Studying interactions among anthropogenic stressors in freshwater ecosystems: A systematic review of 2396 multiple-stressor experiments.

Understanding the interactions among anthropogenic stressors is critical for effective conservation and management of ecosystems. Freshwater scientists have invested considerable resources in conducting factorial experiments to disentangle stressor interactions by testing their individual and combined effects. However, the diversity of stressors and systems studied has hindered previous syntheses of this body of research. To overcome this challenge, we used a novel machine learning framework to identify relevant studies from over 235,000 publications. Our synthesis resulted in a new dataset of 2396 multiple-stressor experiments in freshwater systems. By summarizing the methods used in these studies, quantifying trends in the popularity of the investigated stressors, and performing co-occurrence analysis, we produce the most comprehensive overview of this diverse field of research to date. We provide both a taxonomy grouping the 909 investigated stressors into 31 classes and an open-source and interactive version of the dataset (https://jamesaorr.shinyapps.io/freshwater-multiple-stressors/). Inspired by our results, we provide a framework to help clarify whether statistical interactions detected by factorial experiments align with stressor interactions of interest, and we outline general guidelines for the design of multiple-stressor experiments relevant to any system. We conclude by highlighting the research directions required to better understand freshwater ecosystems facing multiple stressors.

The combined effects of treated sewage discharge and land use on rivers.

Freshwater ecosystems are increasingly threatened by multiple anthropogenic stressors. Release of treated sewage effluent and pollution from agricultural or urban sources can independently reduce water quality with implications for ecological communities. However, our knowledge of the combined effects of these stressors is limited. We performed a field study to quantify the combined effect of treated sewage discharge and land use on nutrient concentrations, sewage fungus presence and communities of macroinvertebrates and benthic algae. Over three seasons in four rivers we found that a model which included an interaction between sewage pollution and time of the year (i.e. months) was the best predictor of nutrient concentrations and the abundance of algae and sewage fungus. Both macroinvertebrate and algae communities shifted downstream of sewage input. Specifically, more tolerant groups, such as cyanobacteria and oligochaetes, were more abundant. The EPT (Ephemeroptera, Plecoptera and Tricoptera) water quality score was best explained by an interaction between month and agriculture in the surrounding landscape. Overall, our results show that sewage discharge has a significant impact on water quality and benthic riverine communities, regardless of the surrounding land uses. Agricultural inputs, however, could be more important than treated sewage discharge in reducing the abundance of sensitive invertebrate taxa. We need both improvements to wastewater treatment processes and reductions in agricultural pollution to reduce threats to vulnerable freshwater communities.

Prediction of Tinnitus Treatment Outcomes Based on EEG Sensors and TFI Score Using Deep Learning.

Tinnitus is a hearing disorder that is characterized by the perception of sounds in the absence of an external source. Currently, there is no pharmaceutical cure for tinnitus, however, multiple therapies and interventions have been developed that improve or control associated distress and anxiety. We propose a new Artificial Intelligence (AI) algorithm as a digital prognostic health system that models electroencephalographic (EEG) data in order to predict patients' responses to tinnitus therapies. The EEG data was collected from patients prior to treatment and 3-months following a sound-based therapy. Feature selection techniques were utilised to identify predictive EEG variables with the best accuracy. The patients' EEG features from both the frequency and functional connectivity domains were entered as inputs that carry knowledge extracted from EEG into AI algorithms for training and predicting therapy outcomes. The AI models differentiated the patients' outcomes into either therapy responder or non-responder, as defined by their Tinnitus Functional Index (TFI) scores, with accuracies ranging from 98%-100%. Our findings demonstrate the potential use of AI, including deep learning, for predicting therapy outcomes in tinnitus. The research suggests an optimal configuration of the EEG sensors that are involved in measuring brain functional changes in response to tinnitus treatments. It identified which EEG electrodes are the most informative sensors and how the EEG frequency and functional connectivity can better classify patients into the responder and non-responder groups. This has potential for real-time monitoring of patient therapy outcomes at home.

A randomized single-blind controlled trial of a prototype digital polytherapeutic for tinnitus.

Objective: This randomized single-blind controlled trial tested the hypothesis that a prototype digital therapeutic developed to provide goal-based counseling with personalized passive and active game-based sound therapy would provide superior tinnitus outcomes, and similar usability, to a popular passive sound therapy app over a 12 week trial period. Methods: The digital therapeutic consisted of an app for iPhone or Android smartphone, Bluetooth bone conduction headphones, neck pillow speaker, and a cloud-based clinician dashboard to enable messaging and app personalization. The control app was a popular self-help passive sound therapy app called White Noise Lite (WN). The primary outcome measure was clinically meaningful change in Tinnitus Functional Index (TFI) between baseline and 12 weeks of therapy. Secondary tinnitus measures were the TFI total score and subscales across sessions, rating scales and the Client Oriented Scale of Improvement in Tinnitus (COSIT). Usability of the US and WN interventions were assessed using the System Usability Scale (SUS) and the mHealth App Usability Questionnaire (MAUQ). Ninety-eight participants who were smartphone app users and had chronic moderate-severe tinnitus (>6 months, TFI score > 40) were enrolled and were randomly allocated to one of the intervention groups. Thirty-one participants in the USL group and 30 in the WN group completed 12 weeks of trial. Results: Mean changes in TFI for the USL group at 6 (16.36, SD 17.96) and 12 weeks (17.83 points, SD 19.87) were clinically meaningful (>13 points reduction), the mean change in WN scores were not clinically meaningful (6 weeks 10.77, SD 18.53; 12 weeks 10.12 points, SD 21.36). A statistically higher proportion of USL participants achieved meaningful TFI change at 6 weeks (55%) and 12 weeks (65%) than the WN group at 6 weeks (33%) and 12 weeks (43%). Mean TFI, rating and COSIT scores favored the US group but were not statistically different from WN. Usability measures were similar for both groups. Conclusions: The USL group demonstrated a higher proportion of responders than the WN group. The usability of the USL therapeutic was similar to the established WN app. The digital polytherapeutic demonstrated significant benefit for tinnitus reduction supporting further development.

Activated B Cells and Plasma Cells Are Resistant to Radiation Therapy.

PURPOSE: B cells play a key role in outcomes of cancer patients and responses to checkpoint blockade immunotherapies. However, the effect of radiation therapy on B cell populations is poorly understood. Here we characterize the effects of radiation on the development, survival, and phenotype of physiological B-cell subsets. METHODS AND MATERIALS: Naïve and immunized tumor bearing and nontumor bearing mice were treated with large-field or focal stereotactic radiation and distinct B-cell subsets of varying developmental stages were analyzed by flow cytometry and real-time reverse transcription polymerase chain reaction. RESULTS: We first report that focal stereotactic radiation is highly superior to large-field radiation at inducing tumor infiltration of B cells, CD8+ T cells, and macrophages. We observed that radiation affects B cell development in the bone marrow, increasing frequencies of early pro-B cells and late pro-B cells while inducing upregulation of programmed cell death protein 1. We then demonstrate that class switched B cells and plasma cells are highly resistant to radiation therapy compared with naïve B cells and upregulate activation markers programmed cell death 1 ligand 2 and major histocompatibility complex class II) after radiation. Mechanistically, radiation upregulates Xbp1 and Bcl6 in plasma cells, conferring radioresistance. Furthermore, using an immunization approach, we demonstrate that radiation enhances activation-induced cytidine deaminase mediated class switching and somatic hypermutation in primed B cells. CONCLUSIONS: These data demonstrate that stereotactic radiation is superior to large-field radiation at inducing infiltration of immune cells into tumors and that plasma cells and class switched B cells are highly resistant to radiation therapy. These results represent the most comprehensive analysis of the effects of radiation on B cells to date and identify novel mechanisms by which radiation modulates immune cells within the tumor microenvironment.

A State-of-Art Review of Digital Technologies for the Next Generation of Tinnitus Therapeutics.

Background: Digital processing has enabled the development of several generations of technology for tinnitus therapy. The first digital generation was comprised of digital Hearing Aids (HAs) and personal digital music players implementing already established sound-based therapies, as well as text based information on the internet. In the second generation Smart-phone applications (apps) alone or in conjunction with HAs resulted in more therapy options for users to select from. The 3rd generation of digital tinnitus technologies began with the emergence of many novel, largely neurophysiologically-inspired, treatment theories that drove development of processing; enabled through HAs, apps, the internet and stand-alone devices. We are now of the cusp of a 4th generation that will incorporate physiological sensors, multiple transducers and AI to personalize therapies. Aim: To review technologies that will enable the next generations of digital therapies for tinnitus. Methods: A "state-of-the-art" review was undertaken to answer the question: what digital technology could be applied to tinnitus therapy in the next 10 years? Google Scholar and PubMed were searched for the 10-year period 2011-2021. The search strategy used the following key words: "tinnitus" and ["HA," "personalized therapy," "AI" (and "methods" or "applications"), "Virtual reality," "Games," "Sensors" and "Transducers"], and "Hearables." Snowballing was used to expand the search from the identified papers. The results of the review were cataloged and organized into themes. Results: This paper identified digital technologies and research on the development of smart therapies for tinnitus. AI methods that could have tinnitus applications are identified and discussed. The potential of personalized treatments and the benefits of being able to gather data in ecologically valid settings are outlined. Conclusions: There is a huge scope for the application of digital technology to tinnitus therapy, but the uncertain mechanisms underpinning tinnitus present a challenge and many posited therapeutic approaches may not be successful. Personalized AI modeling based on biometric measures obtained through various sensor types, and assessments of individual psychology and lifestyles should result in the development of smart therapy platforms for tinnitus.

Behavioral Outcomes and Neural Network Modeling of a Novel, Putative, Recategorization Sound Therapy.

The mechanisms underlying sound's effect on tinnitus perception are unclear. Tinnitus activity appears to conflict with perceptual expectations of "real" sound, resulting in it being a salient signal. Attention diverted towards tinnitus during the later stages of object processing potentially disrupts high-order auditory streaming, and its uncertain nature results in negative psychological responses. This study investigated the benefits and neurophysiological basis of passive perceptual training and informational counseling to recategorize phantom perception as a more real auditory object. Specifically, it examined underlying psychoacoustic correlates of tinnitus and the neural activities associated with tinnitus auditory streaming and how malleable these are to change with targeted intervention. Eighteen participants (8 females, 10 males, mean age = 61.6 years) completed the study. The study consisted of 2 parts: (1) An acute exposure over 30 min to a sound that matched the person's tinnitus (Tinnitus Avatar) that was cross-faded to a selected nature sound (Cicadas, Fan, Water Sound/Rain, Birds, Water and Bird). (2) A chronic exposure for 3 months to the same "morphed" sound. A brain-inspired spiking neural network (SNN) architecture was used to model and compare differences between electroencephalography (EEG) patterns recorded prior to morphing sound presentation, during, after (3-month), and post-follow-up. Results showed that the tinnitus avatar generated was a good match to an individual's tinnitus as rated on likeness scales and was not rated as unpleasant. The five environmental sounds selected for this study were also rated as being appropriate matches to individuals' tinnitus and largely pleasant to listen to. There was a significant reduction in the Tinnitus Functional Index score and subscales of intrusiveness of the tinnitus signal and ability to concentrate with the tinnitus trial end compared to baseline. There was a significant decrease in how strong the tinnitus signal was rated as well as ratings of how easy it was to ignore the tinnitus signal on severity rating scales. Qualitative analysis found that the environmental sound interacted with the tinnitus in a positive way, but participants did not experience change in severity, however, characteristics of tinnitus, including pitch and uniformity of sound, were reported to change. The results indicate the feasibility of the computational SNN method and preliminary evidence that the sound exposure may change activation of neural tinnitus networks and greater bilateral hemispheric involvement as the sound morphs over time into natural environmental sound; particularly relating to attention and discriminatory judgments (dorsal attention network, precentral gyrus, ventral anterior network). This is the first study that attempts to recategorize tinnitus using passive auditory training to a sound that morphs from resembling the person's tinnitus to a natural sound. These findings will be used to design future-controlled trials to elucidate whether the approach used differs in effect and mechanism from conventional Broadband Noise (BBN) sound therapy.

Prediction of tinnitus masking benefit within a case series using a spiking neural network model.

Masking has been widely used as a tinnitus therapy, with large individual differences in its effectiveness. The basis of this variation is unknown. We examined individual tinnitus and psychological responses to three masking types, energetic masking (bilateral broadband static or rain noise [BBN]), informational masking (BBN with a notch at tinnitus pitch and 3-dimensional cues) and a masker combining both effects (BBN with spatial cues). Eleven participants with chronic tinnitus were followed for 12 months, each person used each masking approach for 3 months with a 1 month washout-baseline. The Tinnitus Functional Index (TFI), Tinnitus Rating Scales, Positive and Negative Affect Scale and Depression Anxiety Stress Scales, were measured every month of treatment. Electroencephalography (EEG) and psychoacoustic assessment was undertaken at baseline and following 3 months of each masking sound. The computational modeling of EEG data was based on the framework of brain-inspired Spiking Neural Network (SNN) architecture called NeuCube, designed for this study for mapping, learning, visualizing and classifying of brain activity patterns. EEG was related to clinically significant change in the TFI using the SNN model. The SNN framework was able to predict sound therapy responders (93% accuracy) from non-responders (100% accuracy) using baseline EEG recordings. The combination of energetic and informational masking was an effective treatment sound in more individuals than the other sounds used. Although the findings are promising, they are preliminary and require confirmation in independent and larger samples.

Prediction of Acoustic Residual Inhibition of Tinnitus Using a Brain-Inspired Spiking Neural Network Model.

Auditory Residual Inhibition (ARI) is a temporary suppression of tinnitus that occurs in some people following the presentation of masking sounds. Differences in neural response to ARI stimuli may enable classification of tinnitus and a tailored approach to intervention in the future. In an exploratory study, we investigated the use of a brain-inspired artificial neural network to examine the effects of ARI on electroencephalographic function, as well as the predictive ability of the model. Ten tinnitus patients underwent two auditory stimulation conditions (constant and amplitude modulated broadband noise) at two time points and were then characterised as responders or non-responders, based on whether they experienced ARI or not. Using a spiking neural network model, we evaluated concurrent neural patterns generated across space and time from features of electroencephalographic data, capturing the neural dynamic changes before and after stimulation. Results indicated that the model may be used to predict the effect of auditory stimulation on tinnitus on an individual basis. This approach may aid in the development of predictive models for treatment selection.

Distal lung epithelial progenitor cell function declines with age.

Tissue stem cell exhaustion is a key hallmark of aging, and in this study, we characterised its manifestation in the distal lung. We compared the lungs of 3- and 22-month old mice. We examined the gross morphological changes in these lungs, the density and function of epithelial progenitor populations and the epithelial gene expression profile. Bronchioles became smaller in their cross-sectional area and diameter. Using long-term EdU incorporation analysis and immunohistochemistry, we found that bronchiolar cell density remained stable with aging, but inferred rates of bronchiolar club progenitor cell self-renewal and differentiation were reduced, indicative of an overall slowdown in cellular turnover. Alveolar Type II progenitor cell density and self-renewal were maintained per unit tissue area with aging, but rates of inferred differentiation into Type I cells, and indeed overall density of Type I cells was reduced. Microarray analysis revealed age-related changes in multiple genes, including some with roles in proliferation and differentiation, and in IGF and TGFß signalling pathways. By characterising how lung stem cell dynamics change with aging, this study will elucidate how they contribute to age-related loss of pulmonary function, and pathogenesis of common age-related pulmonary diseases.

On the Timing of Signals in Multisensory Integration and Crossmodal Interactions: a Scoping Review.

A scoping review was undertaken to explore research investigating early interactions and integration of auditory and visual stimuli in the human brain. The focus was on methods used to study low-level multisensory temporal processing using simple stimuli in humans, and how this research has informed our understanding of multisensory perception. The study of multisensory temporal processing probes how the relative timing between signals affects perception. Several tasks, illusions, computational models, and neuroimaging techniques were identified in the literature search. Research into early audiovisual temporal processing in special populations was also reviewed. Recent research has continued to provide support for early integration of crossmodal information. These early interactions can influence higher-level factors, and vice versa. Temporal relationships between auditory and visual stimuli influence multisensory perception, and likely play a substantial role in solving the 'correspondence problem' (how the brain determines which sensory signals belong together, and which should be segregated).

A review of plasticity induced by auditory and visual tetanic stimulation in humans.

Long-term potentiation is a form of synaptic plasticity thought to play an important role in learning and memory. Recently noninvasive methods have been developed to induce and measure activity similar to long-term potentiation in humans. Sensory tetani (trains of quickly repeating auditory or visual stimuli) alter the electroencephalogram in a manner similar to electrical stimulation that results in long-term potentiation. This review briefly covers the development of long-term potentiation research before focusing on in vivo human studies that produce long-term potentiation-like effects using auditory and visual stimulation. Similarities and differences between traditional (animal and brain tissue) long-term potentiation studies and human sensory tetanization studies will be discussed, as well as implications for perceptual learning. Although evidence for functional consequences of sensory tetanization remains scarce, studies involving clinical populations indicate that sensory induced plasticity paradigms may be developed into diagnostic and research tools in clinical settings. Individual differences in the effects of sensory tetanization are not well-understood and provide an interesting avenue for future research. Differences in effects found between research groups that have emerged as the field has progressed are also yet to be resolved.

Interleukin-33 is activated by allergen- and necrosis-associated proteolytic activities to regulate its alarmin activity during epithelial damage.

Interleukin (IL)-33 is an IL-1 family alarmin released from damaged epithelial and endothelial barriers to elicit immune responses and allergic inflammation via its receptor ST2. Serine proteases released from neutrophils, mast cells and cytotoxic lymphocytes have been proposed to process the N-terminus of IL-33 to enhance its activity. Here we report that processing of full length IL-33 can occur in mice deficient in these immune cell protease activities. We sought alternative mechanisms for the proteolytic activation of IL-33 and discovered that exogenous allergen proteases and endogenous calpains, from damaged airway epithelial cells, can process full length IL-33 and increase its alarmin activity up to ~60-fold. Processed forms of IL-33 of apparent molecular weights ~18, 20, 22 and 23 kDa, were detected in human lungs consistent with some, but not all, proposed processing sites. Furthermore, allergen proteases degraded processed forms of IL-33 after cysteine residue oxidation. We suggest that IL-33 can sense the proteolytic and oxidative microenvironment during tissue injury that facilitate its rapid activation and inactivation to regulate the duration of its alarmin function.

Exceptional Response to Nivolumab and Stereotactic Body Radiation Therapy (SBRT) in Neuroendocrine Cervical Carcinoma with High Tumor Mutational Burden: Management Considerations from the Center For Personalized Cancer Therapy at UC San Diego Moores Cancer Center.

Neuroendocrine carcinoma of the cervix is an ultra-rare malignancy with a poor prognosis and limited treatment options. Checkpoint blockade immunotherapy has rapidly developed into an emerging standard of care for several common disease types. Interestingly, in preclinical and retrospective clinical data, radiation therapy has been demonstrated to synergize with checkpoint inhibitors. Here we report a patient with metastatic, chemotherapy-refractory neuroendocrine carcinoma who presented with partial bowel obstruction due to a large tumor burden. Genomic analysis demonstrated a high number of alterations on liquid biopsy (circulating tumor DNA [ctDNA]), which prompted treatment with stereotactic body radiation therapy (SBRT) combined with anti-programmed cell death protein 1 antibody. Tissue rebiopsy and comprehensive genomic profiling confirmed high tumor mutational burden and a mismatch repair gene defect. The patient manifested near-complete systemic resolution of disease, ongoing at 10+ months. We discuss the novel treatment modality of SBRT combined with a checkpoint inhibitor and the implications of molecular profiling and tumor mutational burden as potential predictors of response. KEY POINTS: High-grade, large-cell neuroendocrine carcinoma of the cervix is an ultra-rare malignancy that carries a grim prognosis.Next-generation sequencing may reveal key mutations in MSH2 genes amongst others. MSH2 mutations target the DNA mismatch repair process and can predispose patients to malignancies with high mutational burdens.Immunotherapy combined with radiation therapy can elicit a significant response, both within and outside the field of radiation. The latter is termed the "abscopal" effect, perhaps mediated by radiation-induced cross presentation of tumor antigens resulting in immune activation.Sequencing of blood-derived ctDNA showed a high number of alterations, and tissue sequencing confirmed a high tumor mutational burden as a consequence of a mismatch repair gene defect. This observation led to a therapeutic "match" with an anti- programmed cell death protein 1 antibody combined with SBRT, resulting in a durable (10+ months), near-complete remission in a patient with advanced chemotherapy-refractory disease.

Diacylglycerol kinase ε deficiency preserves glucose tolerance and modulates lipid metabolism in obese mice.

Diacylglycerol kinases (DGKs) catalyze the phosphorylation and conversion of diacylglycerol (DAG) into phosphatidic acid. DGK isozymes have unique primary structures, expression patterns, subcellular localizations, regulatory mechanisms, and DAG preferences. DGKε has a hydrophobic segment that promotes its attachment to membranes and shows substrate specificity for DAG with an arachidonoyl acyl chain in the sn-2 position of the substrate. We determined the role of DGKε in the regulation of energy and glucose homeostasis in relation to diet-induced insulin resistance and obesity using DGKε-KO and wild-type mice. Lipidomic analysis revealed elevated unsaturated and saturated DAG species in skeletal muscle of DGKε KO mice, which was paradoxically associated with increased glucose tolerance. Although skeletal muscle insulin sensitivity was unaltered, whole-body respiratory exchange ratio was reduced, and abundance of mitochondrial markers was increased, indicating a greater reliance on fat oxidation and intracellular lipid metabolism in DGKε KO mice. Thus, the increased intracellular lipids in skeletal muscle from DGKε KO mice may undergo rapid turnover because of increased mitochondrial function and lipid oxidation, rather than storage, which in turn may preserve insulin sensitivity. In conclusion, DGKε plays a role in glucose and energy homeostasis by modulating lipid metabolism in skeletal muscle.

Forced cell cycle exit and modulation of GABAA, CREB, and GSK3ß signaling promote functional maturation of induced pluripotent stem cell-derived neurons.

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.

Mitochondrial-targeted antioxidant therapy decreases transforming growth factor-ß-mediated collagen production in a murine asthma model.

Asthma is a disease of acute and chronic inflammation in which cytokines play a critical role in orchestrating the allergic inflammatory response. IL-13 and transforming growth factor (TGF)-ß promote fibrotic airway remodeling, a major contributor to disease severity. Improved understanding is needed, because current therapies are inadequate for suppressing development of airway fibrosis. IL-13 is known to stimulate respiratory epithelial cells to produce TGF-ß, but the mechanism through which this occurs is unknown. Here, we tested the hypothesis that reactive oxygen species (ROS) are a critical signaling intermediary between IL-13 or allergen stimulation and TGF-ß-dependent airway remodeling. We used cultured human bronchial epithelial cells and an in vivo mouse model of allergic asthma to map a pathway where allergens enhanced mitochondrial ROS, which is an essential upstream signal for TGF-ß activation and enhanced collagen production and deposition in airway fibroblasts. We show that mitochondria in airway epithelium are an essential source of ROS that activate TGF-ß expression and activity. TGF-ß from airway epithelium stimulates collagen expression in fibroblasts, contributing to an early fibrotic response to allergen exposure in cultured human airway cells and in ovalbumin-challenged mice. Treatment with the mitochondrial-targeted antioxidant, (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO), significantly attenuated mitochondrial ROS, TGF-ß, and collagen deposition in OVA-challenged mice and in cultured human epithelial cells. Our findings suggest that mitochondria are a critical source of ROS for promoting TGF-ß activity that contributes to airway remodeling in allergic asthma. Mitochondrial-targeted antioxidants may be a novel approach for future asthma therapies.

CaMKII is essential for the proasthmatic effects of oxidation.

Increased reactive oxygen species (ROS) contribute to asthma, but little is known about the molecular mechanisms connecting increased ROS with characteristic features of asthma. We show that enhanced oxidative activation of the Ca(2+)/calmodulin-dependent protein kinase (ox-CaMKII) in bronchial epithelium positively correlates with asthma severity and that epithelial ox-CaMKII increases in response to inhaled allergens in patients. We used mouse models of allergic airway disease induced by ovalbumin (OVA) or Aspergillus fumigatus (Asp) and found that bronchial epithelial ox-CaMKII was required to increase a ROS- and picrotoxin-sensitive Cl(-) current (ICl) and MUC5AC expression, upstream events in asthma progression. Allergen challenge increased epithelial ROS by activating NADPH oxidases. Mice lacking functional NADPH oxidases due to knockout of p47 and mice with epithelial-targeted transgenic expression of a CaMKII inhibitory peptide or wild-type mice treated with inhaled KN-93, an experimental small-molecule CaMKII antagonist, were protected against increases in ICl, MUC5AC expression, and airway hyperreactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent proasthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.

Lysophosphatidylcholine acyltransferase 3 knockdown-mediated liver lysophosphatidylcholine accumulation promotes very low density lipoprotein production by enhancing microsomal triglyceride transfer protein expression.

After de novo biosynthesis phospholipids undergo extensive remodeling by the Lands' cycle. Enzymes involved in phospholipid biosynthesis have been studied extensively but not those involved in reacylation of lysophosphopholipids. One key enzyme in the Lands' cycle is fatty acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which utilizes lysophosphatidylcholine (LysoPC) and fatty acyl-CoA to produce various phosphatidylcholine (PC) species. Four isoforms of LPCAT have been identified. In this study we found that LPCAT3 is the major hepatic isoform, and its knockdown significantly reduces hepatic LPCAT activity. Moreover, we report that hepatic LPCAT3 knockdown increases certain species of LysoPCs and decreases certain species of PC. A surprising observation was that LPCAT3 knockdown significantly reduces hepatic triglycerides. Despite this, these mice had higher plasma triglyceride and apoB levels. Lipoprotein production studies indicated that reductions in LPCAT3 enhanced assembly and secretion of triglyceride-rich apoB-containing lipoproteins. Furthermore, these mice had higher microsomal triglyceride transfer protein (MTP) mRNA and protein levels. Mechanistic studies in hepatoma cells revealed that LysoPC enhances secretion of apoB but not apoA-I in a concentration-dependent manner. Moreover, LysoPC increased MTP mRNA, protein, and activity. In short, these results indicate that hepatic LPCAT3 modulates VLDL production by regulating LysoPC levels and MTP expression.

The multifunctional Ca2+/calmodulin-dependent kinase II regulates vascular smooth muscle migration through matrix metalloproteinase 9.

The multifunctional CaMKII has been implicated in vascular smooth muscle cell (VSMC) migration, but little is known regarding its downstream targets that mediate migration. Here, we examined whether CaMKII regulates migration through modulation of matrix metalloproteinase 9 (MMP9). Using CaMKIIδ(-/-) mice as a model system, we evaluated migration and MMP9 regulation in vitro and in vivo. After ligation of the common carotid artery, CaMKII was activated in the neointima as determined by oxidation and autophosphorylation. We found that MMP9 was robustly expressed in the neointima and adventitia of carotid-ligated wild-type (WT) mice but was barely detectable in CaMKIIδ(-/-) mice. The perimeter of the external elastic lamina, a correlate of migration-related outward remodeling, was increased in WT but not in CaMKIIδ(-/-) mice. Migration induced by serum, platelet-derived growth factor, and tumor necrosis factor-α (TNF-α) was significantly decreased in CaMKIIδ(-/-) as compared with WT VSMCs, but migration was rescued with adenoviral overexpression of MMP9 in CaMKIIδ(-/-) VSMCs. Likewise, overexpression of CaMKIIδ in CaMKIIδ(-/-) VSMCs increased migration, whereas an oxidation-resistant mutant of CaMKIIδ did not. TNF-α strongly induced CaMKII oxidation and autophosphorylation as well as MMP9 activity, mRNA, and protein levels in WT, but not in CaMKIIδ(-/-) VSMC. Surprisingly, TNF-α strongly induced MMP9 promoter activity in WT and CaMKIIδ(-/-) VSMC. However, the MMP9 mRNA stability was significantly decreased in CaMKIIδ(-/-) VSMC. Our data demonstrate that CaMKII promotes VSMC migration through posttranscriptional regulation of MMP9 and suggest that CaMKII effects on MMP9 expression may be a therapeutic pathway in vascular injury.