Abstract: Under the instruction of cell-fate–determining, DNA-binding transcription factors, chromatin-modifying enzymes mediate and maintain cell states throughout development in multicellular organisms. Currently, small molecules modulating the activity of several classes of chromatin-modifying enzymes are available, including clinically approved histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors. We describe the genome-wide expression changes induced by 29 compounds targeting HDACs, DNMTs, histone lysine methyltransferases (HKMTs), and protein arginine methyltransferases (PRMTs) in pancreatic α- and β-cell lines. HDAC inhibitors regulate several hundred transcripts irrespective of the cell type, with distinct clusters of dissimilar activity for hydroxamic acids and orthoamino anilides. In contrast, compounds targeting histone methyltransferases modulate the expression of restricted gene sets in distinct cell types. For example, we find that G9a/GLP methyltransferase inhibitors selectively up-regulate the cholesterol biosynthetic pathway in pancreatic but not liver cells. These data suggest that, despite their conservation across the entire genome and in different cell types, chromatin pathways can be targeted to modulate the expression of selected transcripts.

Abstract: The pathological end-state of Parkinson disease is well described from postmortem tissue, but there remains a pressing need to define early functional changes to susceptible neurons and circuits. In particular, mechanisms underlying the vulnerability of the dopamine neurons of the substantia nigra pars compacta (SNc) and the importance of protein aggregation in driving the disease process remain to be determined. To better understand the sequence of events occurring in familial and sporadic Parkinson disease, we generated bacterial artificial chromosome transgenic mice ( SNCA -OVX) that express wild-type α-synuclein from the complete human SNCA locus at disease-relevant levels and display a transgene expression profile that recapitulates that of endogenous α-synuclein. SNCA -OVX mice display age-dependent loss of nigrostriatal dopamine neurons and motor impairments characteristic of Parkinson disease. This phenotype is preceded by early deficits in dopamine release from terminals in the dorsal, but not ventral, striatum. Such neurotransmission deficits are not seen at either noradrenergic or serotoninergic terminals. Dopamine release deficits are associated with an altered distribution of vesicles in dopaminergic axons in the dorsal striatum. Aged SNCA -OVX mice exhibit reduced firing of SNc dopamine neurons in vivo measured by juxtacellular recording of neurochemically identified neurons. These progressive changes in vulnerable SNc neurons were observed independently of overt protein aggregation, suggesting neurophysiological changes precede, and are not driven by, aggregate formation. This longitudinal phenotyping strategy in SNCA -OVX mice thus provides insights into the region-specific neuronal disturbances preceding and accompanying Parkinson disease.

Abstract: Active Src localization at focal adhesions (FAs) is essential for cell migration. How this pool is linked mechanistically to the large pool of Src at late endosomes (LEs)/lysosomes (LY) is not well understood. Here, we used inducible Tsg101 gene deletion, TSG101 knockdown, and dominant-negative VPS4 expression to demonstrate that the localization of activated cellular Src and viral Src at FAs requires the endosomal-sorting complexes required for transport (ESCRT) pathway. Tsg101 deletion also led to impaired Src-dependent activation of STAT3 and focal adhesion kinase and reduced cell migration. Impairment of the ESCRT pathway or Rab7 function led to the accumulation of active Src at aberrant LE/LY compartments followed by its loss. Analyses using fluorescence recovery after photo-bleaching show that dynamic mobility of Src in endosomes is ESCRT pathway-dependent. These results reveal a critical role for an ESCRT pathway-dependent LE/LY trafficking step in Src function by promoting localization of active Src to FAs.

Abstract: We showed here that the radiochemical and radiobiological mechanisms and outcomes of fs IR laser-induced filamentation are equivalent to conventional ionizing radiation. Moreover, we have shown here that this (to our knowledge) conceptually unique approach to cancer therapy ( 5 ) may bring us one step closer to achieving the ultimate goal of radiation therapy: maximizing the energy dose inside the tumor volume while not exposing healthy tissue. Our IR laser pulses produce high-density avalanches of low-energy electrons via laser filamentation ( 3 , 4 ), a phenomenon that results in a spatial energy density and temporal dose rate that both exceed by several orders of magnitude any values previously reported even for the most intense clinical radiotherapy systems. We compared the effect of the laser filamentation with those of gamma radiation on thymidine decomposition, DNA damage, and a tumor in vivo. Our results show that ( a ) the type of final damage and its mechanisms in aqueous media are comparable to those of conventional ionizing radiation, and ( b ) laser irradiation method was more effective in treating tumors with respect to reducing their size and even eliminating them. Femtosecond laser filamentation paves the way for new applications of photonic processes in radiotherapy. By using intense ultra-short infrared (IR) laser pulses, a very large energy dose can now be deposited at unprecedented microscopic dose rates (up to 10 11  Gy/s) deep inside an adjustable, well-controlled macroscopic volume, without any dose deposit in front or behind the target volume. The macroscopic dose rate of the laser-induced filamentation process was determined here using two different chemical dosimeters. The spatial dose distribution, and hence the microscopic energy density and dose rate, of the laser-induced filamentation process was captured by a polymer gel dosimeter and visualized by magnetic resonance imaging (MRI). Our results clearly show that changing the duration of the laser pulse enables precise control of the distance in the medium over which the entrance dose is zero. The two counterbalanced processes keep the filament core intensity almost constant below the optical breakdown threshold, yielding a self-regulated generation of spatially homogenous low-density plasma spots along the propagation axis of the laser beam. This plasma makes it possible to produce a high rate of ionizations in the heart of such filaments while minimizing thermo-mechanical effects related to “hot” high-density plasmas. We propose that these ionizing properties of laser-induced filamentation give rise to changes in the medium that are equivalent to conventional therapeutic ionizing radiation. More importantly, the filamentation process deposits a large dose of energy localized deep inside a well-controlled macroscopic volume, because the depth at which filamentation begins and ends is regulated by operator-controlled laser pulse parameters. This process is related to the self-focusing of an intense laser pulse induced by the Kerr effect that is counterbalanced by the induction of self-defocusing induced by subsequent laser-plasma interactions. The Kerr effect is characterized by a nonlinear change in the refractive index of a medium such as water, which varies proportionately with the square of the electric field strength. Applied to intense laser pulses with Gaussian intensity profiles, this local, nonlinear change of refractive index acts as a convex lens and results in intense self-focusing of the laser beam along its propagation axis. Thus, after some propagation distance, the laser intensity becomes so high that it overcomes the thresholds for nonlinear ionization, generating local plasma. The interaction of the laser light with the highly defocusing environment created by this plasma will now diverge the laser beam, such that the resulting decrease in light intensity will arrest the process of plasma generation, thus allowing the laser pulse to undergo self-focusing again at a slightly deeper position along the laser pulse propagation axis. This dynamic equilibrium continues as long as the magnitude of the laser pulse intensity, which slowly decreases due to such energy transfers to the medium, allows nonlinear processes, such as the Kerr effect. Numerous biophotonics applications of ultra-fast lasers are available; however, they are not used for radiotherapy of tumors that reside within macroscopic distances inside human tissue. This is of interest, because many of the modern, long-wavelength, high-power lasers can deliver high-energy-density pulses (doses and dose rates), which, in principle, surpass those of any clinical radiation source. This raises fundamental questions: ( i ) Can lasers, non-linear optics, and near-visible photons (IR, visible) generate a true radiotherapeutic effect, not upon entry in the tissue, but at a macroscopic distance within a tissue? and (ii) Can IR lasers, in particular, which emit non-ionizing radiation, replace ionizing radiation (viz radioactive materials) in certain cancer treatments? Nonlinear energy deposition can occur during the propagation of a powerful femtosecond (fs) laser pulse in a medium such as water, when the intensity of the light is sufficiently high so that its electromagnetic field will strongly perturb and change the optical properties of the medium. Here, we propose that a nonlinear photonic process called filamentation ( Fig. P1 ) ( 3 , 4 ) can solve the main problem of radiotherapy, which is the undesirable dose distribution upon tissue entry. Fig. P1. Photographic illustration of the femtosecond laser pulses filamentation in water. Here, a scattering product (milk) was diluted at a weak concentration in water to visualize multiple filamentation, which results in an elongated sparkly glowing region in the figure. Scientifically speaking, due to the presence of the scattering product, multiple filamentation can be observed through the scattering of the supercontinuum white light generated by self-phase modulation and self-steepening of the laser pulses. The direction of laser beam propagation is from top to bottom. The camera was positioned at an angle of elevation of approximately 45° from the on-axis direction in order to capture both the filaments and the transmission of the supercontinuum strong spectral broadening at the rear side of a screen oriented perpendicularly across the laser propagation axis (circular spectral pattern at the bottom of the picture). Since the invention of cancer radiotherapy, its primary goal has been to maximize lethal radiation doses to the tumor volume while keeping the dose to surrounding healthy tissues at zero. Sadly, conventional radiation sources (γ- or X-rays, electrons) and methods used over the decades, including multiple or modulated beams, inevitably deposit the majority of their dose in front of or behind the tumor, thus damaging healthy tissue and causing secondary cancers years after treatment ( 1 ). Even the most recent pioneering advances in costly proton or carbon ion therapies can not completely avoid dose buildup in front of the tumor volume ( 2 ).

Abstract: Using a diverse collection of small molecules we recently found that compound sets from different sources (commercial; academic; natural) have different protein-binding behaviors, and these behaviors correlate with trends in stereochemical complexity for these compound sets. These results lend insight into structural features that synthetic chemists might target when synthesizing screening collections for biological discovery. We report extensive characterization of structural properties and diversity of biological performance for these compounds and expand comparative analyses to include physicochemical properties and three-dimensional shapes of predicted conformers. The results highlight additional similarities and differences between the sets, but also the dependence of such comparisons on the choice of molecular descriptors. Using a protein-binding dataset, we introduce an information-theoretic measure to assess diversity of performance with a constraint on specificity. Rather than relying on finding individual active compounds, this measure allows rational judgment of compound subsets as groups. We also apply this measure to publicly available data from ChemBank for the same compound sets across a diverse group of functional assays. We find that performance diversity of compound sets is relatively stable across a range of property values as judged by this measure, both in protein-binding studies and functional assays. Because building screening collections with improved performance depends on efficient use of synthetic organic chemistry resources, these studies illustrate an important quantitative framework to help prioritize choices made in building such collections.

Abstract: Using a diverse collection of small molecules generated from a variety of sources, we measured protein-binding activities of each individual compound against each of 100 diverse (sequence-unrelated) proteins using small-molecule microarrays. We also analyzed structural features, including complexity, of the small molecules. We found that compounds from different sources (commercial, academic, natural) have different protein-binding behaviors and that these behaviors correlate with general trends in stereochemical and shape descriptors for these compound collections. Increasing the content of sp 3 -hybridized and stereogenic atoms relative to compounds from commercial sources, which comprise the majority of current screening collections, improved binding selectivity and frequency. The results suggest structural features that synthetic chemists can target when synthesizing screening collections for biological discovery. Because binding proteins selectively can be a key feature of high-value probes and drugs, synthesizing compounds having features identified in this study may result in improved performance of screening collections.

Abstract: High-content screening for small-molecule inducers of insulin expression identified the compound BRD7389, which caused α-cells to adopt several morphological and gene expression features of a β-cell state. Assay-performance profile analysis suggests kinase inhibition as a mechanism of action, and we show that biochemical and cellular inhibition of the RSK kinase family by BRD7389 is likely related to its ability induce a β-cell-like state. BRD7389 also increases the endocrine cell content and function of donor human pancreatic islets in culture.

Abstract: Angiogenin (ANG) is a stress-activated ribonuclease that promotes the survival of motor neurons. Ribonuclease inactivating point mutations are found in a subset of patients with ALS, a fatal neurodegenerative disease with no cure. We recently showed that ANG cleaves tRNA within anticodon loops to produce 5′- and 3′-fragments known as tRNA-derived, stress-induced RNAs (tiRNAs). Selected 5′-tiRNAs (e.g., tiRNA Ala , tiRNA Cys ) cooperate with the translational repressor Y-box binding protein 1 (YB-1) to displace the cap-binding complex eIF4F from capped mRNA, inhibit translation initiation, and induce the assembly of stress granules (SGs). Here, we show that translationally active tiRNAs assemble unique G-quadruplex (G4) structures that are required for translation inhibition. We show that tiRNA Ala binds the cold shock domain of YB-1 to activate these translational reprogramming events. We discovered that 5′-tiDNA Ala (the DNA equivalent of 5′-tiRNA Ala ) is a stable tiRNA analog that displaces eIF4F from capped mRNA, inhibits translation initiation, and induces the assembly of SGs. The 5′-tiDNA Ala also assembles a G4 structure that allows it to enter motor neurons spontaneously and trigger a neuroprotective response in a YB-1–dependent manner. Remarkably, the ability of 5′-tiRNA Ala to induce SG assembly is inhibited by G4 structures formed by pathological GGGGCC repeats found in C9ORF72, the most common genetic cause of ALS, suggesting that functional interactions between G4 RNAs may contribute to neurodegenerative disease.

Abstract: Removal of the parametrial fat pads (partial lipectomy) from female SKH-1 mice fed a high-fat diet inhibited UVB-induced carcinogenesis, but this was not observed in mice fed a low-fat chow diet. Partial lipectomy in high-fat–fed mice decreased the number of keratoacanthomas and squamous cell carcinomas per mouse by 76 and 79%, respectively, compared with sham-operated control mice irradiated with UVB for 33 wk. Immunohistochemical analysis indicated that partial lipectomy increased caspase 3 (active form) positive cells by 48% in precancerous epidermis away from tumors, by 68% in keratoacanthomas, and by 224% in squamous cell carcinomas compared with sham-operated control mice. In addition, partial lipectomy decreased cell proliferation away from tumors and in tumors. RT-PCR analysis for adipokines revealed that mRNAs for TIMP1, MCP1, and SerpinE1 (proinflammatory/antiapoptotic cytokines) in the parametrial fat pads of sham-operated control mice were 54- to 83-fold higher than levels in compensatory fat that returned after surgery in partially lipectomized mice at the end of the tumor study. Feeding mice high-fat diets for 2 wk increased levels of TIMP1 and other adipokines in serum and epidermis, and these increases were inhibited by removal of the parametrial fat pads. Our results are a unique demonstration that surgical removal of a specific tissue fat results in inhibition of carcinogenesis in obese mice. This inhibition was associated with an increase in apoptosis and a decrease in proliferation in tumors and in precancerous areas away from tumors.