Inflammatory pain resolution by mouse serum-derived small extracellular vesicles.

Chronic pain is a significant public health issue. Current treatments have limited efficacy and significant side effects, warranting research on alternative strategies for pain management. One approach involves using small extracellular vesicles (sEVs) to transport beneficial biomolecular cargo to aid pain resolution. Exosomes are 30-150 nm sEVs that can carry RNAs, proteins, and lipid mediators to recipient cells via circulation. Exosomes can be beneficial or harmful depending on their source and contents. To investigate the short and long-term effects of mouse serum-derived sEVs in pain modulation, sEVs from naïve control or spared nerve injury (SNI) model donor mice were injected intrathecally into naïve recipient mice. Basal mechanical thresholds transiently increased in recipient mice. This effect was mediated by opioid signaling as this outcome was blocked by naltrexone. Mass Spectrometry of sEVs detected endogenous opioid peptide leu-enkephalin. A single prophylactic intrathecal injection of sEVs two weeks prior to induction of the pain model in recipient mice delayed mechanical allodynia in SNI model mice and accelerated recovery from inflammatory pain after complete Freund's adjuvant (CFA) injection. ChipCytometry of spinal cord and dorsal root ganglion (DRG) from sEV treated mice showed that prophylactic sEV treatment reduced the number of natural killer (NK) and NKT cells in spinal cord and increased CD206+ anti-inflammatory macrophages in (DRG) after CFA injection. Further characterization of sEVs showed the presence of immune markers suggesting that sEVs can exert immunomodulatory effects in recipient mice to promote the resolution of inflammatory pain. Collectively, these studies demonstrate multiple mechanisms by which sEVs can attenuate pain.

Locus folding mechanisms determine modes of antigen receptor gene assembly.

The dynamic folding of genomes regulates numerous biological processes, including antigen receptor (AgR) gene assembly. We show that, unlike other AgR loci, homotypic chromatin interactions and bidirectional chromosome looping both contribute to structuring Tcrb for efficient long-range V(D)J recombination. Inactivation of the CTCF binding element (CBE) or promoter at the most 5'Vß segment (Trbv1) impaired loop extrusion originating locally and extending to DßJß CBEs at the opposite end of Tcrb. Promoter or CBE mutation nearly eliminated Trbv1 contacts and decreased RAG endonuclease-mediated Trbv1 recombination. Importantly, Trbv1 rearrangement can proceed independent of substrate orientation, ruling out scanning by DßJß-bound RAG as the sole mechanism of Vß recombination, distinguishing it from Igh. Our data indicate that CBE-dependent generation of loops cooperates with promoter-mediated activation of chromatin to juxtapose Vß and DßJß segments for recombination through diffusion-based synapsis. Thus, the mechanisms that fold a genomic region can influence molecular processes occurring in that space, which may include recombination, repair, and transcriptional programming.

Development of parallel forms of a brief smell identification test useful for longitudinal testing.

Although there are numerous brief odor identification tests available for quantifying the ability to smell, none are available in multiple parallel forms that can be longitudinally administered without potential confounding from knowledge of prior test items. Moreover, empirical algorithms for establishing optimal test lengths have not been generally applied. In this study, we employed and compared eight machine learning algorithms to develop a set of four brief parallel smell tests employing items from the University of Pennsylvania Smell Identification Test that optimally differentiated 100 COVID-19 patients from 132 healthy controls. Among the algorithms, linear discriminant analysis (LDA) achieved the best overall performance. The minimum number of odorant test items needed to differentiate smell loss accurately was identified as eight. We validated the sensitivity of the four developed tests, whose means and variances did not differ from one another (Bradley-Blackwood test), by sequential testing an independent group of 32 subjects that included persons with smell dysfunction not due to COVID-19. These eight-item tests clearly differentiated the olfactory compromised subjects from normosmics, with areas under the ROC curve ranging from 0.79 to 0.83. Each test was correlated with the overall UPSIT scores from which they were derived. These brief smell tests can be used separately or sequentially over multiple days in a variety of contexts where longitudinal olfactory testing is needed.

Gene signature reveals decreased SOX10-dependent transcripts in malignant cells from immune checkpoint inhibitor-resistant cutaneous melanomas.

Evidence is mounting for cross-resistance between immune checkpoint and targeted kinase inhibitor therapies in cutaneous melanoma patients. Since the loss of the transcription factor, SOX10, causes tolerance to MAPK pathway inhibitors, we used bioinformatic techniques to determine if reduced SOX10 expression/activity is associated with immune checkpoint inhibitor resistance. We integrated SOX10 ChIP-seq, knockout RNA-seq, and knockdown ATAC-seq data from melanoma cell models to develop a robust SOX10 gene signature. We used computational methods to validate this signature as a measure of SOX10-dependent activity in independent single-cell and bulk RNA-seq SOX10 knockdown, cell line panel, and MAPK inhibitor drug-resistant datasets. Evaluation of patient single-cell RNA-seq data revealed lower levels of SOX10-dependent transcripts in immune checkpoint inhibitor-resistant tumors. Our results suggest that SOX10-deficient melanoma cells are associated with cross-resistance between targeted and immune checkpoint inhibitors and highlight the need to identify therapeutic strategies that target this subpopulation.

Small Extracellular Vesicles From Spared Nerve Injury Model and Sham Control Mice Differentially Regulate Gene Expression in Primary Microglia.

Nerve injury outcomes might be predicted by examining small extracellular vesicles (sEVs) in circulation, as their biomolecular cargo facilitates cellular communication and can alter transcriptional state and behavior of recipient cells. We found that sEVs from the serum of spared nerve injury (SNI) model male mice had 7 differentially expressed miRNAs compared to sEVs from sham-operated control mice 4 weeks postsurgery. We investigated how these sEVs alter transcription in primary cortical microglia, a crucial mediator of neuropathic pain, using RNA sequencing. While the uptake of sEVs from both SNI model and sham groups changed gene expression in microglia compared to PBS treatment, sEVs from the sham group induced a more drastic change, particularly in genes involved in immune response. This was recapitulated by increased levels of pro-inflammatory cytokines and chemokines in microglia incubated with sEVs from sham control compared to sEVs from SNI model, naïve mice, or PBS. However, treating microglia with sEVs from female mice showed that serum sEVs derived from female SNI mice but not from female sham mice induced a more pronounced microglial secretion of pro-inflammatory mediators. Our data demonstrate that the molecular changes induced by sham surgery injuring skin and muscles are reflected in circulating sEVs in male mice 4 weeks later. Thus, when using sEVs from sham mice as control in comparative mechanistic studies after nerve injury, sex of mice should be taken into consideration. PERSPECTIVE: Microglial uptake of sEVs from male sham control mice induces higher pro-inflammatory responses compared to SNI sEVs but the reverse was observed upon treatment with sEVs from female mice. Wound healing may have a long-term impact on sEVs in male mice and should be considered for comparative studies using sEVs.

Circulating microRNAs from the mouse tibia fracture model reflect the signature from patients with complex regional pain syndrome.

INTRODUCTION: Complex regional pain syndrome (CRPS) often results from an initial trauma that later produces a disproportionate amount of pain. The mechanisms underlying CRPS have been studied using a tibia fracture model (TFM) in rodents because this model closely mimics symptoms and has several molecular correlates observed in patients with CRPS. OBJECTIVE: Here, we determined whether the TFM has alterations in circulating microRNAs (miRNAs) and cytokines transported by small extracellular vesicles (sEVs) that faithfully model previously reported miRNA alterations from patients with CRPS. METHODS: We isolated and characterized serum-derived sEVs from mice 3 weeks after fracture when symptoms such as pain hypersensitivity develop. Whole-transcriptome profiling was used to determine sEV miRNAs, and Bio-Plex Pro Mouse Cytokine 23-plex assay was used to measure cytokines. Differentially expressed miRNAs from TFM were compared with previously reported circulating miRNA alterations from patients with CRPS. RESULTS: Although sEV cytokine levels were unchanged, there were significant changes in sEV miRNA profiles. Differentially expressed miRNAs from TFM sEVs significantly overlapped with those previously reported in patients with CRPS. Of the 57 sEV miRNAs dysregulated in the TFM, 30 were previously reported in patients with CRPS compared with healthy control donors both in sEVs and 23 in whole blood. CONCLUSIONS: These findings enhance the validity of TFM as a model for CRPS and suggest that specific miRNA dysregulation may be a shared feature of CRPS and the TFM. These dysregulated miRNAs could help identify mechanistic targets or serve as biomarker candidates for both diagnosis and treatment responses in clinical trials.

Differential RNA packaging into small extracellular vesicles by neurons and astrocytes.

BACKGROUND: Small extracellular vesicles (sEVs) mediate intercellular communication by transferring RNA, proteins, and lipids to recipient cells. These cargo molecules are selectively loaded into sEVs and mirror the physiological state of the donor cells. Given that sEVs can cross the blood-brain barrier and their composition can change in neurological disorders, the molecular signatures of sEVs in circulation can be potential disease biomarkers. Characterizing the molecular composition of sEVs from different cell types is an important first step in determining which donor cells contribute to the circulating sEVs. METHODS: Cell culture supernatants from primary mouse cortical neurons and astrocytes were used to purify sEVs by differential ultracentrifugation and sEVs were characterized using nanoparticle tracking analysis, transmission electron microscopy and western blot. RNA sequencing was used to determine differential expression and loading patterns of miRNAs in sEVs released by primary neurons and astrocytes. Motif analysis was conducted on enriched miRNAs in sEVs and their respective donor cells. RESULTS: Sequencing total cellular RNA, and miRNAs from sEVs isolated from culture media of postnatal mouse cortical neurons and astrocytes revealed a distinct profile between sEVs and their corresponding cells. Though the total number of detected miRNAs in astrocytes was greater than neurons, neurons expressed more sEV-associated miRNAs than astrocytes. Only 20.7% of astrocytic miRNAs were loaded into sEVs, while 41.0% of neuronal miRNAs were loaded into sEVs, suggesting differences in the cellular sorting mechanisms. We identified short RNA sequence motifs, or EXOmotifs, on the miRNAs that were differentially loaded or excluded from sEVs. A sequence motif GUAC was enriched in astrocytic sEVs. miRNAs preferably retained in neurons or astrocytes had a similar RNA motif CACACA, suggesting a cell-type-independent mechanism to maintain cellular miRNAs. mRNAs of five RNA-binding proteins associated with passive or active RNA sorting into sEVs were differentially expressed between neurons and astrocytes, one of which, major vault protein was higher in astrocytes than in neurons and detected in astrocytic sEVs. CONCLUSIONS: Our studies suggest differences in RNA sorting into sEVs. These differences in miRNA signatures can be used for determining the cellular sources of sEVs altered in neurological disorders. Video abstract.

Therapeutic and prophylactic effects of macrophage-derived small extracellular vesicles in the attenuation of inflammatory pain.

Small extracellular vesicles (sEVs) derived from antigen-presenting cells such as macrophages can induce therapeutically relevant immune responses. Anti-inflammatory miRNAs are elevated in sEVs secreted by RAW 264.7 mouse macrophages after lipopolysaccharide (LPS) stimulation. We observed uptake of these sEVs by primary mouse cortical neurons, microglia and astrocytes followed by downregulation of proinflammatory miRNA target genes in recipient cells. Pre-treating primary microglia with these sEVs decreased pro-inflammatory gene expression. A single intrathecal injection of sEVs derived from LPS stimulated RAW 264.7 cells attenuated mechanical hyperalgesia in the complete Freund's adjuvant (CFA) mouse model of inflammatory pain and formalin induced acute pain. Importantly, sEVs did not alter the normal pain threshold in control mice. RNA sequencing of dorsal horn of the spinal cord showed sEVs-induced modulation of immune regulatory pathways. Further, a single prophylactic intrathecal injection of sEVs two weeks prior, attenuated CFA-induced pain hypersensitivity and was ineffective in formalin model. This indicates that prophylactic sEVs administration can be beneficial in attenuating chronic pain without impacting responses to the protective physiological and acute inflammatory pain. Prophylactic administration of sEVs could form the basis for a safe and novel vaccine-like therapy for chronic pain or as an adjuvant, potentially reducing the dose of drugs needed for pain relief.

Xist attenuates acute inflammatory response by female cells.

Biological sex influences inflammatory response, as there is a greater incidence of acute inflammation in men and chronic inflammation in women. Here, we report that acute inflammation is attenuated by X-inactive specific transcript (Xist), a female cell-specific nuclear long noncoding RNA crucial for X-chromosome inactivation. Lipopolysaccharide-mediated acute inflammation increased Xist levels in the cytoplasm of female mouse J774A.1 macrophages and human AML193 monocytes. In both cell types, cytoplasmic Xist colocalizes with the p65 subunit of NF-κB. This interaction was associated with reduced NF-κB nuclear migration, suggesting a novel mechanism to suppress acute inflammation. Further supporting this hypothesis, expression of 5' XIST in male cells significantly reduced IL-6 and NF-κB activity. Adoptive transfer of male splenocytes expressing Xist reduced acute paw swelling in male mice indicating that Xist can have a protective anti-inflammatory effect. These findings show that XIST has functions beyond X chromosome inactivation and suggest that XIST can contribute to sex-specific differences underlying inflammatory response by attenuating acute inflammation in women.

Proteome characterization of small extracellular vesicles from spared nerve injury model of neuropathic pain.

Exosomes are 30-150 nm extracellular vesicles mediating intercellular communication. Disease states can alter exosome composition affecting the message carried and thereby, its functional impact. The objective of this study was to identify proteins present in these vesicles in a mouse model of neuropathic pain induced by spared nerve injury (SNI). Small extracellular vesicles (sEVs) were purified from serum four weeks after SNI surgery and the protein composition was determined using tandem mass spectrometry and cytokine array. Proteomic analysis detected 274 gene products within sEVs. Of these, 24 were unique to SNI model, 100 to sham surgery control and five to naïve control samples. In addition to commonly expressed sEVs proteins, multiple members of serpin and complement family were detected in sEVs. Cytokine profiling using a membrane-based antibody array showed significant upregulation of complement component 5a (C5a) and Intercellular Adhesion Molecule 1 (ICAM-1) in sEVs from SNI model compared to sham control. We observed a differential distribution of C5a and ICAM-1 within sEVs and serum between sham and SNI, indicating changes from local or paracrine to long distance signaling under neuropathic pain. Our studies suggest critical roles for cargo sorting of vesicular proteins in mediating signaling mechanisms underlying neuropathic pain. SIGNIFICANCE: Approximately 100 million U.S. adults are burdened by chronic pain. Neuropathic pain resulting from injury or dysfunction of the nervous system is challenging to treat. Unlike acute pain that resolves over time, chronic pain persists resulting in changes in the peripheral and central nervous system. The transport of biomolecular cargo comprised of proteins and RNAs by small extracellular vesicles (sEVs) including exosomes has been proposed to be a fundamental mode of intercellular communication. To obtain insights on the role of exosome-mediated information transfer in the context of neuropathic pain, we investigated alterations in protein composition of sEVs in a mouse model of neuropathic pain induced by spared nerve injury (SNI). Our studies using mass spectrometry and cytokine array show that sEVs from SNI model harbor unique proteins. We observed an upregulation of C5a and ICAM-1 in exosomes from SNI model compared to control. There was a differential distribution of C5a and ICAM-1 within exosomes and serum, between control and SNI suggesting a switch from local to long distance signaling. Our studies suggest critical roles for cargo sorting of vesicular proteins in mediating signaling under neuropathic pain.

Inflammation potentiates miR-939 expression and packaging into small extracellular vesicles.

Extracellular RNA in circulation mediates intercellular communication in normal and pathological processes. One mode of circulating miRNA transport in bodily fluids is within 30-150 nm small extracellular vesicles (sEVs) or exosomes. Uptake of sEVs can regulate gene expression in recipient cells enabling circulating miRNAs to exert paracrine and systemic effects. Complex regional pain syndrome (CRPS) is a debilitating pain disorder characterized by chronic inflammation. Our previous investigations identified a significant decrease of hsa-miR-939 in whole blood from CRPS patients compared to control; we also observed that overexpression of miR-939 can negatively regulate several proinflammatory genes in vitro. Though downregulated in whole blood, miR-939 was significantly upregulated in sEVs isolated from patient serum. Here we investigated miR-939 packaging into sEVs in vitro under inflammation induced by monocyte chemoattractant protein-1 (MCP-1), a chemokine that is upregulated in CRPS patients. Stimulation of THP-1 monocytes by MCP-1 led to elevated levels of miR-939 in sEVs, which was abrogated using inhibitors of exosome secretion. miRNAs loaded into exosomes largely contain short miRNA sequence motifs called EXOmotifs. Mutation analysis of miR-939 showed that EXOmotif is one of the possible cellular mechanisms responsible for packaging miR-939 into sEVs. We confirmed gene expression changes in recipient cells following the uptake of sEVs enriched in miR-939 using RNA sequencing. Additionally, our data from primary immune cell-derived sEVs of CRPS patients and controls demonstrate that while the relative expression of miR-939 is higher in sEVs derived from B cells, T cells and NK cells relative to monocyte-derived sEVs in controls, only the B cell-derived sEVs showed a significantly higher level of miR-939 in CRPS patients. Differential miRNA sorting into exosomes and its functional impact on recipient cells may contribute to the underlying pathophysiology of CRPS.

Exosome microRNA signatures in patients with complex regional pain syndrome undergoing plasma exchange.

BACKGROUND: Therapeutic plasma exchange (PE) or plasmapheresis is an extracorporeal procedure employed to treat immunological disorders. Exosomes, nanosized vesicles of endosomal origin, mediate intercellular communication by transferring cargo proteins and nucleic acids and regulate many pathophysiological processes. Exosomal miRNAs are potential biomarkers due to their stability and dysregulation in diseases including complex regional pain syndrome (CRPS), a chronic pain disorder with persistent inflammation. A previous study showed that a subset of CRPS patients responded to PE. METHODS: As a proof-of-concept, we investigated the PE-induced exosomal miRNA changes in six CRPS patients. Plasma cytokine levels were measured by HPLC and correlated with miRNA expression. Luciferase assay following co-transfection of HEK293 cells with target 3'UTR constructs and miRNA mimics was used to evaluate miRNA mediated gene regulation of target mRNA. Transient transfection of THP-1 cells with miRNA mimics followed by estimation of target gene and protein expression was used to validate the findings. RESULTS: Comparison of miRNAs in exosomes from the serum of three responders and three poor-responders showed that 17 miRNAs differed significantly before and after therapy. Of these, poor responders had lower exosomal hsa-miR-338-5p. We show that miR-338-5p can bind to the interleukin 6 (IL-6) 3' untranslated region and can regulate IL-6 mRNA and protein levels in vitro. PE resulted in a significant reduction of IL-6 in CRPS patients. CONCLUSIONS: We propose that lower pretreatment levels of miR-338-5p in poor responders are linked to IL-6 levels and inflammation in CRPS. Our data suggests the feasibility of exploring exosomal miRNAs as a strategy in patient stratification for maximizing therapeutic outcome of PE.

Investigation of data-driven optical neuromonitoring approach during general anesthesia with sevoflurane.

Anesthesia monitoring currently needs a reliable method to evaluate the effects of the anesthetics on its primary target, the brain. This study focuses on investigating the clinical usability of a functional near-infrared spectroscopy (fNIRS)-derived machine learning classifier to perform automated and real-time classification of maintenance and emergence states during sevoflurane anesthesia. For 19 surgical procedures, we examine the entire continuum of the maintenance-transition-emergence phases and evaluate the predictive capability of a support vector machine (SVM) classifier during these phases. We demonstrate the robustness of the predictions made by the SVM classifier and compare its performance with that of minimum alveolar concentration (MAC) and bispectral (BIS) index-based predictions. The fNIRS-SVM investigated in this study provides evidence to the usability of the fNIRS signal for anesthesia monitoring. The method presented enables classification of the signal as maintenance or emergence automatically as well as in real-time with high accuracy, sensitivity, and specificity. The features local mean HbTotal, std [Formula: see text], local min Hb and [Formula: see text], and range Hb and [Formula: see text] were found to be robust biomarkers of this binary classification task. Furthermore, fNIRS-SVM was capable of identifying emergence before movement in a larger number of patients than BIS and MAC.

Regulation of proinflammatory genes by the circulating microRNA hsa-miR-939.

Circulating microRNAs are beneficial biomarkers because of their stability and dysregulation in diseases. Here we sought to determine the role of miR-939, a miRNA downregulated in patients with complex regional pain syndrome (CRPS). Hsa-miR-939 is predicted to target several proinflammatory genes, including IL-6, VEGFA, TNFα, NFκB2, and nitric oxide synthase 2 (NOS2A). Binding of miR-939 to the 3' untranslated region of these genes was confirmed by reporter assay. Overexpression of miR-939 in vitro resulted in reduction of IL-6, NOS2A and NFκB2 mRNAs, IL-6, VEGFA, and NOS2 proteins and NFκB activation. We observed a significant decrease in the NOS substrate l-arginine in plasma from CRPS patients, suggesting reduced miR-939 levels may contribute to an increase in endogenous NOS2A levels and NO, and thereby to pain and inflammation. Pathway analysis showed that miR-939 represents a critical regulatory node in a network of inflammatory mediators. Collectively, our data suggest that miR-939 may regulate multiple proinflammatory genes and that downregulation of miR-939 in CRPS patients may increase expression of these genes, resulting in amplification of the inflammatory pain signal transduction cascade. Circulating miRNAs may function as crucial signaling nodes, and small changes in miRNA levels may influence target gene expression and thus disease.

Circulating microRNA Signatures in Rodent Models of Pain.

MicroRNAs (miRNAs) remain stable in circulation and have been identified as potential biomarkers for a variety of conditions. We report miRNA changes in blood from multiple rodent models of pain, including spinal nerve ligation and spared nerve injury models of neuropathic pain; a complete Freund's adjuvant (CFA) model of inflammatory pain; and a chemotherapy-induced model of pain using the histone deacetylase inhibitor JNJ-26481585. The effect of celecoxib, a cyclooxygenase-2-selective nonsteroidal anti-inflammatory drug, was investigated in the CFA model as proof of principle for assessing the utility of circulating miRNAs as biomarkers in determining treatment response. Each study resulted in a unique miRNA expression profile. Despite differences in miRNAs identified from various models, computational target prediction and functional enrichment have identified biological pathways common among different models. The Wnt signaling pathway was affected in all models, suggesting a crucial role for this pathway in the pathogenesis of pain. Our studies demonstrate the utility of circulating miRNAs as pain biomarkers and suggest the potential for rigorous forward and reverse translational approaches. Evaluating alterations in miRNA fingerprints under different pain conditions and after administering therapeutic agents may be beneficial in evaluating clinical trial outcomes, predicting treatment response, and developing correlational outcomes between preclinical and human studies.

Analgesic Response to Intravenous Ketamine Is Linked to a Circulating microRNA Signature in Female Patients With Complex Regional Pain Syndrome.

Although ketamine is beneficial in treating complex regional pain syndrome (CRPS), a subset of patients respond poorly to therapy. We investigated treatment-induced microRNA (miRNA) changes and their predictive validity in determining treatment outcome by assessing miRNA changes in whole blood from patients with CRPS. Blood samples from female patients were collected before and after 5 days of intravenous ketamine administration. Seven patients were responders and 6 were poor responders. Differential miRNA expression was observed in whole blood before and after treatment. In addition, 33 miRNAs differed between responders and poor responders before therapy, suggesting the predictive utility of miRNAs as biomarkers. Investigation of the mechanistic significance of hsa-miR-548d-5p downregulation in poor responders showed that this miRNA can downregulate UDP-glucuronosyltransferase UGT1A1 mRNA. Poor responders had a higher conjugated/unconjugated bilirubin ratio, indicating increased UGT1A1 activity. We propose that lower pretreatment levels of miR-548d-5p may result in higher UDP-GT activity, leading to higher levels of inactive glucuronide conjugates, thereby minimizing the therapeutic efficacy of ketamine in poor responders. Differences in miRNA signatures can provide molecular insights distinguishing responders from poor responders. Extending this approach to other treatment and outcome assessments might permit stratification of patients for maximal therapeutic outcome. Perspective: This study suggests the usefulness of circulating miRNAs as potential biomarkers. Assessing miRNA signatures before and after treatment demonstrated miRNA alterations from therapy; differences in miRNA signature in responders and poor responders before therapy indicate prognostic value. Mechanistic studies on altered miRNAs can provide new insights into disease.

UniAlign: protein structure alignment meets evolution.

MOTIVATION: During the evolution, functional sites on the surface of the protein as well as the hydrophobic core maintaining the structural integrity are well-conserved. However, available protein structure alignment methods align protein structures based solely on the 3D geometric similarity, limiting their ability to detect functionally relevant correspondences between the residues of the proteins, especially for distantly related homologous proteins. RESULTS: In this article, we propose a new protein pairwise structure alignment algorithm (UniAlign) that incorporates additional evolutionary information captured in the form of sequence similarity, sequence profiles and residue conservation. We define a per-residue score (UniScore) as a weighted sum of these and other features and develop an iterative optimization procedure to search for an alignment with the best overall UniScore. Our extensive experiments on CDD, HOMSTRAD and BAliBASE benchmark datasets show that UniAlign outperforms commonly used structure alignment methods. We further demonstrate UniAlign's ability to develop family-specific models to drastically improve the quality of the alignments. AVAILABILITY AND IMPLEMENTATION: UniAlign is available as a web service at: http://sacan.biomed.drexel.edu/unialign CONTACT: ahmet.sacan@drexel.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Effect of histone deacetylase inhibitor JNJ-26481585 in pain.

Recent studies have shown that histone deacetylase (HDAC) inhibitors can alleviate inflammatory and neuropathic pain. We investigated the effects of JNJ-26481585, a pan-HDAC inhibitor on basal mechanical sensitivity. Unlike previous reports for HDAC inhibitors, JNJ-26481585 induced mechanical hypersensitivity in mice. This effect was reversible with gabapentin. Voltage-dependent calcium channel subunit alpha-2/delta-1, one of the putative targets for gabapentin, was upregulated in the spinal cord from JNJ-26481585-treated mice. Transcriptional profiling of spinal cord from JNJ-26481585-treated mice showed significant alterations in pathways involved in axon guidance, suggesting overlap in mechanisms underlying neurotoxicity caused by other known chemotherapeutic agents. To investigate the mechanisms underlying the development of pain, RAW 264.7 mouse macrophage cells were treated with JNJ-26481585. There was a dose- and time-dependent activation of nuclear factor-kappaB and interleukin-1ß increase. Thus, alterations in the axon guidance pathway, increase in voltage-dependent calcium channel alpha(2)delta-1 subunit, and the induction of proinflammatory mediators by JNJ-26481585 could all contribute to increased mechanical sensitivity. Our data indicate that the effect of HDAC inhibitors may be unique to the compound studied and highlights the potential to develop chemotherapy-induced peripheral neuropathy with the use of a pan-HDAC inhibitor for cancer treatment, and this pain may be alleviated by gabapentin.

Functional significance of macrophage-derived exosomes in inflammation and pain.

Exosomes, secreted microvesicles transporting microRNAs (miRNAs), mRNAs, and proteins through bodily fluids, facilitate intercellular communication and elicit immune responses. Exosomal contents vary, depending on the source and the physiological conditions of cells, and can provide insights into how cells and systems cope with physiological perturbations. Previous analysis of circulating miRNAs in patients with complex regional pain syndrome (CRPS), a debilitating chronic pain disorder, revealed a subset of miRNAs in whole blood that are altered in the disease. To determine functional consequences of alterations in exosomal biomolecules in inflammation and pain, we investigated exosome-mediated information transfer in vitro, in a rodent model of inflammatory pain, and in exosomes from patients with CRPS. Mouse macrophage cells stimulated with lipopolysaccharides secrete exosomes containing elevated levels of cytokines and miRNAs that mediate inflammation. Transcriptome sequencing of exosomal RNA revealed global alterations in both innate and adaptive immune pathways. Exosomes from lipopolysaccharide-stimulated cells were sufficient to cause nuclear factor-κB activation in naive cells, indicating functionality in recipient cells. A single injection of exosomes attenuated thermal hyperalgesia in a murine model of inflammatory pain, suggesting an immunoprotective role for macrophage-derived exosomes. Macrophage-derived exosomes carry a protective signature that is altered when secreting cells are exposed to an inflammatory stimulus. We also show that circulating miRNAs altered in patients with complex regional pain syndrome are trafficked by exosomes. With their systemic signaling capabilities, exosomes can induce pleiotropic effects potentially mediating the multifactorial pathology underlying chronic pain, and should be explored for their therapeutic utility.

A novel method for the normalization of microRNA RT-PCR data.

BACKGROUND: MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate mRNA transcript levels and translation. Deregulation of microRNAs is indicated in a number of diseases and microRNAs are seen as a promising target for biomarker identification and drug development. miRNA expression is commonly measured by microarray or real-time polymerase chain reaction (RT-PCR). The findings of RT-PCR data are highly dependent on the normalization techniques used during preprocessing of the Cycle Threshold readings from RT-PCR. Some of the commonly used endogenous controls themselves have been discovered to be differentially expressed in various conditions such as cancer, making them inappropriate internal controls. METHODS: We demonstrate that RT-PCR data contains a systematic bias resulting in large variations in the Cycle Threshold (CT) values of the low-abundant miRNA samples. We propose a new data normalization method that considers all available microRNAs as endogenous controls. A weighted normalization approach is utilized to allow contribution from all microRNAs, weighted by their empirical stability. RESULTS: The systematic bias in RT-PCR data is illustrated on a microRNA dataset obtained from primary cutaneous melanocytic neoplasms. We show that through a single control parameter, this method is able to emulate other commonly used normalization methods and thus provides a more general approach. We explore the consistency of RT-PCR expression data with microarray expression by utilizing a dataset where both RT-PCR and microarray profiling data is available for the same miRNA samples. CONCLUSIONS: A weighted normalization method allows the contribution of all of the miRNAs, whether they are highly abundant or have low expression levels. Our findings further suggest that the normalization of a particular miRNA should rely on only miRNAs that have comparable expression levels.