Molecular subtyping based on immune cell marker genes predicts prognosis and therapeutic response in patients with lung adenocarcinoma.

OBJECTIVE: Lung adenocarcinoma (LA) is one of the most common malignancies and is responsible for the greatest number of tumor-related deaths. Our research aimed to explore the molecular subtype signatures of LA to clarify the correlation among the immune microenvironment, clinical outcomes, and therapeutic response. METHODS: The LA immune cell marker genes (LICMGs) identified by single-cell RNA sequencing (scRNA-seq) analysis were used to discriminate the molecular subtypes and homologous immune and metabolic traits of GSE72094 LA cases. In addition, the model-building genes were identified from 1441 LICMGs by Cox-regression analysis, and a LA immune difference score (LIDscore) was developed to quantify individual differences in each patient, thereby predicting prognosis and susceptibility to immunotherapy and chemotherapy of LA patients. RESULTS: Patients of the GSE72094 cohort were divided into two distinct molecular subtypes based on LICMGs: immune activating subtype (Cluster-C1) and metabolically activating subtype (cluster-C2). The two molecular subtypes have distinct characteristics regarding prognosis, clinicopathology, genomics, immune microenvironment, and response to immunotherapy. Among the LICMGs, LGR4, GOLM1, CYP24A1, SFTPB, COL1A1, HLA-DQA1, MS4A7, PPARG, and IL7R were enrolled to construct a LIDscore model. Low-LIDscore patients had a higher survival rate due to abundant immune cell infiltration, activated immunity, and lower genetic variation, but probably the higher levels of Treg cells in the immune microenvironment lead to immune cell dysfunction and promote tumor immune escape, thus decreasing the responsiveness to immunotherapy compared with that of the high-LIDscore patients. Overall, high-LIDscore patients had a higher responsiveness to immunotherapy and a higher sensitivity to chemotherapy than the low-LIDscore group. CONCLUSIONS: Molecular subtypes based on LICMGs provided a promising strategy for predicting patient prognosis, biological characteristics, and immune microenvironment features. In addition, they helped identify the patients most likely to benefit from immunotherapy and chemotherapy.

scGCC: Graph Contrastive Clustering With Neighborhood Augmentations for scRNA-Seq Data Analysis.

Single-cell RNA sequencing (scRNA-seq) has rapidly emerged as a powerful technique for analyzing cellular heterogeneity at the individual cell level. In the analysis of scRNA-seq data, cell clustering is a critical step in downstream analysis, as it enables the identification of cell types and the discovery of novel cell subtypes. However, the characteristics of scRNA-seq data, such as high dimensionality and sparsity, dropout events and batch effects, present significant computational challenges for clustering analysis. In this study, we propose scGCC, a novel graph self-supervised contrastive learning model, to address the challenges faced in scRNA-seq data analysis. scGCC comprises two main components: a representation learning module and a clustering module. The scRNA-seq data is first fed into a representation learning module for training, which is then used for data classification through a clustering module. scGCC can learn low-dimensional denoised embeddings, which is advantageous for our clustering task. We introduce Graph Attention Networks (GAT) for cell representation learning, which enables better feature extraction and improved clustering accuracy. Additionally, we propose five data augmentation methods to improve clustering performance by increasing data diversity and reducing overfitting. These methods enhance the robustness of clustering results. Our experimental study on 14 real-world datasets has demonstrated that our model achieves extraordinary accuracy and robustness. We also perform downstream tasks, including batch effect removal, trajectory inference, and marker genes analysis, to verify the biological effectiveness of our model.

Selective removal of misfolded SOD1 delays disease onset in a mouse model of amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. There is no cure currently. The discovery that mutations in the gene SOD1 are a cause of ALS marks a breakthrough in the search for effective treatments for ALS. SOD1 is an antioxidant that is highly expressed in motor neurons. Human SOD1 is prone to aberrant modifications. Familial ALS-linked SOD1 variants are particularly susceptible to aberrant modifications. Once modified, SOD1 undergoes conformational changes and becomes misfolded. This study aims to determine the effect of selective removal of misfolded SOD1 on the pathogenesis of ALS. METHODS: Based on the chaperone-mediated protein degradation pathway, we designed a fusion peptide named CT4 and tested its efficiency in knocking down intracellularly misfolded SOD1 and its efficacy in modifying the pathogenesis of ALS. RESULTS: Expression of the plasmid carrying the CT4 sequence in human HEK cells resulted in robust removal of misfolded SOD1 induced by serum deprivation. Co-transfection of the CT4 and the G93A-hSOD1 plasmids at various ratios demonstrated a dose-dependent knockdown efficiency on G93A-hSOD1, which could be further increased when misfolding of SOD1 was enhanced by serum deprivation. Application of the full-length CT4 peptide to primary cultures of neurons expressing the G93A variant of human SOD1 revealed a time course of the degradation of misfolded SOD1; misfolded SOD1 started to decrease by 2 h after the application of CT4 and disappeared by 7 h. Intravenous administration of the CT4 peptide at 10 mg/kg to the G93A-hSOD1 reduced human SOD1 in spinal cord tissue by 68% in 24 h and 54% in 48 h in presymptomatic ALS mice. Intraperitoneal administration of the CT4 peptide starting from 60 days of age significantly delayed the onset of ALS and prolonged the lifespan of the G93A-hSOD1 mice. CONCLUSIONS: The CT4 peptide directs the degradation of misfolded SOD1 in high efficiency and specificity. Selective removal of misfolded SOD1 significantly delays the onset of ALS, demonstrating that misfolded SOD1 is the toxic form of SOD1 that causes motor neuron death. The study proves that selective removal of misfolded SOD1 is a promising treatment for ALS.

Inhibiting NLRP3 inflammasome signaling pathway promotes neurological recovery following hypoxic-ischemic brain damage by increasing p97-mediated surface GluA1-containing AMPA receptors.

BACKGROUND: The nucleotide-binding oligomeric domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is believed to be a key mediator of neuroinflammation and subsequent secondary brain injury induced by ischemic stroke. However, the role and underlying mechanism of the NLRP3 inflammasome in neonates with hypoxic-ischemic encephalopathy (HIE) are still unclear. METHODS: The protein expressions of the NLRP3 inflammasome including NLRP3, cysteinyl aspartate specific proteinase-1 (caspase-1) and interleukin-1ß (IL-1ß), the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid receptor (AMPAR) subunit, and the ATPase valosin-containing protein (VCP/p97), were determined by Western blotting. The interaction between p97 and AMPA glutamate receptor 1 (GluA1) was determined by co-immunoprecipitation. The histopathological level of hypoxic-ischemic brain damage (HIBD) was determined by triphenyltetrazolium chloride (TTC) staining. Polymerase chain reaction (PCR) and Western blotting were used to confirm the genotype of the knockout mice. Motor functions, including myodynamia and coordination, were evaluated by using grasping and rotarod tests. Hippocampus-dependent spatial cognitive function was measured by using the Morris-water maze (MWM). RESULTS: We reported that the NLRP3 inflammasome signaling pathway, such as NLRP3, caspase-1 and IL-1ß, was activated in rats with HIBD and oxygen-glucose deprivation (OGD)-treated cultured primary neurons. Further studies showed that the protein level of the AMPAR GluA1 subunit on the hippocampal postsynaptic membrane was significantly decreased in rats with HIBD, and it could be restored to control levels after treatment with the specific caspase-1 inhibitor AC-YVAD-CMK. Similarly, in vitro studies showed that OGD reduced GluA1 protein levels on the plasma membrane in cultured primary neurons, whereas AC-YVAD-CMK treatment restored this reduction. Importantly, we showed that OGD treatment obviously enhanced the interaction between p97 and GluA1, while AC-YVAD-CMK treatment promoted the dissociation of p97 from the GluA1 complex and consequently facilitated the localization of GluA1 on the plasma membrane of cultured primary neurons. Finally, we reported that the deficits in motor function, learning and memory in animals with HIBD, were ameliorated by pharmacological intervention or genetic ablation of caspase-1. CONCLUSION: Inhibiting the NLRP3 inflammasome signaling pathway promotes neurological recovery in animals with HIBD by increasing p97-mediated surface GluA1 expression, thereby providing new insight into HIE therapy.

Cryo-EM structures of ClC-2 chloride channel reveal the blocking mechanism of its specific inhibitor AK-42.

ClC-2 transports chloride ions across plasma membranes and plays critical roles in cellular homeostasis. Its dysfunction is involved in diseases including leukodystrophy and primary aldosteronism. AK-42 was recently reported as a specific inhibitor of ClC-2. However, experimental structures are still missing to decipher its inhibition mechanism. Here, we present cryo-EM structures of apo ClC-2 and its complex with AK-42, both at 3.5 Å resolution. Residues S162, E205 and Y553 are involved in chloride binding and contribute to the ion selectivity. The side-chain of the gating glutamate E205 occupies the putative central chloride-binding site, indicating that our structure represents a closed state. Structural analysis, molecular dynamics and electrophysiological recordings identify key residues to interact with AK-42. Several AK-42 interacting residues are present in ClC-2 but not in other ClCs, providing a possible explanation for AK-42 specificity. Taken together, our results experimentally reveal the potential inhibition mechanism of ClC-2 inhibitor AK-42.

Isomerization of bioactive acylhydrazones triggered by light or thiols.

The acylhydrazone unit is well represented in screening databases used to find ligands for biological targets, and numerous bioactive acylhydrazones have been reported. However, potential E/Z isomerization of the C=N bond in these compounds is rarely examined when bioactivity is assayed. Here we analysed two ortho-hydroxylated acylhydrazones discovered in a virtual drug screen for modulators of N-methyl-D-aspartate receptors and other bioactive hydroxylated acylhydrazones with structurally defined targets reported in the Protein Data Bank. We found that ionized forms of these compounds, which are populated under laboratory conditions, photoisomerize readily and the isomeric forms have markedly different bioactivity. Furthermore, we show that glutathione, a tripeptide involved with cellular redox balance, catalyses dynamic E⇄Z isomerization of acylhydrazones. The ratio of E to Z isomers in cells is determined by the relative stabilities of the isomers regardless of which isomer was applied. We conclude that E/Z isomerization may be a common feature of the bioactivity observed with acylhydrazones and should be routinely analysed.

GluN2B-containing NMDARs in the mammalian brain: pharmacology, physiology, and pathology.

Glutamate N-methyl-D-aspartate receptor (NMDAR) is critical for promoting physiological synaptic plasticity and neuronal viability. As a major subpopulation of the NMDAR, the GluN2B subunit-containing NMDARs have distinct pharmacological properties, physiological functions, and pathological relevance to neurological diseases compared with other NMDAR subtypes. In mature neurons, GluN2B-containing NMDARs are likely expressed as both diheteromeric and triheteromeric receptors, though the functional importance of each subpopulation has yet to be disentangled. Moreover, the C-terminal region of the GluN2B subunit forms structural complexes with multiple intracellular signaling proteins. These protein complexes play critical roles in both activity-dependent synaptic plasticity and neuronal survival and death signaling, thus serving as the molecular substrates underlying multiple physiological functions. Accordingly, dysregulation of GluN2B-containing NMDARs and/or their downstream signaling pathways has been implicated in neurological diseases, and various strategies to reverse these deficits have been investigated. In this article, we provide an overview of GluN2B-containing NMDAR pharmacology and its key physiological functions, highlighting the importance of this receptor subtype during both health and disease states.

Genetic inhibition of glutamate allosteric potentiation of GABAARs in mice results in hyperexcitability, leading to neurobehavioral abnormalities.

The imbalance between neuronal excitation and inhibition (E/I) in neural circuit has been considered to be at the root of numerous brain disorders. We recently reported a novel feedback crosstalk between the excitatory neurotransmitter glutamate and inhibitory γ-aminobutyric acid type A receptor (GABAAR)-glutamate allosteric potentiation of GABAAR functions through a direct binding of glutamate to the GABAAR itself. Here, we investigated the physiological significance and pathological implications of this cross-talk by generating the ß3E182G knock-in (KI) mice. We found that ß3E182G KI, while had little effect on basal GABAAR-mediated synaptic transmission, significantly reduced glutamate potentiation of GABAAR-mediated responses. These KI mice displayed lower thresholds for noxious stimuli, higher susceptibility to seizures and enhanced hippocampus-related learning and memory. Additionally, the KI mice exhibited impaired social interactions and decreased anxiety-like behaviors. Importantly, hippocampal overexpression of wild-type ß3-containing GABAARs was sufficient to rescue the deficits of glutamate potentiation of GABAAR-mediated responses, hippocampus-related behavioral abnormalities of increased epileptic susceptibility, and impaired social interactions. Our data indicate that the novel crosstalk among excitatory glutamate and inhibitory GABAAR functions as a homeostatic mechanism in fine-tuning neuronal E/I balance, thereby playing an essential role in ensuring normal brain functioning.

Potential circRNA-Disease Association Prediction Using DeepWalk and Nonnegative Matrix Factorization.

Circular RNAs (circRNAs) are a category of noncoding RNAs that exist in great numbers in eukaryotes. They have recently been discovered to be crucial in the growth of tumors. Therefore, it is important to explore the association of circRNAs with disease. This paper proposes a new method based on DeepWalk and nonnegative matrix factorization (DWNMF) to predict circRNA-disease association. Based on the known circRNA-disease association, we calculate the topological similarity of circRNA and disease via the DeepWalk-based method to learn the node features on the association network. Next, the functional similarity of the circRNAs and the semantic similarity of the diseases are fused with their respective topological similarities at different scales. Then, we use the improved weighted K-nearest neighbor (IWKNN) method to preprocess the circRNA-disease association network and correct nonnegative associations by setting different parameters K1 and K2 in the circRNA and disease matrices. Finally, the L2,1-norm, dual-graph regularization term and Frobenius norm regularization term are introduced into the nonnegative matrix factorization model to predict the circRNA-disease correlation. We perform cross-validation on circR2Disease, circRNADisease, and MNDR. The numerical results show that DWNMF is an efficient tool for forecasting potential circRNA-disease relationships, outperforming other state-of-the-art approaches in terms of predictive performance.

Timely insertion of AMPA receptor in developing vestibular circuits is required for manifestation of righting reflexes and effective navigation.

Vestibular information processed first by the brainstem vestibular nucleus (VN), and further by cerebellum and thalamus, underlies diverse brain function. These include the righting reflexes and spatial cognitive behaviour. While the cerebellar and thalamic circuits that decode vestibular information are known, the importance of VN neurons and the temporal requirements for their maturation that allow developmental consolidation of the aforementioned circuits remains unclear. We show that timely unsilencing of glutamatergic circuits in the VN by NMDA receptor-mediated insertion of AMPAR receptor type 1 (GluA1) subunits is critical for maturation of VN and successful consolidation of higher circuits that process vestibular information. Delayed unsilencing of NMDA receptor-only synapses of neonatal VN neurons permanently decreased their functional connectivity with inferior olive circuits. This was accompanied by delayed pruning of the inferior olive inputs to Purkinje cells and permanent reduction in their plasticity. These derangements led to deficits in associated vestibular righting reflexes and motor co-ordination during voluntary movement. Vestibular-dependent recruitment of thalamic neurons was similarly reduced, resulting in permanently decreased efficiency of spatial navigation. The findings thus show that well-choreographed maturation of the nascent vestibular circuitry is prerequisite for functional integration of vestibular signals into ascending pathways for diverse vestibular-related behaviours.

Convolution Neural Networks Using Deep Matrix Factorization for Predicting Circrna-Disease Association.

CircRNAs have a stable structure, which gives them a higher tolerance to nucleases. Therefore, the properties of circular RNAs are beneficial in disease diagnosis. However, there are few known associations between circRNAs and disease. Biological experiments identify new associations is time-consuming and high-cost. As a result, there is a need of building efficient and achievable computation models to predict potential circRNA-disease associations. In this paper, we design a novel convolution neural networks framework(DMFCNNCD) to learn features from deep matrix factorization to predict circRNA-disease associations. Firstly, we decompose the circRNA-disease association matrix to obtain the original features of the disease and circRNA, and use the mapping module to extract potential nonlinear features. Then, we integrate it with the similarity information to form a training set. Finally, we apply convolution neural networks to predict the unknown association between circRNAs and diseases. The five-fold cross-validation on various experiments shows that our method can predict circRNA-disease association and outperforms state of the art methods.

Allosteric potentiation of GABAA receptor single-channel conductance by netrin-1 during neuronal-excitation-induced inhibitory synaptic homeostasis.

As the principal receptor that mediates both synaptic and tonic inhibition of neurons in the brain, the A-type gamma-aminobutyric acid receptor (GABAAR) is functionally important for maintaining the balance between neuronal excitation and inhibition. Here, we report the identification of netrin-1 as an endogenous allosteric modulator of GABAARs. Following increased neuronal excitability, netrin-1 is secreted and binds to the extracellular domains of GABAAR subunits, thereby inducing homeostatic upscaling of GABAAR-mediated synaptic efficacy and currents. Surprisingly, this homeostatic plasticity is primarily mediated by increasing GABAAR single-channel conductance. Our study reveals an important role of netrin-1 as an endogenous GABAAR allosteric modulator in maintaining neuronal excitation-inhibition balance, a fundamental process for brain function and dysfunction.

Glutamate and GABAA receptor crosstalk mediates homeostatic regulation of neuronal excitation in the mammalian brain.

Maintaining a proper balance between the glutamate receptor-mediated neuronal excitation and the A type of GABA receptor (GABAAR) mediated inhibition is essential for brain functioning; and its imbalance contributes to the pathogenesis of many brain disorders including neurodegenerative diseases and mental illnesses. Here we identify a novel glutamate-GABAAR interaction mediated by a direct glutamate binding of the GABAAR. In HEK293 cells overexpressing recombinant GABAARs, glutamate and its analog ligands, while producing no current on their own, potentiate GABA-evoked currents. This potentiation is mediated by a direct binding at a novel glutamate binding pocket located at the α+/ß- subunit interface of the GABAAR. Moreover, the potentiation does not require the presence of a γ subunit, and in fact, the presence of γ subunit significantly reduces the potency of the glutamate potentiation. In addition, the glutamate-mediated allosteric potentiation occurs on native GABAARs in rat neurons maintained in culture, as evidenced by the potentiation of GABAAR-mediated inhibitory postsynaptic currents and tonic currents. Most importantly, we found that genetic impairment of this glutamate potentiation in knock-in mice resulted in phenotypes of increased neuronal excitability, including decreased thresholds to noxious stimuli and increased seizure susceptibility. These results demonstrate a novel cross-talk between excitatory transmitter glutamate and inhibitory GABAAR. Such a rapid and short feedback loop between the two principal excitatory and inhibitory neurotransmission systems may play a critical homeostatic role in fine-tuning the excitation-inhibition balance (E/I balance), thereby maintaining neuronal excitability in the mammalian brain under both physiological and pathological conditions.

Distinct but overlapping roles of LRRTM1 and LRRTM2 in developing and mature hippocampal circuits.

LRRTMs are postsynaptic cell adhesion proteins that have region-restricted expression in the brain. To determine their role in the molecular organization of synapses in vivo, we studied synapse development and plasticity in hippocampal neuronal circuits in mice lacking both Lrrtm1 and Lrrtm2. We found that LRRTM1 and LRRTM2 regulate the density and morphological integrity of excitatory synapses on CA1 pyramidal neurons in the developing brain but are not essential for these roles in the mature circuit. Further, they are required for long-term-potentiation in the CA3-CA1 pathway and the dentate gyrus, and for enduring fear memory in both the developing and mature brain. Our data show that LRRTM1 and LRRTM2 regulate synapse development and function in a cell-type and developmental-stage-specific manner, and thereby contribute to the fine-tuning of hippocampal circuit connectivity and plasticity.

Postsynaptic signaling at glutamatergic synapses as therapeutic targets.

Dysregulation of glutamatergic synapses plays an important role in the pathogenesis of neurological diseases. In addition to mediating excitatory synaptic transmission, postsynaptic glutamate receptors interact with various membrane and intracellular proteins. They form structural and/or signaling synaptic protein complexes and thereby play diverse postsynaptic functions. Recently, several postsynaptic protein complexes have been associated with various neurological diseases and hence, have been characterized as important therapeutic targets. Moreover, novel small molecules and therapeutic peptides targeting and modulating the activities of these protein complexes have been discovered, some of which have advanced through preclinical translational research and/or clinical studies. This article describes the recent investigation of eight key protein complexes associated with the postsynaptic ionotropic and metabotropic glutamate receptors as therapeutic targets for central nervous system diseases.

Distinct Functional Alterations and Therapeutic Options of Two Pathological De Novo Variants of the T292 Residue of GABRA1 Identified in Children with Epileptic Encephalopathy and Neurodevelopmental Disorders.

Mutations of GABAAR have reportedly led to epileptic encephalopathy and neurodevelopmental disorders. We have identified a novel de novo T292S missense variant of GABRA1 from a pediatric patient with grievous global developmental delay but without obvious epileptic activity. This mutation coincidentally occurs at the same residue as that of a previously reported GABRA1 variant T292I identified from a pediatric patient with severe epilepsy. The distinct phenotypes of these two patients prompted us to compare the impacts of the two mutants on the receptor function and to search for suitable therapeutics. In this study, we used biochemical techniques and patch-clamp recordings in HEK293 cells overexpressing either wild-type or mutated rat recombinant GABAARs. We found that the α1T292S variant significantly increased GABA-evoked whole-cell currents, shifting the dose-response curve to the left without altering the maximal response. In contrast, the α1T292I variant significantly reduced GABA-evoked currents, shifting the dose-response curve to the right with a severely diminished maximum response. Single-channel recordings further revealed that the α1T292S variant increased, while the α1T292I variant decreased the GABAAR single-channel open time and open probability. Importantly, we found that the T292S mutation-induced increase in GABAAR function could be fully normalized by the negative GABAAR modulator thiocolchicoside, whereas the T292I mutation-induced impairment of GABAAR function was largely rescued with a combination of the GABAAR positive modulators diazepam and verapamil. Our study demonstrated that α1T292 is a critical residue for controlling GABAAR channel gating, and mutations at this residue may produce opposite impacts on the function of the receptors. Thus, the present work highlights the importance of functionally characterizing each individual GABAAR mutation for ensuring precision medicine.

The selective dopamine D1 receptor agonist SKF81297 modulates NMDA receptor currents independently of D1 receptors.

Dopamine D1 receptor (D1R) agonists are frequently used to study the role of D1Rs in neurotransmission and behaviour. They have been repeatedly shown to modulate glutamatergic NMDAR currents in the prefrontal cortex (PFC), giving rise to the idea that D1R activation tunes glutamatergic networks by regulating NMDAR activity. We report that the widely used D1R agonist SKF81297 potentiates NMDAR currents in a dose-dependent manner, independently of D1R activation in mPFC slices, cortical neuron cultures and NMDAR-expressing recombinant HEK293 cells. SKF81297 potentiated NMDAR currents through both GluN2A and GluN2B subtypes in the absence of D1R expression, while inhibiting NMDAR currents through GluN2C and GluN2D subtypes. In contrast, the D1R ligands SKF38393, dopamine and SCH23390 inhibited GluN2A- and GluN2B-containing NMDAR currents. SKF81297 also inhibited GluN2A- and GluN2B-containing NMDAR currents at higher concentrations and when glutamate/glycine levels were high, exhibiting bidirectional modulation. To our knowledge, these findings are the first report of a D1R-independent positive modulatory effect of a D1R ligand on NMDA receptors. Importantly, our results further emphasize the possibility of off-target effects of many D1R ligands, which has significant implications for interpreting the large body of research relying on these compounds to examine dopamine functions.

A New Method Based on Matrix Completion and Non-Negative Matrix Factorization for Predicting Disease-Associated miRNAs.

Numerous studies have shown that microRNAs are associated with the occurrence and development of human diseases. Thus, studying disease-associated miRNAs is significantly valuable to the prevention, diagnosis and treatment of diseases. In this paper, we proposed a novel method based on matrix completion and non-negative matrix factorization (MCNMF)for predicting disease-associated miRNAs. Due to the information inadequacy on miRNA similarities and disease similarities, we calculated the latter via two models, and introduced the Gaussian interaction profile kernel similarity. In addition, the matrix completion (MC)was employed to further replenish the miRNA and disease similarities to improve the prediction performance. And to reduce the sparsity of miRNA-disease association matrix, the method of weighted K nearest neighbor (WKNKN)was used, which is a pre-processing step. We also utilized non-negative matrix factorization (NMF)using dual L2,1-norm, graph Laplacian regularization, and Tikhonov regularization to effectively avoid the overfitting during the prediction. Finally, several experiments and a case study were implemented to evaluate the effectiveness and performance of the proposed MCNMF model. The results indicated that our method could reliably and effectively predict disease-associated miRNAs.

Pharmacological properties of TRPM3 isoforms are determined by the length of the pore loop.

BACKGROUND AND PURPOSE: Transient receptor potential melastatin 3 (TRPM3) is a non-selective cation channel that plays a pivotal role in the peripheral nervous system as a transducer of painful heat signals. Alternative splicing gives rise to several TRPM3 variants. The functional consequences of these splice isoforms are poorly understood. Here, the pharmacological properties of TRPM3 variants arising from alternative splicing in the pore-forming region were compared. EXPERIMENTAL APPROACH: Calcium microfluorimetry and patch clamp recordings were used to compare the properties of heterologously expressed TRPM3α1 (long pore variant) and TRPM3α2-α6 (short pore variants). Furthermore, site-directed mutagenesis was done to investigate the influence of the length of the pore loop on the channel function. KEY RESULTS: All short pore loop TRPM3α variants (TRPM3α2-α6) were activated by the neurosteroid pregnenolone sulphate (PS) and by nifedipine, whereas the long pore loop variant TRPM3α1 was insensitive to either compound. In contrast, TRPM3α1 was robustly activated by clotrimazole, a compound that does not directly activate the short pore variants but potentiates their responses to PS. Clotrimazole-activated TRPM3α1 currents were largely insensitive to established TRPM3α2 antagonists and were only partially inhibited upon activation of the µ opioid receptor. Finally, by creating a set of mutant channels with pore loops of intermediate length, we showed that the length of the pore loop dictates differential channel activation by PS and clotrimazole. CONCLUSION AND IMPLICATIONS: Alternative splicing in the pore-forming region of TRPM3 defines the channel's pharmacological properties, which depend critically on the length of the pore-forming loop. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.

Predicting miRNA-Disease Association Based on Improved Graph Regression.

Recently, as a growing number of associations between microRNAs (miRNAs) and diseases are discovered, researchers gradually realize that miRNAs are closely related to several complicated biological processes and human diseases. Hence, it is especially important to construct availably models to infer associations between miRNAs and diseases. In this study, we presented Improved Graph Regression for miRNA-Disease Association Prediction (IGRMDA) to observe potential relationship between miRNAs and diseases. In order to reduce the inherent noise existing in the acquired biological datasets, we utilized matrix decomposition algorithm to process miRNA functional similarity and disease semantic similarity and then combining them with existing similarity information to obtain final miRNA similarity data and disease similarity data. Then, we applied miRNA-disease association data, miRNA similarity data and disease similarity data to form corresponding latent spaces. Furthermore, we performed improved graph regression algorithm in latent spaces, which included miRNA-disease association space, miRNA similarity space and disease similarity space. Non-negative matrix factorization and partial least squares were used in the graph regression process to obtain important related attributes. The cross validation experiments and case studies were also implemented to prove the effectiveness of IGRMDA, which showed that IGRMDA could predict potential associations between miRNAs and diseases.