Zarko Manojlovic

Nicotine Exposure During Adolescence Induces a Depression-Like State in Adulthood

There is a strong link between tobacco consumption and mood disorders. It has been suggested that afflicted individuals smoke to manage mood, however, there is evidence indicating that tobacco consumption can induce negative mood. This study was designed to investigate whether nicotine exposure during adolescence influences emotionality/behavioral functioning later in life. Adolescent (postnatal days, PD 30–44) male rats were treated with twice-daily injections of nicotine (0, 0.16, 0.32, or 0.64 mg/kg) for 15 consecutive days, and their behavioral reactivity to various behavioral paradigms (the elevated plus maze (EPM), sucrose preference, locomotor activity in the open field, and forced swim test (FST) was assessed 24 h (short term) or 1-month (long term) after exposure. Separate groups of adult rats received nicotine (0.32 mg/kg) to control for age-dependent effects. We report that nicotine exposure during adolescence—but not adulthood—leads to a depression-like state manifested in decreased sensitivity to natural reward (sucrose), and enhanced sensitivity to stress- (FST) and anxiety-eliciting situations (EPM) later in life. Our data show that behavioral dysregulation can emerge 1 week after drug cessation, and that a single day of nicotine exposure during adolescence can be sufficient to precipitate a depression-like state in adulthood. We further demonstrate that these deficits can be normalized by subsequent nicotine (0.32 mg/kg) or antidepressant (ie fluoxetine or bupropion; 10 mg/kg) treatment in adulthood. These data suggest that adolescent exposure to nicotine results in a negative emotional state rendering the organism significantly more vulnerable to the adverse effects of stress. Within this context, our findings, together with others indicating that nicotine exposure during adolescence enhances risk for addiction later in life, could serve as a potential model of comorbidity.

Extracellular Signal-Regulated Kinase-2 within the Ventral Tegmental Area Regulates Responses to Stress

Neurotrophic factors and their signaling pathways have been implicated in the neurobiological adaptations in response to stress and the regulation of mood-related behaviors. A candidate signaling molecule implicated in mediating these cellular responses is the extracellular signal-regulated kinase (ERK1/2), although its functional role in mood regulation remains to be fully elucidated. Here we show that acute (1 d) or chronic (4 weeks) exposure to unpredictable stress increases phosphorylation of ERK1/2 and of two downstream targets (ribosomal S6 kinase and mitogen- and stress-activated protein kinase 1) within the ventral tegmental area (VTA), an important substrate for motivated behavior and mood regulation. Using herpes simplex virus-mediated gene transfer to assess the functional significance of this ERK induction, we show that overexpressing ERK2 within the VTA increases susceptibility to stress as measured in the forced swim test, responses to unconditioned nociceptive stimuli, and elevated plus maze in Sprague Dawley male rats, and in the tail suspension test and chronic social defeat stress procedure in C57BL/6 male mice. In contrast, blocking ERK2 activity in the VTA produces stress-resistant behavioral responses in these same assays and also blocks a chronic stress-induced reduction in sucrose preference. The effects induced by ERK2 blockade were accompanied by decreases in the firing frequency of VTA dopamine neurons, an important electrophysiological hallmark of resilient-like behavior. Together, these results strongly implicate a role for ERK2 signaling in the VTA as a key modulator of responsiveness to stress and mood-related behaviors.

RFX7 is required for the formation of cilia in the neural tube.

Regulatory Factor X (RFX) transcription factors are important for development and are likely involved in the pathogenesis of serious human diseases including ciliopathies. While seven RFX genes have been identified in vertebrates and several RFX transcription factors have been reported to be regulators of ciliogenesis, the role of RFX7 in development including ciliogenesis is not known. Here we show that RFX7 in Xenopus laevis is expressed in the neural tube, eye, otic vesicles, and somites. Knockdown of RFX7 in Xenopus embryos resulted in a defect of ciliogenesis in the neural tube and failure of neural tube closure. RFX7 controlled the formation of cilia by regulating the expression of RFX4 gene, which has been reported to be required for ciliogenesis in the neural tube. Moreover, ectopic expression of Foxj1, which is a master regulator of motile cilia formation, suppressed the expression of RFX4 but not RFX7. Taken together, RFX7 plays an important role in the process of neural tube closure at the top of the molecular cascade which controls ciliogenesis in the neural tube.

Serine-threonine kinase receptor associated protein (STRAP) regulates translation of type I collagen mRNAs

ABSTRACT Type I collagen is the most abundant protein in the human body and is composed of two α1(I) and one α2(I) polypeptides which assemble into a triple helix. For the proper assembly of the collagen triple helix, the individual polypeptides must be translated in coordination. Here, we show that serine-threonine kinase receptor-associated protein (STRAP) is tethered to collagen mRNAs by interaction with LARP6. LARP6 is a protein which directly binds the 5′ stem-loop (5′SL) present in collagen α1(I) and α2(I) mRNAs, but it interacts with STRAP with its C-terminal domain, which is not involved in binding 5′SL. Being tethered to collagen mRNAs, STRAP prevents unrestricted translation, primarily that of collagen α2(I) mRNAs, by interacting with eukaryotic translation initiation factor 4A (eIF4A). In the absence of STRAP, more collagen α2(I) mRNA can be pulled down with eIF4A, and collagen α2(I) mRNA is unrestrictedly loaded onto the polysomes. This results in an imbalance of synthesis of α1(I) and α2(I) polypeptides, in hypermodifications of α1(I) polypeptide, and in inefficient assembly of the polypeptides into a collagen trimer and their secretion as monomers. These defects can be partially restored by supplementing STRAP. Thus, we discovered STRAP as a novel regulator of the coordinated translation of collagen mRNAs.

Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma.

Genomic analyses of cutaneous melanoma (CM) have yielded biological and therapeutic insights, but understanding of non-ultraviolet (UV)-derived CMs remains limited. Deeper analysis of acral lentiginous melanoma (ALM), a rare sun-shielded melanoma subtype associated with worse survival than CM, is needed to delineate non-UV oncogenic mechanisms. We thus performed comprehensive genomic and transcriptomic analysis of 34 ALM patients. Unlike CM, somatic alterations were dominated by structural variation and absence of UV-derived mutation signatures. Only 38% of patients demonstrated driver BRAF/NRAS/NF1 mutations. In contrast with CM, we observed PAK1 copy gains in 15% of patients, and somatic TERT translocations, copy gains, and missense and promoter mutations, or germline events, in 41% of patients. We further show that in vitro TERT inhibition has cytotoxic effects on primary ALM cells. These findings provide insight into the role of TERT in ALM tumorigenesis and reveal preliminary evidence that TERT inhibition represents a potential therapeutic strategy in ALM.

Tacrolimus (FK506) Prevents Early Stages of Ethanol Induced Hepatic Fibrosis by Targeting LARP6 Dependent Mechanism of Collagen Synthesis

Tacrolimus (FK506) is a widely used immunosuppressive drug. Its effects on hepatic fibrosis have been controversial and attributed to immunosuppression. We show that in vitro FK506, inhibited synthesis of type I collagen polypeptides, without affecting expression of collagen mRNAs. In vivo, administration of FK506 at a dose of 4 mg/kg completely prevented development of alcohol/carbon tetrachloride induced liver fibrosis in rats. Activation of hepatic stellate cells (HSCs) was absent in the FK506 treated livers and expression of collagen α2(I) mRNA was at normal levels. Collagen α1(I) mRNA was increased in the FK506 treated livers, but this mRNA was not translated into α1(I) polypeptide. No significant inflammation was associated with the fibrosis model used. FK506 binding protein 3 (FKBP3) is one of cellular proteins which binds FK506 with high affinity. We discovered that FKBP3 interacts with LARP6 and LARP6 is the major regulator of translation and stability of collagen mRNAs. In the presence of FK506 the interaction between FKBP3 and LARP6 is weakened and so is the pull down of collagen mRNAs with FKBP3. We postulate that FK506 inactivates FKBP3 and that lack of interaction of LARP6 and FKBP3 results in aberrant translation of collagen mRNAs and prevention of fibrosis. This is the first report of such activity of FK506 and may renew the interest in using this drug to alleviate hepatic fibrosis.

Comprehensive molecular profiling of 718 Multiple Myelomas reveals significant differences in mutation frequencies between African and European descent cases

Multiple Myeloma (MM) is a plasma cell malignancy with significantly greater incidence and mortality rates among African Americans (AA) compared to Caucasians (CA). The overall goal of this study is to elucidate differences in molecular alterations in MM as a function of self-reported race and genetic ancestry. Our study utilized somatic whole exome, RNA-sequencing, and correlated clinical data from 718 MM patients from the Multiple Myeloma Research Foundation CoMMpass study Interim Analysis 9. Somatic mutational analyses based upon self-reported race corrected for ancestry revealed significant differences in mutation frequency between groups. Of interest, BCL7A, BRWD3, and AUTS2 demonstrate significantly higher mutation frequencies among AA cases. These genes are all involved in translocations in B-cell malignancies. Moreover, we detected a significant difference in mutation frequency of TP53 and IRF4 with frequencies higher among CA cases. Our study provides rationale for interrogating diverse tumor cohorts to best understand tumor genomics across populations.

A method to reduce ancestry related germline false positives in tumor only somatic variant calling

BackgroundSignificant clinical and research applications are driving large scale adoption of individualized tumor sequencing in cancer in order to identify tumors-specific mutations. When a matched germline sample is available, somatic mutations may be identified using comparative callers. However, matched germline samples are frequently not available such as with archival tissues, which makes it difficult to distinguish somatic from germline variants. While population databases may be used to filter out known germline variants, recent studies have shown private germline variants result in an inflated false positive rate in unmatched tumor samples, and the number germline false positives in an individual may be related to ancestry.MethodsFirst, we examined the relationship between the germline false positives and ancestry. Then we developed and implemented a tumor only caller (LumosVar) that leverages differences in allelic frequency between somatic and germline variants in impure tumors. We used simulated data to systematically examine how copy number alterations, tumor purity, and sequencing depth should affect the sensitivity of our caller. Finally, we evaluated the caller on real data.ResultsWe find the germline false-positive rate is significantly higher for individuals of non-European Ancestry largely due to the limited diversity in public polymorphism databases and due to population-specific characteristics such as admixture or recent expansions. Our Bayesian tumor only caller (LumosVar) is able to greatly reduce false positives from private germline variants, and our sensitivity is similar to predictions based on simulated data.ConclusionsTaken together, our results suggest that studies of individuals of non-European ancestry would most benefit from our approach. However, high sensitivity requires sufficiently impure tumors and adequate sequencing depth. Even in impure tumors, there are copy number alterations that result in germline and somatic variants having similar allele frequencies, limiting the sensitivity of the approach. We believe our approach could greatly improve the analysis of archival samples in a research setting where the normal is not available.

Histologic Validation of 11Carbon-Acetate Positron Emission Tomography/Computer Tomography in Detecting Lymph Node Metastases in Prostate Cancer

Purpose: Conventional imaging cannot definitively detect nodal metastases of prostate cancer. We histologically validated 11C-acetate positron emission tomography/computerized tomography to identify nodal metastases, examining prostate cancer factors that influence detection rates. Materials and Methods: Patients with 11C-acetate avid positron emission tomography/computerized tomography imaged pelvic/retroperitoneal lymph nodes underwent high extended robotic lymphadenectomy. A standardized mapping template comprising 8 predetermined anatomical regions was dissected during lymphadenectomy, allowing for matched, region based analysis and comparison of imaging and histological data. Results: In 25 patients a total of 2,149 lymph nodes were excised (mean 86 per patient, range 27 to 136) and 528 (22%) harbored metastases (mean 21 positive nodes per patient, range 0 to 109). A total of 174 anatomical regions had matching imaging histological data. 11C-acetate positron emission tomography/computerized tomography accurately identified 48 node-positive regions and accurately ruled out 88 regions as metastasis-free. 11C-acetate sensitivity, specificity, and positive and negative predictive values were 67%, 84%, 74% and 79%, respectively. An increasing, histologically measured metastatic lesion size in long axis diameter of 5 or less, 6 to 10, 11 to 15, 16 to 20 and 21 mm or greater correlated with improved 11C-acetate detection rates of 45%, 62%, 81%, 89% and 100%, respectively. Each standard uptake value unit increase correlated with a 1.9 mm increase in nodal long axis diameter (p <0.001) and a 1.2 mm increase in short axis diameter (p <0.001). Positive 11C-acetate positron emission tomography/computerized tomography findings correlated with histological lymph node size (long axis diameter 12 mm and short axis diameter 6 mm), metastatic lesion size (long axis diameter 11 mm and short axis diameter 6 mm) and extranodal extension (positive 88% vs false-negative 58%, p = 0.005). Conclusions: 11C-acetate positron emission tomography/computerized tomography can identify prostate cancer metastatic nodal disease. However, it underestimates the true cephalad extent of nodal involvement, performing better in the pelvis than in the retroperitoneum. Standard uptake value, histological nodal size, intranodal metastasis size and extranodal extension correlate with cancer bearing nodes.

Lack of APC somatic mutation is associated with early-onset colorectal cancer in African Americans

African Americans (AAs) have higher incidence and mortality rates of colorectal cancer (CRC) compared with other US populations. They present with more right-sided, microsatellite stable disease and are diagnosed at earlier ages compared with non-Hispanic Whites (NHWs). To gain insight into these trends, we conducted exome sequencing (n = 45), copy number (n = 33) and methylation analysis (n = 11) of microsatellite stable AA CRCs. Results were compared with data from The Cancer Genome Atlas (TCGA). Two of the 45 tumors contained POLE mutations. In the remaining 43 tumors, only 27 (63%) contained loss-of-function mutations in APC compared with 80% of TCGA NHW CRCs. APC-mutation-negative CRCs were associated with an earlier onset of CRC (P = 0.01). They were also associated with lower overall mutation burden, fewer copy number variants and a DNA methylation signature that was distinct from the CpG island methylator phenotype characterized in microsatellite unstable disease. Three of the APC-mutation-negative CRCs had loss-of-function mutations in BCL9L. Mutations in driver genes identified by TCGA exome analysis were less frequent in AA CRC cases than TCGA NHWs. Genes that regulate the WNT signaling pathway, including SOX9, GATA6, TET1, GLIS1 and FAT1, were differentially hypermethylated in APC-mutation-negative CRCs, suggesting a novel mechanism for cancer development in these tumors. In summary, we have identified a subtype of CRC that is associated with younger age of diagnosis, lack of APC mutation, microsatellite and chromosome stability, lower mutation burden and distinctive methylation changes.