A Neural Circuit for the Suppression of Pain by a Competing Need State.

Hunger and pain are two competing signals that individuals must resolve to ensure survival. However, the neural processes that prioritize conflicting survival needs are poorly understood. We discovered that hunger attenuates behavioral responses and affective properties of inflammatory pain without altering acute nociceptive responses. This effect is centrally controlled, as activity in hunger-sensitive agouti-related protein (AgRP)-expressing neurons abrogates inflammatory pain. Systematic analysis of AgRP projection subpopulations revealed that the neural processing of hunger and inflammatory pain converge in the hindbrain parabrachial nucleus (PBN). Strikingly, activity in AgRP → PBN neurons blocked the behavioral response to inflammatory pain as effectively as hunger or analgesics. The anti-nociceptive effect of hunger is mediated by neuropeptide Y (NPY) signaling in the PBN. By investigating the intersection between hunger and pain, we have identified a neural circuit that mediates competing survival needs and uncovered NPY Y1 receptor signaling in the PBN as a target for pain suppression.

Novel nanomedicines to overcome cancer multidrug resistance.

Chemotherapy remains a powerful tool to eliminate malignant cells. However, the efficacy of chemotherapy is compromised by the frequent emergence of intrinsic and acquired multidrug resistance (MDR). These chemoresistance modalities are based on a multiplicity of molecular mechanisms of drug resistance, including : 1) Impaired drug uptake into cancer cells; 2) Increased expression of ATP-binding cassette efflux transporters; 3) Loss of function of pro-apoptotic factors; 4) Enhanced DNA repair capacity; 5) Qualitative or quantitative alterations of specific cellular targets; 6) Alterations that allow cancer cells to tolerate adverse or stressful conditions; 7) Increased biotransformation or metabolism of anticancer drugs to less active or completely inactive metabolites; and 8) Intracellular and intercellular drug sequestration in well-defined organelles away from the cellular target. Hence, one of the major aims of cancer research is to develop novel strategies to overcome cancer drug resistance. Over the last decades, nanomedicine, which focuses on targeted delivery of therapeutic drugs into tumor tissues using nano-sized formulations, has emerged as a promising tool for cancer treatment. Therefore, nanomedicine has been introduced as a reliable approach to improve treatment efficacy and minimize detrimental adverse effects as well as overcome cancer drug resistance. With rationally designed strategies including passively targeted delivery, actively targeted delivery, delivery of multidrug combinations, as well as multimodal combination therapy, nanomedicine paves the way towards efficacious cancer treatment and hold great promise in overcoming cancer drug resistance. Herein, we review the recent progress of nanomaterials used in medicine, including liposomal nanoparticles, polymeric nanoparticles, inorganic nanoparticles and hybrid nanoparticles, to surmount cancer multidrug resistance. Finally, the future perspectives of the application of nanomedicine to reverse cancer drug resistance will be addressed.

Codelivery of Anti-PD-1 Antibody and Paclitaxel with Matrix Metalloproteinase and pH Dual-Sensitive Micelles for Enhanced Tumor Chemoimmunotherapy.

Immune checkpoint blockade (ICB) is demonstrating great potential in cancer immunotherapy nowadays. Yet, the low response rate to ICB remains an urgent challenge for tumor immunotherapy. A pH and matrix metalloproteinase dual-sensitive micellar nanocarrier showing spatio-temporally controlled release of anti-PD-1 antibody (aPD-1) and paclitaxel (PTX) in solid tumors is prepared to realize synergistic cancer chemoimmunotherapy. Antitumor immunity can be activated by PTX-induced immunogenic cell death (ICD), while aPD-1 blocks the PD-1/PD-L1 axis to suppress the immune escape due to PTX-induced PD-L1 up-regulation, thus resulting in a synergistic antitumor chemoimmunotherapy. Through decoration with a sheddable polyethylene glycol (PEG) shell, the nanodrug may better accumulate in tumors to boost the synergistic antitumor treatment in a mouse melanoma model. The present study demonstrates a potent antitumor chemoimmunotherapy utilizing tumor microenvironment-sensitive micelles bearing a sheddable PEG layer to mediate site-specific sequential release of aPD-1 and PTX.

Novel cell-free high-throughput screening method for pharmacological tools targeting K+ channels.

K(+) channels, a superfamily of ∼80 members, control cell excitability, ion homeostasis, and many forms of cell signaling. Their malfunctions cause numerous diseases including neuronal disorders, cardiac arrhythmia, diabetes, and asthma. Here we present a novel liposome flux assay (LFA) that is applicable to most K(+) channels. It is robust, low cost, and high throughput. Using LFA, we performed small molecule screens on three different K(+) channels and identified new activators and inhibitors for biological research on channel function and for medicinal development. We further engineered a hERG (human ether-à-go-go-related gene) channel, which, when used in LFA, provides a highly sensitive (zero false negatives on 50 hERG-sensitive drugs) and highly specific (zero false positives on 50 hERG-insensitive drugs), low-cost hERG safety assay.

Mechanosensitivity is mediated directly by the lipid membrane in TRAAK and TREK1 K+ channels.

Mechanosensitive ion channels underlie neuronal responses to physical forces in the sensation of touch, hearing, and other mechanical stimuli. The fundamental basis of force transduction in eukaryotic mechanosensitive ion channels is unknown. Are mechanical forces transmitted directly from membrane to channel as in prokaryotic mechanosensors or are they mediated through macromolecular tethers attached to the channel? Here we show in cells that the K(+) channel TRAAK (K2P4.1) is responsive to mechanical forces similar to the ion channel Piezo1 and that mechanical activation of TRAAK can electrically counter Piezo1 activation. We then show that the biophysical origins of force transduction in TRAAK and TREK1 (K2P2.1) two-pore domain K(+) (K2P) channels come from the lipid membrane, not from attached tethers. These findings extend the "force-from-lipid" principle established for prokaryotic mechanosensitive channels MscL and MscS to these eukaryotic mechanosensitive K(+) channels.

Microfluidics-enabled Serial Assembly of Lipid-siRNA-sorafenib Nanoparticles for Synergetic Hepatocellular Carcinoma Therapy.

Multi-component nanoparticles (mNPs) hold great potential for disease prevention and treatment. However, a major barrier is the lack of versatile platforms to accommodate steps of assembly processes of mNPs. Here the microfluidics-enabled serial assembly (MESA) of mNPs is presented. The microfluidic chip, as a mini-conveyor of initial materials, sequentially enables the assembly of sorafenib supramolecule, electrostatic adsorption of siRNA, and surface assembly of protective lipids. The produced lipid-siRNA-sorafenib nanoparticles (LSS NPs) have ultrahigh encapsulation efficiencies for sorafenib (≈100%) and siRNA (≈95%), which benefit from the accommodation of both fast and slow processes on the chip. Although carrying negative charges, LSS NPs enable cytosolic delivery of agents and high gene silencing efficiency within tumor cells. In vivo, the LSS NPs delivering hypoxia-induced factor (HIF1α)-targeted siRNA efficiently regress tumors of Hep3B xenograft and hepatocellular carcinoma patient-derived primary cells xenograft (PDCX) and finally extend the average survival of PDCX mice to 68 days. Thus, this strategy is promising as a sorafenib/siRNA combination therapy, and MESA can be a universal platform for fabricating complex nanosystems.

Microfluidics-Enabled Nanovesicle Delivers CD47/PD-L1 Antibodies to Enhance Antitumor Immunity and Reduce Immunotoxicity in Lung Adenocarcinoma.

The CD47/PD-L1 antibodies combination exhibits durable antitumor immunity but also elicits excessive immune-related adverse events (IRAEs) caused by the on-target off-tumor immunotoxicity, hindering their clinical benefits greatly. Here, a microfluidics-enabled nanovesicle using ultra-pH-sensitive polymer mannose-poly(carboxybetaine methacrylate)-poly(hydroxyethyl piperidine methacrylate) (Man-PCB-PHEP) is developed to deliver CD47/PD-L1 antibodies (NCPA) for tumor-acidity-activated immunotherapy. The NCPA can specifically release antibodies in acidic environment, thereby stimulating the phagocytosis of bone marrow-derived macrophages. In mice bearing Lewis lung carcinoma, NCPA shows significantly improved intratumoral CD47/PD-L1 antibodies accumulation, promoted tumor-associated macrophages remodeling to antitumoral status, and increased infiltration of dendritic cells and cytotoxic T lymphocytes, resulting in more favorable treatment effect compared to those of free antibodies. Additionally, NCPA also shows less IRAEs, including anemia, pneumonia, hepatitis, and small intestinal inflammation in vivo. Altogether, a potent dual checkpoint blockade immunotherapy utilizing NCPA with enhanced antitumor immunity and reduced IRAEs is demonstrated.

Studies with neutralizing antibodies suggest CXCL8-mediated neutrophil activation is independent of C-C motif chemokine receptor-like 2 (CCRL2) ligand binding function.

C-C motif chemokine receptor-like 2 (CCRL2) is a non-signaling 7 transmembrane receptor that binds chemotactic ligands to shape leukocyte recruitment to sites of inflammation. However, there is a lack of consensus on the ligands that directly bind CCRL2 or their functional impact. Studies with CCRL2 knockout mice have demonstrated that neutrophils have impaired degranulation and migration in response to CXCL8, where the underlying molecular mechanism is proposed to be due to the formation of CCRL2 heterodimers with the chemokine receptor CXCR2. Herein, we characterized the ligands that bind directly to CCRL2 and interrogated the impact of CCRL2 neutralization on CXCL8 signaling in neutrophils using pharmacological antibody tools. Using flow cytometry and Surface Plasmon Resonance microscopy (SPRm) cell binding experiments, we confirmed that chemerin, but not previously reported C-C chemokines, binds CCRL2. Furthermore, we identified human and mouse CCRL2 antibodies that neutralized chemerin binding to CCRL2. Unexpectedly, we found that neutralization of CCRL2 with these antibodies did not attenuate CXCL8-induced human neutrophil degranulation nor CXCL8-induced murine neutrophil recruitment to the peritoneum. Based on the observed differences in modulating CCRL2 function with neutralizing antibodies compared to the reported CCRL2 deficient murine models, we hypothesize that the ligand binding function of CCRL2 is dispensable for CXCL8 signaling in neutrophils. Finally, extensive profiling of CCRL2 expression on peripheral blood leukocytes revealed monocytes, dendritic cells (DC), and subpopulations of natural killer T (NKT) cells as additional targets, highlighting potential roles for CCRL2 in human cell types beyond neutrophils that warrants future investigation.

Forward genetic analysis reveals multiple gating mechanisms of TRPV4.

TRPV4 is a polymodal cation channel gain-of-function (GOF) allele which causes skeletal dysplasia in humans. To better understand its gating, we screened for additional GOF alleles based on their ability to block yeast proliferation. Repeatedly, only a limited number of such growth-blocking mutations were isolated. Expressed in oocytes, wild-type channels can be strongly activated by either hypotonicity or exposure to the potent agonist 4alphaPDD, although the GOF channels behaved as if they were fully prestimulated as well as lacking a previously uncharacterized voltage-dependent inactivation. Five of six mutations occurred at or near the inner ends of the predicted core helices, giving further direct evidence that this region indeed forms the main intracellular gate in TRP channels. Surprisingly, both wild-type channels as well as these GOF channels maintain strong steady-state outward rectification that is not due to a Ca(2+) block, as has been proposed elsewhere. We conclude that TRPV4 contains an additional voltage-dependent gating mechanism in series with the main intracellular gate.

Wild-type and brachyolmia-causing mutant TRPV4 channels respond directly to stretch force.

Whether animal ion channels functioning as mechanosensors are directly activated by stretch force or indirectly by ligands produced by the stretch is a crucial question. TRPV4, a key molecular model, can be activated by hypotonicity, but the mechanism of activation is unclear. One model has this channel being activated by a downstream product of phospholipase A(2), relegating mechanosensitivity to the enzymes or their regulators. We expressed rat TRPV4 in Xenopus oocytes and repeatedly examined >200 excised patches bathed in a simple buffer. We found that TRPV4 can be activated by tens of mm Hg pipette suctions with open probability rising with suction even in the presence of relevant enzyme inhibitors. Mechanosensitivity of TRPV4 provides the simplest explanation of its various force-related physiological roles, one of which is in the sensing of weight load during bone development. Gain-of-function mutants cause heritable skeletal dysplasias in human. We therefore examined the brachyolmia-causing R616Q gain-of-function channel and found increased whole-cell current densities compared with wild-type channels. Single-channel analysis revealed that R616Q channels maintain mechanosensitivity but have greater constitutive activity and no change in unitary conductance or rectification.

Dual-Sensitive PEG-Sheddable Nanodrug Hierarchically Incorporating PD-L1 Antibody and Zinc Phthalocyanine for Improved Immuno-Photodynamic Therapy.

Tumor immunotherapy like immune checkpoint blockade (ICB) shows great success nowadays but is severely limited by low response rates and immune-related adverse events (IRAEs). While photodynamic therapy (PDT) could efficiently eradicate tumor cells and further induce immune responses to promote activating of T lymphocytes. Herein a nanodrug hierarchically incorporating photosensitizer and PD-L1 antibody was developed for synergistic tumor immuno-photodynamic therapy. A pH/enzyme dual-sensitive polymeric micelle with sheddable PEG coating was designed for codelivery of PD-L1 antibody and zinc phthalocyanine (ZnPc) in the tumor. The tumor microenvironment featuring low pH and high matrix metallopeptidase 2 (MMP-2) sequentially triggered the shedding of PEG and the release of PD-L1 antibody to exert local ICB in tumor tissue, after which the remaining nanodrug with ZnPc undergoing charge reversal was readily delivered into tumor cells. With light irradiation, the photodynamic therapy effect of sAMPc induced immunogenic cell death of tumor cells and further promoted intratumor recruitment of CD8+ T cells, thus resulting in a synergistic immuno-photodynamic therapy with ICB. Moreover, the PEG-sheddable strategy endowed the nanodrug with stealth properties in blood circulation, making the IRAEs of PD-L1 antibody significantly reduced. This pH/MMP-2 dual-sensitive PEG sheddable nanodrug provids a promising strategy for well-combined ICB therapy and PDT to achieve improved anticancer immuno-photodynamic therapy with reduced adverse effects.

Yeast gain-of-function mutations reveal structure-function relationships conserved among different subfamilies of transient receptor potential channels.

Transient receptor potential (TRP) channels found in animals, protists, and fungi are primary chemo-, thermo-, or mechanosensors. Current research emphasizes the characteristics of individual channels in each animal TRP subfamily but not the mechanisms common across subfamilies. A forward genetic screen of the TrpY1, the yeast TRP channel, recovered gain-of-function (GOF) mutations with phenotype in vivo and in vitro. Single-channel patch-clamp analyses of these GOF-mutant channels show prominent aberrations in open probability and channel kinetics. These mutations revealed functionally important aromatic amino acid residues in four locations: at the intracellular end of the fifth transmembrane helix (TM5), at both ends of TM6, and at the immediate extension of TM6. These aromatics have counterparts in most TRP subfamilies. The one in TM5 (F380L) aligns precisely with an exceptional Drosophila mutant allele (F550I) that causes constitutive activity in the canonical TRP channel, resulting in rapid and severe retinal degeneration beyond mere loss of phototaxis. Thus, this phenylalanine maintains the balance of various functional states (conformations) of a channel for insect phototransduction as well as one for fungal mechanotransduction. This residue is among a small cluster of phenylalanines found in all known subfamilies of TRP channels. This unique case illustrates that GOF mutations can reveal structure-function principles that can be generalized across different TRP subfamilies. It appears that the conserved aromatics in the four locations have conserved functions in most TRP channels. The possible mechanistic roles of these aromatics and the further use of yeast genetics to dissect TRP channels are discussed.

Near-Infrared Light Laser-Triggered Release of Doxorubicin and Sorafenib from TemperatureSensitive Liposomes for Synergistic Therapy of Hepatocellular Carcinoma.

Chemotherapy of hepatocellular carcinoma (HCC) is facing drug resistance, which leads to unsatisfactory therapeutic effect. Thus, a combination therapy using multiple drugs may overcome this challenge. The current study aims to realize a synergistic chemotherapy of HCC by using a near-infrared light (NIR) responsive nanocarrier to co-deliver the chemotherapeutic drug Doxorubicin (DOX) and molecular targeting agent Sorafenib (SF). The nanocarrier, which could effectively load DOX in its aqueous core while SF and IR-780 in its lipid bilayer, is fabricated from a temperature-sensitive liposome (TSL) modified with PF127. An efficient SF and DOX co-loading was achieved, and meanwhile the effective photothermal conversion of IR-780 under NIR laser may cause a disassembly of the liposome structure which may trigger a rapid drug release in tumor site, greatly boosting the synergetic chemotherapeutic effect. The NIR laser-triggered drug release and the synergistic anti-tumor effect were evaluated both in cell and animal experiments, which revealed that the PF127-modified TSL is a potent nanoplatform to improve the HCC treatment through co-delivering a drug combination.

Dual pH-sensitive nanodrug blocks PD-1 immune checkpoint and uses T cells to deliver NF-κB inhibitor for antitumor immunotherapy.

The response to programmed cell death protein-1 (PD-1)/programmed death ligand-1 (PD-L1) blockade in cancer immunotherapy is limited because of multiple immune evasion mechanisms. Here, a previously unknown strategy is proposed to synergize the nuclear factor κB (NF-κB) inhibition and PD-1 blockade for antitumor immunotherapy. A dual pH-sensitive nanocarrier loading curcumin (CUR) and anti-PD-1 monoclonal antibody (aPD-1) may bind to circulating PD-1+ T cells and then follow their infiltration into the tumor. Furthermore, the nanodrug bound to PD-1+ T cells may be released in the tumor microenvironment, leaving aPD-1 to block PD-1 on T cells and generating a CUR-encapsulated cationic nanodrug that can be easily taken up by tumor cells/tumor associated macrophages (TAMs). Thus, not only the antitumor T cells mediate efficient CUR delivery to tumor but also the efficient CUR delivery promotes the tumor infiltration of antitumor T cells, thereby resulting in effective activation of antitumor immunity.

The use of yeast to understand TRP-channel mechanosensitivity.

Mechanosensitive (MS) ion channels likely underlie myriad force-sensing processes, from basic osmotic regulation to specified sensations of animal hearing and touch. Albeit important, the molecular identities of many eukaryotic MS channels remain elusive, let alone their working mechanisms. This is in stark contrast to our advanced knowledge on voltage- or ligand-sensitive channels. Several members of transient receptor potential (TRP) ion channel family have been implicated to function in mechanosensation and are recognized as promising candidate MS channels. The yeast TRP homolog, TRPY1, is clearly a first-line force transducer. It can be activated by hypertonic shock in vivo and by membrane stretch force in excised patches under patch clamp, making it a useful model for understanding TRP channel mechanosensitivity in general. TRPY1 offers two additional research advantages: (1) It has a large ( approximately 300 pS) unitary conductance and therefore a favorable S/N ratio. (2) Budding yeast allows convenient and efficient genetic and molecular manipulations. In this review, we focus on the current research of TRPY1 and discuss its prospect. We also describe the use of yeast as a system to express and characterize animal TRP channels.

Mechanical force and cytoplasmic Ca(2+) activate yeast TRPY1 in parallel.

The ability to sense mechanical and osmotic stimuli is vital to all organisms from mammals to bacteria. Members of the transient receptor potential (TRP) ion-channel family have attracted intense attention for their involvement in mechanosensation. The yeast homologue TRPY1 can clearly be activated by hypertonic shock in vivo and by stretch force under patch clamp. Like its animal counterparts, TRPY1 is polymodal, being gated by membrane stretch force and by cytoplasmic Ca(2+). Here, we investigated how these two gating principles interact. We found that stretch force can induce some channel activation without cytoplasmic Ca(2+). Tens of micromolar Ca(2+) greatly enhance the observed force-induced activities, with open probabilities following well the Boltzmann distribution, in which the two gating energies are summed as exponents. To map this formalism to structures, we found Ca(2+)-binding proteins such as calmodulin or calcineurin to be unnecessary. However, removing a dense cluster of negative charges in the C-terminal cytoplasmic domain of TRPY1 greatly diminishes the Ca(2+) activation as well as its influence on force activation. We also found a strategic point upstream of this charge cluster, at which insertion of amino acids weakens Ca(2+) activation considerably but leaves the mechanosensitivity nearly intact. These results led to a structure-function model in which Ca(2+) binding to the cytoplasmic domain and stretching of the membrane-embedded domain both generate gating force, reaching the gate in parallel.

A pH and reduction dual-sensitive polymeric nanomicelle for tumor microenvironment triggered cellular uptake and controlled intracellular drug release.

Minimal drug leakage during blood circulation and intracellular drug delivery in tumor sites are of great significance in chemotherapeutics. Herein we propose an interlayer crosslinked polymeric micelle with tumor acidity and reduction dual sensitivity for highly efficient drug delivery to cancer cells. A novel copolymer mPEG-C[double bond, length as m-dash]N-PAsp(MEA)-CA was synthesized and self-assembled into a dual-sensitive interlayer-crosslinked micelle (ICM). The micelle was composed of a tumor acidity sheddable PEG outer layer, a reduction-sensitive disulfide-crosslinked interlayer (PAsp(MEA)) and a hydrophobic core of cholic acid (CA) for doxorubicin (DOX) delivery. The nano-sized ICM was stable and showed little drug leakage in a neutral physiological environment. In tumor microenvironments (TMEs) with mild acidity, the PEG outer layer was readily detached due to the hydrolysis of the Schiff base linker, and the surface of the ICM was switched to positively charged to enhance the cellular uptake. Furthermore, inside tumor cells DOX was rapidly released due to the reduction of disulfide bonds by glutathione (GSH). The DOX-loaded ICM exhibited an effective anticancer effect against C6 glioma and reduced side effects both in vitro and in vivo. The study reveals that this pH and reduction dual-sensitive micelle may have great potential to mediate effective anticancer therapy.

Indole and other aromatic compounds activate the yeast TRPY1 channel.

The yeast TRPY1 (Yvc1p) channel is activated by membrane stretch to release vacuolar Ca2+ into the cytoplasm upon osmotic upshock. Exogenously added indole greatly enhances the upshock-induced Ca2+ release in vivo. Indole also reversibly activates the channels under patch clamp. A minimum of 10(-6)M Ca2+ is needed for membrane stretch force to open TPRY1, but indole activation appears to be Ca2+ independent. A deletion of 30 residues at the predicted cytoplasmic domain, 570-600Delta, renders TRPY1 insensitive to stretch force upto 10(-3)M Ca2+. Nonetheless, indole readily activates this mutant channel. Several other aromatic compounds, e.g. the antimicrobial parabens, also activate TRPY1. These compounds likely alter the innate forces in the lipid bilayer received by the channel.

Nutritive, Post-ingestive Signals Are the Primary Regulators of AgRP Neuron Activity.

The brain regulates food intake by processing sensory cues and peripheral physiological signals, but the neural basis of this integration remains unclear. Hypothalamic, agouti-related protein (AgRP)-expressing neurons are critical regulators of food intake. AgRP neuron activity is high during hunger and is rapidly reduced by the sight and smell of food. Here, we reveal two distinct components of AgRP neuron activity regulation: a rapid but transient sensory-driven signal and a slower, sustained calorie-dependent signal. We discovered that nutrients are necessary and sufficient for sustained reductions in AgRP neuron activity and that activity reductions are proportional to the calories obtained. This change in activity is recapitulated by exogenous administration of gut-derived satiation signals. Furthermore, we showed that the nutritive value of food trains sensory systems-in a single trial-to drive rapid, anticipatory AgRP neuron activity inhibition. Together, these data demonstrate that nutrients are the primary regulators of AgRP neuron activity.

Construction of negatively charged and environment-sensitive nanomedicine for tumor-targeted efficient siRNA delivery.

A novel siRNA delivery system based on a triblock copolymer with pH and reduction dual-sensitivity was introduced. The polyplex, having high delivery efficiency not dependent on surface charge reversion in response to the pH value of tumor tissue, was used for target gene silencing in cancer therapy.