Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems

Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels.Simons Foundation. Postdoctoral FellowshipLife Sciences Research FoundationBurroughs Wellcome Fund (Career Award at the Scientific Interface)Searle Scholars ProgramMichael J. Fox Foundation for Parkinson's ResearchUnited States. Defense Advanced Research Projects AgencyNational Institutes of Health (U.S.) (1-U01-NS090473-01

Inhibitory Activity of Bevacizumab to Differentiation of Retinoblastoma Cells

Vascular endothelial growth factor (VEGF) is a major regulator in retinal and choroidal angiogenesis, which are common causes of blindness in all age groups. Recently anti-VEGF treatment using anti-VEGF antibody has revolutionarily improved the visual outcome in patients with vaso-proliferative retinopathies. Herein, we demonstrated that bevacizumab as an anti-VEGF antibody could inhibit differentiation of retinoblastoma cells without affection to cellular viability, which would be mediated via blockade of extracellular signal-regulated kinase (ERK) 1/2 activation. The retinoblastoma cells expressed VEGFR-2 as well as TrkA which is a neurotrophin receptor associated with differentiation of retinoblastoma cells. TrkA in retinoblastoma cells was activated with VEGF treatment. Interestingly even in the concentration of no cellular death, bevascizumab significantly attenuated the neurite formation of differentiated retinoblastoma cells, which was accompanied by inhibition of neurofilament and shank2 expression. Furthermore, bevacizumab inhibited differentiation of retinoblastoma cells by blockade of ERK 1/2 activation. Therefore, based on that the differentiated retinoblastoma cells are mostly photoreceptors, our results suggest that anti-VEGF therapies would affect to the maintenance or function of photoreceptors in mature retina

Acute Iron Deprivation Reprograms Human Macrophage Metabolism and Reduces Inflammation In Vivo.

Iron is an essential metal that fine-tunes the innate immune response by regulating macrophage function, but an integrative view of transcriptional and metabolic responses to iron perturbation in macrophages is lacking. Here, we induced acute iron chelation in primary human macrophages and measured their transcriptional and metabolic responses. Acute iron deprivation causes an anti-proliferative Warburg transcriptome, characterized by an ATF4-dependent signature. Iron-deprived human macrophages show an inhibition of oxidative phosphorylation and a concomitant increase in glycolysis, a large increase in glucose-derived citrate pools associated with lipid droplet accumulation, and modest levels of itaconate production. LPS polarization increases the itaconate:succinate ratio and decreases pro-inflammatory cytokine production. In rats, acute iron deprivation reduces the severity of macrophage-dependent crescentic glomerulonephritis by limiting glomerular cell proliferation and inducing lipid accumulation in the renal cortex. These results suggest that acute iron deprivation has in vivo protective effects mediated by an anti-inflammatory immunometabolic switch in macrophages.This work was supported by the Medical Research Council (MR/M004716/1 and MR/N01121X/1, to J.B., and MRC_MC_UU_12022/6, to C.F.

Derepression of CLDN3 and CLDN4 during ovarian tumorigenesis is associated with loss of repressive histone modifications

Unlike epigenetic silencing of tumor suppressor genes, the role of epigenetic derepression of cancer-promoting genes or oncogenes in carcinogenesis remains less well understood. The tight junction proteins claudin-3 and claudin-4 are frequently overexpressed in ovarian cancer and their overexpression was previously reported to promote the migration and invasion of ovarian epithelial cells. Here, we show that the expression of claudin-3 and claudin-4 is repressed in ovarian epithelial cells in association with promoter ‘bivalent’ histone modifications, containing both the activating trimethylated histone H3 lysine 4 (H3K4me3) mark and the repressive mark of trimethylated histone H3 lysine 27 (H3K27me3). During ovarian tumorigenesis, derepression of CLDN3 and CLDN4 expression correlates with loss of H3K27me3 in addition to trimethylated histone H4 lysine 20 (H4K20me3), another repressive histone modification. Although CLDN4 repression was accompanied by both DNA hypermethylation and repressive histone modifications, DNA methylation was not required for CLDN3 repression in immortalized ovarian epithelial cells. Moreover, activation of both CLDN3 and CLDN4 in ovarian cancer cells was associated with simultaneous changes in multiple histone modifications, whereas H3K27me3 loss alone was insufficient for their derepression. CLDN4 repression was robustly reversed by combined treatment targeting both DNA demethylation and histone acetylation. Our study strongly suggests that in addition to the well-known chromatin-associated silencing of tumor suppressor genes, epigenetic derepression by the conversely related loss of repressive chromatin modifications also contributes to ovarian tumorigenesis via activation of cancer-promoting genes or candidate oncogenes

Airborne formaldehyde and volatile organic compound measurements over the Daesan petrochemical complex on Korea’s northwest coast during the Korea-United States Air Quality study

The U.S. National Aeronautics and Space Administration in partnership with Korea’s National Institute of Environmental Research embarked on the Korea-United States Air Quality (KORUS-AQ) study to address air quality issues over the Korean peninsula. Underestimation of volatile organic compound (VOC) emissions from various large facilities on South Korea’s northwest coast may contribute to this problem, and this study focuses on quantifying top-down emissions of formaldehyde (CH₂O) and VOCs from the largest of these facilities, the Daesan petrochemical complex, and comparisons with the latest emission inventories. To accomplish this and additional goals discussed herein, this study employed a number of measurements acquired during KORUS-AQ onboard the NASA DC-8 aircraft during three Daesan overflights on June 2, 3, and 5, 2016, in conjunction with a mass balance approach. The measurements included fast airborne measurements of CH₂O and ethane from an infrared spectrometer, additional fast measurements from other instruments, and a suite of 33 VOC measurements acquired by the whole air sampler. The mass balance approach resulted in consistent top-down yearly Daesan VOC emission flux estimates, which averaged (61 ± 14) × 10³ MT/year for the 33 VOC compounds, a factor of 2.9 ± 0.6 (±1.0) higher than the bottom-up inventory value. The top-down Daesan emission estimate for CH₂O and its four primary precursors averaged a factor of 4.3 ± 1.5 (± 1.9) times higher than the bottom-up inventory value. The uncertainty values in parentheses reflect upper limits for total uncertainty estimates. The resulting averaged top-down Daesan emission estimate for sulfur dioxide (SO₂) yielded a ratio of 0.81–1.0 times the bottom-up SO₂ inventory, and this provides an important cross-check on the accuracy of our mass balance analysis

Intravitreally injected anti-VEGF antibody reduces brown fat in neonatal mice

Anti-vascular endothelial growth factor (VEGF) agents are the mainstay treatment for various angiogenesis-related retinal diseases. Currently, bevacizumab, a recombinant humanized anti-VEGF antibody, is trailed in retinopathy of prematurity, a vasoproliferative retinal disorder in premature infants. However, the risks of systemic complications after intravitreal injection of anti-VEGF antibody in infants are not well understood. In this study, we show that intravitreally injected anti-VEGF antibody is transported into the systemic circulation into the periphery where it reduces brown fat in neonatal C57BL/6 mice. A considerable amount of anti-VEGF antibody was detected in serum after intravitreal injection. Furthermore, in interscapular brown adipose tissue, we found lipid droplet accumulation, decreased VEGF levels, loss of vascular network, and decreased expression of mitochondriarelated genes, Ppargc1a and Ucp1, all of which are characteristics of "whitening" of brown fat. With increasing age and body weight, brown fat restored its morphology and vascularity. Our results show that there is a transient, but significant impact of intravitreally administered anti-VEGF antibody on brown adipose tissue in neonatal mice. We suggest that more attention should be focused on the metabolic and developmental significance of brown adipose tissue in bevacizumab treated retinopathy of prematurity infants. Copyright

Co-Crystal Structures of PKG Iβ (92–227) with cGMP and cAMP Reveal the Molecular Details of Cyclic-Nucleotide Binding

Cyclic GMP-dependent protein kinases (PKGs) are central mediators of the NO-cGMP signaling pathway and phosphorylate downstream substrates that are crucial for regulating smooth muscle tone, platelet activation, nociception and memory formation. As one of the main receptors for cGMP, PKGs mediate most of the effects of cGMP elevating drugs, such as nitric oxide-releasing agents and phosphodiesterase inhibitors which are used for the treatment of angina pectoris and erectile dysfunction, respectively. configuration, with a conserved threonine residue anchoring both cyclic phosphate and guanine moieties. The structure of CNBD-A in the absence of bound cyclic nucleotide was similar to that of the cyclic nucleotide bound structures. Surprisingly, isothermal titration calorimetry experiments demonstrated that CNBD-A binds both cGMP and cAMP with a relatively high affinity, showing an approximately two-fold preference for cGMP. conformation through its interaction with Thr193 and an unusual cis-peptide forming residues Leu172 and Cys173. Although these studies provide the first structural insights into cyclic nucleotide binding to PKG, our ITC results show only a two-fold preference for cGMP, indicating that other domains are required for the previously reported cyclic nucleotide selectivity

Antileishmanial High-Throughput Drug Screening Reveals Drug Candidates with New Scaffolds

Drugs currently available for leishmaniasis treatment often show parasite resistance, highly toxic side effects and prohibitive costs commonly incompatible with patients from the tropical endemic countries. In this sense, there is an urgent need for new drugs as a treatment solution for this neglected disease. Here we show the development and implementation of an automated high-throughput viability screening assay for the discovery of new drugs against Leishmania. Assay validation was done with Leishmania promastigote forms, including the screening of 4,000 compounds with known pharmacological properties. In an attempt to find new compounds with leishmanicidal properties, 26,500 structurally diverse chemical compounds were screened. A cut-off of 70% growth inhibition in the primary screening led to the identification of 567 active compounds. Cellular toxicity and selectivity were responsible for the exclusion of 78% of the pre-selected compounds. The activity of the remaining 124 compounds was confirmed against the intramacrophagic amastigote form of the parasite. In vitro microsomal stability and cytochrome P450 (CYP) inhibition of the two most active compounds from this screening effort were assessed to obtain preliminary information on their metabolism in the host. The HTS approach employed here resulted in the discovery of two new antileishmanial compounds, bringing promising candidates to the leishmaniasis drug discovery pipeline

Peptide Substrates for Rho-Associated Kinase 2 (Rho-Kinase 2/ROCK2)

Peptide substrates sensitive for a certain protein kinase could be important for new-drug development and to understand the mechanism of diseases. Rho-associated kinase (Rho-kinase/ROCK) is a serine/threonine kinase, and plays an important part in cardiovascular disease, migration and invasion of tumor cells, and in neurological disorders. The purpose of this study was to find substrates with high affinity and sensitivity for ROCK2. We synthesized 136 peptide substrates from protein substrates for ROCK2 with different lengths and charged peptides. Incorporation of 32P [counts per minute (CPM)] for each peptide substrate was determined by the radiolabel assay using [γ-32P]ATP. When the top five peptide substrates showing high CPMs (R4, R22, R133, R134, and R135) were phosphorylated by other enzymes (PKA, PKCα, and ERK1), R22, R133, and R135 displayed the highest CPM level for ROCK2 compared with other enzymes, whereas R4 and R134 showed similar CPM levels for ROCK2 and PKCα. We hypothesize that R22, R133, and R135 can be useful peptide substrates for ROCK2