Alterations In Key Genes Research Articles

Unraveling the socio-cognitive consequences of KCC2 disruption in zebrafish: implications for neurodevelopmental disorders and therapeutic interventions.

During postnatal brain development, maintaining a delicate balance between excitation and inhibition (E/I) is essential for the precise formation of neuronal circuits. The K+/cl- cotransporter 2 (KCC2) is instrumental in this process, and its dysregulation is implicated in various neurological disorders. This study utilized zebrafish (Danio rerio) to investigate the socio-cognitive consequences of KCC2 disruption. Through CRISPR-Cas9 technology, biallelic kcc2a knockout zebrafish larvae were generated, revealing behavioral abnormalities, including impaired social interactions and memory deficits. Molecular analyses unveiled alterations in key genes associated with the GABAergic and glutamatergic systems, potentially contributing to E/I imbalance. Additionally, KCC2 disruption influenced the expression of oxytocin and BDNF, crucial regulators of social behaviors, synaptic plasticity, and memory formation. The study also explored the therapeutic potential of KCC2 modulation using pharmaceuticals, showing the rescuing effects of CLP-290 and LIT-001 on social abnormalities. However, the selective impact of LIT-001 on social behaviors, not memory, highlights the complexity of neurobehavioral modulation. In summary, this study sheds light on the pivotal role of KCC2 in shaping socio-cognitive functions and suggests potential therapeutic avenues for KCC2-related neurological disorders.

Exploration of Response Mechanisms in the Gills of Pacific Oyster (Crassostrea gigas) to Cadmium Exposure through Integrative Metabolomic and Transcriptomic Analyses.

Marine mollusks, including oysters, are highly tolerant to high levels of cadmium (Cd), but the molecular mechanisms underlying their molecular response to acute Cd exposure remain unclear. In this study, the Pacific oyster Crassostrea gigas was used as a biological model, exposed to acute Cd stress for 96 h. Transcriptomic analyses of their gills were performed, and metabolomic analyses further validated these results. In our study, a total of 111 differentially expressed metabolites (DEMs) and 2108 differentially expressed genes (DEGs) were identified under acute Cd exposure. Further analyses revealed alterations in key genes and metabolic pathways associated with heavy metal stress response. Cd exposure triggered physiological and metabolic responses in oysters, including enhanced oxidative stress and disturbances in energy metabolism, and these changes revealed the biological response of oysters to acute Cd stress. Moreover, oysters could effectively enhance the tolerance and detoxification ability to acute Cd exposure through activating ABC transporters, enhancing glutathione metabolism and sulfur relay system in gill cells, and regulating energy metabolism. This study reveals the molecular mechanism of acute Cd stress in oysters and explores the molecular mechanism of high tolerance to Cd in oysters by using combined metabolomics and transcriptome analysis.

Single-cell RNA sequencing reveals the epithelial cell, fibroblast, and key gene alterations in chronic rhinosinusitis with nasal polyps

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the nasal mucosa, and epithelial–mesenchymal transition (EMT) is thought to be an essential process in the pathogenesis of CRSwNP. However, the mechanisms of epithelial and fibroblastic changes at the single-cell level are unclear. In this study, we investigated the epithelial cell, fibroblast, and key gene alterations in the development of CRSwNP. We revealed major cell types involved in CRSwNP and nasal mucosal inflammation formation, then mapped epithelial and fibroblast subpopulations. We showed that the apical and glandular epithelial cells and the ADGRB3+ and POSTN+ fibroblasts were the key cell subtypes in the progression of CRSwNP. Pseudotime and cell cycle analysis identified dynamic changes between epithelial cells and fibroblasts during its development. WFDC2 and CCL26 were identified as the key marker genes involved in the development of CRSwNP and were validated by IHC staining, which may provide a potential novel target for future CRSwNP therapy. ScRNA-seq data provided insights into the cellular landscape and the relationship between epithelial cells and fibroblasts in the progression of CRSwNP. WFDC2 and CCL26 were identified as the key genes involved in the development of CRSwNP and may be the potential markers for gene therapy.

Analyzing the impact and mechanism of bisphenol A on testicular lipid metabolism in Gobiocypris rarus through integrated lipidomics and transcriptomics

Bisphenol A (BPA) is one of the most common environmental endocrine chemicals, known for its estrogenic effects that can interfere with male spermatogenesis. Lipids play crucial roles in sperm production, capacitation, and motility as important components of the sperm plasma membrane. However, limited research has explored whether BPA affects lipid metabolism in the testes of male fish and subsequently impacts spermatogenesis. In this study, we employed Gobiocypris rarus rare minnow as a research model and exposed them to environmentally relevant concentrations of BPA (15 μg/L) for 5 weeks. We assessed sperm morphology and function and analyzed changes in testicular lipid composition and transcriptomics. The results demonstrated a significant increase in the sperm head membrane damage rate, along with reduced sperm motility and fertilization ability due to BPA exposure. Lipidomics analysis revealed that BPA increased the content of 11 lipids while decreasing the content of 6 lipids in the testes, particularly within glycerophospholipids, glycerolipids, and sphingolipid subclasses. Transcriptomics results indicated significant up-regulation in pathways such as cholesterol metabolism, peroxisome proliferator-activated receptor signaling, and fat digestion and absorption, with significant alterations in key genes related to lipid metabolism, including apolipoprotein A-I, apolipoprotein C-I, and translocator protein. These findings suggest that BPA exposure can induce testicular lipid metabolism disruption in rare minnows, potentially resulting in abnormalities in rare minnow spermatogenesis.

Elucidating the molecular mechanisms of ozone therapy for neuropathic pain management by integrated transcriptomic and metabolomic approach.

Introduction: Neuropathic pain remains a prevalent and challenging condition to treat, with current therapies often providing inadequate relief. Ozone therapy has emerged as a promising treatment option; however, its mechanisms of action in neuropathic pain remain poorly understood. Methods: In this study, we investigated the effects of ozone treatment on gene expression and metabolite levels in the brainstem and hypothalamus of a rat model, using a combined transcriptomic and metabolomic approach. Results: Our findings revealed significant alterations in key genes, including DCST1 and AIF1L, and metabolites such as Aconitic acid, L-Glutamic acid, UDP-glucose, and Tyrosine. These changes suggest a complex interplay of molecular pathways and region-specific mechanisms underlying the analgesic effects of ozone therapy. Discussion: Our study provides insights into the molecular targets of ozone treatment for neuropathic pain, laying the groundwork for future research on validating these targets and developing novel therapeutic strategies.

Hepatic consequences of a mixture of endocrine-disrupting chemicals in male mice

The global epidemic of metabolic syndrome has been partially linked to ubiquitous exposure to endocrine-disrupting chemicals (EDCs). Although the impacts of exposure to single EDCs have been thoroughly studied, the consequences of simultaneous uncontrolled exposure to multiple EDCs require further investigations. Therefore, in this study, we evaluated how exposure to mixtures containing bisphenol A and seven phthalates impacts liver functions and metabolic homeostasis. Male mice were gavaged with either EDCs at four different dose combinations or corn oil (control) for six weeks. The results showed that exposure to EDCs at the human daily exposure limit had a negligible impact on liver function. However, EDC at ≥ 25 orders of magnitude of human-relevant doses had detrimental impacts on overall liver function, leading to metabolic abnormalities, steatohepatitis, and hepatic fibrosis via the activation of both genomic and non-genomic pathways. The metabolic phenotype was linked to alterations in key genes involved in hepatic lipid and glucose metabolism. In contrast, alterations in cytokine expression, oxidative stress, and apoptosis impacted steatohepatitis and fibrosis. Because EDC exposure does not occur independently, the findings of the combined effects of exposure to multiple EDCs have significant relevance for public health.

R-Score: A New Parameter to Assess the Quality of Variants' Calls Assessed by NGS Using Liquid Biopsies.

Simple SummaryCirculating tumor DNA profiling by next-generation sequencing (NGS) is becoming essential for guiding targeted therapies. However, it remains challenging. Here, we show that variant allele fraction and the median of absolute values of all pairwise differences impact the agreement between digital PCR and NGS calls. Therefore, we propose a new parameter, named R-score, which integrates both variables, and we evaluate its usefulness for optimizing NGS variant calling.Next-generation sequencing (NGS) has enabled a deeper knowledge of the molecular landscape in non-small cell lung cancer (NSCLC), identifying a growing number of targetable molecular alterations in key genes. However, NGS profiling of liquid biopsies risk for false positive and false negative calls and parameters assessing the quality of NGS calls remains lacking. In this study, we have evaluated the positive percent agreement (PPA) between NGS and digital PCR calls when assessing EGFR mutation status using 85 plasma samples from 82 EGFR-positive NSCLC patients. According to our data, variant allele fraction (VAF) was significantly lower in discordant calls and the median of the absolute values of all pairwise differences (MAPD) was significantly higher in discordant calls (p < 0.001 in both cases). Based on these results, we propose a new parameter that integrates both variables, named R-score. Next, we sought to evaluate the PPA for EGFR mutation calls between two independent NGS platforms using a subset of 40 samples from the same cohort. Remarkably, there was a significant linear correlation between the PPA and the R-score (r = 0.97; p < 0.001). Specifically, the PPA of samples with an R-score ≤ −1.25 was 95.83%, whereas PPA falls to 81.63% in samples with R-score ≤ 0.25. In conclusion, R-score significantly correlates with PPA and can assist laboratory medicine specialists and data scientists to select reliable variants detected by NGS.

998 - Potential therapeutic role of the combination of biguanides and statins in prostate cancer: Association with alterations in key genes and miRNAs levels

998 - Potential therapeutic role of the combination of biguanides and statins in prostate cancer: Association with alterations in key genes and miRNAs levels

Functional Responses to the Chronic Activation of 5-HT1A Receptors in Mice with Genetic Predisposition to Catalepsy

The effects of chronic 5-HT1A receptor activation on the behavior, functional activity of 5-HT1A receptors, and expression of key genes of the brain 5-HT system were studied in mice of the catalepsy-prone CBA strain and the catalepsy-resistant C57BL/6 strain. Chronic treatment with 8-Hydroxy-2-(di-n-propyl-amino)tetralin (8-OH-DPAT) (1.0 mg/kg i.p., 14 days) led to a significant decrease in the hypothermic response to acute administration of 8-OH-DPAT in CBA and C57BL/6 mice, which indicates the desensiti-zation of 5-HT1A receptors in both strains. Pretreatment with the 5-HT7 receptor agonist SB 269970 did not affect the hypothermic response to the acute administration of 8-OH-DPAT, which suggests an independent functional response of 5-HT1A receptors. The treatment did not induce any changes in the behavior in the open field paradigm in CBA mice, but significantly increased the total path, the time spent in the center, and the number of rearings in C57BL/6 mice, which indicates the enhancement of locomotor and exploratory activity in C57BL/6 mice. The chronic activation of 5-HT1A receptor downregulated 5-HT1A gene expression, as well as the expression of the gene that encodes tryptophan hydroxylase 2, a key enzyme of 5-HT biosynthesis, in the midbrain and the expression of the gene that encodes the 5-HT2A receptor in the frontal cortex of CBA, but not C57BL/6 mice. The obtained data provide a new evidence on the receptor-gene cross talk in the brain 5-HT system that may underlie the loss of pharmacological efficacy of 5-HT1A receptor agonists. In turn, the loss of the behavioral response and compensatory alterations in key genes of the brain 5-HT system in CBA mice suggests that catalepsy-prone and -resistant genotypes demonstrate different sensibility to the effects of drugs.

Team work and cytopathology molecular diagnosis of solid pancreatic lesions.

Pancreatic ductal adenocarcinoma (PDAC) is expected to become the second leading cause of cancer-associated death in the next decade or so. It is widely accepted that tumorigenesis is linked to specific alterations in key genes and pancreatic neoplasms are some of the best characterized at the genomic level. Recent whole-exome and whole-genome sequencing analyses confirmed that PDAC is frequently characterized by mutations in a set of four genes among others: KRAS, TP53, CDKN2A/p16, and SMAD4. Sequencing, for example, is the preferable technique available for detecting KRAS mutations, whereas in situ immunochemistry is the main approach for detecting TP53 gene alteration. Nevertheless, the diagnosis of PDAC is still a clinical challenge, involving adequate acquisition of endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) and specific pathological assessment from tissue architecture to specific biomolecular tests. The aim of the present review is to provide a complete overview of the current knowledge of the biology of pancreatic cancer as detected by the latest biomolecular techniques and, moreover, to propose a paradigm for strict teamwork collaboration in order to improve the correct use of diagnostic sources.

OA06.01 Clinical Utility of Circulating Tumor DNA (ctDNA) Analysis by Digital next Generation Sequencing of over 5,000 Advanced NSCLC Patients

Detection of actionable genomic alterations is now required for NCCN guideline-compliant work-up of NSCLC adenocarcinoma. Next-generation sequencing (NGS) of ctDNA, if sufficiently sensitive and specific, could provide a non-invasive, comprehensive genotyping platform relevant to clinical decision-making when tissue is insufficient or at time of progression on targeted therapies. A highly accurate, deep-coverage (15,000x) ctDNA plasma NGS test targeting 54-70 genes (Guardant360) was used to genotype 5,206 advanced-stage NSCLC patients accrued between 6/2014 – 4/2016. The frequency and distribution of somatic alterations in key genes were compared to those described in TCGA (Pearson and Spearman correlations). The clinical impact of ctDNA testing was evaluated by identification of resistance mechanisms emergent at progression on targeted therapies, and through analysis of additional driver mutations detected by ctDNA at baseline in 362 consecutive NSCLC patients with tissue mutation data available. The positive predictive value (PPV) of ctDNA sequencing was assessed in 229 patients with known tumor driver alterations. ctDNA alterations were detected in 86% of cases; EGFR mutations in 25%, KRAS mutations in 17%, MET amplification in 4%, BRAF mutations in 3% and other rare but potentially actionable alterations in 9%. Mutation patterns among driver oncogenes were highly consistent with those from TCGA (Pearson r=0.92, 0.99, 0.99 for EGFR, KRAS, and fusion breakpoint location). PPV of ctDNA-detected variants was 100% for EGFRL858R, 98% for EGFRE19del, 96% for ALK, RET, or ROS1 fusions, and 100% for KRASG12/G13/Q61 mutations. In 362 cases with tissue information available, 63% (229/362) were tissue quantity-insufficient or undergenotyped (QNS/UG). ctDNA analysis identified driver mutations in 51 of the 229 QNS/UG cases, a 38% increase in detection rate over tissue alone. Among 1,111 EGFR-mutant cases, resistance mutations were identified at progression at frequencies consistent with published literature: EGFRT790M 47%, MET amp 5%, ERBB2 amp 5%, FGFR3 fusions 0.4%, ALK/other fusions 1%, BRAF mutations 1.8%, PTEN inactivation 2.5%, NF1 inactivation 3%, RB1 inactivation 3%, KRAS mutations 1.9%. In 143 consecutive NSCLC patients with detailed follow-up and serial analysis seen at the UC Davis Cancer Center, informative driver mutations were observed in 48 (34%). This series represents the largest NSCLC ctDNA study to date. Genotypic patterns of truncal mutations were highly consistent with TCGA in terms of frequency and distribution. At baseline, ctDNA augmented tissue analysis by identifying additional, actionable mutations when tissue was QNS/UG. ctDNA NGS conducted at progression identified emergent resistance mutations that could inform subsequent courses of therapy.

Abstract B40: Mouse clinical trials: integrating PDX models of sarcoma subtypes with genomics to replicate patient responses to cancer therapeutics

Abstract Objective: Sarcomas are clinically and genetically heterogeneous tumors that are often difficult to treat. Patient-derived xenograft (PDX or TumorGraft) models have been shown to accurately reflect the characteristics of patient tumors and may be useful tools for developing personalized treatment strategies and deployment in mouse clinical trials assessing novel therapies. We evaluated the accuracy of PDX models in reproducing clinical responses to standard and experimental drugs used for sarcoma treatment. Methods: Fresh tumor tissue (comprising 172 distinct explants) was collected by surgery or biopsy from 150 patients with sarcoma and implanted into immunodeficient mice. Tumors successfully engrafting were screened using next-generation sequencing technology to identify key genomic alterations with therapeutic implications. PDX sensitivity to standard of care and experimental agents was evaluated and tumor growth inhibition/regression values and clinical RECIST outcomes determined. Drug screening results were correlated with individual patient outcomes. Results: Of the 172 implanted tumors, 145 have completed the implantation process, with 86 (59%) successfully establishing a PDX model. Engraftment rate depended on sarcoma subtype and specimen origin (surgical explant versus biopsy). Next generation sequencing of models from major sarcoma subtypes (Ewing sarcoma, leiomyosarcoma, liposarcoma, osteosarcoma, and rhabdomyosarcoma) highlighted alterations in 454 genes, including those informing treatment selection such as PIK3CA, MET, and CDK4. A total of 26 PDX models from 25 patients across the major sarcoma subtypes were screened in 148 drug tests employing 64 FDA-approved drugs/combinations such as ifosfamide, and gemcitabine/docetaxel, and 26 experimental therapies in clinical trial. In 13/13 (100%) cases with available data, a significant correlation between patient clinical response and PDX model outcome was noted (p=0.0004; Fisher's exact test). Conclusions: Given the close match between patient clinical responses and PDX model outcomes, these results validate the concept of mouse clinical trials for determining the efficacy of novel therapies in sarcoma prior to broad application in expensive human trials. Moreover, the retention of alterations in key genes influencing therapeutic decision-making suggests a use for PDX models in functionally validating genomic hypotheses in a pre-clinical setting. Citation Format: Amanda Katz, Raphael E. Pollock, Leonard H. Wexler, Carlos Rodriguez-Galindo, Jonathan C. Trent, Robert Maki, Jennifer Jaskowiak, Lindsay Ryland, Daniel Ciznadija, Angela Davies, Keren Paz. Mouse clinical trials: integrating PDX models of sarcoma subtypes with genomics to replicate patient responses to cancer therapeutics. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B40.

Effects of valproic acid and pioglitazone on cell cycle progression and proliferation of T-cell acute lymphoblastic leukemia Jurkat cells.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignant tumor. Administration of chemical compounds influencing apoptosis and T cell development has been discussed as promising novel therapeutic strategies. Valproic acid (VPA) as a recently emerged anti-neoplastic histone deacetylase (HDAC) inhibitor and pioglitazone (PGZ) as a high-affinity peroxisome proliferator-activated receptor-gamma (PPARγ) agonist have been shown to induce apoptosis and cell cycle arrest in different studies. Here, we aimed to investigate the underlying molecular mechanisms involved in anti-proliferative effects of these compounds on human Jurkat cells. Treated cells were evaluated for cell cycle progression and apoptosis using flowcytometry and MTT viability assay. Real-time RT-PCR was carried out to measure the alterations in key genes associated with cell death and cell cycle arrest. Our findings illustrated that both VPA and PGZ can inhibit Jurkat E6.1 cells in vitro after 24 hr; however, PGZ 400 μM presents the most anti-proliferative effect. Interestingly, treated cells have been arrested in G2/M with deregulated cell division cycle 25A (Cdc25A) phosphatase and cyclin-dependent kinase inhibitor 1B (CDKN1B or p27) expression. Expression of cyclin D1 gene was inhibited when DNA synthesis entry was declined. Cell cycle deregulation in PGZ and VPA-exposed cells generated an increase in the proportion of aneuploid cell population, which has not reported before. These findings define that anti-proliferative effects of PGZ and VPA on Jurkat cell line are mediated by cell cycle deregulation. Thus, we suggest PGZ and VPA may relieve potential therapeutic application against apoptosis-resistant malignancies.

Causes and Pathophysiology of Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) results from the neoplastic transformation of pleural mesothelial cells. Asbestos exposure is a major risk factor for MPM, but epidemiologic studies demonstrated the occurrence of MPM in populations exposed to other fibers, and an excess of MPM in populations occupationally exposed to man-made vitreous fibers and previously to asbestos. The development of nanotechnologies also raises some concern about the potential health effects of new particles of high aspect ration, such as carbon nanotubes. Toxicological studies investigated the mechanism of asbestos-induced transformation of mesothelial cells, and molecular analyses defined the genomic and physiopathological changes in MPM. These findings allowed identifying some key events accounting for the neoplastic process. This article summarizes the known and suspected causes of MPM, the cellular events and responses of mesothelial cells to asbestos fibers and the alterations of key genes and regulatory pathways involved in the pathological mechanism.

Downregulation of Rab25 activates Akt1 in head and neck squamous cell carcinoma.

Several studies have suggested that Ras-associated binding 25 protein (Rab25) is involved in the pathogenesis of human cancer. Although it has been demonstrated that the development of head and neck squamous cell carcinoma (HNSCC) is the result of an accumulation of multiple sequential genetic and epigenetic alterations in key genes with important functions in cell growth and the cell cycle, recent studies have indicated that HNSCC is a complex and heterogenous disease. To the best of our knowledge, there is no data regarding the regulation of the Rab25 gene at the mRNA or protein level in HNSCC. Furthermore, available data on Rab25 expression in other types of cancer are conflicting. The aim of the present study was to investigate whether Rab25 is involved in the development and/or progression of HNSCC, and to analyze the mechanisms underlying its effects in this type of cancer. The expression of Rab25 mRNA in HNSCC tissues and adjacent non-tumor tissue samples was measured using reverse transcription-quantitative polymerase chain reaction, while the level of the Rab25, Akt1 and phosphorylated-Akt1 proteins was measured using western blotting. Expression of Rab25 mRNA and protein was downregulated in 69.1% and 56.1% of tumor tissue samples, respectively. This downregulation was associated with an increase in p-Akt1 expression, in the absence of a change in total Akt1 protein levels, in tumor tissues compared with normal tissues. The current findings suggest that Rab25 acts as a tumor suppressor in HNSCC.

Cryopreservation Causes Genetic and Epigenetic Changes in Zebrafish Genital Ridges

Cryopreservation is an important tool routinely employed in Assisted Reproduction Technologies (ARTs) and germplasm banking. For several years, the assessment of global DNA fragmentation seemed to be enough to ensure the integrity of genetic material. However, cryopreservation can produce molecular alterations in key genes and transcripts undetectable by traditional assays, such modifications could interfere with normal embryo development. We used zebrafish as a model to study the effect of cryopreservation on key transcripts and genes. We employed an optimized cryopreservation protocol for genital ridges (GRs) containing primordial germ cells (PGCs) considered one of the best cell sources for gene banking. Our results indicated that cryopreservation produced a decrease in most of the zebrafish studied transcripts (cxcr4b, pou5f1, vasa and sox2) and upregulation of heat shock proteins (hsp70, hsp90). The observed downregulation could not always be explained by promoter hypermethylation (only the vasa promoter underwent clear hypermethylation). To corroborate this, we used human spermatozoa (transcriptionally inactive cells) obtaining a reduction in some transcripts (eIF2S1, and LHCGR). Our results also demonstrated that this effect was caused by freezing/thawing rather than exposure to cryoprotectants (CPAs). Finally, we employed real-time PCR (qPCR) technology to quantify the number of lesions produced by cryopreservation in the studied zebrafish genes, observing very different vulnerability to damage among them. All these data suggest that molecular alterations caused by cryopreservation should be studied in detail in order to ensure the total safety of the technique.

Identification of De Novo Copy Number Variants Associated with Human Disorders of Sexual Development

Disorders of sexual development (DSD), ranging in severity from genital abnormalities to complete sex reversal, are among the most common human birth defects with incidence rates reaching almost 3%. Although causative alterations in key genes controlling gonad development have been identified, the majority of DSD cases remain unexplained. To improve the diagnosis, we screened 116 children born with idiopathic DSD using a clinically validated array-based comparative genomic hybridization platform. 8951 controls without urogenital defects were used to compare with our cohort of affected patients. Clinically relevant imbalances were found in 21.5% of the analyzed patients. Most anomalies (74.2%) evaded detection by the routinely ordered karyotype and were scattered across the genome in gene-enriched subtelomeric loci. Among these defects, confirmed de novo duplication and deletion events were noted on 1p36.33, 9p24.3 and 19q12-q13.11 for ambiguous genitalia, 10p14 and Xq28 for cryptorchidism and 12p13 and 16p11.2 for hypospadias. These variants were significantly associated with genitourinary defects (P = 6.08×10−12). The causality of defects observed in 5p15.3, 9p24.3, 22q12.1 and Xq28 was supported by the presence of overlapping chromosomal rearrangements in several unrelated patients. In addition to known gonad determining genes including SRY and DMRT1, novel candidate genes such as FGFR2, KANK1, ADCY2 and ZEB2 were encompassed. The identification of risk germline rearrangements for urogenital birth defects may impact diagnosis and genetic counseling and contribute to the elucidation of the molecular mechanisms underlying the pathogenesis of human sexual development.

Genomic stability in clinical scale expanded mesenchymal stem/stromal cells

Owing to their multi ⁄ pluripotency and immunosuppressive properties, mesenchymal stem ⁄ stromal cells (MSCs) are important tools for treatment of immune disorders and tissue repair. The increasing uses of MSCs lead to the development of production processes that need to be in accordance with good manufacturing practices (GMP). In cellular therapy, safety remains one of the main concerns; of these the risk of transformation is a major one and to avoid side effects, accurate production controls have to be implemented. At this time, there are conflicting data regarding the genomic stability of culture expanded MSCs, and the risk of transformation. Conducted on immortalized human bone marrow MSCs, the first study showed that transformation in MSCs was a long and multistep process involving genetic and epigenetic changes in cell experiencing more than 100 population doublings. Moreover, alterations of key genes, as p16, were mandatory for transformation. The first evidence of human MSC transformation during culture was reported for adipose tissue MSCs subjected to long-term culture. If there were karyotype abnormalities, the transformation appeared only after p16 deletion, over-expression of c-myc and re-expression of human telomerase (hTERT). This transformation process could be linked to a mesenchymal–epithelial transition. As recently reported, these features were related to contamination by a cancer cell line. One study on bone marrow MSCs reported that transformation could be a frequent and fast process. On the contrary, using karyotyping, array-comparative genomic hybridization (array-CGH) and fluorescent in situ hybridization (FISH), different studies on clinical-grade cultured MSCs did not show any evidence of genetic instability and transformation. Recently, in two different clinical trials, we showed that aneuploidy could appear whatever the culture process was. However, presenting or not aneuploidy, all cultivated MSCs reached senescence, and they did not exhibit any transforming events. In conclusion, if genetic stability of clinical grade expanded MSCs has to be tested, karyotyping does not seem a relevant control. Focusing on the molecular events involved in transformation, a new approach of controls is needed.

High-resolution mapping of copy-number alterations with massively parallel sequencing

Cancer results from somatic alterations in key genes, including point mutations, copy-number alterations and structural rearrangements. A powerful way to discover cancer-causing genes is to identify genomic regions that show recurrent copy-number alterations (gains and losses) in tumor genomes. Recent advances in sequencing technologies suggest that massively parallel sequencing may provide a feasible alternative to DNA microarrays for detecting copy-number alterations. Here we present: (i) a statistical analysis of the power to detect copy-number alterations of a given size; (ii) SegSeq, an algorithm to segment equal copy numbers from massively parallel sequence data; and (iii) analysis of experimental data from three matched pairs of tumor and normal cell lines. We show that a collection of approximately 14 million aligned sequence reads from human cell lines has comparable power to detect events as the current generation of DNA microarrays and has over twofold better precision for localizing breakpoints (typically, to within approximately 1 kilobase).

Potentiation of Morphine's Antinociception by Group II and Group III Metabotropic Glutamate Receptors Agonists on a Rat Incisional Pain

A postoperative pain is directly associated with peripheral tissue damage during surgery. It is widely recognized that peripheral tissue damage results in increased activation of peripheral nociceptors and an activity-dependent increase in central neuronal excitability, inducing peripheral and central sensitization. 1) Recent research suggests that transcriptional alterations in key genes and induction of sustained plasticity of neuronal activity underlies longer-lasting pain. Accumulating evidence suggests a pivotal role for metabotropic glutamate receptors (mGluRs) in nociceptive processing and they have traditionally been divided into three groups: I (mGluR1 and mGluR5), II (mGluR2 and mGluR3), and III (mGluR4, mGluR6-mGluR8). 4) In particular, group II and group III mGluRs inhibit adenylate cyclase, thereby producing an antinociceptive effect. 5) One study has reported that mGluRs are differentially expressed in the sheep spinal cord following abdominal surgery. 6) These data lead to the hypothesis that agonizing on group II and group III mGluRs activity may contribute to modulation of postoperative pain and hyperalgesia. However, the effects of group II and group III mGluRs agonists in response to surgery have not been determined before. Therefore, the aim of the present study was to increase our understanding of spinal group II and group III mGluRs signaling mechanisms underlying the development and maintenance of postoperative pain in an animal model of incisional pain, that it closely mimics the human postopeartive pain. In addition, we sought to determine the characteristics of the drug interaction between intrathecal group II agonist (APDC) Potentiation of Morphine's Antinociception by Group II and Group III Metabotropic Glutamate Receptors Agonists on a Rat Incisional Pain