LSD1/KDM1A promotes hematopoietic commitment of hemangioblasts through downregulation of Etv2.

The hemangioblast is a progenitor cell with the capacity to give rise to both hematopoietic and endothelial progenitors. Currently, the regulatory mechanisms underlying hemangioblast formation are being elucidated, whereas those controllers for the selection of hematopoietic or endothelial fates still remain a mystery. To answer these questions, we screened for zebrafish mutants that have defects in the hemangioblast expression of Gata1, which is never expressed in endothelial progenitors. One of the isolated mutants, it627, showed not only down-regulation of hematopoietic genes but also up-regulation of endothelial genes. We identified the gene responsible for the it627 mutant as the zebrafish homolog of Lys-specific demethylase 1 (LSD1/KDM1A). Surprisingly, the hematopoietic defects in lsd1(it627) embryos were rescued by the gene knockdown of the Ets variant 2 gene (etv2), an essential regulator for vasculogenesis. Our results suggest that the LSD1-dependent shutdown of Etv2 gene expression may be a significant event required for hemangioblasts to initiate hematopoietic differentiation.

Mathematical modeling for meshwork formation of endothelial cells in fibrin gels.

Vasculogenesis is the earliest process in development for spontaneous formation of a primitive capillary network from endothelial progenitor cells. When human umbilical vein endothelial cells (HUVECs) are cultured on Matrigel, they spontaneously form a network structure which is widely used as an in vitro model of vasculogenesis. Previous studies indicated that chemotaxis or gel deformation was involved in spontaneous pattern formation. In our study, we analyzed the mechanism of vascular pattern formation using a different system, meshwork formation by HUVECs embedded in fibrin gels. Unlike the others, this experimental system resulted in a perfusable endothelial network in vitro. We quantitatively observed the dynamics of endothelial cell protrusion and developed a mathematical model for one-dimensional dynamics. We then extended the one-dimensional model to two-dimensions. The model showed that random searching by endothelial cells was sufficient to generate the observed network structure in fibrin gels.

Heme-mediated inhibition of Bach1 regulates the liver specificity and transience of the Nrf2-dependent induction of zebrafish heme oxygenase 1.

The induction of the gene encoding heme oxygenase 1 (Hmox1, HO-1) by Nrf2 is unique compared with other Nrf2 targets. We previously showed that the Nrf2a-mediated induction of zebrafish hmox1a was liver specific and transient. We screened transcription factors that could repress the induction of hmox1a but not other Nrf2a targets and concluded that Bach1b was a prime candidate. In bach1b-knocked-down larvae, the induction of hmox1a was observed ectopically in nonliver tissues and persisted longer than normal fish, suggesting that Bach1 is the only regulator for both the liver-specific and transient induction of hmox1a. Co-knockdown of bach1b with its co-ortholog bach1a enhanced these effects. To determine why Bach1 could not repress the hmox1a induction in the liver, we analyzed the effects of a heme biosynthesis inhibitor, succinylacetone, and a heme precursor, hemin. Succinylacetone decreased the Nrf2a-mediated hmox1a induction, whereas pre-treatment with hemin caused ectopic induction of hmox1a in nonliver tissues, implying that the high heme levels in the liver may release the repressive activity of Bach1. Our results suggested that Bach1 regulates the liver specificity and transience of the Nrf2a-dependent induction of hmox1a and that heme mediates this regulation through Bach1 inhibition based on its level in each tissue.

A general scavenging rate constant for reaction of hydroxyl radical with organic carbon in atmospheric waters.

Hydroxyl radical (OH) is an important oxidant in atmospheric aqueous phases such as cloud and fog drops and water-containing aerosol particles. We find that numerical models nearly always overestimate aqueous hydroxyl radical concentrations because they overpredict its rate of formation and, more significantly, underpredict its sinks. To address this latter point, we examined OH sinks in atmospheric drops and aqueous particles using both new samples and an analysis of published data. Although the molecular composition of organic carbon, the dominant sink of OH, is extremely complex and poorly constrained, this sink behaves very similarly in different atmospheric waters and even in surface waters. Thus, the sink for aqueous OH can be estimated as the concentration of dissolved organic carbon multiplied by a general scavenging rate constant [kC,OH = (3.8 ± 1.9) × 10(8) L (mol C)(-1) s(-1)], a simple process that should significantly improve estimates of OH concentrations in atmospheric drops and aqueous particles.

Nitro-fatty acids and cyclopentenone prostaglandins share strategies to activate the Keap1-Nrf2 system: a study using green fluorescent protein transgenic zebrafish.

Nitro-fatty acids are electrophilic fatty acids produced in vivo from nitrogen peroxide that have many physiological activities. We recently demonstrated that nitro-fatty acids activate the Keap1-Nrf2 system, which protects cells from damage owing to electrophilic or oxidative stresses via transactivating an array of cytoprotective genes, although the molecular mechanism how they activate Nrf2 is unclear. A number of chemical compounds with different structures have been reported to activate the Keap1-Nrf2 system, which can be categorized into at least six classes based on their sensing pathways. In this study, we showed that nitro-oleic acid (OA-NO2), one of major nitro-fatty acids, activates Nrf2 in the same manner that of a cyclopentenone prostaglandin 15-deoxy-Δ(12,14) -prostaglandin J2 (15d-PGJ2) using transgenic zebrafish that expresses green fluorescent protein (GFP) in response to Nrf2 activators. In transgenic embryos, GFP was induced in the whole body by treatment with OA-NO2, 15d-PGJ2 or diethylmaleate (DEM), but not with hydrogen peroxide (H2O2), when exogenous Nrf2 and Keap1 were co-overexpressed. Induction by OA-NO2 or 15d-PGJ2 but not DEM was observed, even when a C151S mutation was introduced in Keap1. Our results support the contention that OA-NO2 and 15d-PGJ2 share an analogous cysteine code as electrophiles and also have similar anti-inflammatory roles.

A retrospective analysis of pembrolizumab plus chemotherapy versus pembrolizumab monotherapy for advanced or recurrent non-small cell lung cancer.

BACKGROUND: Although clinical trials have investigated the addition of pembrolizumab to chemotherapy for non-small cell lung cancer, none have investigated the addition of chemotherapy to pembrolizumab. METHODS: We conducted a retrospective study of 71 NSCLC patients including 33 treated with pembrolizumab plus chemotherapy (combination therapy group) and 38 treated with pembrolizumab monotherapy (monotherapy group) from 1 May 2016 to 31 August 2020. RESULTS: Eleven of 33 (33.3%) patients in the combination therapy group and 37 of 38 (97.4%) patients in the monotherapy group had programmed cell death ligand-1 (PD-L1) tumor proportion score (TPS) ≥50%. Objective response rate (ORR) and median overall survival (OS) were not significantly different between the combination therapy group and monotherapy group (54.5% vs. 47.4, p = 0.637 and 16.6 vs. 27.0 months, p = 0.463). In patients with PD-L1 TPS ≥50%, ORR and median OS were not different between the combination therapy group and the monotherapy group (63.6% vs. 48.6%, p = 0.499 and not reached vs. 27.0 months, p = 0.976). Thirty-three (100%) patients experienced adverse events (AEs) in the combination therapy group and 32 (84.2%) in the monotherapy group. Treatment discontinuation at 1 year due to AEs occurred more frequently in the combination therapy group (45.2%) than in the monotherapy group (21.1%). CONCLUSION: There was no significant difference in ORR and OS between the two groups, and treatment discontinuation was more frequent in the combination group. A randomized controlled trial is needed to evaluate the addition of chemotherapy to pembrolizumab for first-line treatment in patients with PD-L1 TPS ≥50%.

OLIVe: A Genetically Encoded Fluorescent Biosensor for Quantitative Imaging of Branched-Chain Amino Acid Levels inside Single Living Cells.

Branched-chain amino acids (BCAAs) are essential amino acids, controlling cellular metabolic processes as signaling molecules; therefore, utilization of intracellular BCAAs may be regulated by the availability of nutrients in the environment. However, spatial and temporal regulation of intracellular BCAA concentration in response to environmental conditions has been unclear due to the lack of suitable methods for measuring BCAA concentrations inside single living cells. Here, we developed a Förster resonance energy transfer (FRET)-based genetically encoded biosensor for BCAAs, termed optical biosensor for leucine-isoleucine-valine (OLIVe). The biosensor showed approximately 2-fold changes in FRET values corresponding to BCAA concentrations. Importantly, FRET signals from HeLa cells expressing OLIVe in the cytoplasm and nucleus correlated with bulk intracellular BCAA concentrations determined from populations of cells by a biochemical method, and were decreased by knockdown of L-type amino acid transporter 1 (LAT1), a transporter for BCAAs, indicating that OLIVe can reliably report intracellular BCAA concentrations inside single living cells. We also succeeded in imaging BCAA concentrations in the mitochondria using mitochondria-targeted OLIVe. Using the BCAA imaging technique, we found apparently correlated concentrations between the cytoplasm and the mitochondria. We also found that extracellular non-BCAA amino acids affected intracellular BCAA concentrations. Of these amino acids, extracellular glutamine markedly increased intracellular BCAA concentrations in a LAT1-dependent manner. Unexpectedly, extracellular pyruvate was also found to have significant positive effects on maintaining intracellular BCAA concentrations, suggesting that the cells have pyruvate-dependent systems to import BCAAs and/or to regulate BCAA metabolism.

[MRI-intracranial compliance analysis in patients with NPH].

We devised a method for non-invasively assessing intracranial compliance with retrospective ECG-triggered phase contrast cine MRI. This method was examined in patients with normal pressure hydrocephalus (NPH) group and those with asymptomatic ventricular dilation or brain atrophy (VD group), and in healthy volunteers (control group). Intracranial volume change (DeltaV(max)) was calculated from arterial inflow, venous outflow, cerebrospinal fluid (CSF) flow, and spinal cord motion at the C2 level during a cardiac cycle. Next, craniospinal CSF pressure gradient change (DeltaPG(max)) was calculated from measured CSF flow velocity using a simplified Navier-Stokes equation. Finally, Ci was obtained by dividing Delta V(max) into DeltaPG(max). Ci in the NPH group was significantly smaller and could be differentiated from other groups. This method makes it possible to non-invasively obtain a more detailed determination of the intracranial state and dynamics in NPH and to assist in its diagnosis.

Sensory systems and ionocytes are targets for silver nanoparticle effects in fish.

Some nanoparticles (NPs) may induce adverse health effects in exposed organisms, but to date the evidence for this in wildlife is very limited. Silver nanoparticles (AgNPs) can be toxic to aquatic organisms, including fish, at concentrations relevant for some environmental exposures. We applied whole mount in-situ hybridisation (WISH) in zebrafish embryos and larvae for a suite of genes involved with detoxifying processes and oxidative stress, including metallothionein (mt2), glutathionine S-transferase pi (gstp), glutathionine S-transferase mu (gstm1), haem oxygenase (hmox1) and ferritin heavy chain 1 (fth1) to identify potential target tissues and effect mechanisms of AgNPs compared with a bulk counterpart and ionic silver (AgNO3). AgNPs caused upregulation in the expression of mt2, gstp and gstm1 and down regulation of expression of both hmox1 and fth1 and there were both life stage and tissue-specific responses. Responding tissues included olfactory bulbs, lateral line neuromasts and ionocytes in the skin with the potential for effects on olfaction, behaviour and maintenance of ion balance. Silver ions induced similar gene responses and affected the same target tissues as AgNPs. AgNPs invoked levels of target gene responses more similar to silver treatments compared to coated AgNPs indicating the responses seen were due to released silver ions. In the Nrf2 zebrafish mutant, expression of mt2 (24 hpf) and gstp (3 dpf) were either non-detectable or were at lower levels compared with wild type zebrafish for exposures to AgNPs, indicating that these gene responses are controlled through the Nrf2-Keap pathway.

Genetic evidence of an evolutionarily conserved role for Nrf2 in the protection against oxidative stress.

Transcription factor Nrf2 is considered a master regulator of antioxidant defense in mammals. However, it is unclear whether this concept is applicable to nonmammalian vertebrates, because no animal model other than Nrf2 knockout mice has been generated to examine the effects of Nrf2 deficiency. Here, we characterized a recessive loss-of-function mutant of Nrf2 (nrf2(fh318)) in a lower vertebrate, the zebrafish (Danio rerio). In keeping with the findings in the mouse model, nrf2(fh318) mutants exhibited reduced induction of the Nrf2 target genes in response to oxidative stress and electrophiles but were viable and fertile, and their embryos developed normally. The nrf2(fh318) larvae displayed enhanced sensitivity to oxidative stress and electrophiles, especially peroxides, and pretreatment with an Nrf2-activating compound, sulforaphane, decreased peroxide-induced lethality in the wild type but not nrf2(fh318) mutants, indicating that resistance to oxidative stress is highly dependent on Nrf2 functions. These results reveal an evolutionarily conserved role of vertebrate Nrf2 in protection against oxidative stress. Interestingly, there were no significant differences between wild-type and nrf2(fh318) larvae with regard to their sensitivity to superoxide and singlet oxygen generators, suggesting that the importance of Nrf2 in oxidative stress protection varies based on the type of reactive oxygen species (ROS).

Tissue-restricted expression of Nrf2 and its target genes in zebrafish with gene-specific variations in the induction profiles.

The Keap1-Nrf2 system serves as a defense mechanism against oxidative stress and electrophilic toxicants by inducing more than one hundred cytoprotective proteins, including antioxidants and phase 2 detoxifying enzymes. Since induction profiles of Nrf2 target genes have been studied exclusively in cultured cells, and not in animal models, their tissue-specificity has not been well characterized. In this paper, we examined and compared the tissue-specific expression of several Nrf2 target genes in zebrafish larvae by whole-mount in situ hybridization (WISH). Seven zebrafish genes (gstp1, mgst3b, prdx1, frrs1c, fthl, gclc and hmox1a) suitable for WISH analysis were selected from candidates for Nrf2 targets identified by microarray analysis. Tissue-restricted induction was observed in the nose, gill, and/or liver for all seven genes in response to Nrf2-activating compounds, diethylmaleate (DEM) and sulforaphane. The Nrf2 gene itself was dominantly expressed in these three tissues, implying that tissue-restricted induction of Nrf2 target genes is defined by tissue-specific expression of Nrf2. Interestingly, the induction of frrs1c and gclc in liver and nose, respectively, was quite low and that of hmox1a was restricted in the liver. These results indicate the existence of gene-specific variations in the tissue specificity, which can be controlled by factors other than Nrf2.

Contribution of fulvic acid to the photochemical formation of Fe(II) in acidic Suwannee River fulvic acid solutions.

We investigated the contribution of fulvic acid to the photoformation of Fe(II) using aqueous Suwannee River fulvic acid (SRFA) as a surrogate for the humic-like substances (HULIS) found in atmospheric condensed phases. The effects of pH (3.2, 4.1, and 5.0) and wavelength (313, 334, 366, and 405nm) on Fe(II) photoformation were studied using monochromatic radiation at 20 degrees C. We calculated the wavelength-dependent Fe(II) photoformation efficiency values ("E-value"), defined here as a weighted sum of the product of the quantum yield and molar absorptivity of each Fe(II)-forming chemical species, and found that the E-values of acidic SRFA solutions were similar to those of Fe(OH)(2+). In addition, a comparison showed that the acidic SRFA solutions did not form Fe(II) fast enough to account for the observed Fe(II) formation efficiencies of the aqueous extracts of authentic aerosol samples. It was observed that 17-73% of Fe(III) had been reduced to Fe(II) in the dark in acidic SRFA solutions with added Fe(III) ranging from 0.5 to 10muM. The results of this study suggest that HULIS is unlikely to be the major reducing ligand in the process of photochemical formation of Fe(II) in acidic atmospheric drops. However, HULIS could reduce Fe(III) to Fe(II) in the dark, which in turn, could be important for night-time ()OH formation via the reaction between Fe(II) and H(2)O(2) (the Fenton reaction).