Dynamic genetic regulation of gene expression during cellular differentiation.

Genetic regulation of gene expression is dynamic, as transcription can change during cell differentiation and across cell types. We mapped expression quantitative trait loci (eQTLs) throughout differentiation to elucidate the dynamics of genetic effects on cell type-specific gene expression. We generated time-series RNA sequencing data, capturing 16 time points during the differentiation of induced pluripotent stem cells to cardiomyocytes, in 19 human cell lines. We identified hundreds of dynamic eQTLs that change over time, with enrichment in enhancers of relevant cell types. We also found nonlinear dynamic eQTLs, which affect only intermediate stages of differentiation and cannot be found by using data from mature tissues. These fleeting genetic associations with gene regulation may explain some of the components of complex traits and disease. We highlight one example of a nonlinear eQTL that is associated with body mass index.

Discovery of potent molecular chimera (CM358) to treat human metastatic melanoma.

The resistance of cancer cells to chemotherapeutic agents, whether through intrinsic mechanisms or developed resistance, motivates the search for new chemotherapeutic strategies. In the present report, we demonstrate a facile synthetic strategy towards the discovery of new anti-cancer substances. This strategy is based on simple covalent coupling between known anti-cancer drugs, which results in novel 'chimeric' small molecules. One of these novel compounds, CM358, is the product of an amide bond formation between the known Topoisomerase II (Topo II) inhibitor amonafide (AM) and the known DNA mustard alkylator chlorambucil (CLB). It demonstrates significant enhanced cytotoxicity over an equimolar mixture of AM and CLB in various cancer cell lines and in a xenograft model of human metastatic melanoma. Topo II inhibition as well as in silico docking studies suggest that CM358 is a stronger Topo II binder than AM. This may be attributed, at least partially, to the placement of the CLB moiety in a favorable orientation with respect to DNA cross-linking with nearby guanines. In a human metastatic melanoma (WM 266-4) xenograft model, this compound was profoundly superior to a mixture of AM and CLB in reduction of tumor growth, maintenance of body weight and extension of overall survival.

Synthesis, biological studies and molecular dynamics of new anticancer RGD-based peptide conjugates for targeted drug delivery.

New cyclic RGD peptide-anticancer agent conjugates, with different chemical functionalities attached to the parent peptide were synthesized in order to evaluate their biological activities and to provide a comparative study of their drug release profiles. The Integrin binding c(RGDfK) penta-peptide was used for the synthesis of Camptothecin (CPT) carbamate and Chlorambucil (CLB) amide conjugates. Substitution of the amino acid Lys with Ser resulted in a modified c(RGDfS) with a new attachment site, which enabled the synthesis of an ester CLB conjugate. Functional versatility of the conjugates was reflected in the variability of their drug release profiles, while the conserved RGD sequence of a selective binding to the αv integrin family, likely preserved their recognition by the Integrin and consequently their favorable toxicity towards targeted cancer cells. This hypothesis was supported by a computational analysis suggesting that all conjugates occupy conformational spaces similar to that of the Integrin bound bio-active parent peptide.

Dual-drug RGD conjugates provide enhanced cytotoxicity to melanoma and non-small lung cancer cells.

To enhance the efficacy of targeted drug delivery, four new peptide-ligand conjugates were synthesized, each consisting of a cyclic RGDfK penta-peptide loaded with two anticancer drugs. The drug release profiles in different media of these new compounds and their cytotoxic activity against melanoma and non-small lung cancer cell lines were evaluated and compared with those of their singly loaded analogs. The cyclic RGDfK penta-peptide was selected as a targeting moiety because of its high affinity and selectivity to the αv ß3 integrin receptor, which is frequently over-expressed in various types of cancer cells. The peptide's core was modified at the side chain of its Lys residue by coupling it with a sixth amino acid (AA) - either Lys (5a) or Ser (5b) (Lys/Ser splitter), resulting in two functional sites which enabled the loading of two therapeutic equivalents onto a single targeting carrier. Using Lys as a splitter resulted in two primary amines. Consequently, conjugates 1a and 1b were synthesized by coupling of 2 Chlorambucils (CLBs) or 2 Camptothecins (CPTs), respectively, to the primary amines of 5a. Conjugate 1c was synthesized from 5b by loading two equivalents of CLB on the amine and the hydroxyl of the Ser splitter, resulting in a homodimeric system with two distinct conjugation sites - amide and ester. The heterodimeric conjugate 1d of CLB and CPT was synthesized by loading each one of the primary amines of 5a with two different drugs - CLB and CPT. The doubling of drug equivalents loaded onto the targeting peptide correlated with enhanced cytotoxic efficacy of the conjugates towards cancer cells. The versatility of chemical linkages of the drugs to the peptides resulted in conjugates with different drug release profiles. Molecular dynamics simulations performed on conjugate 1d demonstrated that this compound occupies a conformational space similar to the bio-active conformation of an integrin-bound cyclic RGD peptide reference peptide (c(RGDf(NMe)V). The modified position in 1d (relative to the reference peptide) points away from the integrin, leading us to hypothesize that this peptide binds the integrin in a manner similar to that of the reference peptide thereby fulfilling a crucial requirement for targeted delivery. The strategy of dual drug loading on a single peptide carrier, gives rise to drugs with different mechanisms of action and release profiles, thus substantially increasing the efficacy of selective killing of tumor cells and while reducing the risk of the development of drug resistance. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 160-171, 2016.

DAPK2 is a novel regulator of mTORC1 activity and autophagy.

Autophagy is a tightly regulated catabolic process, which is upregulated in cells in response to many different stress signals. Inhibition of mammalian target of rapmaycin complex 1 (mTORC1) is a crucial step in induction of autophagy, yet the mechanisms regulating the fine tuning of its activity are not fully understood. Here we show that death-associated protein kinase 2 (DAPK2), a Ca(2+)-regulated serine/threonine kinase, directly interacts with and phosphorylates mTORC1, and has a part in suppressing mTOR activity to promote autophagy induction. DAPK2 knockdown reduced autophagy triggered either by amino acid deprivation or by increases in intracellular Ca(2+) levels. At the molecular level, DAPK2 depletion interfered with mTORC1 inhibition caused by these two stresses, as reflected by the phosphorylation status of mTORC1 substrates, ULK1 (unc-51-like kinase 1), p70 ribosomal S6 kinase and eukaryotic initiation factor 4E-binding protein 1. An increase in mTORC1 kinase activity was also apparent in unstressed cells that were depleted of DAPK2. Immunoprecipitated mTORC1 from DAPK2-depleted cells showed increased kinase activity in vitro, an indication that DAPK2 regulation of mTORC1 is inherent to the complex itself. Indeed, we found that DAPK2 associates with components of mTORC1, as demonstrated by co-immunoprecipitation with mTOR and its complex partners, raptor (regulatory-associated protein of mTOR) and ULK1. DAPK2 was also able to interact directly with raptor, as shown by recombinant protein-binding assay. Finally, DAPK2 was shown to phosphorylate raptor in vitro. This phosphorylation was mapped to Ser721, a site located within a highly phosphorylated region of raptor that has previously been shown to regulate mTORC1 activity. Thus, DAPK2 is a novel kinase of mTORC1 and is a potential new member of this multiprotein complex, modulating mTORC1 activity and autophagy levels under stress and steady-state conditions.

The interference of medical radionuclides with occupational in vivo gamma spectrometry.

Radiation workers undergo routine monitoring for the evaluation of external and internal radiation exposures. The monitoring of internal exposures involves gamma spectrometry of the whole body (whole body counting) and measurements of excreta samples. Medical procedures involving internal administration of radioactive radionuclides are widely and commonly used. Medical radionuclides are typically short-lived, but high activities are generally administered, whereas occupational radionuclides are mostly long-lived and, if present, are found generally in relatively smaller quantities. The aim of the present work was to study the interference of some common medical radionuclides (201Tl, 9mTc, 57Co, and 131I) with the detection of internal occupational exposures to natural uranium and to 137Cs. Workers having undergone a medical procedure with one of the radionuclides mentioned above were asked to give frequent urine samples and to undergo whole body and thyroid counting with phoswich detectors operated at the Nuclear Research Center Negev. Urine and whole body counting monitoring were continued as long as radioactivity was detectable by gamma spectrometry. The results indicate that the activity of medical radionuclides may interfere with interpretation of occupational intakes for months after administration.

Mechanisms for evolving hypervariability: the case of conopeptides.

Hypervariability is a prominent feature of large gene families that mediate interactions between organisms, such as venom-derived toxins or immunoglobulins. In order to study mechanisms for evolution of hypervariability, we examined an EST-generated assemblage of 170 distinct conopeptide sequences from the venoms of five species of marine Conus snails. These sequences were assigned to eight gene families, defined by conserved elements in the signal domain and untranslated regions. Order-of-magnitude differences were observed in the expression levels of individual conopeptides, with five to seven transcripts typically comprising over 50% of the sequenced clones in a given species. The conopeptide precursor alignments revealed four striking features peculiar to the mature peptide domain: (1) an accelerated rate of nucleotide substitution, (2) a bias for transversions over transitions in nucleotide substitutions, (3) a position-specific conservation of cysteine codons within the hypervariable region, and (4) a preponderance of nonsynonymous substitutions over synonymous substitutions. We propose that the first three observations argue for a mutator mechanism targeted to mature domains in conopeptide genes, combining a protective activity specific for cysteine codons and a mutagenic polymerase that exhibits transversion bias, such as DNA polymerase V. The high D:(n)/D:(s) ratio is consistent with positive or diversifying selection, and further analyses by intraspecific/interspecific gene tree contingency tests weakly support recent diversifying selection in the evolution of conopeptides. Since only the most highly expressed transcripts segregate in gene trees according to the feeding specificity of the species, diversifying selection might be acting primarily on these sequences. The combination of a targeted mutator mechanism to generate high variability with the subsequent action of diversifying selection on highly expressed variants might explain both the hypervariability of conopeptides and the large number of unique sequences per species.

Mouse-human orthology relationships in an olfactory receptor gene cluster.

The olfactory receptor (OR) subgenome harbors the largest known gene family in mammals, disposed in clusters on numerous chromosomes. One of the best characterized OR clusters, located at human chromosome 17p13.3, has previously been studied by us in human and in other primates, revealing a conserved set of 17 OR genes. Here, we report the identification of a syntenic OR cluster in the mouse and the partial DNA sequence of many of its OR genes. A probe for the mouse M5 gene, orthologous to one of the OR genes in the human cluster (OR17-25), was used to isolate six PAC clones, all mapping by in situ hybridization to mouse chromosome 11B3-11B5, a region of shared synteny with human chromosome 17p13.3. Thirteen mouse OR sequences amplified and sequenced from these PACs allowed us to construct a putative physical map of the OR gene cluster at the mouse Olfr1 locus. Several points of evidence, including a strong similarity in subfamily composition and at least four cases of gene orthology, suggest that the mouse Olfr1 and the human 17p13.3 clusters are orthologous. A detailed comparison of the OR sequences within the two clusters helps trace their independent evolutionary history in the two species. Two types of evolutionary scenarios are discerned: cases of "true orthologous genes" in which high sequence similarity suggests a shared conserved function, as opposed to instances in which orthologous genes may have undergone independent diversification in the realm of "free reign" repertoire expansion.

Dichotomy of single-nucleotide polymorphism haplotypes in olfactory receptor genes and pseudogenes.

Substantial efforts are focused on identifying single-nucleotide polymorphisms (SNPs) throughout the human genome, particularly in coding regions (cSNPs), for both linkage disequilibrium and association studies. Less attention, however, has been directed to the clarification of evolutionary processes that are responsible for the variability in nucleotide diversity among different regions of the genome. We report here the population sequence diversity of genomic segments within a 450-kb cluster of olfactory receptor (OR) genes on human chromosome 17. We found a dichotomy in the pattern of nucleotide diversity between OR pseudogenes and introns on the one hand and the closely interspersed intact genes on the other. We suggest that weak positive selection is responsible for the observed patterns of genetic variation. This is inferred from a lower ratio of polymorphism to divergence in genes compared with pseudogenes or introns, high non-synonymous substitution rates in OR genes, and a small but significant overall reduction in variability in the entire OR gene cluster compared with other genomic regions. The dichotomy among functionally different segments within a short genomic distance requires high recombination rates within this OR cluster. Our work demonstrates the impact of weak positive selection on human nucleotide diversity, and has implications for the evolution of the olfactory repertoire.

Identification and characterization of coding single-nucleotide polymorphisms within a human olfactory receptor gene cluster.

Single-nucleotide polymorphisms (SNPs) were studied in 15 olfactory receptor (OR) coding regions, one control region and two noncoding sequences all residing within a 412 kb OR gene cluster on human chromosome 17p13.3, as well as in other G-protein coupled receptors (GPCRs). A total of 26 SNPs were identified in ORs, 21 of which are coding SNPs (cSNPs). The mean nucleotide diversity of OR coding regions was 0.078% (ranging from 0 to 0.16%), which is about twice higher than that of other GPCRs, and similar to the nucleotide diversity levels of noncoding regions along the human genome. The high polymorphism level in the OR coding regions might be due to a weak positive selection pressure acting on the OR genes. In two cases, OR genes have been found to share the same cSNP. This could be explained by recent gene conversion events, which might be a part of a concerted evolution mechanism acting on the OR superfamily. Using the genotype data of 85 unrelated individuals in 15 SNPs, we found linkage disequilibrium (LD) between pairs of SNPs located on the centromeric part of the cluster. On the other hand, no LD was found between SNPs located on the telomeric part of the cluster, suggesting the presence of several hot-spots for recombination within this cluster. Thus, different regions of this gene cluster may have been subject to different recombination rates.

An outbreak of shigellosis in an ultra-orthodox Jewish community.

An outbreak of Shigellosis due to Shigella sonnei, S. flexnerii and S. boydii in an ultra-religious Jewish community in Southern Israel is described. The source of epidemic was traced to vegetables bought from a single source. The importance of epidemic and endemic modes of spread of Shigella is discussed.