Lower Maternal Chronic Physiological Stress and Better Child Behavior at 18 Months: Follow-Up of a Cluster Randomized Trial of Neonatal Intensive Care Unit Family Integrated Care.

OBJECTIVE: To assess whether Family Integrated Care (FICare) in the neonatal intensive care unit improves maternal chronic physiological stress and child behavior at 18 months of corrected age for infants born preterm. STUDY DESIGN: Follow-up of a multicenter, prospective cluster-randomized controlled trial comparing FICare and standard care of children born at <33 weeks of gestation and parents, stratified by tertiary neonatal intensive care units, across Canada. Primary outcomes at 18 months of corrected age were maternal stress hormones (cortisol, ie, hair cumulative cortisol [HCC], dehydroepiandrosterone [DHEA]) assayed from hair samples. Secondary outcomes included maternal reports of parenting stress, child behaviors (Internalizing, Externalizing, Dysregulation), and observer-rated caregiving behaviors. Outcomes were analyzed using multilevel modeling. RESULTS: We included 126 mother-child dyads from 12 sites (6 FICare sites, n = 83; 6 standard care sites, n = 43). FICare intervention significantly lowered maternal physiological stress as indicated by HCC (B = -0.22 [-0.41, -0.04]) and cortisol/DHEA ratio (B = -0.25 [-0.48, -0.02]), but not DHEA (B = 0.01 [-0.11, 0.14]). Enrollment in FICare led to lower child Internalizing (B = -0.93 [-2.33, 0.02]) and Externalizing behavior T scores (B = -0.91 [-2.25, -0.01]) via improvements to maternal HCC (mediation). FICare buffered the negative effects of high maternal HCC on child Dysregulation T scores (B = -11.40 [-23.01, 0.21]; moderation). For mothers reporting high parenting stress at 18 months, FICare was related to lower Dysregulation T scores via maternal HCC; moderated mediation = -0.17 (-0.41, -0.01). CONCLUSIONS: FICare has long-term beneficial effects for mother and child, attenuating maternal chronic physiological stress, and improving child behavior in toddlerhood. CLINICAL TRIAL REGISTRATION: NCT01852695.

Structure and Dynamics of Stacking Interactions in an Antibody Binding Site.

For years, antibodies (Abs) have been used as a paradigm for understanding how protein structure contributes to molecular recognition. However, with the ability to evolve Abs that recognize specific chromophores, they also have great potential as models for how protein dynamics contribute to molecular recognition. We previously raised murine Abs to different chromophores and, with the use of three-pulse photon echo peak shift spectroscopy, demonstrated that the immune system is capable of producing Abs with widely varying flexibility. We now report the characterization of the complexes formed between two Abs, 5D11 and 10A6, and the chromophoric ligand that they were evolved to recognize, 8-methoxypyrene-1,3,6-trisulfonic acid (MPTS). The sequences of the Ab genes indicate that they evolved from a common precursor. We also used a variety of spectroscopic methods to probe the photophysics and dynamics of the Ab-MPTS complexes and found that they are similar to each other but distinct from previously characterized anti-MPTS Abs. Structural studies revealed that this difference likely results from a unique mode of binding in which MPTS is sandwiched between the side chain of PheH98, which interacts with the chromophore via T-stacking, and the side chain of TrpL91, which interacts with the chromophore via parallel stacking. The T-stacking interaction appears to mediate relaxation on the picosecond time scale, while the parallel stacking appears to mediate relaxation on an ultrafast, femtosecond time scale, which dominates the response. The anti-MPTS Abs thus not only demonstrate the simultaneous use of the two limiting modes of stacking for molecular recognition, but also provide a unique opportunity to characterize how dynamics might contribute to molecular recognition. Both types of stacking are common in proteins and protein complexes where they may similarly contribute to dynamics and molecular recognition.

DNA methylation genome-wide analysis in remnant and primary gastric cancers.

BACKGROUND: Although primary (PGC) and remnant gastric cancers (RGC) both originate from the same gastrointestinal organ, they have very distinct clinicopathological behaviors. We hypothesized that there would be distinct differences in DNA methylation patterns that would occur during carcinogenesis of RGC and PGC, and that the differences in methylation patterns may help identify the primary factor contributing to chronic inflammation in patients with RGC. METHODS: We investigated the genome-wide DNA methylation patterns of PGC and RGC tissues from 48 patients using the Infinium HumanMethylation450 Beadchip assay. The results were validated by quantitative methylation-specific PCR (qMSP) in separate, independent cohorts. RESULTS: We found that in our training cohort of 48 patients, the most variable genes from the gastric cancer tissues identified by the Infinium HumanMethylation450 Beadchip clustered the resultant heatmap into high and low methylation groups. On multivariate analysis, PGCs contributed significantly to the high methylation group (p = 0.004, OR 12.33), which suggested that the promoter methylation status in PGC is higher than that in RGC. Supporting this conclusion was the finding that in a separate qMSP analysis in a test cohort, the EPB41L3 gene, chosen because of its high ß value on microarray analysis in the gastric cancer tissues, had significantly higher DNA promoter methylation in cancer tissues in the validation PGC tissues than in RGC. CONCLUSIONS: This study demonstrated that promoter methylation status in PGC is higher than in RGC. This result may reflect the effects of the absence of Helicobacter pylori on the reduced DNA methylation in the remnant stomach.

MicroRNA profiling of low-grade glial and glioneuronal tumors shows an independent role for cluster 14q32.31 member miR-487b.

Low-grade (WHO I-II) gliomas and glioneuronal tumors represent the most frequent primary tumors of the central nervous system in children. They often have a good prognosis following total resection, however they can create many neurological complications due to mass effect, and may be difficult to resect depending on anatomic location. MicroRNAs have been identified as molecular regulators of protein expression/translation that can repress multiple mRNAs concurrently through base pairing, and have an important role in cancer, including brain tumors. Using the NanoString digital counting system, we analyzed the expression levels of 800 microRNAs in nine low-grade glial and glioneuronal tumor types (n=45). A set of 61 of these microRNAs were differentially expressed in tumors compared with the brain, and several showed levels varying by tumor type. The expression differences were more accentuated in subependymal giant cell astrocytoma, compared with other groups, and demonstrated the highest degree of microRNA repression validated by RT-PCR, including miR-129-2-3p, miR-219-5p, miR-338-3p, miR-487b, miR-885-5p, and miR-323a-3p. Conversely, miR-4488 and miR-1246 were overexpressed in dysembryoplastic neuroepithelial tumors compared with the brain and other tumors. The cluster 14q32.31 member miR-487b was variably under-expressed in pediatric glioma lines compared with human neural stem cells. Overexpression of miR-487b in a pediatric glioma cell line (KNS42) using lentiviral vectors led to a decrease in colony formation in soft agar (30%) (P<0.05), and decreased expression of known predicted targets PROM1 and Nestin (but not WNT5A). miR-487b overexpression had no significant effect on cell growth, proliferation, sensitivity to temozolomide, migration, or invasion. In summary, microRNA regulation appears to have a role in the biology of glial and glioneuronal tumor subtypes, a finding that deserves further investigation.

Hypoxia inhibits growth, proliferation, and increases response to chemotherapy in retinoblastoma cells.

Retinoblastoma is a malignant tumor of the retina and the most frequent intraocular cancer in children. Low oxygen tension (hypoxia) is a common phenomenon in advanced retinoblastomas, but its biological effect on retinoblastoma growth is not clearly understood. Here we studied how hypoxia altered retinoblastoma gene expression and modulated growth and response to chemotherapy. The hypoxic marker lysyl oxidase (LOX) was expressed in 8 of 12 human retinoblastomas analyzed by immunohistochemistry, suggesting that a hypoxic microenvironment is present in up to two thirds of the cases. WERI Rb1 and Y79 retinoblastoma lines were exposed to 1% or 5% pO2, cobalt chloride (CoCl2), or to normoxia (21% pO2) for up to 8 days. Both 1% and 5% pO2 inhibited growth of both lines by more than 50%. Proliferation was reduced by 25-50% when retinoblastoma cells were exposed to 1% vs 21% pO2, as determined by Ki67 assay. Surprisingly, Melphalan, Carboplatin, and Etoposide produced greater reduction in growth and survival of hypoxic cells than normoxic ones. Gene expression profile analysis of both lines, exposed for 48 h to 1%, 5%, or 21% pO2, showed that glycolysis and glucose transport were the most up-regulated pathways, whereas oxidative phosphorylation was the most down-regulated pathway in hypoxia as compared to normoxia. These data support a role for hypoxia in suppressing growth, proliferation, and enhancing response of retinoblastoma cells to chemotherapy, possibly by impairing energy production through activation of glycolysis and inhibition of mitochondrial respiration. Targeting glucose metabolism or enhancing delivery of chemotherapeutic agents to hypoxic regions may improve treatment of advanced retinoblastomas.

Altered gene expression in conjunctival squamous cell carcinoma.

Conjunctival squamous cell carcinoma is a malignancy of the ocular surface. The molecular drivers responsible for the development and progression of this disease are not well understood. We therefore compared the transcriptional profiles of eight snap-frozen conjunctival squamous cell carcinomas and one in situ lesion with normal conjunctival specimens in order to identify diagnostic markers or therapeutic targets. RNA was analyzed using oligonucleotide microarrays, and a wide range of transcripts with altered expression identified, including many dysregulated in carcinomas arising at other sites. Among the upregulated genes, we observed more than 30-fold induction of the matrix metalloproteinases, MMP-9 and MMP-11, as well as a prominent increase in the mRNA level of a calcium-binding protein important for the intracellular calcium signaling, S100A2, which was induced over 20-fold in the tumor cohort. Clusterin was the most downregulated gene, with an approximately 180-fold reduction in the mRNA expression. These alterations were all confirmed by qPCR in the samples used for initial microarray analysis. In addition, immunohistochemical analysis confirmed the overexpression of MMP-11 and S100A2, as well as reductions in clusterin, in several independent in situ carcinomas of conjunctiva. These data identify a number of alterations, including upregulation of MMP-9, MMP-11, and S100A2, as well as downregulation of clusterin, associated with epithelial tumorigenesis in the ocular surface.

Characterization of aldehyde dehydrogenase 1 high ovarian cancer cells: Towards targeted stem cell therapy.

OBJECTIVE: The cancer stem cell (CSC) paradigm hypothesizes that successful clinical eradication of CSCs may lead to durable remission for patients with ovarian cancer. Despite mounting evidence in support of ovarian CSCs, their phenotype and clinical relevance remain unclear. We and others have found high aldehyde dehydrogenase 1 (ALDH(high)) expression in a variety of normal and malignant stem cells, and sought to better characterize ALDH(high) cells in ovarian cancer. METHODS: We compared ALDH(high) to ALDH(low) cells in two ovarian cancer models representing distinct subtypes: FNAR-C1 cells, derived from a spontaneous rat endometrioid carcinoma, and the human SKOV3 cell line (described as both serous and clear cell subtypes). We assessed these populations for stem cell features then analyzed expression by microarray and qPCR. RESULTS: ALDH(high) cells displayed CSC properties, including: smaller size, quiescence, regenerating the phenotypic diversity of the cell lines in vitro, lack of contact inhibition, nonadherent growth, multi-drug resistance, and in vivo tumorigenicity. Microarray and qPCR analysis of the expression of markers reported by others to enrich for ovarian CSCs revealed that ALDH(high) cells of both models showed downregulation of CD24, but inconsistent expression of CD44, KIT and CD133. However, the following druggable targets were consistently expressed in the ALDH(high) cells from both models: mTOR signaling, her-2/neu, CD47 and FGF18/FGFR3. CONCLUSIONS: Based on functional characterization, ALDH(high) ovarian cancer cells represent an ovarian CSC population. Differential gene expression identified druggable targets that have the potential for therapeutic efficacy against ovarian CSCs from multiple subtypes.

A stem cell-like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing.

Adult cancers may derive from stem or early progenitor cells. Epigenetic modulation of gene expression is essential for normal function of these early cells but is highly abnormal in cancers, which often show aberrant promoter CpG island hypermethylation and transcriptional silencing of tumor suppressor genes and pro-differentiation factors. We find that for such genes, both normal and malignant embryonic cells generally lack the hypermethylation of DNA found in adult cancers. In embryonic stem cells, these genes are held in a 'transcription-ready' state mediated by a 'bivalent' promoter chromatin pattern consisting of the repressive mark, histone H3 methylated at Lys27 (H3K27) by Polycomb group proteins, plus the active mark, methylated H3K4. However, embryonic carcinoma cells add two key repressive marks, dimethylated H3K9 and trimethylated H3K9, both associated with DNA hypermethylation in adult cancers. We hypothesize that cell chromatin patterns and transient silencing of these important regulatory genes in stem or progenitor cells may leave these genes vulnerable to aberrant DNA hypermethylation and heritable gene silencing during tumor initiation and progression.

Adaptive mutations alter antibody structure and dynamics during affinity maturation.

While adaptive mutations can bestow new functions on proteins via the introduction or optimization of reactive centers, or other structural changes, a role for the optimization of protein dynamics also seems likely but has been more difficult to evaluate. Antibody (Ab) affinity maturation is an example of adaptive evolution wherein the adaptive mutations may be identified and Abs may be raised to specific targets that facilitate the characterization of protein dynamics. Here, we report the characterization of three affinity matured Abs that evolved from a common germline precursor to bind the chromophoric antigen (Ag), 8-methoxypyrene-1,3,6-trisulfonate (MPTS). In addition to characterizing the sequence, molecular recognition, and structure of each Ab, we characterized the dynamics of each complex by determining their mechanical response to an applied force via three-pulse photon echo peak shift (3PEPS) spectroscopy and deconvoluting the response into elastic, anelastic, and plastic components. We find that for one Ab, affinity maturation was accomplished via the introduction of a single functional group that mediates a direct contact with MPTS and results in a complex with little anelasticity or plasticity. In the other two cases, more mutations were introduced but none directly contact MPTS, and while their effects on structure are subtle, their effects on anelasticity and plasticity are significant, with the level of plasticity correlated with specificity, suggesting that the optimization of protein dynamics may have contributed to affinity maturation. A similar optimization of structure and dynamics may contribute to the evolution of other proteins.

Quantification of Measurable Residual Disease Detection by Next-Generation Sequencing-Based Clonality Testing in B-Cell and Plasma Cell Neoplasms.

Next-generation sequencing (NGS)-based measurable residual disease (MRD) monitoring in post-treatment settings can be crucial for relapse risk stratification in patients with B-cell and plasma cell neoplasms. Prior studies have focused on validation of various technical aspects of the MRD assays, but more studies are warranted to establish the performance characteristics and enable standardization and broad utilization in routine clinical practice. Here, the authors describe an NGS-based IGH MRD quantification assay, incorporating a spike-in calibrator for monitoring B-cell and plasma cell neoplasms based on their unique IGH rearrangement status. Comparison of MRD status (positive or undetectable) by NGS and flow cytometry (FC) assays showed high concordance (91%, 471/519 cases) and overall good linear correlation in MRD quantitation, particularly for chronic lymphocytic leukemia and B-lymphoblastic leukemia/lymphoma (R = 0.85). Quantitative correlation was lower for plasma cell neoplasms, where underestimation by FC is a known limitation. No significant effects on sequencing efficiency by the spike-in calibrator were observed, with excellent inter- and intra-assay reproducibility within the authors' laboratory, and in comparison to an external laboratory, using the same assay and protocols. Assays performed both at internal and external laboratories showed highly concordant MRD detection (100%) and quantitation (R = 0.97). Overall, this NGS-based MRD assay showed highly reproducible results with quantitation that correlated well with FC MRD assessment, particularly for B-cell neoplasms.

Protein dynamics and the diversity of an antibody response.

The immune system is remarkable in its ability to produce antibodies (Abs) with virtually any specificity from a limited repertoire of germ line precursors. Although the contribution of sequence diversity to this molecular recognition has been studied for decades, recent models suggest that protein dynamics may also broaden the range of targets recognized. To characterize the contribution of protein dynamics to immunological molecular recognition, we report the sequence, thermodynamic, and time-resolved spectroscopic characterization of a panel of eight Abs elicited to the chromophoric antigen 8-methoxypyrene-1,3,6-trisulfonate (MPTS). Based on the sequence data, three of the Abs arose from unique germ line Abs, whereas the remaining five comprise two sets of siblings that arose by somatic mutation of a common precursor. The thermodynamic data indicate that the Abs recognize MPTS via a variety of mechanisms. Although the spectroscopic data reveal small differences in protein dynamics, the anti-MPTS Abs generally show similar levels of flexibility and conformational heterogeneity, possibly representing the convergent evolution of the dynamics necessary for function. However, one Ab is significantly more rigid and conformationally homogeneous than the others, including a sibling Ab from which it differs by only five somatic mutations. This example of divergent evolution demonstrates that point mutations are capable of fixing significant differences in protein dynamics. The results provide unique insight into how high affinity Abs may be produced that bind virtually any target and possibly, from a more general perspective, how new protein functions are evolved.

MicroRNA deregulation in polycythemia vera and essential thrombocythemia patients.

Polycythemia vera (PV) and essential thrombocythemia (ET) are the two most common myeloproliferative neoplasms. The same JAK2(V617F) mutation can be found in both disorders and is able to recapitulate many of the phenotypic abnormalities of these diseases in the murine models. The disease phenotype is also influenced by other unknown genetic or epigenetic factors. MicroRNAs (miRNA) are 18-24 nucleotides single-stranded non-protein-coding RNAs that function primarily as gene repressors by binding to their target messenger RNAs. We performed miRNA expression profiling by oligonucleotide microarray analysis in purified peripheral blood CD34+ cells from eight JAK2(V617F)-positive PV patients and six healthy donors. A quantitative reverse-transcription polymerase chain reaction assay was used to verify differential miRNA expression. Since erythrocytosis is the only feature that distinguishes PV from ET, we also compared specific miRNA expression in the nucleated erythroid cells directly descended from the early erythroid progenitor cells of PV and ET patients. Our data indicate that significant miRNA deregulation occurs in PV CD34+ cells and confirm a genetic basis for the gender-specific differences that characterize PV with respect to miRNA. The results of our study also suggest that deregulated miRNAs may represent an important mechanism by which the PV erythrocytosis and ET thrombocytosis phenotypes are determined.

Analytical comparability to evaluate impact of manufacturing changes of ARX788, an Anti-HER2 ADC in late-stage clinical development.

ARX788 is an anti-HER2 antibody drug conjugate (ADC) developed using Ambrx proprietary Engineered Precision Biologics technology. The manufacturing process of ARX788 has been optimized during the course of early to late-phase clinical development. A comprehensive evaluation of side-by-side comparability between pre- and post-change process for ARX788 drug substance and drug product from a quality perspective was conducted based on ICH Q5E guidelines consisting of batch release assays, physicochemical and biophysical characterization, biological characterization, and forced degradation studies. All results have substantiated a high degree of similarity between the pre- and post-change ARX788 drug substance batches and drug product lots, demonstrating that the process manufacturing changes did not impact product quality.

Validation of a High-Sensitivity Assay for Detection of Chimeric Antigen Receptor T-Cell Vectors Using Low-Partition Digital PCR Technology.

Although in vivo engraftment, expansion, and persistence of chimeric antigen receptor (CAR) T cells are pivotal components of treatment efficacy, quantitative monitoring has not been implemented in routine clinical practice. We describe the development and analytical validation of a digital PCR assay for ultrasensitive detection of CAR constructs after treatment, circumventing known technical limitations of low-partitioning platforms. Primers and probes, designed for detection of axicabtagene, brexucabtagene, and Memorial Sloan Kettering CAR constructs, were employed to validate testing on the Bio-Rad digital PCR low-partitioning platform; results were compared with Raindrop, a high-partitioning system, as reference method. Bio-Rad protocols were modified to enable testing of DNA inputs as high as 500 ng. Using dual-input reactions (20 and 500 ng) and a combined analysis approach, the assay demonstrated consistent target detection around 1 × 10-5 (0.001%) with excellent specificity and reproducibility and 100% accuracy compared with the reference method. Dedicated analysis of 53 clinical samples received during validation/implementation phases showed the assay effectively enabled monitoring across multiple time points of early expansion (day 6 to 28) and long-term persistence (up to 479 days). CAR vectors were detected at levels ranging from 0.005% to 74% (vector versus reference gene copies). The highest levels observed in our cohort correlated strongly with the temporal diagnosis of grade 2 and 3 cytokine release syndrome diagnosis (P < 0.005). Only three patients with undetectable constructs had disease progression at the time of sampling.

Clonal Characterization and Somatic Hypermutation Assessment by Next-Generation Sequencing in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma: A Detailed Description of the Technical Performance, Clinical Utility, and Platform Comparison.

Somatic hypermutation status of the IGHV gene is essential for treating patients with chronic lymphocytic leukemia/small lymphocytic lymphoma. Unlike the conventional low-throughput method, assessment of somatic hypermutation by next-generation sequencing (NGS) has potential for uniformity and scalability. However, it lacks standardization or guidelines for routine clinical use. We critically assessed the performance of an amplicon-based NGS assay across 458 samples. Using a validation cohort (35 samples), the comparison of two platforms (Ion Torrent versus Illumina) and two primer sets [leader versus framework region 1 (FR1)] in their ability to identify clonotypic IGHV rearrangement(s) revealed 97% concordance. The mutation rates were identical by both platforms when using the same primer set (FR1), whereas a slight overestimation bias (+0.326%) was found when comparing FR1 with leader primers. However, for nearly all patients this did not affect the stratification into mutated or unmutated categories, suggesting that use of FR1 may provide comparable results if leader sequencing is not available and allowing for a simpler NGS laboratory workflow. In routine clinical practice (423 samples), the productive rearrangement was successfully detected by either primer set (leader, 97.7%; FR1, 94.7%), and a combination of both in problematic cases reduced the failure rate to 1.2%. Higher sensitivity of the NGS-based analysis also detected a higher frequency of double IGHV rearrangements (19.1%) compared with traditional approaches.

Prenatal alcohol exposure alters the course and severity of adjuvant-induced arthritis in female rats.

Prenatal alcohol exposure (PAE) has adverse effects on the development of numerous physiological systems, including the hypothalamic-pituitary-adrenal (HPA) axis and the immune system. HPA hyper-responsiveness and impairments in immune competence have been demonstrated. The present study investigated immune function in PAE females utilizing an adjuvant-induced arthritis (AA) model, widely used as a model of human rheumatoid arthritis. Given the effects of PAE on HPA and immune function, and the known interaction between HPA and immune systems in arthritis, we hypothesized that PAE females would have heightened autoimmune responses, resulting in increased severity of arthritis, compared to controls, and that altered HPA activity might play a role in the immune system changes observed. The data demonstrate, for the first time, an adverse effect of PAE on the course and severity of AA in adulthood, indicating an important long-term alteration in functional immune status. Although overall, across prenatal treatments, adjuvant-injected animals gained less weight, and exhibited decreased thymus and increased adrenal weights, and increased basal levels of corticosterone and adrenocorticotropin, PAE females had a more prolonged course of disease and greater severity of inflammation compared to controls. In addition, PAE females exhibited blunted lymphocyte proliferative responses to concanavalin A and a greater increase in basal ACTH levels compared to controls during the induction phase, before any clinical signs of disease were apparent. These data suggest that prenatal alcohol exposure has both direct and indirect effects on inflammatory processes, altering both immune and HPA function, and likely, the normal interactions between these systems.

Whole genome microarray of the major pelvic ganglion after cavernous nerve injury: new insights into molecular profile changes after nerve injury.

UNLABELLED: What's known on the subject? and What does the study add? With the present study, we aimed to provide a global picture of the molecular processes that are activated by CN injury. The present study used genomic expression profiling to identify candidate genes that might be useful targets in the CN recovery process and, thus, the ultimate preservation of penile erection. Regeneration of the CN and axonal outgrowth clearly involve changes in multiple biochemical pathways that have never been investigated by microarray analysis. We analyzed global gene expression in the major pelvic ganglion at early stages (48 h and 14 days) after CN injury and focused on the detection of changes in genes related to nervous tissue repair and proliferation. The findings of the present study provide important insight into the molecular systems affected by CN injury and identify candidate genes that may be utilized for novel molecular-based therapies for the preservation and protection of the CN during RP. OBJECTIVES: To to examine the complexity of the many molecular systems involved in supporting cavernous nerve (CN) repair and regeneration in a rat model of bilateral crush injury utilizing a microarray analysis approach. Erectile dysfunction (ED) is a common clinical complication after prostate cancer treatment by radical prostatectomy, and recovery of erectile function can take as long as 2 years. There are gaps in our understanding of the autonomic pelvic innervation of the penis that still need to be addressed for the development of an adequate treatment strategy for post-prostatectomy ED. The molecular mechanisms of the intrinsic ability of CN to regenerate after an injury have not been elucidated. MATERIALS AND METHODS: We analyzed global gene expression in the major pelvic ganglion 48 h and 14 days after CN injury. Overall, a comparative analysis showed that 325 genes changed at the 48-h time point and 114 genes changed at 14 days. There were 60 changed genes in common with both time points. Using the Ingenuity Pathway Analysis® system (Ingenuity Systems, Inc., Redwood City, CA, USA), we were able to analyze the significantly changed genes that were unique and common to each time point by biological function. We focused on the detection of changes related to nervous tissue repair and proliferation, molecular networks of neurotrophic factors, stem cell regulation and synaptic transmission. RESULTS: There was strong evidence of the early mobilization of genes involved in repair and neuroprotection mechanisms (SERPINF1, IGF1, PLAU/PLAUR, ARG1). Genes related to nervous system development (ATF3 GJA1, PLAU, SERPINE1), nerve regeneration (SERPINE2, IGF1, ATF3, ARG1) and synaptic transmission (GJC1, GAL) were changed. Several genes related to proliferation as well as apoptosis (A2M, ATF3, C3, EGR4, FN1, GJA1, GAL) were also changed, possibly as part of a protective mechanism or the initiation of remodelling. CONCLUSIONS: The results obtained show that multiple biological processes are associated with injury and repair of the CN and provide a systematic genome-wide screen for neurotrophic and/or inhibitory pathways of nerve regeneration. These data identify the candidate genes that may be utilized in novel molecular-based therapies for the preservation and protection of the CN during radical prostatectomy.

Modulatory role of prenatal alcohol exposure and adolescent stress on the response to arthritis challenge in adult female rats.

BACKGROUND: There are known environmental risk factors associated with rheumatoid arthritis; however, less is known regarding how the prenatal environment impacts later-life risk for rheumatoid arthritis. Based on preliminary clinical data suggesting that individuals with fetal alcohol spectrum disorder (FASD) are at higher risk for autoimmune disorders, this study investigated the modulatory impact of prenatal alcohol exposure (PAE) on the inflammatory disease profile in an adjuvant-induced arthritis rat model. METHODS: Pregnant rats received liquid ethanol or control diet throughout gestation. To model the increased exposure to stressors often experienced by individuals with FASD, adolescent offspring were exposed to chronic mild stress (CMS) or remained undisturbed. In adulthood, experimental arthritis was initiated and rats terminated either at the peak or following resolution from inflammation to assess endocrine, immune, and histopathological outcomes. FINDINGS: PAE rats had an increased incidence and severity of, and impaired recovery from, arthritis. Increased joint damage was observed in PAE animals, even in the face of apparent recovery from the clinical signs of arthritis, while it appeared that oestradiol may have a protective role. Moreover, with the combination of PAE and adolescent stress, increased macrophage density was detected in the synovium of PAE but not control rats. INTERPRETATION: These findings demonstrate that PAE alters the severity and course of arthritis, highlighting the potential immunomodulatory impact of adverse prenatal exposures. In particular, these data have implications for understanding preliminary data that suggest a heightened propensity for autoimmune disorders in individuals with FASD. FUNDING: This work was supported by: National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism [R37 AA007789] and Kids Brain Health Network (KBHN; Canadian Networks of Centres of Excellence) to JW, a Natural Sciences and Engineering Research Council of Canada (NSERC) CGS-D to TSB and NIH/NIAAA R01 AA022460 to JW and TSB.

Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes.

We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15- to 51-fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100- to 200-fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency-associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming.