trans-Endothelial neutrophil migration activates bactericidal function via Piezo1 mechanosensing.

The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and in vivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.

End binding-3 inhibitor activates regenerative program in age-related macular degeneration.

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.

Targeting EB3-IP3R3 Interface with Cognate Peptide Protects from Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a lung disease characterized by acute onset of noncardiogenic pulmonary edema, hypoxemia, and respiratory insufficiency. The current treatment for ARDS is mainly supportive in nature, providing a critical need for targeted pharmacological management. We addressed this medical problem by developing a pharmacological treatment for pulmonary vascular leakage, a culprit of alveolar damage and lung inflammation. Our novel therapeutic target is the microtubule accessory factor EB3 (end binding protein 3), which contributes to pulmonary vascular leakage by amplifying pathological calcium signaling in endothelial cells in response to inflammatory stimuli. EB3 interacts with IP3R3 (inositol 1,4,5-trisphosphate receptor 3) and orchestrates calcium release from endoplasmic reticulum stores. Here, we designed and tested the therapeutic benefits of a 14-aa peptide named CIPRI (cognate IP3 receptor inhibitor), which disrupted EB3-IP3R3 interaction in vitro and in lungs of mice challenged with endotoxin. Treatment with CIPRI or depletion of IP3R3 in lung microvascular endothelial monolayers mitigated calcium release from endoplasmic reticulum stores and prevented a disassembly of vascular endothelial cadherin junctions in response to the proinflammatory mediator α-thrombin. Furthermore, intravenous administration of CIPRI in mice mitigated inflammation-induced lung injury, blocked pulmonary microvascular leakage, prevented activation of NFAT (nuclear factor of activated T cells) signaling, and reduced production of proinflammatory cytokines in the lung tissue. CIPRI also improved survival of mice from endotoxemia and polymicrobial sepsis. Together, these data demonstrate that targeting EB3-IP3R3 interaction with a cognate peptide is a promising strategy to address hyperpermeability of microvessels in inflammatory lung diseases.

Phenolic acids and flavonoids-rich Glechoma hederacea L. (Lamiaceae) water extract against H2 O2 -induced apoptosis in PC12 cells.

Reactive oxygen species (ROS)-induced oxidative stress is reportedly associated with progressive neuronal cell damage. Glechoma hederacea L. (Lamiaceae), belonging to the Labiatae family, has demonstrated several biologic activities including depigmentation, antimelanogenic, antitumor, antioxidative, hepatoprotective, and anti-inflammatory activities. Previously, we reported that rosmarinic acid, chlorogenic acid, caffeic acid, rutin, genistin, and ferulic acids were the most abundant phytochemicals detected in hot water extracts of G. hederacea L. (HWG). This study aimed to study the neuroprotective effects of phenolic acids and flavonoid-rich HWG against hydrogen peroxide (H2 O2 )-induced oxidative damage in PC12 cells and its inhibitory effect on acetylcholinesterase (AChE). The experiment analyzed cytotoxicity, ROS production, mitochondrial transmembrane potential (MMP) level, and caspase-3 activity and used comet assay and antioxidant enzyme activity to determine the redox status of PC12 cells. Results showed that the inhibitory effect of HWG on AChE was in a competitive pattern (IC50 , 23.23 mg/ml). HWG antagonized H2 O2 -mediated cytotoxicity and DNA damage, reduced ROS production, stabilized MMP, and inhibited caspase-3 activity and apoptosis. Furthermore, HWG inhibited the release of cytochrome C and apoptosis-inducing factors (AIF) and decreased the malondialdehyde levels in PC12 cells. Collectively, HWG rich in antioxidant phenolic acids and flavonoids may have neuroprotective effects. PRACTICAL APPLICATIONS: Polyphenolic compounds are one of the most important natural products, known to possess a range of health-promoting effects. In this study, it was found that HWG, which is rich in antioxidant phenolic acids and flavonoids, can protect PC12 cells from oxidative stress induced by H2 O2 and may have neuroprotective effects.

Risk assessment of methylmercury based on internal exposure and fish and seafood consumption estimates in Taiwanese children.

Fish and seafood consumption is a major source of human exposure to methylmercury (MeHg). This study evaluated the potential health risk of MeHg in Taiwanese children from fish and seafood consumption using a toxicokinetic model, hazard quotients and hazard indices (HIs). Two biomonitoring programs provided an important resource for blood specimens for assessing MeHg exposure in human populations. For internal exposures, total mercury (THg) was measured as a biomarker of MeHg in whole blood (WB) and red blood cells using inductively coupled plasma mass spectrometry and cold-vapor atomic absorption spectroscopy, respectively. The THg concentrations were used to estimate MeHg concentrations. Consumption of fish and seafood was assessed using the National Food Consumption database in Taiwan, while mercury concentrations in edible fish and seafood were collected from published studies in Taiwan. Our results indicated that 1) the highest median THg (representing estimated MeHg) daily intakes were found to decrease with increasing age in children consuming saltwater fish for age groups 0-3, 4-6, 7-12, and 13-18 years: 0.03 > 0.02 > 0.017 > 0.007 (µg kg-BW-1 day-1); 2) HI greater than one, based on WB-THg, was found in 28% of 4-6-year-old children and 3) internal exposure estimates based on WB-THg, though slightly higher, were comparable to those based on fish and seafood consumption. The results support the use of dietary intake estimates as surrogates for internal blood MeHg levels in Taiwanese children to assess their exposure.

Discovery of estrogen receptor alpha target genes and response elements in breast tumor cells.

BACKGROUND: Estrogens and their receptors are important in human development, physiology and disease. In this study, we utilized an integrated genome-wide molecular and computational approach to characterize the interaction between the activated estrogen receptor (ER) and the regulatory elements of candidate target genes. RESULTS: Of around 19,000 genes surveyed in this study, we observed 137 ER-regulated genes in T-47D cells, of which only 89 were direct target genes. Meta-analysis of heterogeneous in vitro and in vivo datasets showed that the expression profiles in T-47D and MCF-7 cells are remarkably similar and overlap with genes differentially expressed between ER-positive and ER-negative tumors. Computational analysis revealed a significant enrichment of putative estrogen response elements (EREs) in the cis-regulatory regions of direct target genes. Chromatin immunoprecipitation confirmed ligand-dependent ER binding at the computationally predicted EREs in our highest ranked ER direct target genes, NRIP1, GREB1 and ABCA3. Wider examination of the cis-regulatory regions flanking the transcriptional start sites showed species conservation in mouse-human comparisons in only 6% of predicted EREs. CONCLUSIONS: Only a small core set of human genes, validated across experimental systems and closely associated with ER status in breast tumors, appear to be sufficient to induce ER effects in breast cancer cells. That cis-regulatory regions of these core ER target genes are poorly conserved suggests that different evolutionary mechanisms are operative at transcriptional control elements than at coding regions. These results predict that certain biological effects of estrogen signaling will differ between mouse and human to a larger extent than previously thought.

Tissue-specific gene silencing mediated by a naturally occurring chalcone synthase gene cluster in Glycine max.

Chalcone synthase, a key regulatory enzyme in the flavonoid pathway, constitutes an eight-member gene family in Glycine max (soybean). Three of the chalcone synthase (CHS) gene family members are arranged as inverted repeats in a 10-kb region, corresponding to the I locus (inhibitor). Spontaneous mutations of a dominant allele (I or i(i)) to a recessive allele (i) have been shown to delete promoter sequences, paradoxically increasing total CHS transcript levels and resulting in black seed coats. However, it is not known which of the gene family members contribute toward pigmentation and how this locus affects CHS expression in other tissues. We investigated the unusual nature of the I locus using four pairs of isogenic lines differing with respect to alleles of the I locus. RNA gel blots using a generic open reading frame CHS probe detected similar CHS transcript levels in stems, roots, leaves, young pods, and cotyledons of the yellow and black isolines but not in the seed coats, which is consistent with the dominant I and i(i) alleles mediating CHS gene silencing in a tissue-specific manner. Using real-time RT-PCR, a variable pattern of expression of CHS genes in different tissues was demonstrated. However, increase in pigmentation in the black seed coats was associated with release of the silencing effect specifically on CHS7/CHS8, which occurred at all stages of seed coat development. These expression changes were linked to structural changes taking place at the I locus, shown to encompass a much wider region of at least 27 kb, comprising two identical 10.91-kb stretches of CHS gene duplications. The suppressive effect of this 27-kb I locus in a specific tissue of the G. max plant represents a unique endogenous gene silencing mechanism.