Systematic assessment of ISWI subunits shows that NURF creates local accessibility for CTCF.

Catalytic activity of the imitation switch (ISWI) family of remodelers is critical for nucleosomal organization and DNA binding of certain transcription factors, including the insulator protein CTCF. Here we define the contribution of individual subcomplexes by deriving a panel of isogenic mouse stem cell lines, each lacking one of six ISWI accessory subunits. Individual deletions of subunits of either CERF, RSF, ACF, WICH or NoRC subcomplexes only moderately affect the chromatin landscape, while removal of the NURF-specific subunit BPTF leads to a strong reduction in chromatin accessibility and SNF2H ATPase localization around CTCF sites. This affects adjacent nucleosome occupancy and CTCF binding. At a group of sites with reduced chromatin accessibility, CTCF binding persists but cohesin occupancy is reduced, resulting in decreased insulation. These results suggest that CTCF binding can be separated from its function as an insulator in nuclear organization and identify a specific role for NURF in mediating SNF2H localization and chromatin opening at bound CTCF sites.

SS18-SSX Antibody: A Useful Tool to Save Time and Reduce Costs in Synovial Sarcoma Diagnosis. Proposal of a Novel Diagnostic Algorithm.

Synovial sarcoma is a rare malignant mesenchymal neoplasm mostly affecting young adults, characterized by a specific translocation which results in the fusion of the SS18 gene on chromosome 18 with one of the three highly homologous SSX genes on chromosome X. Its morphological diagnosis, especially in monophasic or poorly differentiated variants, can be challenging because histological features often overlap with other malignant mesenchymal tumors. Until recently, the differential diagnosis mostly relied on the use of cytogenetic or molecular analyses to detect the specific t(X;18)(p11;q11) translocation, thus virtually restricting its correct identification to referral centers with a high histological and molecular pathology workflow. The recently commercialized highly sensitive and fusion-specific SS18-SSX antibody has significantly improved the approach to these tumors, representing a relatively cheap and easy to access tool for synovial sarcoma diagnosis. Through a retrospective analysis of 79 synovial sarcomas and histological mimickers, this study confirms the usefulness of the SS18-SSX antibody in the diagnosis of synovial sarcoma, particularly focusing on its application in the pathological response evaluation after neoadjuvant treatment as well as its time- and cost-saving advantages. Finally, we here propose a new diagnostic algorithm to apply into the routine practice.

Stabilization of ß-catenin promotes melanocyte specification at the expense of the Schwann cell lineage.

The canonical Wnt/ß-catenin pathway governs a multitude of developmental processes in various cell lineages, including the melanocyte lineage. Indeed, ß-catenin regulates transcription of Mitf-M, the master regulator of this lineage. The first wave of melanocytes to colonize the skin is directly derived from neural crest cells, whereas the second wave of melanocytes is derived from Schwann cell precursors (SCPs). We investigated the influence of ß-catenin in the development of melanocytes of the first and second waves by generating mice expressing a constitutively active form of ß-catenin in cells expressing tyrosinase. Constitutive activation of ß-catenin did not affect the development of truncal melanoblasts but led to marked hyperpigmentation of the paws. By activating ß-catenin at various stages of development (E8.5-E11.5), we showed that the activation of ß-catenin in bipotent SCPs favored melanoblast specification at the expense of Schwann cells in the limbs within a specific temporal window. Furthermore, in vitro hyperactivation of the Wnt/ß-catenin pathway, which is required for melanocyte development, induces activation of Mitf-M, in turn repressing FoxD3 expression. In conclusion, ß-catenin overexpression promotes SCP cell fate decisions towards the melanocyte lineage.

Assessment of BCOR Internal Tandem Duplications in Pediatric Cancers by Targeted RNA Sequencing.

Alterations in the BCOR gene, including internal tandem duplications (ITDs) of exon 15 have emerged as important oncogenic changes that define several diagnostic entities. In pediatric cancers, BCOR ITDs have recurrently been described in clear cell sarcoma of kidney (CCSK), primitive myxoid mesenchymal tumor of infancy (PMMTI), and central nervous system high-grade neuroepithelial tumor with BCOR ITD in exon 15 (HGNET-BCOR ITDex15). In adults, BCOR ITDs are also reported in endometrial and other sarcomas. The utility of multiplex targeted RNA sequencing for the identification of BCOR ITD in pediatric cancers was investigated. All available archival cases of CCSK, PMMTI, and HGNET-BCOR ITDex15 were collected. Each case underwent anchored multiplex PCR library preparation with a custom-designed panel, with BCOR targeted for both fusions and ITDs. BCOR ITD was detected in all cases across three histologic subtypes using the RNA panel, with no other fusions identified in any of the cases. All BCOR ITDs occurred in the final exon, within 16 codons from the stop sequence. Multiplex targeted RNA sequencing from formalin-fixed, paraffin-embedded tissue is successful at identifying BCOR internal tandem duplications. This analysis supports the use of anchored multiplex PCR targeted RNA next-generation sequencing panels for identification of BCOR ITDs in pediatric tumors. The use of post-analytic algorithms to improve the detection of BCOR ITD using DNA panels was also explored.

Assessment of MGMT methylation status using high-performance liquid chromatography in newly diagnosed glioblastoma.

BACKGROUND: The utility of O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status as a prognostic marker in patients with glioblastoma (GBM) has been established. However, the number of CpG sites that must be methylated to cause transcriptional silencing remains unclear, and no significant consensus exists on the optimal method of assessing MGMT methylation. We developed a new high-performance liquid chromatography (HPLC) method that enables accurate analysis of DNA methylation levels using long PCR products. In the present study, we analyzed the MGMT methylation status of 28 isocitrate dehydrogenase-wild-type GBMs treated with temozolomide using ion-exchange HPLC and set the optimal cutoff values. RESULTS: We designed three primers for separate regions (regions 1-3) that had 21 to 38 CpGs for PCR and validated the MGMT promoter methylation status using frozen samples. There was a strong correlation between HPLC and bisulfite sequencing results (R = 0.794). The optimal cutoff values for MGMT methylation in HPLC were determined to allow differentiation of patient prognosis by receiver operating characteristic curve analysis. The cutoff values were 34.15% for region 1, 8.84% for region 2, and 36.72% for region 3. Kaplan-Meyer curve analysis estimated that the most differentiated prognosis was enabled in the setting of 8.84% methylation of MGMT in region 2. Progression-free survival and overall survival were significantly longer for patients in this setting of region 2 methylation (p = 0.00365 and p = 0.00258, respectively). CONCLUSIONS: The combination of our HPLC method and the original primer setting provides a new standard method for determination of MGMT methylation status in patients with GBM and is useful for refining MGMT-based drug selection.

The utility of a methylation panel in the assessment of clinical response to radiofrequency ablation for Barrett's esophagus.

BACKGROUND: Radiofrequency ablation (RFA) is an effective treatment for dysplastic Barrett's esophagus (BE), but recurrence can occur after initial response. Currently there is uncertainty about how to best define histological remission. A DNA methylation panel on esophageal samples was previously shown to have high diagnostic accuracy for BE. We aimed to investigate this biomarker panel in the assessment of response to RFA treatment. METHODS: We retrospectively analyzed esophageal and gastroesophageal junction (GEJ) biopsies from patients with BE before and after RFA treatment. We quantified the extent of intestinal metaplasia (IM) based on number of glands with goblet cells (IM-Score) and expression of the intestinal factor trefoil factor-3 (TFF3-Score). Promoter methylation of 3 genes (ZNF345, TFP12, ZNF569) was measured by methylight (Meth-Score) throughout the RFA treatment pathway. FINDINGS: We included 45 patients (11 non-dysplastic BE, 14 low-grade dysplasia, 20 high-grade dysplasia/intramucosal cancer). Meth-Scores were significantly higher in BE with and without dysplasia and GEJ with IM compared to GEJ without IM (P<·001). Meth-scores significantly correlated with the extent of IM at the GEJ measured both with IM-Scores (rho=66·0%, P<·001), and TFF3-Scores (rho=75·6%, P<·001). In patients with residual IM at the GEJ, RFA re-treatment brought about a 7·6-fold reduction in the methylation levels. The Meth-score had an area under the ROC curve of 95·1% (95%CI 91·1% - 99·1%) differentiating BE from normal GEJ. INTERPRETATION: A DNA methylation panel can discriminate between the extent of histological IM in esophageal and junctional biopsies and could be used to objectively quantify residual disease following RFA.

Myeloid-specific Asxl2 deletion limits diet-induced obesity by regulating energy expenditure.

We previously established that global deletion of the enhancer of trithorax and polycomb (ETP) gene, Asxl2, prevents weight gain. Because proinflammatory macrophages recruited to adipose tissue are central to the metabolic complications of obesity, we explored the role of ASXL2 in myeloid lineage cells. Unexpectedly, mice without Asxl2 only in myeloid cells (Asxl2ΔLysM) were completely resistant to diet-induced weight gain and metabolically normal despite increased food intake, comparable activity, and equivalent fecal fat. Asxl2ΔLysM mice resisted HFD-induced adipose tissue macrophage infiltration and inflammatory cytokine gene expression. Energy expenditure and brown adipose tissue metabolism in Asxl2ΔLysM mice were protected from the suppressive effects of HFD, a phenomenon associated with relatively increased catecholamines likely due to their suppressed degradation by macrophages. White adipose tissue of HFD-fed Asxl2ΔLysM mice also exhibited none of the pathological remodeling extant in their control counterparts. Suppression of macrophage Asxl2 expression, via nanoparticle-based siRNA delivery, prevented HFD-induced obesity. Thus, ASXL2 controlled the response of macrophages to dietary factors to regulate metabolic homeostasis, suggesting modulation of the cells' inflammatory phenotype may impact obesity and its complications.

Integrative comparison of the genomic and transcriptomic landscape between prostate cancer patients of predominantly African or European genetic ancestry.

Men of predominantly African Ancestry (AA) have higher prostate cancer (CaP) incidence and worse survival than men of predominantly European Ancestry (EA). While socioeconomic factors drive this disparity, genomic factors may also contribute to differences in the incidence and mortality rates. To compare the prevalence of prostate tumor genomic alterations and transcriptomic profiles by patient genetic ancestry, we evaluated genomic profiles from The Cancer Genome Atlas (TCGA) CaP cohort (n = 498). Patient global and local genetic ancestry were estimated by computational algorithms using genotyping data; 414 (83.1%) were EA, 61 (12.2%) were AA, 11 (2.2%) were East Asian Ancestry (EAA), 10 (2.0%) were Native American (NA), and 2 (0.4%) were other ancestry. Genetic ancestry was highly concordant with self-identified race/ethnicity. Subsequent analyses were limited to 61 AA and 414 EA cases. Significant differences were observed by ancestry in the frequency of SPOP mutations (20.3% AA vs. 10.0% EA; p = 5.6×10-03), TMPRSS2-ERG fusions (29.3% AA vs. 39.6% EA; p = 4.4×10-02), and PTEN deletions/losses (11.5% AA vs. 30.2% EA; p = 3.5×10-03). Differentially expressed genes (DEGs) between AAs and EAs showed significant enrichment for prostate eQTL target genes (p = 8.09×10-48). Enrichment of highly expressed DEGs for immune-related pathways was observed in AAs, and for PTEN/PI3K signaling in EAs. Nearly one-third of DEGs (31.3%) were long non-coding RNAs (DE-lncRNAs). The proportion of DE-lncRNAs with higher expression in AAs greatly exceeded that with lower expression in AAs (p = 1.2×10-125). Both ChIP-seq and RNA-seq data suggested a stronger regulatory role for AR signaling pathways in DE-lncRNAs vs. non-DE-lncRNAs. CaP-related oncogenic lncRNAs, such as PVT1, PCAT1 and PCAT10/CTBP1-AS, were found to be more highly expressed in AAs. We report substantial heterogeneity in the prostate tumor genome and transcriptome between EA and AA. These differences may be biological contributors to racial disparities in CaP incidence and outcomes.

Gold-Hybridized Zinc Oxide Nanorods as Real-Time Low-Cost NanoBiosensors for Detection of virulent DNA signature of HPV-16 in Cervical Carcinoma.

Detection of host integrated viral oncogenes are critical for early and point-of-care molecular diagnostics of virus-induced carcinoma. However, available diagnostic approaches are incapable of combining both cost-efficient medical diagnosis and high analytical performances. To circumvent this, we have developed an improved IDE-based nanobiosensor for biorecognition of HPV-16 infected cervical cancer cells through electrochemical impedance spectroscopy. The system is fabricated by coating gold (Au) doped zinc oxide (ZnO) nanorods interfaced with HPV-16 viral DNA bioreceptors on top of the Interdigitated Electrode (IDE) chips surface. Due to the concurrently improved sensitivity and biocompatibility of the designed nanohybrid film, Au decorated ZnO-Nanorod biosensors demonstrate exceptional detection of HPV-16 E6 oncogene, the cancer biomarker for HPV infected cervical cancers. This sensor displayed high levels of sensitivity by detecting as low as 1fM of viral E6 gene target. The sensor also exhibited a stable functional life span of more than 5 weeks, good reproducibility and high discriminatory properties against HPV-16. Sensor current responses are obtained from cultured cervical cancer cells which are close to clinical cancer samples. Hence, the developed sensor is an adaptable tool with high potential for clinical diagnosis especially useful for economically challenged countries/regions.

Method and its Composition for encapsulation, stabilization, and delivery of siRNA in Anionic polymeric nanoplex: An In vitro- In vivo Assessment.

Small interfering RNA (siRNA) are synthetic RNA duplex designed to specifically knockdown the abnormal gene to treat a disease at cellular and molecular levels. In spite of their high potency, specificity, and therapeutic potential, the full-fledged utility of siRNA is predominantly limited to in vitro set-up. Till date, Onpattro is the only USFDA approved siRNA therapeutics available in the clinic. The lack of a reliable in vivo siRNA delivery carrier remains a foremost obstacle towards the clinical translation of siRNA therapeutics. To address the obstacles associated with siRNA delivery, we tested a dendrimer-templated polymeric approach involving a USFDA approved carrier (albumin) for in vitro as well as in vivo delivery of siRNA. The developed approach is simple in application, enhances the serum stability, avoids in vivo RNase-degradation and mediates cytosolic delivery of siRNA following the endosomal escape process. The successful in vitro and in vivo delivery of siRNA, as well as targeted gene knockdown potential, was demonstrated by HDAC4 inhibition in vitro diabetic nephropathy (DN) podocyte model as well as in vivo DN C57BL/6 mice model. The developed approach has been tested using HDAC4 siRNA as a model therapeutics, while the application can also be extended to other gene therapeutics including micro RNA (miRNA), plasmids oligonucleotides, etc.

Laying the foundation for genomically-based risk assessment in chronic myeloid leukemia.

Outcomes for patients with chronic myeloid leukemia (CML) have substantially improved due to advances in drug development and rational treatment intervention strategies. Despite these significant advances there are still unanswered questions on patient management regarding how to more reliably predict treatment failure at the time of diagnosis and how to select frontline tyrosine kinase inhibitor (TKI) therapy for optimal outcome. The BCR-ABL1 transcript level at diagnosis has no established prognostic impact and cannot guide frontline TKI selection. BCR-ABL1 mutations are detected in ~50% of TKI resistant patients but are rarely responsible for primary resistance. Other resistance mechanisms are largely uncharacterized and there are no other routine molecular testing strategies to facilitate the evaluation and further stratification of TKI resistance. Advances in next-generation sequencing technology has aided the management of a growing number of other malignancies, enabling the incorporation of somatic mutation profiles in diagnosis, classification, and prognostication. A largely unexplored area in CML research is whether expanded genomic analysis at diagnosis, resistance, and disease transformation can enhance patient management decisions, as has occurred for other cancers. The aim of this article is to review publications that reported mutated cancer-associated genes in CML patients at various disease phases. We discuss the frequency and type of such variants at initial diagnosis and at the time of treatment failure and transformation. Current limitations in the evaluation of mutants and recommendations for future reporting are outlined. The collective evaluation of mutational studies over more than a decade suggests a limited set of cancer-associated genes are indeed recurrently mutated in CML and some at a relatively high frequency. Genomic studies have the potential to lay the foundation for improved diagnostic risk classification according to clinical and genomic risk, and to enable more precise early identification of TKI resistance.

Tobacco transcription repressors NtJAZ: Potential involvement in abiotic stress response and glandular trichome induction.

Members of the Jasmonate ZIM domain (JAZ) proteins act as transcriptional repressors in the jasmonate (JA) hormonal response. To characterize the potential roles of JAZ gene family in plant development and abiotic stress response, fifteen JAZs were identified based on the genome of Nicotiana tabacum. Structural analysis confirmed the presence of single Jas and TIFY motif. Tissue expression pattern analysis indicated that NtJAZ-2, -3, -5, and -10 were highly expressed in roots and NtJAZ-11 was expressed only in the cotyledons. The transcript level of NtJAZ-3, -5, -9, and -10 in the stem epidermis was higher than that in the stem without epidermis. Dynamic expression of NtJAZs exposed to abiotic stress and phytohormone indicated that the expression of most NtJAZs was activated by salicylic acid, methyl jasmonate, gibberellic acid, cold, salt, and heat stresses. With abscisic acid treatment, NtJAZ-1, -2, and -3 were not activated; NtJAZ-4, -5, and -6 were up-regulated; and the remaining NtJAZ genes were inhibited. With drought stress, the expression of NtJAZ-1, -2, -3, -4, -5, -6, -7, and -8 was up-regulated, whereas the transcript of the remaining genes was inhibited. Moreover, high concentration MeJA (more than 1 mM MeJA) had an effect on secreting trichome induction, but inhabited the plant growth. Nine NtJAZs may play important role in secreting trichome induction. These results indicate that the JAZ proteins are convergence points for various phytohormone signal networks, which are involved in abiotic stress responses.

Assessment of the interaction between the flux-signaling metabolite fructose-1,6-bisphosphate and the bacterial transcription factors CggR and Cra.

Bacteria regulate cell physiology in response to extra- and intracellular cues. Recent work showed that metabolic fluxes are reported by specific metabolites, whose concentrations correlate with flux through the respective metabolic pathway. An example of a flux-signaling metabolite is fructose-1,6-bisphosphate (FBP). In turn, FBP was proposed to allosterically regulate master regulators of carbon metabolism, Cra in Escherichia coli and CggR in Bacillus subtilis. However, a number of questions on the FBP-mediated regulation of these transcription factors is still open. Here, using thermal shift assays and microscale thermophoresis we demonstrate that FBP does not bind Cra, even at millimolar physiological concentration, and with electrophoretic mobility shift assays we also did not find FBP-mediated impairment of Cra's affinity for its operator site, while fructose-1-phosphate does. Furthermore, we show for the first time that FBP binds CggR within the millimolar physiological concentration range of the metabolite, and decreases DNA-binding activity of this transcription factor. Molecular docking experiments only identified a single FBP binding site CggR. Our results provide the long thought after clarity with regards to regulation of Cra activity in E. coli and reveals that E. coli and B. subtilis use distinct cellular mechanism to transduce glycolytic flux signals into transcriptional regulation.

Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP.

Two major transcriptional regulators of carbon metabolism in bacteria are Cra and CRP. CRP is considered to be the main mediator of catabolite repression. Unlike for CRP, in vivo DNA binding information of Cra is scarce. Here we generate and integrate ChIP-exo and RNA-seq data to identify 39 binding sites for Cra and 97 regulon genes that are regulated by Cra in Escherichia coli. An integrated metabolic-regulatory network was formed by including experimentally-derived regulatory information and a genome-scale metabolic network reconstruction. Applying analysis methods of systems biology to this integrated network showed that Cra enables optimal bacterial growth on poor carbon sources by redirecting and repressing glycolysis flux, by activating the glyoxylate shunt pathway, and by activating the respiratory pathway. In these regulatory mechanisms, the overriding regulatory activity of Cra over CRP is fundamental. Thus, elucidation of interacting transcriptional regulation of core carbon metabolism in bacteria by two key transcription factors was possible by combining genome-wide experimental measurement and simulation with a genome-scale metabolic model.

Fitness cost constrains the spectrum of marR mutations in ciprofloxacin-resistant Escherichia coli.

OBJECTIVES: To determine whether the spectrum of mutations in marR in ciprofloxacin-resistant clinical isolates of Escherichia coli shows evidence of selection bias, either to reduce fitness costs, or to increase drug resistance. MarR is a repressor protein that regulates, via MarA, expression of the Mar regulon, including the multidrug efflux pump AcrAB-TolC. METHODS: Isogenic strains carrying 36 different marR alleles identified in resistant clinical isolates, or selected for resistance in vitro, were constructed. Drug susceptibility and relative fitness in growth competition assays were measured for all strains. The expression level of marA, and of various efflux pump components, as a function of specific mutations in marR, was measured by qPCR. RESULTS: The spectrum of genetic alterations in marR in clinical isolates is strongly biased against inactivating mutations. In general, the alleles found in clinical isolates conferred a lower level of resistance and imposed a lower growth fitness cost than mutations selected in vitro. The level of expression of MarA correlated well with the MIC of ciprofloxacin. This supports the functional connection between mutations in marR and reduced susceptibility to ciprofloxacin. CONCLUSIONS: Mutations in marR selected in ciprofloxacin-resistant clinical isolates are strongly biased against inactivating mutations. Selection favours mutant alleles that have the lowest fitness costs, even though these cause only modest reductions in drug susceptibility. This suggests that selection for high relative fitness is more important than selection for increased resistance in determining which alleles of marR will be selected in resistant clinical isolates.

EMP3 is induced by TWIST1/2 and regulates epithelial-to-mesenchymal transition of gastric cancer cells.

In this study, we aimed to explore new downstream effectors of TWIST1/2 in inducing epithelial-to-mesenchymal transition in gastric cancer. Bioinformatic data mining was performed using data in The Cancer Genome Atlas Stomach Adenocarcinoma. Survival curves were generated using Kaplan-Meier plotter. Gastric cancer cell lines (AGS and SGC-7901) were used as in vitro cell model to investigate the regulative effect of TWIST1/2 on epithelial membrane protein 3 expression and the progression of epithelial-to-mesenchymal transition. Results showed that TWIST1 and TWIST2 are usually co-upregulated in patients with primary gastric cancer. High TWIST1 expression is associated with worse overall survival (hazard ratio = 1.26; 95% confidence interval = 1.06-1.49; p = 0.007) and also worse first progression-free survival (hazard ratio = 1.47; 95% confidence interval = 1.18-1.82; p < 0.0001). Similarly, high TWIST2 expression is associated with unfavorable overall survival (hazard ratio = 1.71; 95% confidence interval = 1.32-2.22; p < 0.0001) and progression-free survival (hazard ratio = 1.99; 95% confidence interval = 1.45-2.72; p < 0.0001). Epithelial membrane protein 3 is negatively correlated to CDH1 expression (Pearson's r = -0.46) but is positively correlated to VIM expression (Pearson's r = 0.83). Knockdown of epithelial membrane protein 3 significantly increased E-cadherin but significantly decreased Vimentin expression in AGS cells. Gastric cancer patients with metastasis have significantly higher epithelial membrane protein 3 expression than the cases without metastasis. In addition, high epithelial membrane protein 3 expression is associated with worse overall survival (hazard ratio = 2.59; 95% confidence interval = 2.06-3.26; p < 0.0001) and also worse progression-free survival (hazard ratio = 2.21; 95% confidence interval = 1.78-2.74; p < 0.0001). In conclusion, epithelial membrane protein 3 is a downstream effector of TWIST1/2 in inducing epithelial-to-mesenchymal transition in gastric cancer. Epithelial membrane protein 3 upregulation might be associated with gastric cancer metastasis and is a potential indicator of unfavorable overall survival and progression-free survival in gastric cancer patients.

Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice.

The zinc finger protein CTCF has been invoked in establishing boundaries between genes, thereby controlling spatial and temporal enhancer activities. However, there is limited genetic evidence to support the concept that these boundaries restrict the search space of enhancers. We have addressed this question in the casein locus containing five mammary and two non-mammary genes under the control of at least seven putative enhancers. We have identified two CTCF binding sites flanking the locus and two associated with a super-enhancer. Individual deletion of these sites from the mouse genome did not alter expression of any of the genes. However, deletion of the border CTCF site separating the Csn1s1 mammary enhancer from neighboring genes resulted in the activation of Sult1d1 at a distance of more than 95 kb but not the more proximal and silent Sult1e1 gene. Loss of this CTCF site led to de novo interactions between the Sult1d1 promoter and several enhancers in the casein locus. Our study demonstrates that only one out of the four CTCF sites in the casein locus had a measurable in vivo activity. Studies on additional loci are needed to determine the biological role of CTCF sites associated with enhancers.

High binding affinity of repressor IolR avoids costs of untimely induction of myo-inositol utilization by Salmonella Typhimurium.

Growth of Salmonella enterica serovar Typhimurium strain 14028 with myo-inositol (MI) is characterized by a bistable phenotype that manifests with an extraordinarily long (34 h) and variable lag phase. When cells were pre-grown in minimal medium with MI, however, the lag phase shortened drastically to eight hours, and to six hours in the absence of the regulator IolR. To unravel the molecular mechanism behind this phenomenon, we investigated this repressor in more detail. Flow cytometry analysis of the iolR promoter at a single cell level demonstrated bistability of its transcriptional activation. Electrophoretic mobility shift assays were used to narrow the potential binding region of IolR and identified at least two binding sites in most iol gene promoters. Surface plasmon resonance spectroscopy quantified IolR binding and indicated its putative oligomerization and high binding affinity towards specific iol gene promoters. In competitive assays, the iolR deletion mutant, in which iol gene repression is abolished, showed a severe growth disadvantage of ~15% relative to the parental strain in rich medium. We hypothesize that the strong repression of iol gene transcription is required to maintain a balance between metabolic flexibility and fitness costs, which follow the inopportune induction of an unusual metabolic pathway.

Assessment of mutations in KCNN2 and ZNF135 to patient neurological symptoms.

Exome sequencing from a patient with neurological and developmental symptoms revealed two mutations in separate genes. One was a homozygous transition mutation that results in an in-frame, premature translational stop codon in the ZNF135 gene predicted to encode a transcriptional repressor. Another mutation was heterozygous, a single nucleotide duplication in the KCNN2 gene that encodes a Ca-activated K channel, SK2, and leads to a translational frame shift and a premature stop codon. Heterologous expression studies, brain slice recordings, and coordination tests from a transgenic mouse line carrying the SK2 mutation suggest that it does not contribute to the patient's symptoms. ZNF135 is expressed in human brain and it is likely that the homozygous mutation underlies the human phenotype.

Decreased zinc in the development and progression of malignancy: an important common relationship and potential for prevention and treatment of carcinomas.

INTRODUCTION: Efficacious chemotherapy does not exist for treatment or prevention of prostate, liver, and pancreatic carcinomas, and some other cancers that exhibit decreased zinc in malignancy. Zinc treatment offers a potential solution; but its support has been deterred by adverse bias. Areas covered: 1. The clinical and experimental evidence for the common ZIP transporter/Zn down regulation in these cancers. 2. The evidence for a zinc approach to prevent and/or treat these carcinomas. 3. The issues that introduce bias against support for the zinc approach. Expert opinion: ZIP/Zn downregulation is a clinically established common event in prostate, hepatocellular and pancreatic cancers. 2. Compelling evidence supports the plausibility that a zinc treatment regimen will prevent development of malignancy and termination of progressing malignancy in these cancers; and likely other carcinomas that exhibit decreased zinc. 3. Scientifically-unfounded issues that oppose this ZIP/Zn relationship have introduced bias against support for research and funding of a zinc treatment approach. 4. The clinically-established and supporting experimental evidence provide the scientific credibility that should dictate the support for research and funding of a zinc approach for the treatment and possible prevention of these cancers. 5. This is in the best interest of the medical community and the public-at-large.