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Cheng-Jun Chen,1 Zhi-Xia Zhao,1 Jian-Cheng Wang,1 En-Yu Zhao,1 Ling-Yan Gao,1 Shu-Feng Zhou,2 Xiao-Yan Liu,1 Wan-Liang Lu,1 Qiang Zhang11State Key Laboratory of Natural and Biomimetic Drugs, Department of Pharmaceutics, School of Pharmaceutical Science, Peking University, Beijing, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USAAbstract: In this study, a novel redox-responsive hyperbranched poly(amido amine) (named PCD) was synthesized and used as a cationic polymer to form a ternary complex with small interfering RNA (siRNA) and hyaluronic acid (HA) for siRNA delivery. Here, it is hypothesized that different mixing orders result in different assembly structures, which may affect the siRNA delivery efficiency. To investigate the effects of mixing orders on siRNA delivery efficiency in two human breast cancer cell lines, three ternary complexes with different mixing orders of siRNA/PCD/HA were prepared and characterized: mixing order I (initially prepared siRNA/PCD binary complex further coated by negatively charged HA), mixing order II (initially prepared HA/PCD binary complex further incubated with siRNA), and mixing order III (initially prepared siRNA/HA mixture further electrostatically compacted by positively charged PCD). With an optimized siRNA/PCD/HA charge ratio of 1/20/16, the particle sizes and zeta potentials of these ternary complexes were 124.8 nm and 27.3 mV (mixing order I), 147.5 nm and 29.9 mV (mixing order II), and 128.8 nm and 19.4 mV (mixing order III). Also, the effects on stability, cellular uptake, and gene silencing efficiency of siRNA formulated in ternary complexes with different mixing orders were investigated. The results showed that mixing orders I and III displayed better siRNA transfection and protection than mixing order II in human breast cancer MCF-7 and MDA-MB-231 cells. More interesting, at the siRNA/PCD/HA charge ratio of 1/20/16, the gene silencing effects on vascular endothelial growth factor expression in MDA-MB-231 cells were as follows: mixing order III > mixing order I > mixing order II. Based on these results, a likely explanation for the difference in functionality dependent on mixing orders is the formation of different assembly structures. These results may help future optimization of siRNA ternary complexes for achieving better delivery efficiencies, especially for target-specific siRNA delivery to cells with HA receptor overexpression.Keywords: small interfering RNA, poly(amido amine)s, siRNA ternary complex, mixing order, hyaluronic acid, siRNA delivery

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) in rice, a serious bacterial disease of rice in Asia and parts of Africa. The virulence mechanisms of Xoc are not entirely clear and control measures for BLS are poorly developed. The solo LuxR proteins are widespread and shown to be involved in virulence in some plant associated bacteria (PAB). Here, we have cloned and characterized a PAB LuxR solo from Xoc, named as XocR. Mutation of xocR almost completely impaired the virulence ability of Xoc on host rice, but did not alter the ability to trigger HR (hypersensitive response, a programmed cell death) on non-host (plant) tobacco, suggesting the diversity of function of xocR in host and non-host plants. We also provide evidence to show that xocR is involved in the regulation of growth-independent cell motility in response to a yet-to-be-identified rice signal, as mutation of xocR impaired cell swimming motility of wild-type Rs105 in the presence but not absence of rice macerate. We further found that xocR regulated the transcription of two characterized virulence-associated genes (recN and trpE) in the presence of rice macerate. The promoter regions of recN and trpE possessed a potential binding motif (an imperfect pip box-like element) of XocR, raising the possibility that XocR might directly bind the promoter regions of these two genes to regulate their transcriptional activity. Our studies add a new member of PAB LuxR solos and also provide new insights into the role of PAB LuxR solo in the virulence of Xanthomonas species.

The cost of next-generation sequencing is now approaching that of early GWAS panels, but is still out of reach for large epidemiologic studies and the millions of rare variants expected poses challenges for distinguishing causal from non-causal variants. We review two types of designs for sequencing studies: two-phase designs for targeted follow-up of genomewide association studies using unrelated individuals; and family-based designs exploiting co-segregation for prioritizing variants and genes.Two-phase designs subsample subjects for sequencing from a larger case-control study jointly on the basis of their disease and carrier status; the discovered variants are then tested for association in the parent study. The analysis combines the full sequence data from the substudy with the more limited SNP data from the main study. We discuss various methods for selecting this subset of variants and describe the expected yield of true positive associations in the context of an on-going study of second breast cancers following radiotherapy.While the sharing of variants within families means that family-based designs are less efficient for discovery than sequencing unrelated individuals, the ability to exploit co-segregation of variants with disease within families helps distinguish causal from non-causal ones. Furthermore, by enriching for family history, the yield of causal variants can be improved and use of identity-by-descent information improves imputation of genotypes for other family members. We compare the relative efficiency of these designs with those using unrelated individuals for discovering and prioritizing variants or genes for testing association in larger studies. While associations can be tested with single variants, power is low for rare ones. Recent generalizations of burden or kernel tests for gene-level associations to family-based data are appealing. These approaches are illustrated in the context of a family-based study of colorectal cancer.

Early kinetics of circulating tumor DNA (ctDNA) in plasma predict response to pembrolizumab but typically requires sequencing of matched tumor tissue or fixed gene panels. We analyzed genome-wide methylation and fragment-length profiles using cell-free methylated DNA immunoprecipitation and sequencing (cfMeDIP-seq) in 204 plasma samples from 87 patients before and during treatment with pembrolizumab from a pan-cancer phase II investigator-initiated trial (INSPIRE). We trained a pan-cancer methylation signature using independent methylation array data from The Cancer Genome Atlas to quantify cancer-specific methylation (CSM) and fragment-length score (FLS) for each sample. CSM and FLS are strongly correlated with tumor-informed ctDNA levels. Early kinetics of CSM predict overall survival and progression-free survival, independently of tumor type, PD-L1, and tumor mutation burden. Early kinetics of FLS are associated with overall survival independently of CSM. Our tumor-naïve mutation-agnostic ctDNA approach integrating methylomics and fragmentomics could predict outcomes in patients treated with pembrolizumab., Significance: Analysis of methylation and fragment length in plasma using cfMeDIP-seq provides a tumor-naive approach to measure ctDNA with results comparable with a tumor-informed bespoke ctDNA. Early kinetics within the first weeks of treatment in methylation and fragment quantity can predict outcomes with pembrolizumab in patients with various advanced solid tumors. This article is featured in Selected Articles from This Issue, p. 897., (©2024 The Authors; Published by the American Association for Cancer Research.)

People with Li-Fraumeni syndrome (LFS) harbor a germline pathogenic variant in the TP53 tumor suppressor gene, face a near 100% lifetime risk of cancer, and routinely undergo intensive surveillance protocols. Liquid biopsy has become an attractive tool for a range of clinical applications, including early cancer detection. Here, we provide a proof-of-principle for a multimodal liquid biopsy assay that integrates a targeted gene panel, shallow whole-genome, and cell-free methylated DNA immunoprecipitation sequencing for the early detection of cancer in a longitudinal cohort of 89 LFS patients. Multimodal analysis increased our detection rate in patients with an active cancer diagnosis over uni-modal analysis and was able to detect cancer-associated signal(s) in carriers prior to diagnosis with conventional screening (positive predictive value = 67.6%, negative predictive value = 96.5%). Although adoption of liquid biopsy into current surveillance will require further clinical validation, this study provides a framework for individuals with LFS., Significance: By utilizing an integrated cell-free DNA approach, liquid biopsy shows earlier detection of cancer in patients with LFS compared with current clinical surveillance methods such as imaging. Liquid biopsy provides improved accessibility and sensitivity, complementing current clinical surveillance methods to provide better care for these patients. See related commentary by Latham et al., p. 23. This article is featured in Selected Articles from This Issue, p. 5., (©2023 The Authors; Published by the American Association for Cancer Research.)

Summary: Cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) has emerged as a promising liquid biopsy technology to detect cancers and monitor treatments. While several bioinformatics tools for DNA methylation analysis have been adapted for cfMeDIP-seq data, an end-to-end pipeline and quality control framework specifically for this data type is still lacking. Here, we present the MEDIPIPE, which provides a one-stop solution for cfMeDIP-seq data quality control, methylation quantification, and sample aggregation. The major advantages of MEDIPIPE are: (i) ease of implementation and reproducibility with Snakemake containerized execution environments that will be automatically deployed via Conda; (ii) flexibility to handle different experimental settings with a single configuration file; and (iii) computationally efficiency for large-scale cfMeDIP-seq profiling data analysis and aggregation., Availability and Implementation: This pipeline is an open-source software under the MIT license and it is freely available at https://github.com/pughlab/MEDIPIPE., (© The Author(s) 2023. Published by Oxford University Press.)

Uveal melanomas are rare tumors arising from melanocytes that reside in the eye. Despite surgical or radiation treatment, approximately 50% of patients with uveal melanoma will progress to metastatic disease, most often to the liver. Cell-free DNA (cfDNA) sequencing is a promising technology due to the minimally invasive sample collection and ability to infer multiple aspects of tumor response. We analyzed 46 serial cfDNA samples from 11 patients with uveal melanoma over a 1-year period following enucleation or brachytherapy ( n = ∼4/patient) using targeted panel, shallow whole genome, and cell-free methylated DNA immunoprecipitation sequencing. We found detection of relapse was highly variable using independent analyses ( P = 0.06-0.46), whereas a logistic regression model integrating all cfDNA profiles significantly improved relapse detection ( P = 0.02), with greatest power derived from fragmentomic profiles. This work provides support for the use of integrated analyses to improve the sensitivity of circulating tumor DNA detection using multi-modal cfDNA sequencing., Significance: Here, we demonstrate integrated, longitudinal cfDNA sequencing using multi-omic approaches is more effective than unimodal analysis. This approach supports the use of frequent blood testing using comprehensive genomic, fragmentomic, and epigenomic techniques., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Objectives: To study the association of methylenetetrahydrofolate reductase ( MTHFR ) gene polymorphism with susceptibility to bronchial asthma and glucocorticoid (GC) efficacy in children., Methods: A total of 173 children with bronchial asthma who were hospitalized between June 2018 and December 2020 were selected as the observation group. The children received aerosol inhalation of GC for three consecutive months. A total of 178 healthy children who underwent physical examination during the same period were selected as the control group. PCR was used to detect the genotypes of the MTHFR C677T for the two groups. The differences in genotype distribution between the two groups were analyzed. Children with different genotypes in the observation group were compared in terms of immunoglobulin E (IgE), interleukin-8 (IL-8), leukotriene B4 (LTB4), lung function, and clinical outcome before and after treatment., Results: TT genotype and T allele were significantly more frequent in the observation group than in the control group ( P <0.001). TT/CT genotypes and T allele were independent risk factors for bronchial asthma ( OR =6.615 and 7.055 respectively; P <0.001). After GC treatment, the children with CC, CT or TT genotypes experienced significantly decreased levels of IgE, IL-8, and LTB4 and significantly increased forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), and FEV1/FVC ratio ( P <0.001). The children with TT genotype showed significantly lower levels of IL-8 and LTB4 than those with CC genotype, a significantly lower level of LTB4 than those with CT genotype, significantly higher FVC than those with CT genotype, and a significantly higher FEV1/FVC ratio than those with CC genotype ( P <0.05). The children with TT genotype had better GC efficacy compared with those with CC genotype ( P <0.05). TT genotype was an independent factor for good GC efficacy ( OR =2.111, P =0.018)., Conclusions: MTHFR gene polymorphism is associated with asthma susceptibility and GC efficacy in children. Children carrying TT/CT genotypes have a higher risk of developing asthma, and those with TT genotype are more sensitive to GC treatment.

Reestablishing an appropriate balance between T effector cells (Teff) and Tregs is essential for correcting autoimmunity. Multiple sclerosis (MS) is an immune-mediated chronic CNS disease characterized by neuroinflammation, demyelination, and neuronal degeneration, in which the Teff:Treg balance is skewed toward pathogenic Teffs Th1 and Th17 cells. STAT3 is a key regulator of Teff:Treg balance. Using the structure-based design, we have developed a potentially novel small-molecule prodrug LLL12b that specifically inhibits STAT3 and suppresses Th17 differentiation and expansion. Moreover, LLL12b regulates the fate decision between Th17 and Tregs in an inflammatory environment, shifting Th17:Treg balance toward Tregs and favoring the resolution of inflammation. Therapeutic administration of LLL12b after disease onset significantly suppresses disease progression in adoptively transferred, chronic, and relapsing-remitting experimental autoimmune encephalomyelitis. Disease relapses were also significantly suppressed by LLL12b given during the remission phase. Additionally, LLL12b shifts Th17:Treg balance of CD4+ T cells from MS patients toward Tregs and increases Teff sensitivity to Treg-mediated suppression. These data suggest that selective inhibition of STAT3 by the small molecule LLL12b recalibrates the effector and regulatory arms of CD4+ T responses, representing a potentially clinically translatable therapeutic strategy for MS.

Intervertebral disc degeneration (IDD) remains a leading cause of adult disability. High mobility group box l (HMGB1) is a nuclear DNA-binding protein and acts as a central mediator of inflammation. The purpose of this study was to investigate the effect of HMGB1 in IDD. In our study, IDD intervertebral disc tissues were collected and nucleus pulposus cells (NPCs) were primarily cultured. The HMGB1 expression and the effect of HMGB1 on NPCs and extracellular matrix and autophagy were all evaluated. Results showed that HMGB1 was markedly overexpressed in IDD ( P <0.05), and upregulated expression of HMGB1 can inhibit NPC proliferation and promote NPC apoptosis ( P <0.05), promote extracellular matrix degradation, and activate cell autophagy ( P <0.05). Therefore, we concluded that HMGB1 was up-regulated in IDD and HMGB1-induced autophagy can promotes extracellular matrix degradation and thus lead to intervertebral disc degeneration. In brief, HMGB1 may serve as a novel diagnostic biomarker and therapeutic target for IDD., Competing Interests: None., (IJCEP Copyright © 2020.)

Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, "Circular RNA hsa&#95;circ&#95;0011946 promotes cell growth, migration, and invasion of oral squamous cell carcinoma by upregulating PCNA, by Y. Meng, E.-Y. Zhao, Y. Zhou, D.-X. Qiang, S. Wang, L. Shi, L.-Y. Jiang, L.-J. Bi, published in Eur Rev Med Pharmacol Sci 2020; 24 (3): 1226-1232-DOI: 10.26355/eurrev&#95;202002&#95;20175-PMID: 32096152" has been withdrawn. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/20175.

Advanced and metastatic tumors with complex treatment histories drive cancer mortality. Here we describe the POG570 cohort, a comprehensive whole-genome, transcriptome and clinical dataset, amenable for exploration of the impacts of therapies on genomic landscapes. Previous exposure to DNA-damaging chemotherapies and mutations affecting DNA repair genes, including POLQ and genes encoding Polζ, were associated with genome-wide, therapy-induced mutagenesis. Exposure to platinum therapies coincided with signatures SBS31 and DSB5 and, when combined with DNA synthesis inhibitors, signature SBS17b. Alterations in ESR1, EGFR, CTNNB1, FGFR1, VEGFA and DPYD were consistent with drug resistance and sensitivity. Recurrent noncoding events were found in regulatory region hotspots of genes including TERT, PLEKHS1, AP2A1 and ADGRG6. Mutation burden and immune signatures corresponded with overall survival and response to immunotherapy. Our data offer a rich resource for investigation of advanced cancers and interpretation of whole-genome and transcriptome sequencing in the context of a cancer clinic., (© 2020. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Purpose: This study investigated therapeutic potential of integrated genome and transcriptome profiling of metastatic sarcoma, a rare but extremely heterogeneous group of aggressive mesenchymal malignancies with few systemic therapeutic options., Methods: Forty-three adult patients with advanced or metastatic non-GI stromal tumor sarcomas of various histology subtypes who were enrolled in the Personalized OncoGenomics program at BC Cancer were included in this study. Fresh tumor tissues along with blood samples underwent whole-genome and transcriptome sequencing., Results: The most frequent genomic alterations in this cohort are large-scale structural variation and somatic copy number variation. Outlier RNA expression as well as somatic copy number variations, structural variations, and small mutations together suggest the presence of one or more potential therapeutic targets in the majority of patients in our cohort. Point mutations or deletions in known targetable cancer genes are rare; for example, tuberous sclerosis complex 2 provides a rationale for targeting the mammalian target of rapamycin pathway, resulting in a few patients with exceptional clinical benefit from everolimus. In addition, we observed recurrent 17p11-12 amplifications, which seem to be a sarcoma-specific event. This may suggest that this region harbors an oncogene(s) that is significant for sarcoma tumorigenesis. Furthermore, some sarcoma tumors carrying a distinct mutational signature suggestive of homologous recombination deficiency seem to demonstrate sensitivity to double-strand DNA-damaging agents., Conclusion: Integrated large-scale genomic analysis may provide insights into potential therapeutic targets as well as novel biologic features of metastatic sarcomas that could fuel future experimental and clinical research and help design biomarker-driven basket clinical trials for novel therapeutic strategies.

Glioblastoma multiforme (GBM) is the most deadly brain tumor, and currently lacks effective treatment options. Brain tumor-initiating cells (BTICs) and orthotopic xenografts are widely used in investigating GBM biology and new therapies for this aggressive disease. However, the genomic characteristics and molecular resemblance of these models to GBM tumors remain undetermined. We used massively parallel sequencing technology to decode the genomes and transcriptomes of BTICs and xenografts and their matched tumors in order to delineate the potential impacts of the distinct growth environments. Using data generated from whole-genome sequencing of 201 samples and RNA sequencing of 118 samples, we show that BTICs and xenografts resemble their parental tumor at the genomic level but differ at the mRNA expression and epigenomic levels, likely due to the different growth environment for each sample type. These findings suggest that a comprehensive genomic understanding of in vitro and in vivo GBM model systems is crucial for interpreting data from drug screens, and can help control for biases introduced by cell-culture conditions and the microenvironment in mouse models. We also found that lack of MGMT expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment., Competing Interests: The authors declare no conflict of interest.

Pancreatic neuroendocrine neoplasms (PanNENs) represent a minority of pancreatic neoplasms that exhibit variability in prognosis. Ongoing mutational analyses of PanNENs have found recurrent abnormalities in chromatin remodeling genes (e.g., DAXX and ATRX ), and mTOR pathway genes (e.g., TSC2 , PTEN PIK3CA , and MEN1 ), some of which have relevance to patients with related familial syndromes. Most recently, grade 3 PanNENs have been divided into two groups based on differentiation, creating a new group of well-differentiated grade 3 neuroendocrine tumors (PanNETs) that have had a limited whole-genome level characterization to date. In a patient with a metastatic well-differentiated grade 3 PanNET, our study utilized whole-genome sequencing of liver metastases for the comparative analysis and detection of single-nucleotide variants, insertions and deletions, structural variants, and copy-number variants, with their biologic relevance confirmed by RNA sequencing. We found that this tumor most notably exhibited a TSC1 -disrupting fusion, showed a novel CHD7-BEND2 fusion, and lacked any somatic variants in ATRX , DAXX , and MEN1 ., (© 2019 Williamson et al.; Published by Cold Spring Harbor Laboratory Press.)

Tumors from individuals with cancer are frequently genetically profiled for information about the driving forces behind the disease. We present the CancerMine resource, a text-mined and routinely updated database of drivers, oncogenes and tumor suppressors in different types of cancer. All data are available online ( http://bionlp.bcgsc.ca/cancermine ) and downloadable under a Creative Commons Zero license for ease of use.

Multiple sclerosis (MS) is the leading cause of non-traumatic neurological disability in the United States in young adults, but current treatments are only partially effective, making it necessary to develop new, innovative therapeutic strategies. Myelin-specific interleukin (IL)-17-producing T helper type 17 (Th17) cells are a major subset of CD4 T effector cells (T eff ) that play a critical role in mediating the development and progression of MS and its mouse model, experimental autoimmune encephalomyelitis (EAE), while regulatory T cells (T reg ) CD4 T cells are beneficial for suppressing disease. The IL-6/signal transducer and activator of transcription 3 (STAT-3) signaling pathway is a key regulator of Th17 and T reg cells by promoting Th17 development and suppressing T reg development. Here we show that three novel small molecule IL-6 inhibitors, madindoline-5 (MDL-5), MDL-16 and MDL-101, significantly suppress IL-17 production in myelin-specific CD4 T cells in a dose-dependent manner in vitro. MDL-101 showed superior potency in suppressing IL-17 production compared to MDL-5 and MDL-16. Treatment of myelin-specific CD4 T cells with MDL-101 in vitro reduced their encephalitogenic potential following their subsequent adoptive transfer. Furthermore, MDL-101 significantly suppressed proliferation and IL-17 production of anti-CD3-activated effector/memory CD45RO + CD4 + human CD4 T cells and promoted human T reg development. Together, these data demonstrate that these novel small molecule IL-6 inhibitors have the potential to shift the T eff  : T reg balance, which may provide a novel therapeutic strategy for ameliorating disease progression in MS., (© 2019 British Society for Immunology.)

We report a case of early-onset pancreatic ductal adenocarcinoma in a patient harboring biallelic MUTYH germline mutations, whose tumor featured somatic mutational signatures consistent with defective MUTYH -mediated base excision repair and the associated driver KRAS transversion mutation p.Gly12Cys. Analysis of an additional 730 advanced cancer cases ( N = 731) was undertaken to determine whether the mutational signatures were also present in tumors from germline MUTYH heterozygote carriers or if instead the signatures were only seen in those with biallelic loss of function. We identified two patients with breast cancer each carrying a pathogenic germline MUTYH variant with a somatic MUTYH copy loss leading to the germline variant being homozygous in the tumor and demonstrating the same somatic signatures. Our results suggest that monoallelic inactivation of MUTYH is not sufficient for C:G>A:T transversion signatures previously linked to MUTYH deficiency to arise ( N = 9), but that biallelic complete loss of MUTYH function can cause such signatures to arise even in tumors not classically seen in MUTYH -associated polyposis ( N = 3). Although defective MUTYH is not the only determinant of these signatures, MUTYH germline variants may be present in a subset of patients with tumors demonstrating elevated somatic signatures possibly suggestive of MUTYH deficiency (e.g., COSMIC Signature 18, SigProfiler SBS18/SBS36, SignatureAnalyzer SBS18/SBS36)., (© 2019 Thibodeau et al.; Published by Cold Spring Harbor Laboratory Press.)

Although generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in more than 90% of cases in malaria-endemic regions, and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumor EBV status or geographic origin. Through an integrative analysis of whole-genome and transcriptome data, we show a striking genome-wide increase in aberrant somatic hypermutation in EBV-positive tumors, supporting a link between EBV and activation-induced cytidine deaminase (AICDA) activity. In addition to identifying novel candidate BL genes such as SIN3A , USP7 , and CHD8 , we demonstrate that EBV-positive tumors had significantly fewer driver mutations, especially among genes with roles in apoptosis. We also found immunoglobulin variable region genes that were disproportionally used to encode clonal B-cell receptors (BCRs) in the tumors. These include IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Our results suggest that tumor EBV status defines a specific BL phenotype irrespective of geographic origin, with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply rational use of DNA-damaging chemotherapy in some patients with BL and targeted agents such as the CDK4/6 inhibitor palbociclib in others, whereas the importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk, and Src family kinases among these patients., (© 2019 by The American Society of Hematology.)

Genome sequencing of cancer has fundamentally advanced our understanding of the underlying biology of this disease, and more recently has provided approaches to characterize and monitor tumors in the clinic, guiding and evaluating treatment. Although cancer research is relying more on whole-genome characterization, the clinical application of genomics is largely limited to targeted sequencing approaches, tailored to capture specific clinically relevant biomarkers. However, as sequencing costs reduce, and the tools to effectively analyze complex and large-scale data improve, the ability to effectively characterize whole genomes at scale in a clinically relevant time frame is now being piloted. This ability effectively blurs the line between clinical cancer research and the clinical management of the disease. This leads to a new paradigm in cancer management in which real-time analysis of an individual's disease can have a rapid and lasting impact on our understanding of how clinical practices need to change to exploit novel therapeutic rationales. In this article, we will discuss how whole-genome sequencing (WGS), often combined with transcriptome analysis, has been used to understand cancer and how this approach is uniquely positioned to provide a comprehensive view of an evolving disease in response to therapy., (Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved.)

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3' UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.

Synaptic neurodegeneration is thought to be an early event initiated by soluble β-amyloid (Aβ) aggregates that closely correlates with cognitive decline in Alzheimer disease (AD). Apolipoprotein ε4 (APOE4) is the most common genetic risk factor for both familial AD (FAD) and sporadic AD; it accelerates Aβ aggregation and selectively impairs glutamate receptor function and synaptic plasticity. However, its molecular mechanisms remain elusive and these synaptic deficits are difficult to monitor. AD- and APOE4-dependent plasma biomarkers have been proposed, but synapse-related plasma biomarkers are lacking. We evaluated neuronal pentraxin 1 (NP1), a potential CNS-derived plasma biomarker of excitatory synaptic pathology. NP1 is preferentially expressed in brain and involved in glutamate receptor internalization. NP1 is secreted presynaptically induced by Aβ oligomers, and implicated in excitatory synaptic and mitochondrial deficits. Levels of NP1 and its fragments were increased in a correlated fashion in both brain and plasma of 7-8 month-old E4FAD mice relative to E3FAD mice. NP1 was also found in exosome preparations and reduced by dietary DHA supplementation. Plasma NP1 was higher in E4FAD+ (APOE4 +/+ /FAD +/- ) relative to E4FAD- (non-carrier; APOE4 +/+ /FAD -/- ) mice, suggesting NP1 is modulated by Aβ expression. Finally, relative to normal elderly, plasma NP1 was also elevated in patients with mild cognitive impairment (MCI) and elevated further in the subset who progressed to early-stage AD. In those patients, there was a trend towards increased NP1 levels in APOE4 carriers relative to non-carriers. These findings indicate that NP1 may represent a potential synapse-derived plasma biomarker relevant to early alterations in excitatory synapses in MCI and early-stage AD., (Copyright © 2018. Published by Elsevier Inc.)

Pancreatic neuroendocrine tumors (PNETs) are a genomically and clinically heterogeneous group of pancreatic neoplasms often diagnosed with distant metastases. Recurrent somatic mutations, chromosomal aberrations, and gene expression signatures in PNETs have been described, but the clinical significance of these molecular changes is still poorly understood, and the clinical outcomes of PNET patients remain highly variable. To help identify the molecular factors that contribute to PNET progression and metastasis, and as part of an ongoing clinical trial at the BC Cancer Agency (clinicaltrials.gov ID: NCT02155621), the genomic and transcriptomic profiles of liver metastases from five patients (four PNETs and one neuroendocrine carcinoma) were analyzed. In four of the five cases, we identified biallelic loss of MEN1 and DAXX as well as recurrent regions with loss of heterozygosity. Several novel findings were observed, including focal amplification of MYCN concomitant with loss of APC and TP53 in one sample with wild-type MEN1 and DAXX Transcriptome analyses revealed up-regulation of MYCN target genes in this sample, confirming a MYCN -driven gene expression signature. We also identified a germline NTHL1 fusion event in one sample that resulted in a striking C>T mutation signature profile not previously reported in PNETs. These varying molecular alterations suggest different cellular pathways may contribute to PNET progression, consistent with the heterogeneous clinical nature of this disease. Furthermore, genomic profiles appeared to correlate well with treatment response, lending support to the role of prospective genotyping efforts to guide therapy in PNETs., (© 2018 Wong et al.; Published by Cold Spring Harbor Laboratory Press.)

Purpose: Recent studies have identified mutation signatures of homologous recombination deficiency (HRD) in over 20% of breast cancers, as well as pancreatic, ovarian, and gastric cancers. There is an urgent need to understand the clinical implications of HRD signatures. Whereas BRCA1/2 mutations confer sensitivity to platinum-based chemotherapies, it is not yet clear whether mutation signatures can independently predict platinum response. Experimental Design: In this observational study, we sequenced tumor whole genomes (100× depth) and matched normals (60×) of 93 advanced-stage breast cancers (33 platinum-treated). We computed a published metric called HRDetect, independently trained to predict BRCA1/2 status, and assessed its capacity to predict outcomes on platinum-based chemotherapies. Clinical endpoints were overall survival (OS), total duration on platinum-based therapy (TDT), and radiographic evidence of clinical improvement (CI). Results: HRDetect predicted BRCA1/2 status with an area under the curve (AUC) of 0.94 and optimal threshold of 0.7. Elevated HRDetect was also significantly associated with CI on platinum-based therapy (AUC = 0.89; P = 0.006) with the same optimal threshold, even after adjusting for BRCA1/2 mutation status and treatment timing. HRDetect scores over 0.7 were associated with a 3-month extended median TDT ( P = 0.0003) and 1.3-year extended median OS ( P = 0.04). Conclusions: Our findings not only independently validate HRDetect, but also provide the first evidence of its association with platinum response in advanced breast cancer. We demonstrate that HRD mutation signatures may offer clinically relevant information independently of BRCA1/2 mutation status and hope this work will guide the development of clinical trials. Clin Cancer Res; 23(24); 7521-30. ©2017 AACR ., (©2017 American Association for Cancer Research.)

BRCA1 and BRCA2 germline mutation-associated breast cancers are known to be deficient in the process of homologous recombination and often respond favorably to drugs targeting this important DNA repair pathway. There is emerging evidence that a significant proportion of patients with BRCA1 / BRCA2 wild-type breast cancer are also deficient in homologous recombination, and it is hypothesized that these patients may derive similar benefit from drugs targeting this pathway. Current research has focused on the development of a companion diagnostic to identify these sporadic BRCA-like tumors. This review outlines the various approaches that researchers have taken to predict homologous recombination deficiency as part of correlative biomarker work in various studies and clinical trials in breast cancer. As some of these tests of homologous recombination deficiency move closer to clinical use, understanding the approach and limitations of each is of relevance to clinicians who treat patients with breast cancer.

The effectiveness of the rehabilitative benefits of physical exercise appears to be contingent upon when the exercise is initiated after stroke. The present study assessed the hypothesis that very early exercise increases the extent of apoptotic cell death via increased expression of proapoptotic proteins in a rat stroke model. Adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2 hr using an intraluminal filament and assigned to four nonexercise and three exercise groups. Exercise on a Rota-Rod was initiated for 30 min at 6 hr (considered very early), at 24 hr (early), and at 3 days (relatively late) after reperfusion. At 24 hr after exercise, apoptotic cell death was determined. At 3 and 24 hr after exercise, the expression of pro- and antiapoptotic proteins was evaluated through Western blotting. As expected, ischemic stroke significantly increased the levels of apoptotic cell death. Compared with the stroke group without exercise, apoptotic cell death was further increased (P < 0.05) at 6 hr but not at 24 hr or 3 days with exercise. This exacerbated cell injury was associated with increased expression of proapoptotic proteins (BAX and caspase-3). The expression of Bcl-2, an antiapoptotic protein, was not affected by exercise. In ischemic stroke, apoptotic cell death was enhanced by very early exercise in association with increased expression of proapoptotic proteins. These results shed light on the time-sensitive effect of exercise in poststroke rehabilitation. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Introduction: Although physical exercise has emerged as a potential therapeutic modality for functional deficits following ischemic stroke, the extent of this effect appears to be contingent upon the time of exercise initiation. In the present study, we assessed how exercise timing affected brain damage through hyperglycolysis-associated NADPH oxidase (NOX) activation., Methods: Using an intraluminal filament, adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2h and assigned to one non-exercise and three exercise groups. Exercise on Rota-rod was initiated for 30min at 6h (considered very early), at 24h (early), and at day 3 (relatively late) after reperfusion. Lactate production was measured 30min after exercise completion, and NOX activity and protein expression of NOX subunits (p47(phox), gp91(phox), p22(phox) and p67(phox)) and glucose transporter 1 and 3 (Glut-1 and -3) were measured at 3 and 24h after exercise. Apoptotic cell death was determined at 24h after exercise., Results: Lactate production and Glut-1 and Glut-3 expression were increased after very early exercise (6h), but not after late exercise (3 days), suggesting hyperglycolysis. NOX activity was increased with the initiation of exercise at 6h (P<0.05), but not 24h or 3 days, following stroke. Early (6 and 24h), but not late (3 days), post-stroke exercise was associated with increased (P<0.05) expression of the NOX protein subunit p47(phox), gp91(phox)and p67(phox). This may have led to the enhanced apoptosis observed after early exercise in ischemic rats., Conclusion: Hyperglycolysis and NOX activation was associated with an elevation in apoptotic cell death after very early exercise, and the detrimental effect of exercise on stroke recovery began to decrease when exercise was initiated 24h after reperfusion., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Congenital heart disease (CHD) is a complex illness with high rates of morbidity and mortality. In embryonic development, the heart is the first formed organ, which is strictly controlled by gene regulatory networks, including transcription factors, signaling pathways, epigenetic factors and microRNAs. Bone morphogenetic protein (BMP)-2 and -4 are essential in cardiogenesis as they can induce the expression of transcription factors, NKX2‑5 and GATA binding protein 4, which are important in the development of the heart. The inhibition of BMP‑2 and ‑4 inhibits the late expression of NKX2-5 and affects cardiac differentiation. The aim of the present study was to investigate whether BMP-2 and ‑4 variations may be associated with CHD in Chinese Han populations. The rs1049007, rs235768 and rs17563 single nucleotide polymorphisms (SNPs), which are genetic variations located within the translated region of the BMP-2 and -4, were evaluated in 230 patients with CHD from the Chinese Han population and 160 non-CHD control individuals. Statistical analyses were performed using the χ2 test, implemented using SPSS software (version 13.0). The Hardy-Weinberg equilibrium test was performed on the population using online Online Encyclopedia for Genetic Epidemiology studies software, and multiple-sequence alignments of the BMP proteins were performed using Vector NTI software. No statistically significant associations were identified between these genetic variations and the risk of CHD (rs1049007, P‑value=0.560; rs235768, P‑value=0.972; rs17563, P‑value=0.787). In addition, no correlation was found between the patients with CHD and the non‑CHD control individuals. Therefore, the rs1049007, rs235768 and rs17563 genetic variations of BMP-2 were not associated with CHD in the Chinese Han population.

Stroke is a leading cause of long-term disability and death in the United States. Currently, tissue plasminogen activator (tPA), is the only Food and Drug Administration-approved treatment for acute ischemic stroke. However, the use of tPA is restricted to a small subset of acute stroke patients due to its limited 3-h therapeutic time window. Given the limited therapeutic options at present and the multi-factorial progression of ischemic stroke, emphasis has been placed on the discovery and use of combination therapies aimed at various molecular targets contributing to ischemic cell death. Protein kinase C (PKC) and Akt (protein kinase B) are serine/threonine kinases that play a critical role in mediating ischemic-reperfusion injury and cellular growth and survival, respectively. The present review will examine the role of PKC and Akt in the cellular response to ischemic-reperfusion injury.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that activates at least five known G-protein-coupled receptors (GPCRs): LPA1-LPA5. The nervous system is a major locus for LPA1 expression. LPA has been shown to regulate neuronal proliferation, migration, and differentiation during central nervous system development as well as neuronal survival. Furthermore, deficient LPA signaling has been implicated in several neurological disorders including neuropathic pain and schizophrenia. Parkinson's disease (PD) is a neurodegenerative movement disorder that results from the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). The specific molecular pathways that lead to DA neuron degeneration, however, are poorly understood. The influence of LPA in the differentiation of mesenchymal stem cells (MSCs) into DA neurons in vitro and LPA1 expression in a 6-hydroxydopamine (6-OHDA) lesion model of PD in vivo were examined in the present study. LPA induced neuronal differentiation in 80.2 % of the MSC population. These MSCs developed characteristic neuronal morphology and expressed the neuronal marker, neuron-specific enolase (NSE), while expression of the glial marker, glial fibrillary acidic protein (GFAP), was absent. Moreover, 27.6 % of differentiated MSCs were positive for tyrosine hydroxylase (TH), a marker for DA neurons. In the 6-OHDA PD rat model, LPA1 expression in the substantia nigra was significantly reduced compared to control. These results suggest LPA signaling via activation of LPA1 may be necessary for DA neuron development and survival. Furthermore, reduced LPA/LPA1 signaling may be involved in DA neuron degeneration thus contributing to the pathogenesis of PD.

Objective: To investigate the effect of p65 gene inhibited by siRNA on neuronic differentiation in the marrow mesenchymal stem cells (MSCs)., Methods: The MSCs were transfected with Rn-p65-siRNA. Fasudil hydrochloride induced MSCs differentiating into neurons. The non-transfected group and negative control group (transfected with negative control siRNA marked by Cy3) were used as controls. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope at 24 h,48 h and 72 h after transfected with negative control siRNA. The viability of MSCs was detected by MTT at 24 h, 48 h and 72 h after transfected with Rn-p65-siRNA. The expressions of p65 mRNA and protein in MSCs were detected by RT-PCR and Western blot respectively. The expressions of p65 protein, NSE, MAP-2 and glial fibrillary acidic protein (GFAP) were detected by immunocytochemical method after transfection for 6 h., Results: The fluorescence of MSCs was mostly displayed after transfection of 72 hours and the efficiency of transfection was up to 83.3% ± 3.8%. Meanwhile, the p65 mRNA and p65 protein expressed by MSCs of transfected group were significantly decreased (P < 0.05); MTT displayed that the viability of MSCs was also significantly reduced (P < 0.05). The best efficiency of induction was observed in the transfected group. There were higher expressions of NSE and MAP-2 than the other group (P < 0.05)., Conclusion: The p65 gene inhibited by siRNA can promote the marrow mesenchymal stem cells to differentiate into neurons.

Background: Ascites, the most frequent complication of cirrhosis, is associated with poor prognosis and reduced quality of life. Recurrent hospital admissions are common and often unplanned, resulting in increased use of hospital services., Aims: To examine use of hospital services by patients with cirrhosis and ascites requiring paracentesis, and to investigate factors associated with early unplanned readmission., Methods: A retrospective review of the medical chart and clinical databases was performed for patients who underwent paracentesis between October 2011 and October 2012. Clinical parameters at index admission were compared between patients with and without early unplanned hospital readmissions., Results: The 41 patients requiring paracentesis had 127 hospital admissions, 1164 occupied bed days and 733 medical imaging services. Most admissions (80.3%) were for management of ascites, of which 41.2% were unplanned. Of those eligible, 69.7% were readmitted and 42.4% had an early unplanned readmission. Twelve patients died and nine developed spontaneous bacterial peritonitis. Of those eligible for readmission, more patients died (P = 0.008) and/or developed spontaneous bacterial peritonitis (P = 0.027) if they had an early unplanned readmission during the study period. Markers of liver disease, as well as haemoglobin (P = 0.029), haematocrit (P = 0.024) and previous heavy alcohol use (P = 0.021) at index admission, were associated with early unplanned readmission., Conclusion: Patients with cirrhosis and ascites comprise a small population who account for substantial use of hospital services. Markers of disease severity may identify patients at increased risk of early readmission. Alternative models of care should be considered to reduce unplanned hospital admissions, healthcare costs and pressure on emergency services., (© 2014 The Authors; Internal Medicine Journal © 2014 Royal Australasian College of Physicians.)

Background: Nodal/TGF-Lefty signaling pathway has important effects at early stages of differentiation of human embryonic stem cells in directing them to differentiate into different embryonic lineages. LEFTY, one of transforming growth factors in the Nodal/TGF-Lefty signaling pathway, plays an important role in the development of heart. The aim of this work was to find evidence on whether Lefty variations are associated with congenital heart diseases (CHD)., Methods: We sequenced the Lefty gene for 230 Chinese Han CHD patients and evaluated SNPs rs2295418, rs360057 and g.G169A, which are located within the translated regions of the genes. The statistical analyses were conducted using Chi-Square Tests as implemented in SPSS (version 13.0). The Hardy-Weinberg equilibrium test of the population was carried out using online software OEGE, and multiple-sequence alignments of LEFTY proteins were carried out using the Vector NTI software., Results: Two heterozygous variants in Lefty1 gene, g.G169A and g.A1035C, and one heterozygous variant in Lefty2 gene, g.C925A, were identified. Statistical analyses showed that the rs2295418 (g.C925A) variant in Lefty2 gene was obviously associated with the risk of CHD (P value = 0.016<0.05). The genotype frequency of rs360057 (g.A1035C) variant in Lefty1 gene was associated with the risk of CHD (P value = 0.007<0.05), but the allele frequency was not (P value = 0.317>0.05)., Conclusions: The SNP rs2295418 in the Lefty2 gene is associated with CHD in Chinese Han populations.

Background: Isolating specific cellular and tissue compartments from 3D image stacks for quantitative distribution analysis is crucial for understanding cellular and tissue physiology under normal and pathological conditions. Current approaches are limited because they are designed to map the distributions of synapses onto the dendrites of stained neurons and/or require specific proprietary software packages for their implementation., New Method: To overcome these obstacles, we developed algorithms to Grow and Shrink Volumes of Interest (GSVI) to isolate specific cellular and tissue compartments from 3D image stacks for quantitative analysis and incorporated these algorithms into a user-friendly computer program that is open source and downloadable at no cost., Results: The GSVI algorithm was used to isolate perivascular regions in the cortex of live animals and cell membrane regions of stained spinal motoneurons in histological sections. We tracked the real-time, intravital biodistribution of injected fluorophores with sub-cellular resolution from the vascular lumen to the perivascular and parenchymal space following a vascular microlesion, and mapped the precise distributions of membrane-associated KCC2 and gephyrin immunolabeling in dendritic and somatic regions of spinal motoneurons., Comparison With Existing Methods: Compared to existing approaches, the GSVI approach is specifically designed for isolating perivascular regions and membrane-associated regions for quantitative analysis, is user-friendly, and free., Conclusions: The GSVI algorithm is useful to quantify regional differences of stained biomarkers (e.g., cell membrane-associated channels) in relation to cell functions, and the effects of therapeutic strategies on the redistributions of biomolecules, drugs, and cells in diseased or injured tissues., (Copyright © 2014 Elsevier B.V. All rights reserved.)

DT104 emerged as a new branch of Salmonella typhimurium with resistance to multiple antimicrobials. To reveal some general genomic features of DT104 for clues of evolutionary events possibly associated with the emergence of this relatively new type of this pathogen, we mapped 11 independent DT104 strains and compared them with non-DT104 S. typhimurium strains. We found that all 11 DT104 strains contained three insertions absent in non-DT104 strains, i.e., the previously reported ST104, ST104B and ST64B. However, SGI-1, a genomic island known to be responsible for DT104 multidrug resistance, was not present in all DT104 strains examined in this study: one DT104 strain did not contain SGI-1 but carried a 144 kb plasmid, suggesting possible evolutionary relationships between the two DNA elements in the development of antimicrobial resistance.

Background: The RP-HPLC resolution of two triterpenic acid isomers was unstable., Objective: To separate the oleanolic acid (OA) and ursolic acid (UA) simply within RP-HPLC., Materials and Methods: The separation ability of five stationary phases was studied with the retention effect of their carbon loads. Also the resolution effects of mobile phase composition and different column temperatures were systematically investigated by using Drylab(®) (Rheodyne LLC.) after evaluating chromatograms automatically., Results: The best available resolution of two bioactive isomers was achieved (r = 3.4) via using PAH (polycyclic aromatic hydrocarbons) polymeric C18 bonded phase column. The chromatographic system was applied to the quantification in ten Chinese medicinal plants and the validation was carried out and the precision (RSD ≤1.34%), the linearity (r ≥ 0.9998) and the recovery (range from 92.1% to 102.6%) were acceptable., Conclusion: It is clear that the method was simple, rapid and reliable for the quantification of two compounds in new HPLC method within PAH polymeric C18.

In this study, a series of bioreducible poly(amidoamine)s grafting different percentages of cholesterol (rPAA-Ch14: 14%, rPAA-Ch29: 29%, rPAA-Ch57: 57% and rPAA-Ch87: 87%) was synthesized and used for siRNA delivery. These amphiphilic polymers were able to self-assemble into cationic nanoparticles in aqueous solution at low concentrations. The nanoparticle formation was evidenced via cryo-transmission electron microscope (Cryo-TEM) and dynamic light scattering analysis. The average hydrodynamic size of rPAA-Ch blank nanoparticles was about 80-160 nm with zeta potential of 50-60 mV. Also, the effects of different percentages of cholesterol grafted onto rPAA on physicochemical characteristics, in vitro cytotoxicity, cellular uptake, VEGF gene silencing efficacy and translocation mechanism of rPAA-Ch/siRNA complexes were investigated. The results showed that rPAA-Ch57 polymer was not only able to form stable nanocomplexes and possess high cell uptake, but also to exhibit the best in vitro VEGF gene silencing efficacy and the best in vivo tumor growth inhibition effect when it was formulated with VEGF-siRNA. Moreover, the observations of confocal laser scanning microscope (CLSM) and the study of cholesterol competitive inhibition demonstrated that endosomal/lysosomal escape and cytoplasmic dissociation of rPAA-Ch57/siRNA complexes were dependent on the "proton sponge effect" and disulfide cleavage, following internalization with cholesterol-related endocytosis pathway and subsequent transportion into endosomes/lysosomes. These findings indicated that the rPAA-Ch57 polymer should be a promising and potent carrier for siRNA delivery., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Due to the absence of safe and effective carriers for in vivo delivery, the applications of small interference RNA (siRNA) in clinic for therapeutic purposes have been limited. In this study, a biodegradable amphiphilic tri-block copolymer (mPEG(2000)-PLA(3000)-b-R(15)) composed of monomethoxy poly(ethylene glycol), poly(d,l-lactide) and polyarginine was synthesized and further self-assembled to cationic polymeric nanomicelles for in vivo siRNA delivery, with an average diameter of 54.30 ± 3.48 nm and a zeta potential of approximately 34.8 ± 1.77 mV. The chemical structures of the copolymers were well characterized by (1)H NMR spectroscopy and FT-IR spectra. In vitro cytotoxicity and hemolysis assays demonstrated that the polymeric nanomicelles showed greater cell viability and haemocompatibility than those of polyethyleneimine (PEI) or R(15) peptide. In vitro experiments demonstrated that EGFR targeted siRNA formulated in micelleplexes exhibited approximately 65% inhibition of EGFR expression on MCF-7 cells in a sequence-specific manner, which was comparable to Lipofectamine™ 2000. The results of intravenous administration showed Micelleplex/EGFR-siRNA significantly inhibited tumor growth in nude mice xenografted MCF-7 tumors, with a remarkable inhibition of EGFR expression. Furthermore, no positive activation of the innate immune responses and no significant body weight loss was observed during treatment suggested that this polymeric micelle delivery system is non-toxic. In conclusion, the present nanomicelles based on cationic mPEG(2000)-PLA(3000)-b-R(15) copolymer would be a safe and efficient nanocarrier for in vivo delivery of therapeutic siRNA., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Hypokalemic periodic paralysis (HypoPP) is an autosomal dominant disorder characterized by periodic attacks of muscle weakness associated with a decrease in the serum potassium level. Several mutations in the skeletal muscle calcium channel α-subunit gene CACNA1S have been documented to be causative for HypoPP, but mutations in other genes have also been implicated in HypoPP. To further reveal the genetic causes of HypoPP, we genotyped members of a five-generational Chinese family with HypoPP patients and identified a novel His916Gln mutation in all male HypoPP patients of the family. Clinical analysis demonstrated that the penetrance of the mutation was complete in male carriers, but we did not find evident clinical features in female carriers. This study expanded the spectrum of CACNA1S mutations associated with HypoPP and demonstrated a gender difference in the penetrance of the disease.

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.

Objective: To investigate the effect of notch signaling on differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons induced by fasudil hydrochloride., Methods: The experiments were divided into non-transfected group, transfected group (transfected with Rn-Notch1-siRNA), positive control group (transfected with Rn-MAPK-1 Control siRNA) and negative control group (transfected with negative control siRNA). Fasudil hydrochloride induced MSCs differentiating into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The expression of notch1 mRNA, Hes1 mRNA and MAPK1 mRNA in MSCs was detected by RT-PCR. The expression of Notch1 protein, NSE, neurofilament M (NF-M) and glial fibrillary acidic protein(GFAP)was detected by immunocytochemical method. The viability of MSCs was detected by MTT., Results: (1) The fluorescence of MSCs was mostly displayed after transfection for 72 h and the efficiency of transfection was up to 91.3% +/- 4.2%. Meanwhile, the notch1 mRNA and Hes1 mRNA expressed by MSCs of transfected group were significantly decreased (P < 0.05) and MTT displayed that the viability of MSCs was also significantly reduced (P < 0.05). (2) Fasudil hydrochloride could induce MSCs differentiate into neurons and the best efficiency of induction was observed in the transfected group. There was higher expression of NSE and neurofilament-M (NF-M) than the other groups (P < 0.05)., Conclusion: There may be notch1 signaling and Rho/Rho GTPase signaling synergy on differentiation of rat bone marrow stromal cell into neurons induced by fasudil hydrochloride and they jointly promote the differentiation of MSCs into neurons.

Objective: To study the changes of microRNA expression in cortex tissues in neonatal rats with hypoxic-ischemic brain damage (HIBD)and the possible roles of microRNA in the pathogenesis of HIBD. METHODS Rat HIBD model was prepared. The cortex tissues were obtained 14 days after the HIBD event. The microRNA expression profiles were measured using microRNA microarray. Expression of 9 microRNAs (miR-126,-26a,-674-5p,-21,-25,-290, miR-124,-125b-5p and microRNA-9a) was determined by quantitative real-time PCR., Results: he results of microRNA expression profiles indicated that 27 pieces of microRNA were up-regulated more than 2 folds and 60 pieces were down-regulated more than 2 folds compared with the normal control group. The results of the 9 microRNAs detected by quantitative real-time PCR were consistent with those detected by microRNA microarray., Conclusions: HIBD rats have significant changes in microRNA expression, suggesting that microRNA expression may play important roles in the pathogenesis of HIBD.

Thrombin-like enzyme (TLE) plays a significant role in vessel injury hemostasis. A novel snake venom TLE (Agacutin) was purified from Agkistrodon Acutus snake venom. Structural analysis indicated that Agacutin is a heterodimer that has a MW of 29,402 Da, a pI value of 5.39, and optimum activity at 35 degrees C and pH 7.5. The N-terminal 15 amino acid sequences of Agacutin are DSSGWSSYEGHEYYV (small subunit) and DCSSGWSSYEEHQYY (large subunit). In vitro studies indicated that the coagulation activity of Agacutin was activated by Ca(+2) or inhibited by phenylmethanesulfonyl fluoride, but not influenced by heparin or hirudin. The arginine esterase activity and fibrinogen hydrolysis result showed that Agacutin only cleaves alpha-subunit and releases fibrinopeptide A. In vivo studies indicated that Agacutin iv (0.01-0.05 U/kg) shortened 30.2-49% of the rabbit blood clotting time, or ip (0.5-2.0 U/kg) shortened 29.7-73.1% of the mouse tail bleeding time. Agacutin does not influence APTT, platelet or euglobulin clotting time, and activate Factor II or XIII. It converts fibrinogen into the soluble fibrin that accelerates hemostasis at wound.

Objective: To study the effects of Down syndrome cellular adhesion molecule (DSCAM) on differentiation of rat marrow mesenchymal stem cells (MSCs) into neurons in vitro., Methods: MSCs from Sprague-Dawley rats were induced into neurons by baicalin. The expression of DSCAM before and after induction was evaluated by immunocytochemical staining and Western blot assay. After knockdown of DSCAM by siRNA transfection, the differentiation rate of neurons derived from MSCs was measured., Results: Before induction, the expression of DSCAM was not detectable in MSCs. After bFGF preinduction for 24 hrs, DSCAM was slightly expressed in MSCs (1.71+/- 0.67%). The DSCAM expression increased 6 hrs after baicalin induction (15.79+/- 4.24%), reached a peak at 3 days (53.16+/- 5.94%) and then decreased gradually. The DSCAM expression 6 days after baicalin induction (28.99+/- 6.72%) was significantly lower than that at 3 days (P<0.01). However, after DSCAM-siRNA transfection, the DSCAM expression in MSCs was significantly reduced. MSCs did not express neuron-specific beta-III-tubulin before induction. After baicalin induction for 6 hrs, 3 days and 6 days, the expression of beta-III-tubulin was 1.40+/- 0.79%, 41.59+/- 3.17% and 59.11+/- 4.76% respectively. But the beta-III-tubulin expression significantly decreased 3 and 6 days after DSCAM-siRNA transfection (28.57+/- 2.91% and 43.90+/- 12.31% respectively)., Conclusions: DSCAM may play an important role in MSCs differentiation into neural cells.

Objective: To investigate the association of gonadotrophin (Gn) dose and ovarian response with the clinical outcome of in vitro fertilization and embryo transfer (IVF-ET)., Methods: Patients undergoing IVF-ET with Gn stimulation for no more than 15 days were enrolled in this study. The patients were divided into 3 groups, namely group A (390 cycles) with total Gn dose :3375 IU and retrieved oocytes:4, group B (64 cycles) with total Gn dose :3375 IU and retrieved oocytes < or =3, and group C (97 cycles) with total Gn dose< or =3300 IU and retrieved oocytes< or =3. The clinical characteristics and outcomes of these 3 groups were comparatively analyzed., Results: The clinical pregnancy rate and delivery rate were 38.8% and 32.5% in group A, 16.7% and 10.4% in group B, and 27.3% and 23.4% in group C, respectively. The follicle number, oocyte number, number of embryo transferred, peak serum E2 level, clinical pregnancy rate and delivery rate were significantly higher in group A than in groups B and C (P<0.05). Groups B and C had similar follicle number, oocyte number, and number of available embryos, but group C had significantly lower total Gn dose (P<0.05); the peak serum E2 level, clinical pregnancy rate and delivery rate were lower in group B than in group C, but the difference was not statistically significant (P>0.05)., Conclusions: In patients receiving a relatively low dose of Gn with smaller number of retrieved oocytes, Gn dose increment can improve the clinical pregnancy rate and delivery rate, suggesting a state of relatively poor ovarian response or mild ovarian reserve decrease; failure of increasing the number of oocytes retrieved with greater Gn dose suggests severely decreased ovarian responsiveness or ovarian reserve and also poor clinical prognosis.

Objective: To investigate the outcome of in vitro fertilization and embryo transfer (IVF-ET) in couples with the husband positive for chronic infection of hepatitis B virus (HBV)., Methods: This study involved 102 infertile couples receiving IVF-ET with the husbands(but not the wives) positive for hepatitis B surface antigen (HBsAg), and another 204 couples negative for HBsAg receiving the treatment served as the control group. The cumulative embryo score, fertilization rate, cleavage rate, rate of good quality embryos, implantation rate, clinical pregnancy rate, first trimester and late miscarriage rates, delivery rate, and neonatal malformation rate were recorded and compared between the two groups., Results: Between the HBsAg-positive and the control groups, the cumulative embryo score (52.8-/+18.7 vs 55.4-/+16.9), insemination rate (66.9% vs 66.1%), cleavage rate (97.6% vs 97.2%), rate of good quality embryos (34.0% vs 37.1%), implantation rate (40.9% vs 34.6%), clinical pregnancy rate (56.9% vs 50%), first trimester miscarriage rate (6.9% vs 5.9%) and late pregnancy miscarriage rate (8.6% vs 4.9%), delivery rate (40.2% vs 43.6%) and neonatal malformation rate (0 vs 0) were all similar (P>0.05;)., Conclusion: Chronic HBV infection in the husband might not affect the outcome of IVF-ET treatment.