WGS). We investigated the overall frequency of chromosomal abnormalities in 149 miscarriage specimens using AnnoCNV., Results: Among 149 fetal miscarriage samples, more than two fifths of them (42.95%, 64) carried at least one chromosomal abnormality, and a subset (40) was identified as autosomal trisomy which account for 26.84% of all samples. We have also developed a robust algorithm in AnnoCNV, which is able to differentiate specifically karyotype 69,XXY from sex chromosomal aneuploidy 45,X, and to identify 45,X/46,XX mosaicism. Lastly, across the whole genome AnnoCNV identifies CNVs, which are associated with both reported symptoms and unknown clinical conditions., Conclusion: This cost-effective strategy reveals genome wide discovery of chromosome aberrations at higher resolution, which are consistent with parallel investigation conducted by SNP based assay., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: To explore the utility of circulating tumor DNA detection in early breast cancer by using next-generation sequencing. Methods: This exploratory study of circulating tumor DNA detection is for early invasive breast cancer patients treated in Breast Disease Center, Peking University First Hospital from December 2015 to July 2016. Plasma samples were collected and were used to isolate plasma cell-free DNA.Exons or hotspots of 247 cancer related genes were sequenced by next-generation sequencing. Mutations and their correlation with clinic-pathological factors were analyzed. The correlation between mutations and clinic-pathological factors was evaluated by χ(2) test or Fisher's exact test. Results: Seventy-five patients were enrolled in this study. All patients were female and aged from 31 to 88 years with median age of 58 years. All patients' clinic-pathological records were complete. Sixty-four mutations in 18 genes (ALK, BCR, ERBB2, ROS1, PDGFRA, EGFR, FGFR2, CYP1B1, CALR, CASP7, BRAF, FGFR1, FGFR3, MET, NRAS, PTEN, KIT, SOD2) were detected in 47 (62.7%) among all 75 patients.Exons were captured in 10 genes, and mutations in 2 of 3 genes analyzed were clustered. Gene mutations were not correlated with menopausal status, histological type, primary tumor (T), regional lymph nodes (N), TNM stage, histological grade, estrogen receptor status, progesterone receptor status, human epidermal growth factor receptor 2 status, Ki-67 and molecular subtype (all P >0.05). Conclusion: Circulating tumor DNA sequencing by next-generation sequencing was useful for detecting breast cancer-related mutations.

Casein kinase 2 (CK2) is a highly conserved, ubiquitously expressed serine/threonine protein kinase with hundreds of substrates. The role of CK2 in the G 2 /M transition of oocytes, zygotes, and 2-cell embryos was studied in mouse by enzyme activity inhibition using the specific inhibitor 4, 5, 6, 7-tetrabromobenzotriazole (TBB). Zygotes and 2-cell embryos were arrested at G 2 phase by TBB treatment, and DNA damage was increased in the female pronucleus of arrested zygotes. Further developmental ability of arrested zygotes was reduced, but that of arrested 2-cell embryos was not affected after releasing from inhibition. By contrast, the G 2 /M transition in oocytes was not affected by TBB. These results indicate that CK2 activity is essential for mitotic G 2 /M transition in early embryos but not for meiotic G 2 /M transition in oocytes.

Background: Increasing evidence suggests that microRNAs are functionally involved in the initiation and maintenance of pain hypersensitivity, including chronic morphine analgesic tolerance, through the posttranscriptional regulation of pain-related genes. We have previously demonstrated that miR-219 regulates inflammatory pain in the spinal cord by targeting calcium/calmodulin-dependent protein kinase II gamma (CaMKIIγ). However, whether miR-219 regulates CaMKIIγ expression in the dorsal root ganglia to mediate morphine tolerance remains unclear., Results: MiR-219 expression was downregulated and CaMKIIγ expression was upregulated in mouse dorsal root ganglia following chronic morphine treatment. The changes in miR-219 and CaMKIIγ expression closely correlated with the development of morphine tolerance, which was measured using the reduction of percentage of maximum potential efficiency to thermal stimuli. Morphine tolerance was markedly delayed by upregulating miR-219 expression using miR-219 mimics or downregulating CaMKIIγ expression using CaMKIIγ small interfering RNA. The protein and mRNA expression of brain-derived neurotrophic factor were also induced in dorsal root ganglia by prolonged morphine exposure in a time-dependent manner, which were transcriptionally regulated by miR-219 and CaMKIIγ. Scavenging brain-derived neurotrophic factor via tyrosine receptor kinase B-Fc partially attenuated morphine tolerance. Moreover, functional inhibition of miR-219 via miR-219-sponge in naive mice elicited thermal hyperalgesia and spinal neuronal sensitization, which were both suppressed by CaMKIIγ small interfering RNA or tyrosine receptor kinase B-Fc., Conclusions: These results demonstrate that miR-219 contributes to the development of chronic tolerance to morphine analgesia in mouse dorsal root ganglia by targeting CaMKIIγ and enhancing CaMKIIγ-dependent brain-derived neurotrophic factor expression., (© The Author(s) 2016.)

Corticotropin-releasing factor (CRF) serves as a neuromodulator in the hypothalamic-pituitary-adrenal axis, playing an essential role in depression, anxiety, and pain regulation. However, its biological role in bone cancer induced pain has not been investigated. In the present study, we aimed to elucidate the expression and distribution of CRF in spinal cord using a rodent model of bone cancer pain. Our study showed that implantation of Walker 256 mammary gland carcinoma cells into the tibia of rats significantly increased CRF expression in the spinal cord in a time-dependent manner. The upregulated expression of CRF mainly expressed in the superficial dorsal horn of spinal cord. Moreover, immunofluorescence double staining showed that CRF was extensively colocalized with neurons, but hardly with astrocytes or microglia. In addition, intrathecal injection of CRF receptor antagonist (α-helical-CRF) significantly inhibited heat hyperalgesia, mechanical allodynia, and the expression of c-Fos in spinal dorsal horn of bone cancer pain rats. In summary, our study demonstrates that CRF plays an important role in the development and maintenance of bone cancer pain via activation of neurons.

The activation of MAPK pathways in spinal cord and subsequent production of proinflammatory cytokines in glial cells contribute to the development of spinal central sensitization, the basic mechanism underlying bone cancer pain (BCP). Our previous study showed that spinal CXCL12 from astrocytes mediates BCP generation by binding to CXCR4 in both astrocyters and microglia. Here, we verified that CXCL12/CXCR4 signaling contributed to BCP through a MAPK-mediated mechanism. In naïve rats, a single intrathecal administration of CXCL12 considerably induced pain hyperalgesia and phosphorylation expression of spinal MAPK members (including extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase), which could be partially prevented by pre-treatment with CXCR4 inhibitor AMD3100. This CXCL12-induced hyperalgesia was also reduced by MAPK inhibitors. In bone cancer rats, tumor cell inoculation into the tibial cavity caused prominent and persistent pain hyperalgesia, and associated with up-regulation of CXCL12 and CXCR4, activation of glial cells, phosphorylation of MAPKs, and production of proinflammatory cytokines in the spinal cord. These tumor cell inoculation-induced behavioral and neurochemical alterations were all suppressed by blocking CXCL12/CXCR4 signaling or MAPK pathways. Taken together, these results demonstrate that spinal MAPK pathways mediated CXCL12/CXCR4-induced pain hypersensitivity in bone cancer rats, which could be druggable targets for alleviating BCP and glia-derived neuroinflammation. Following tumor cell inoculation, chemokine CXCL12 from astrocytes spreads around the spinal environment, resulting in functional activation of CXCR4-expressing astrocytes and microglia. Once glia are activated, they may initiate MAPK (mitogen-activated protein kinase) pathways, and subsequently produce proinflammatory cytokines and chemokines. Among them, CXCL12 could reinforce the astrocytic and microglial activation in autocrine and paracrine manners. Such positive feedback loops sustain perseverant neuroinflammation, facilitate glial activation, and finally lead to bone cancer pain. IL = interleukin; TNF = tumor necrosis factor., (© 2014 International Society for Neurochemistry.)

A full-length genome infectious clone is a powerful tool for functional assays in virology. In this study, using a chemical synthesized complete genome of Japanese encephalitis virus (JEV) strain SA14 (GenBank accession no. U14163), we constructed a full-length genomic cDNA clone of JEV. The recovered virus from the cDNA clone replicated poorly in baby hamster kidney (BHK-21) cells and in suckling mice brain. Following serial passage in BHK-21 cells, adaptive mutations within the NS2B and NS4A proteins were recovered in the passaged viruses leading to viruses with a large-plaque phenotype. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that the adaptive mutations restored replication to different degrees, and the restoration efficiencies were in the order: NS2B-T102M

This study was aimed to investigate the significance of interphase fluorescence in situ hybridization (FISH) in detecting +12, del (13q14), p53 and atm gene deletion in chronic lymphocytic leukemia (CLL). FISH and a panel of probes (CEP 12, LSI D13S319, LSI p53, LSI atm) were used to detect molecular cytogenetic abnormalities in 30 patients with CLL. Cytogenetic aberrations and their relation with some other prognostic factors (peripheral lymphocyte count, Binet stage, LDH level, ZAP-70 and so on) were analyzed. The results indicated that out of the 30 CLL patients, molecular cytogenetic aberrations were found in 19 (63.3%) cases and 7 (23.3%) patients showed more than two kinds of abnormalities. The most frequent abnormality detected was del (13q14) (43.3%), followed by trisomy of chromosome 12 (23.3%), del (atm) (13.3%) and del (p53) (10.0%). There were no significant differences between molecular cytogenetic aberrations and sex, age, Binet stage, peripheral lymphocyte count, or the serum levels of lactate dehydrogenase (LDH), beta(2)-microglobulin (beta(2)-MG), or ZAP-70. The incidence of atm gene deletion was higher in the group of CD38 high expression than that in the group of low expression (p = 0.035). It is concluded that FISH is a rapid and sensitive technique in analysing molecular cytogenetic abnormalities, but its prognostic significance in CLL needs to further investigate.

Porcine circovirus type 2(PCV2) recently emerged as an important infectious pathogen for pigs in the world. Unfortunately, there is no efficient method to deal with PCV2 infection until now. In this study, chimeric porcine circovirus molecular clone (pSK2PCV1-2) was constructed by cloning capsid gene of PCV2 into the backbone of PCV1. PK-15 cells was transfected with pSK2PCV1-2 and then cultivated in plate for five passages. mRNA of PCV1 ORF1 and PCV2 ORF2 were detected in the fifth passage, but mRNA of PCV1 ORF2 and PCV2 ORF1 were not detected in the cells. On the other hand, capsid protein of PCV2 was also detected in the nucleolus of transfected cells by IIF. This study indicated that pSK2PCV1-2 could form infectious virus in transfected cells. It will provide base for further study on biological characteristic of chimeric porcine circovirus.

ORF1 and ORF2 gene of porcine circovirus type 2 were cloned by PCR with the specific primers designed according to genome of PCV2 (AY035820). Following extraction and digestion, PCR products were subsequently inserted into universal transfer vector plECMV (deleted partial gE and gI of pseudorabies virus) to generate recombinant transfer plasmid pIEORF1-ORF2. The genomic DNA of PRV TK-/gE- /LacZ+ strain and pIEORF1-ORF2 were co-transfected into IBRS-2 cells with lipofectin, and recombinant virus TK- /gE- /gI- /ORF1-ORF2+ was selected by PCR with ORF1 gene and ORF2 gene primers respectively. The recombinant virus was analyzed with Southern blotting and Western blotting. The results indicated that ORF1 and ORF2 gene of PCV2 had been inserted into the genome of TK- /gE- /LacZ+ strain and the expressed ORF1-ORF2 fusion protein could react with PCV2 positive sera. Result of virus titers detection showed the insertion of ORF1 and ORF2 gene did not influence propagation of recombinant virus.