The aged brain exhibits a loss in gray matter and a decrease in spines and synaptic densities that may represent a sequela for neurodegenerative diseases such as Alzheimer's. Membrane/lipid rafts (MLR), discrete regions of the plasmalemma enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for the development and stabilization of synapses. Caveolin-1 (Cav-1), a cholesterol binding protein organizes synaptic signaling components within MLR. It is unknown whether loss of synapses is dependent on an age-related loss of Cav-1 expression and whether this has implications for neurodegenerative diseases such as Alzheimer's disease.

Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process.

Patients experiencing large thermal injuries are susceptible to opportunistic infections that can delay recovery and lead to sepsis. Dendritic cells (DC) are important for the detection of pathogens and activation of the innate and acquired immune responses. DCs are significantly decreased in burn patients early after injury, and the development of sepsis is associated with persistent DC depletion. In a murine model of burn wound infection, the enhancement of DCs after injury by treatment with the DC growth factor Fms-like tyrosine kinase-3 ligand (FL) enhances neutrophil migration to infection, improves bacterial clearance, and increases survival in a DC-dependent manner. FL expands the production of both conventional DCs (cDC) and plasmacytoid DCs (pDC). It has been established that cDCs are required for some of the protective effects of FL after burn injury. This study was designed to determine the contribution of the pDC subset.

Studies have confirmed that activation of the neurons of primary somatosensory cortex (S1) is involved in the process of remifentanil (Remi)-induced hyperalgesia (RIH), which can be suppressed by lidocaine (Lido). A total intravenous anesthesia model of rats mimicking clinical Remi-based anesthesia was set up to explore the release of amino acid neurotransmitters of S1 cortex in RIH and its inhibition by Lido in this study.

Prescription opioids are powerful pain-controlling medications that have both benefits and potentially serious risks. Morphine is one of the preferred analgesics that are widely used to treat chronic pain. However, chronic morphine exposure has been found to cause both functional and structural changes in several brain regions, including the medial prefrontal cortex (mPFC), ventral tegmental area (VTA), and hippocampus (HPC), which lead to addictive behavior. Caveolin-1 (Cav-1), a scaffolding protein of membrane lipid rafts (MLRs), has been shown to organize GPCRs and multiple synaptic signaling proteins within the MLRs to regulate synaptic signaling and neuroplasticity. Previously, we showed that in vitro morphine treatment significantly elevates Cav-1 expression and causes neuroplasticity changes. In this study, we confirmed that chronic morphine exposure can significantly increase Cav-1 expression (P < 0.05) and microtubule-associated protein (MAP-2)-positive neuronal dendritic growth in the hippocampus. Moreover, the rewarding effect and dendritic growth in the HPC induced by chronic morphine exposure were significantly inhibited by hippocampal Cav-1 knockdown. Together, these data suggest that Cav-1 in the hippocampus plays an essential role in the neuroplasticity changes that underlie morphine addiction behaviors.

Three-dimensional cell agglomerates are broadly useful in tissue engineering and drug testing. We report a well-free method to form large (1.4-mm) multicellular clusters using 100-MHz surface acoustic waves (SAW) without direct contact with the media or cells. A fluid couplant is used to transform the SAW into acoustic streaming in the cell-laden media held in a petri dish. The couplant transmits longitudinal sound waves, forming a Lamb wave in the petri dish that, in turn, produces longitudinal sound in the media. Due to recirculation, human embryonic kidney (HEK293) cells in the dish are carried to the center of the coupling location, forming a cluster in less than 10 min. A few minutes later, these clusters may then be translated and merged to form large agglomerations, and even repeatedly folded to produce a roughly spherical shape of over 1.4 mm in diameter for incubation-without damaging the existing intercellular bonds. Calcium ion signaling through these clusters and confocal images of multiprotein junctional complexes suggest a continuous tissue construct: intercellular communication. They may be formed at will, and the method is feasibly useful for formation of numerous agglomerates in a single petri dish.

The noble gas helium (He) induces cardioprotection in vivo through unknown molecular mechanisms. He can interact with and modify cellular membranes. Caveolae are cholesterol and sphingolipid-enriched invaginations of the plasma-membrane-containing caveolin (Cav) proteins that are critical in protection of the heart. Mice (C57BL/6J) inhaled either He gas or adjusted room air. Functional measurements were performed in the isolated Langendorff perfused heart at 24 h post He inhalation. Electron paramagnetic resonance spectrometry (EPR) of samples was carried out at 24 h post He inhalation. Immunoblotting was used to detect Cav-1/3 expression in whole-heart tissue, exosomes isolated from platelet free plasma (PFP) and membrane fractions. Additionally, transmission electron microscopy analysis of cardiac tissue and serum function and metabolomic analysis were performed. In contrast to cardioprotection observed in in vivo models, the isolated Langendorff perfused heart revealed no protection after He inhalation. However, levels of Cav-1/3 were reduced 24 h after He inhalation in whole-heart tissue, and Cav-3 was increased in exosomes from PFP. Addition of serum to muscle cells in culture or naïve ventricular tissue increased mitochondrial metabolism without increasing reactive oxygen species generation. Primary and lipid metabolites determined potential changes in ceramide by He exposure. In addition to direct effects on myocardium, He likely induces the release of secreted membrane factors enriched in caveolae. Our results suggest a critical role for such circulating factors in He-induced organ protection.

Translation of preclinical treatments for ischaemia-reperfusion injury into clinical therapies has been limited by a number of factors. This review will focus on a single mode of cardiac protection related to a membrane scaffolding protein, caveolin, which regulates protective signalling as well as myocyte ultrastructure in the setting of ischaemic stress. Factors that have limited the clinical translation of protection will be considered specifically in terms of signalling and structural defects. The potential of caveolin to overcome barriers to protection with the ultimate hope of clinical translation will be discussed.

Traumatic brain injury (TBI) enhances pro-inflammatory responses, neuronal loss and long-term behavioral deficits. Caveolins (Cavs) are regulators of neuronal and glial survival signaling. Previously we showed that astrocyte and microglial activation is increased in Cav-1 knock-out (KO) mice and that Cav-1 and Cav-3 modulate microglial morphology. We hypothesized that Cavs may regulate cytokine production after TBI.

The local delivery of therapeutic small interfering RNA or siRNA to the lungs has the potential to improve the prognosis for patients suffering debilitating lung diseases. Recent advances in materials science have been aimed at addressing delivery challenges including biodistribution, bioavailability and cell internalization, but an equally important challenge to overcome is the development of an inhalation device that can deliver the siRNA effectively to the lung, without degrading the therapeutic itself. Here, we report the nebulization of siRNA, either naked siRNA or complexed with polyethyleneimine (PEI) or a commercial transfection agent, using a miniaturizable acoustomicrofluidic nebulization device. The siRNA solution could be nebulised without significant degradation into an aerosol mist with tunable mean aerodynamic diameters of approximately 3 µm, which is appropriate for deep lung deposition via inhalation. The nebulized siRNA was tested for its stability, as well as its toxicity and gene silencing properties using the mammalian lung carcinoma cell line A549, which demonstrated that the gene silencing capability of siRNA is retained after nebulization. This highlights the potential application of the acoustomicrofluidic device for the delivery of efficacious siRNA via inhalation, either for systemic delivery via the alveolar epithelium or local therapeutic delivery to the lung.

The nucleotide oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) in dorsal root ganglion (DRG) contributes to pain hypersensitivity in multiple neuropathic pain models, but the function of the NLRP3 in diabetic neuropathic pain (DNP) and the regulation mechanism are still largely unknown. Epigenetic regulation plays a vital role in the controlling of gene expression. Ten-eleven translocation methylcytosine dioxygenase 2 (TET2) is a DNA demethylase that contributes to transcriptional activation. TET2 is also involved in high glucose (HG)-induced pathology.

Congenital heart disease (CHD) is the most frequent birth defect worldwide. The number of adult patients with CHD, now referred to as ACHD, is increasing with improved surgical and treatment interventions. However the mechanisms whereby ACHD predisposes patients to heart dysfunction are still unclear. ACHD is strongly associated with metabolic syndrome, but how ACHD interacts with poor modern lifestyle choices and other comorbidities, such as hypertension, obesity, and diabetes, is mostly unknown.

Ultrasound has been used to manipulate cells in both humans and animal models. While intramembrane cavitation and lipid clustering have been suggested as likely mechanisms, they lack experimental evidence. Here, high-speed digital holographic microscopy (kiloHertz order) is used to visualize the cellular membrane dynamics. It is shown that neuronal and fibroblast membranes deflect about 150 nm upon ultrasound stimulation. Next, a biomechanical model that predicts changes in membrane voltage after ultrasound exposure is developed. Finally, the model predictions are validated using whole-cell patch clamp electrophysiology on primary neurons. Collectively, it is shown that ultrasound stimulation directly defects the neuronal membrane leading to a change in membrane voltage and subsequent depolarization. The model is consistent with existing data and provides a mechanism for both ultrasound-evoked neurostimulation and sonogenetic control.

Ultrasound has been used to non-invasively manipulate neuronal functions in humans and other animals. However, this approach is limited as it has been challenging to target specific cells within the brain or body. Here, we identify human Transient Receptor Potential A1 (hsTRPA1) as a candidate that confers ultrasound sensitivity to mammalian cells. Ultrasound-evoked gating of hsTRPA1 specifically requires its N-terminal tip region and cholesterol interactions; and target cells with an intact actin cytoskeleton, revealing elements of the sonogenetic mechanism. Next, we use calcium imaging and electrophysiology to show that hsTRPA1 potentiates ultrasound-evoked responses in primary neurons. Furthermore, unilateral expression of hsTRPA1 in mouse layer V motor cortical neurons leads to c-fos expression and contralateral limb responses in response to ultrasound delivered through an intact skull. Collectively, we demonstrate that hsTRPA1-based sonogenetics can effectively manipulate neurons within the intact mammalian brain, a method that could be used across species.

Studies in vitro demonstrate that neuronal membrane/lipid rafts (MLRs) establish cell polarity by clustering progrowth receptors and tethering cytoskeletal machinery necessary for neuronal sprouting. However, the effect of MLR and MLR-associated proteins on neuronal aging is unknown.

Long-term repeated application of morphine induces plasticity changes in sensitive neurons, which represents the neurobiological basis of morphine addiction. Caveolin-1 (Cav-1) is an important scaffolding protein that plays a key role in the regulation of neurostructural plasticity. However, in-depth studies focused on the role of Cav-1 in morphine-induced changes in neurostructural plasticity are lacking. The present study explored the effects of Cav-1 on the expression levels of 2 markers of neurite outgrowth, growth association protein 43 (GAP-43) and microtubule-associated protein 2 (MAP-2), during the process of morphine-induced changes in the structural plasticity. A primary mouse cerebral cortical neuron culture was established, and Cav-1 knockdown was achieved. The results showed that morphine at a concentration of 10.0μmol/L had no adverse effect on neuronal viability, but enhanced the Cav-1 and GAP-43 levels and induced the outgrowth of MAP-2-labeled neurites. The qRT-PCR data showed that 10.0μmol/L of morphine increased the Cav-1 mRNA level and promoted Cav-1 expression at the transcriptional level. Moreover, Cav-1 knockdown inhibited the morphine-induced upregulation of GAP-43 expression and the prolongation of MAP-2-labeled neurites. In conclusion, morphine was capable of inducing changes in the structural plasticity of primary cultured mouse cortical neurons, and Cav-1 played an important role in this process. Inhibition of Cav-1 expression reduced the morphine-induced increase in the neuronal growth markers GAP-43 and MAP-2. Thus, Cav-1 may be a potential molecular target for inhibiting morphine-induced neuroplasticity.

While obesity blunts the ability of muscle to mount a protein synthetic response to an amino acid infusion in older adults, it is unclear if this insensitivity to nutrition persists long term and in response to complete foods is unknown. To address this, young (2 months old) and old (17-20 months old) Brown Norway rats were randomized to receive either chow or a 12 wk diet of calorie-dense human foods. At wk 10 drinking water was supplemented with 2% heavy water, followed 2 weeks later by a flooding dose of [2H5]-phenylalanine and an oral leucine bolus, allowing the short and long-term effects of age and diet on muscle protein synthesis rates to be determined. The experimental diet increased energy intake in both young (7.4 ± 0.9%) and old (18.2 ± 1.8%) animals (P < 0.01), but only led to significant increases in body weight in the former (young: 10.2 ± 3.0% (P < 0.05) and old: 3.1 ± 5.1% (NS) vs. age-matched controls). Notably, energy expenditure in response to the cafeteria diet was increased in old animals only (chow: 5.1 ± 0.4; cafe: 8.2 ± 1.6 kcal.kg b.w-1.h-1; P < 0.05). Gastrocnemius protein fractional synthetic rates in response to either an acute leucine bolus or two weeks of feeding were equivalent across groups irrespective of age or diet. Rodents in old age appear capable of preventing weight gain in response to a calorie-dense diet by increasing energy expenditure while maintaining the anabolic sensitivity of muscle to nutrition; the mechanisms of which could have important implications for the aging obese human.

Remarkably, the interface of a fluid droplet will produce visible capillary waves when exposed to acoustic waves. For example, a small (∼1 μL) sessile droplet will oscillate at a low ∼102 Hz frequency when weakly driven by acoustic waves at ∼106 Hz frequency and beyond. We measured such a droplet's interfacial response to 6.6 MHz ultrasound to gain insight into the energy transfer mechanism that spans these vastly different time scales, using high-speed microscopic digital transmission holography, a unique method to capture three-dimensional surface dynamics at nanometer space and microsecond time resolutions. We show that low-frequency capillary waves are driven into existence via a feedback mechanism between the acoustic radiation pressure and the evolving shape of the fluid interface. The acoustic pressure is distributed in the standing wave cavity of the droplet, and as the shape of the fluid interface changes in response to the distributed pressure present on the interface, the standing wave field also changes shape, feeding back to produce changes in the acoustic radiation pressure distribution in the cavity. A physical model explicitly based upon this proposed mechanism is provided, and simulations using it were verified against direct observations of both the microscale droplet interface dynamics from holography and internal pressure distributions using microparticle image velocimetry. The pressure-interface feedback model accurately predicts the vibration amplitude threshold at which capillary waves appear, the subsequent amplitude and frequency of the capillary waves, and the distribution of the standing wave pressure field within the sessile droplet responsible for the capillary waves.

Ultrasound has been shown to affect the function of both neurons and non-neuronal cells, but, the underlying molecular machinery has been poorly understood. Here, we show that at least two mechanosensitive proteins act together to generate C. elegans behavioral responses to ultrasound stimuli. We first show that these animals generate reversals in response to a single 10 msec pulse from a 2.25 MHz ultrasound transducer. Next, we show that the pore-forming subunit of the mechanosensitive channel TRP-4, and a DEG/ENaC/ASIC ion channel MEC-4, are both required for this ultrasound-evoked reversal response. Further, the trp-4;mec-4 double mutant shows a stronger behavioral deficit compared to either single mutant. Finally, overexpressing TRP-4 in specific chemosensory neurons can rescue the ultrasound-triggered behavioral deficit in the mec-4 null mutant, suggesting that both TRP-4 and MEC-4 act together in affecting behavior. Together, we demonstrate that multiple mechanosensitive proteins likely cooperate to transform ultrasound stimuli into behavioral changes.

Fms-like tyrosine kinase-3 ligand (Flt3L) is a hemopoietic cytokine and dendritic cell (DC) growth factor that promotes the proliferation and differentiation of progenitor cells into DCs. We have previously found that treatment of severely burned mice with recombinant Flt3L significantly enhances DC production and bacterial clearance from infected burn wounds, and increases global immune cell activation and survival in response to a burn wound infection. These significant benefits of Flt3L treatment after burn injury have prompted the question of whether or not severe burn injury induces deficits in endogenous Flt3L levels that could affect DCs and subsequent responses to infection.