Trisulfide Bond-Mediated Molecular Phototheranostic Platform for "Activatable" NIR-II Imaging-Guided Enhanced Gas/Chemo-Hypothermal Photothermal Therapy.

Tumor microenvironment (TME)-triggered phototheranostic platform offers a feasible strategy to improve cancer diagnosis accuracy and minimize treatment side effects. Developing a stable and biocompatible molecular phototheranostic platform for TME-activated second near-infrared (NIR-II) fluorescence imaging-guided multimodal cascade therapy is a promising strategy for creating desirable anticancer agents. Herein, a new NIR-II fluorescence imaging-guided activatable molecular phototheranostic platform (IR-FEP-RGD-S-S-S-Fc) is presented for actively targeted tumor imaging and hydrogen sulfide (H2 S) gas-enhanced chemodynamic-hypothermal photothermal combined therapy (CDT/HPTT). It is revealed for the first time that the coupling distance between IR-FE and ferrocene is proportional to the photoinduced electron transfer (PET), and the aqueous environment is favorable for PET generation. The part of Cyclic-RGDfK (cRGDfk) peptides can target the tumor and benefit the endocytosis of nanoparticles. The high-concentration glutathione (GSH) in the TME will separate the fluorescence molecule and ferrocene by the GSH-sensitive trisulfide bond, realizing light-up NIR-II fluorescence imaging and a cascade of trimodal synergistic CDT/HPTT/gas therapy (GT). In addition, the accumulation of hydroxyl radicals (•OH) and down-regulation of glutathione peroxidase 4 (GPX4) can produce excessive harmful lipid hydroperoxides, ultimately leading to ferroptosis.

Gelidocalamuszixingensis (Poaceae, Bambusoideae, Arundinarieae), a new species from southern China revealed by morphological and molecular evidence.

The genus Gelidocalamus T. H. Wen, endemic to southern China, is a small but taxonomically problematic genus of Arundinarieae (Poaceae, Bambusoideae). During field work, a population of Gelidocalamus from Zixing, Hunan, was discovered, appearing to be distinct from our previously identified collection. Comparisons of the population of Zixing were performed by using scanning electron microscopy (SEM) and a plastid genome-based phylogeny. Morphologically, it was mostly similar to G.multifolius, but differed by culm leaf erect with densely white pubescence, apical branch sheath much longer than the internodes and foliage leaf larger. Phylogenetically, the new species was well-supported as a sister to the clade of G.multifolius + G.tessellatus, and the above three taxa were clustered in the Shibataea clade (IV) of Arundinarieae. Thus, the new species, formally named as Gelidocalamuszixingensis W.G.Zhang, G.Y.Yang & C.K.Wang, was described and illustrated herein.

Antecedents and Consequences of Green Mindfulness: A Conceptual Model.

While many companies take the environmental environment as a fundamental part of their business strategies, managers are facing the challenges to explore the integration of environmental concepts and business operations. Although there are an amount of studies about environmental management in the literature, only a few of them applied the concept of mindfulness to environmental management. Mindfulness is regarded as a way of operation marked by the willingness to consider alternative perspectives, focus on the present, attention to operational detail, and interest in exploring and understanding failures. This study suggests that companies require keeping mindfulness in environmental management implementation. Therefore, this paper aims to explore the application of mindfulness theory to environmental management, and propose a conceptual model of antecedents and consequences of green mindfulness. The proposed multilevel model describes the influences of organizational and individual antecedents on green mindfulness, and the organizational and individual consequences of green mindfulness.

Effect of low-dose Levamlodipine Besylate in the treatment of vascular dementia.

Vascular dementia (VaD) is a complex disorder caused by reduced blood flow in the brain. However, there is no effective pharmacological treatment option available until now. Here, we reported that low-dose levamlodipine besylate could reverse the cognitive impairment in VaD mice model of right unilateral common carotid arteries occlusion (rUCCAO). Oral administration of levamlodipine besylate (0.1 mg/kg) could reduce the latency to find the hidden platform in the MWM test as compared to the vehicle group. Furthermore, vehicle-treated mice revealed reduced phospho-CaMKII (Thr286) levels in the hippocampus, which can be partially restored by levamlodipine besylate (0.1 mg/kg and 0.5 mg/kg) treatment. No significant outcome on microglia and astrocytes were observed following levamlodipine besylate treatment. This data reveal novel findings of the therapeutic potential of low-dose levamlodipine besylate that could considerably enhance the cognitive function in VaD mice.

Visualizing Autophagic Flux during Endothelial Injury with a Pathway-Inspired Tandem-Reaction Based Fluorogenic Probe.

Autophagy is a dynamic and complicated catabolic process. Imaging autophagic flux can clearly advance knowledge of its pathophysiology significance. While the most common way autophagy is imaged relies on fluorescent protein-based probes, this method requires substantial genetic manipulation that severely restricts the application. Small fluorescent probes capable of tracking autophagic flux with good spatiotemporal resolution are highly demanable. Methods: In this study, we developed a small-molecule fluorogenic probe (AFG-1) that facilitates real-time imaging of autophagic flux in both intact cells and live mice. AFG-1 is inspired by the cascading nitrosative and acidic microenvironments evolving during autophagy. It operates over two sequential steps. In the first step, AFG-1 responds to the up-regulated peroxynitrite at the initiation of autophagy by its diphenylamino group being oxidatively dearylated to yield a daughter probe. In the second step, the daughter probe responds to the acidic autolysosomes at the late stage of autophagy by being protonated. Results: This pathway-dependent mechanism has been confirmed first by sequentially sensing ONOO- and acid in aqueous solution, and then by imaging autophagic flux in live cells. Furthermore, AFG-1 has been successfully applied to visualize autophagic flux in real-time in live mice following brain ischemic injury, justifying its robustness. Conclusion: Due to the specificity, easy operation, and the dynamic information yielded, AFG-1 should serve as a potential tool to explore the roles of autophagy under various pathological settings.

GPR124 facilitates pericyte polarization and migration by regulating the formation of filopodia during ischemic injury.

Prolonged occlusion of multiple microvessels causes microvascular injury. G protein-coupled receptor 124 (GPR124) has been reported to be required for maintaining central nervous system (CNS) angiogenesis and blood-brain barrier integrity. However, the molecular mechanisms by which GPR124 regulates pericytes during ischemia have remained elusive. Methods: A microsphere embolism-induced ischemia model was used to evaluate the expression of GPR124 following microsphere embolism. Immunocytochemistry and stochastic optical reconstruction microscopy imaging were used to assess the expression and distribution of GPR124 in human brain vascular pericytes (HBVPs) and after the treatment with 3-morpholino-sydnonimine (SIN-1) or oxygen-glucose deprivation (OGD). The effect of GPR124 knockdown or overexpression on HBVP migration was analyzed in vitro using wound healing assays and a microfluidic device. GPR124 loss-of-function studies were performed in HBVPs and HEK293 cells using CRISPR-Cas9-mediated gene deletion. Time-lapse imaging was used to assess dynamic changes in the formation of filopodia in an individual cell. Finally, to explore the functional domains required for GPR124 activity, deletion mutants were constructed for each of the N-terminal domains. Results: GPR124 expression was increased in pericytes following microsphere embolism. Morphological analysis showed localization of GPR124 to focal adhesions where GPR124 bound directly to the actin binding protein vinculin and upregulated Cdc42. SIN-1 or OGD treatment redistributed GPR124 to the leading edges of HBVPs where GPR124 signaling was required for pericyte filopodia formation and directional migration. Partial deletion of GPR124 domains decreased SIN-1-induced filopodia formation and cell migration. Conclusion: Taken together, our results provide the first evidence for a role of GPR124 in pericyte migration under ischemic conditions and suggest that GPR124 was essential for Cdc42 activation and filopodia formation.

Functional coupling of Tmem74 and HCN1 channels regulates anxiety-like behavior in BLA neurons.

Anxiety disorders are the most prevalent psychiatric disorders, but their pathogenic mechanism remains poorly understood. Here, we report that transmembrane protein 74 (TMEM74), which contains two putative transmembrane domains and exhibits high levels of mRNA in the brain, is closely associated with the pathogenesis of anxiety disorders. TMEM74 was decreased in the serum of patients with anxiety and the basolateral amygdaloid nucleus (BLA) in chronic stress mice. Furthermore, genetic deletion of Tmem74 or selective knockdown of Tmem74 in BLA pyramidal neurons resulted in anxiety-like behaviors in mice. Whole-cell recordings in BLA pyramidal neurons revealed lower hyperpolarization-activated cation current (Ih) and greater input resistance and excitability in Tmem74-/- neurons than in wild-type neurons. Accordingly, surface expression of hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels was also lower in the BLA of Tmem74-/- mice. The Ih current blocker ZD7288 mimicked these effects in BLA pyramidal neurons in wild-type mice but not in Tmem74-/- mice. Consistent with the improvement in anxiety-like behaviors, Tmem74 overexpression restored HCN1 channel trafficking and pyramidal neuron excitability in the BLA of Tmem74-/- and chronic stress mice. Mechanistically, we demonstrate that interactions between Tmem74 and HCN1 are physiologically relevant and that transmembrane domain 1 (TM1) is essential for the cellular membrane localization of Tmem74 to enhance Ih. Together, our findings suggest that Tmem74 coupling with HCN1 acts as a critical component in the pathophysiology of anxiety and is a potential target for new treatments of anxiety disorders.

Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2/PlexinA4.

The proper interactions between blood vessels and neurons are critical for maintaining the strength of neural circuits and cognitive function. However, the precise molecular events underlying these interactions remain largely unknown. Here, we report that the selective knockout of semaphorin 3G (Sema3G) in endothelial cells impaired hippocampal-dependent memory and reduced dendritic spine density in CA1 neurons in mice; these effects were reversed after restoration of Sema3G levels in the hippocampus by AAV transfection. We further show that Sema3G increased excitatory synapse density via neuropilin-2/PlexinA4 signaling and through activation of Rac1. These results provide the first evidence that, in the central nervous system, endothelial Sema3G serves as a vascular-derived synaptic organizer that regulates synaptic plasticity and hippocampal-dependent memory. Our findings highlight the role of vascular endothelial cells in regulating cognitive function through intercellular communication with neurons in the hippocampus.

Cathepsin B inhibition ameliorates leukocyte-endothelial adhesion in the BTBR mouse model of autism.

AIMS: Autism spectrum disorder (ASD) is a wide range of neurodevelopmental disorders involving deficits in social interaction and communication. Unfortunately, autism remains a scientific and clinical challenge owing to the lack of understanding the cellular and molecular mechanisms underlying it. This study aimed to investigate the pathophysiological mechanism underlying leukocyte-endothelial adhesion in autism-related neurovascular inflammation. METHODS: Male BTBR T+tf/J mice were used as an autism model. The dynamic pattern of leukocyte-endothelial adhesion in mouse cerebral vessels was detected by two-photon laser scanning microscopy (TPLSM). Using FACS, RT-PCR, and Western blotting, we explored the expression of cell adhesion molecules, the mRNA expression of endothelial chemokine, the protein levels of cathepsin B, and inflammatory mediators. RESULTS: We found a significant increase in leukocyte-endothelial adhesion in BTBR mice, accompanied by elevated expression of the adhesion molecule neutrophils CD11b and endothelial ICAM-1. Our data further indicate that elevated neutrophil cathepsin B levels contribute to elevated endothelial chemokine CXCL7 levels in BTBR mice. The pharmacological inhibition of cathepsin B reverses the enhanced leukocyte-endothelial adhesion in the cerebral vessels of autistic mice. CONCLUSION: Our results revealed the prominent role of cathepsin B in modulating leukocyte-endothelial adhesion during autism-related neurovascular inflammation and identified a promising novel approach for autism treatment.

Bis-reaction-trigger as a strategy to improve the selectivity of fluorescent probes.

By the strategy of equipping a fluorophore with two reaction triggers that are tailored to the specific chemistry of peroxynitrite, we have developed a highly selective probe for detecting peroxynitrite in live cells. Sequential response by the two triggers enabled the probe to reveal various degrees of nitrosative stress in live cells via a sensitive emission colour change.

Myt1l induced direct reprogramming of pericytes into cholinergic neurons.

OBJECTIVE: The cholinergic deficit is thought to underlie progressed cognitive decline in Alzheimer Disease. The lineage reprogramming of somatic cells into cholinergic neurons may provide strategies toward cell-based therapy of neurodegenerative diseases. METHODS AND RESULTS: Here, we found that a combination of neuronal transcription factors, including Ascl1, Myt1l, Brn2, Tlx3, and miR124 (5Fs) were capable of directly converting human brain vascular pericytes (HBVPs) into cholinergic neuronal cells. Intriguingly, the inducible effect screening of reprogramming factors showed that a single reprogramming factor, Myt1l, induced cells to exhibit similarly positive staining for Tuj1, MAP2, ChAT, and VAChT upon lentivirus infection with the 5Fs after 30 days. HBVP-converted neurons were rarely labeled even after long-term incubation with BrdU staining, suggesting that induced neurons were directly converted from HBVPs rather than passing through a proliferative state. In addition, the overexpression of Myt1l induced the elevation of Ascl1, Brn2, and Ngn2 levels that contributed to reprogramming. CONCLUSIONS: Our findings provided proof of the principle that cholinergic neurons could be produced from HBVPs by reprogramming factor-mediated fate instruction. Myt1l was a critical mediator of induced neuron cell reprogramming. HBVPs represent another excellent alternative cell resource for cell-based therapy to treat neurodegenerative disease.

A fluorescent peptidyl substrate for visualizing peptidyl-prolyl cis/trans isomerase activity in live cells.

This communication reports on a fluorescent probe (PPI-P) for imaging active peptidyl-prolyl cis/trans isomerases in live cells. PPI-P is capable of responding to both recombinant and cellular PPIases fluorogenically, and has been shown to specifically image active PPIases in live cells.

Cholinergic Grb2-Associated-Binding Protein 1 Regulates Cognitive Function.

Grb2-associated-binding protein 1 (Gab1) is a docking/scaffolding molecule known to play an important role in cell growth and survival. Here, we report that Gab1 is decreased in cholinergic neurons in Alzheimer's disease (AD) patients and in a mouse model of AD. In mice, selective ablation of Gab1 in cholinergic neurons in the medial septum impaired learning and memory and hippocampal long-term potentiation. Gab1 ablation also inhibited SK channels, leading to an increase in firing in septal cholinergic neurons. Gab1 overexpression, on the other hand, improved cognitive function and restored hippocampal CaMKII autorphosphorylation in AD mice. These results suggest that Gab1 plays an important role in the pathophysiology of AD and may represent a novel therapeutic target for diseases involving cholinergic dysfunction.

A H2O2-Responsive Theranostic Probe for Endothelial Injury Imaging and Protection.

Overproduction of H2O2 causes oxidative stress and is the hallmark of vascular diseases. Tracking native H2O2 in the endothelium is therefore indispensable to gain fundamental insights into this pathogenesis. Previous fluorescent probes for H2O2 imaging were generally arylboronates which were decomposed to emissive arylphenols in response to H2O2. Except the issue of specificity challenged by peroxynitrite, boric acid by-produced in this process is actually a waste with unknown biological effects. Therefore, improvements could be envisioned if a therapeutic agent is by-produced instead. Herein, we came up with a "click-to-release-two" strategy and demonstrate that dual functional probes could be devised by linking a fluorophore with a therapeutic agent via a H2O2-responsive bond. As a proof of concept, probe AP consisting of a 2-(2'-hydroxyphenyl) benzothiazole fluorophore and an aspirin moiety has been prepared and confirmed for its theranostic effects. This probe features high specificity towards H2O2 than other reactive species including peroxynitrite. Its capability to image and ameliorate endothelial injury has been verified both in vitro and in vivo. Noteworthy, as a result of its endothelial-protective effect, AP also works well to reduce thrombosis formation in zebrafish model.

Melatonin ameliorates hypoglycemic stress-induced brain endothelial tight junction injury by inhibiting protein nitration of TP53-induced glycolysis and apoptosis regulator.

Severe hypoglycemia has a detrimental impact on the cerebrovasculature, but the molecular events that lead to the disruption of the integrity of the tight junctions remain unclear. Here, we report that the microvessel integrity was dramatically compromised (59.41% of wild-type mice) in TP53-induced glycolysis and apoptosis regulator (TIGAR) transgenic mice stressed by hypoglycemia. Melatonin, a potent antioxidant, protects against hypoglycemic stress-induced brain endothelial tight junction injury in the dosage of 400 nmol/L in vitro. FRET (fluorescence resonance energy transfer) imaging data of endothelial cells stressed by low glucose revealed that TIGAR couples with calmodulin to promote TIGAR tyrosine nitration. A tyrosine 92 mutation interferes with the TIGAR-dependent NADPH generation (55.60% decreased) and abolishes its protective effect on tight junctions in human brain microvascular endothelial cells. We further demonstrate that the low-glucose-induced disruption of occludin and Caludin5 as well as activation of autophagy was abrogated by melatonin-mediated blockade of nitrosative stress in vitro. Collectively, we provide information on the detailed molecular mechanisms for the protective actions of melatonin on brain endothelial tight junctions and suggest that this indole has translational potential for severe hypoglycemia-induced neurovascular damage.

Valproic Acid Influences MTNR1A Intracellular Trafficking and Signaling in a ß-Arrestin 2-Dependent Manner.

Valproate exposure is associated with increased risks of autism spectrum disorder. To date, the mechanistic details of disturbance of melatonin receptor subtype 1 (MTNR1A) internalization upon valproate exposure remain elusive. By expressing epitope-tagged receptors (MTNR1A-EGFP) in HEK-293 and Neuro-2a cells, we recorded the dynamic changes of MTNR1A intracellular trafficking after melatonin treatment. Using time-lapse confocal microscopy, we showed in living cells that valproic acid interfered with the internalization kinetics of MTNR1A in the presence of melatonin. This attenuating effect was associated with a decrease in the phosphorylation of PKA (Thr197) and ERK (Thr202/Tyr204). VPA treatment did not alter the whole-cell currents of cells with or without melatonin. Furthermore, fluorescence resonance energy transfer imaging data demonstrated that valproic acid reduced the melatonin-initiated association between YFP-labeled ß-arrestin 2 and CFP-labeled MTNR1A. Together, we suggest that valproic acid influences MTNR1A intracellular trafficking and signaling in a ß-arrestin 2-dependent manner.

P2RX7 sensitizes Mac-1/ICAM-1-dependent leukocyte-endothelial adhesion and promotes neurovascular injury during septic encephalopathy.

Septic encephalopathy (SE) is a critical factor determining sepsis mortality. Vascular inflammation is known to be involved in SE, but the molecular events that lead to the development of encephalopathy remain unclear. Using time-lapse in vivo two-photon laser scanning microscopy, we provide the first direct evidence that cecal ligation and puncture in septic mice induces microglial trafficking to sites adjacent to leukocyte adhesion on inflamed cerebral microvessels. Our data further demonstrate that septic injury increased the chemokine CXCL1 level in brain endothelial cells by activating endothelial P2RX7 and eventually enhanced the binding of Mac-1 (CD11b/CD18)-expressing leukocytes to endothelial ICAM-1. In turn, leukocyte adhesion upregulated endothelial CX3CL1, thereby triggering microglia trafficking to the injured site. The sepsis-induced increase in endothelial CX3CL1 was abolished in CD18 hypomorphic mutant mice. Inhibition of the P2RX7 pathway not only decreased endothelial ICAM-1 expression and leukocyte adhesion but also prevented microglia overactivation, reduced brain injury, and consequently doubled the early survival of septic mice. These results demonstrate the role of the P2RX7 pathway in linking neurovascular inflammation to brain damage in vivo and provide a rationale for targeting endothelial P2RX7 for neurovascular protection during SE.

Effects of VBMDMP on the reversal of cisplatin resistance in human lung cancer A549/DDP cells.

Tumor drug resistance is a major obstacle to cancer chemotherapy. We previously constructed a fusion protein based on two tumstatin-derived sequences named recombinant VBMDM (rVBMDMP). We preliminarily confirmed its inhibition of HUVEC and colon cancer cell growth. The present study further systematically observed the inhibitory effect of rVBMDMP on lung cancer cell growth and analyzed a possible mechanism to provide a theoretical basis for the development of new antitumor protein drugs. The effect of rVBMDMP on human lung adenocarcinoma (A549) and cisplatin-resistant human lung adenocarcinoma (A549/DDP) cell proliferation was evaluated by MTS assay. Hoechst 33342 staining performed together with fluorescence microscopy and immunoblot analysis were used to examine the effects of rVBMDMP on the apoptosis of A549/DDP cells. A protein phosphorylation chip was used to identify changes in rVBMDMP-induced signaling protein phosphorylation. Changes in the phosphatidylinositol 3 kinase (PI3K)/Akt signal transduction pathway and expression of multidrug resistance protein (MRP-2)-related molecules following rVBMDMP treatment in A549/DDP cells were evaluated by western blot analysis. A lung cancer xenograft model was used to evaluate the reversal effect of rVBMDMP on drug-resistance of A549/DDP cell tumors to cisplatin in vivo. The results demonstrated that rVBMDMP increased the phosphorylation of 79 signaling proteins, including focal adhesion kinase (FAK), caspase-6, Fas, FasL and FAF1 and downregulated 30 signaling proteins, including integrin αV, integrin ß3, PI3K/Akt, NF-κB and MRP-2 compared with the controls. rVBMDMP also increased the sensitivity of A549 and A549/DDP cells to cisplatin and directly induced apoptosis, which may be related to MRP-2 and Bcl-2 downregulation. The effects of growth inhibition and apoptosis induction of rVBMDMP on A549/DDP cells may be related to the inhibition of integrin αVß3 and PI3K/Akt protein phosphorylation. Finally, we observed an increase in cancer cell sensitivity to cisplatin by rVBMDMP using the A549/DDP cell xenograft model in nude mice. Our study suggests that rVBMDMP may be an effective potential chemotherapy sensitizer and may be a viable drug candidate in anticancer therapies.

Acacetin inhibits the invasion and migration of human non-small cell lung cancer A549 cells by suppressing the p38α MAPK signaling pathway.

Lung cancer is one of the most common malignancies in the world and its metastasis is the major cause of death in cancer patients. Acacetin (5,7-dihydroxy-4'-methoxyflavone), a flavonoid compound, has anti-peroxidative and anti-inflammatory effects. The effect of acacetin on invasion and migration in human NSCLC A549 cells was investigated. First, the result demonstrated acacetin could exhibit an inhibitory effect on the abilities of the adhesion, morphology/actin cytoskeleton arrangement, invasion, and migration by cell-matrix adhesion assay, immunofluorescence assay, Boyden chamber assay, and wound-healing assay. Molecular data showed that the effect of acacetin in A549 cells might be mediated via sustained inactivation of the phosphorylation of mixed-lineage protein kinase 3 (MLK3), mitogen-activated protein kinase kinases 3/6 (MKK3/6), and p38α MAPK signal involved in the downregulation of the expressions of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and urokinase-type plasminogen activator (u-PA). Next, acacetin significantly decreased in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), and the nuclear levels of nuclear factor kappa B (NF-κB), c-Fos, and c-Jun. Also, the treatment with acacetin to A549 cells also leads to a concentration-dependent inhibition on the binding abilities of NF-κB and activator protein-1 (AP-1). Furthermore, the treatment of specific inhibitor for p38 MAPK (SB203580) to A549 cells could cause reduced activities of MMP-2/9 and u-PA. In addition, acacetin significantly decreased the levels of phospho-p38α MAPK, MMP-2/9, and u-PA in p38α-cDNA-transfected cells concomitantly with a marked reduction on cell invasion and migration. Our results revealed the anti-migration and anti-invasion effects of acacetin, which may act as a promising therapeutic agent for the treatment of lung cancer.