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Agriculture plays a vital role in Bangladesh's economy. It is essential to ensure the proper growth and health of crops for the development of the agricultural sector. In the context of Bangladesh, crop diseases pose a significant threat to agricultural output and, consequently, food security. This necessitates the timely and precise identification of such diseases to ensure the sustainability of food production. This study focuses on building a hybrid deep learning model for the identification of three specific diseases affecting three major crops: late blight in potatoes, brown spot in rice, and common rust in corn. The proposed model leverages EfficientNetB0's feature extraction capabilities, known for achieving rapid high learning rates, coupled with the classification proficiency of SVMs, a well-established machine learning algorithm. This unified approach streamlines data processing and feature extraction, potentially improving model generalizability across diverse crops and diseases. It also aims to address the challenges of computational efficiency and accuracy that are often encountered in precision agriculture applications. The proposed hybrid model achieved 97.29% accuracy. A comparative analysis with other models, CNN, VGG16, ResNet50, Xception, Mobilenet V2, Autoencoders, Inception v3, and EfficientNetB0 each achieving an accuracy of 86.57%, 83.29%, 68.79%, 94.07%, 90.71%, 87.90%, 94.14%, and 96.14% respectively, demonstrated the superior performance of our proposed model.

In this study, we automated the diagnostic procedure of autism spectrum disorder (ASD) with the help of anatomical alterations found in structural magnetic resonance imaging (sMRI) data of the ASD brain and machine learning tools. Initially, the sMRI data was preprocessed using the FreeSurfer toolbox. Further, the brain regions were segmented into 148 regions of interest using the Destrieux atlas. Features such as volume, thickness, surface area, and mean curvature were extracted for each brain region, and the morphological connectivity was computed using Pearson correlation. These morphological connections were fed to XGBoost for feature reduction and to build the diagnostic model. The results showed an average accuracy of 94.16% for the top 18 features. The frontal and limbic regions contributed more features to the classification model. Our proposed method is thus effective for the classification of ASD and can also be useful for the screening of other similar neurological disorders.

Oral cancer patients suffer pain at the site of the cancer. Calcitonin gene related polypeptide (CGRP), a neuropeptide expressed by a subset of primary afferent neurons, promotes oral cancer growth. CGRP also mediates trigeminal pain (migraine) and neurogenic inflammation. The contribution of CGRP to oral cancer pain is investigated in the present study. The findings demonstrate that CGRP-immunoreactive (-ir) neurons and neurites innervate orthotopic oral cancer xenograft tumors in mice. Cancer increases anterograde transport of CGRP in axons innervating the tumor, supporting neurogenic secretion as the source of CGRP in the oral cancer microenvironment. CGRP antagonism reverses oral cancer nociception in preclinical oral cancer pain models. Single-cell RNA-sequencing is used to identify cell types in the cancer microenvironment expressing the CGRP receptor components, receptor activity modifying protein 1 Ramp1 and calcitonin receptor like receptor (CLR, encoded by Calcrl ). Ramp1 and Calcrl transcripts are detected in cells expressing marker genes for Schwann cells, endothelial cells, fibroblasts and immune cells. Ramp1 and Calcrl transcripts are more frequently detected in cells expressing fibroblast and immune cell markers. This work identifies CGRP as mediator of oral cancer pain and suggests the antagonism of CGRP to alleviate oral cancer pain.

Oral squamous cell carcinoma (OSCC) patients suffer from poor survival due to metastasis or locoregional recurrence, processes that are both facilitated by perineural invasion (PNI). OSCC has higher rates of PNI than other cancer subtypes, with PNI present in 80% of tumors. Despite the impact of PNI on oral cancer prognosis and pain, little is known about the genes that drive PNI, which in turn drive pain, invasion, and metastasis. In this study, clinical data, preclinical, and in vitro models are leveraged to elucidate the role of neurotrophins in OSCC metastasis, PNI, and pain. The expression data in OSCC patients with metastasis, PNI, or pain demonstrate dysregulation of neurotrophin genes. TrkA and nerve growth factor receptor (NGFR) are focused, two receptors that are activated by NGF, a neurotrophin expressed at high levels in OSCC. It is demonstrated that targeted knockdown of these two receptors inhibits proliferation and invasion in an in vitro and preclinical model of OSCC, and metastasis, PNI, and pain. It is further determined that TrkA knockdown alone inhibits thermal hyperalgesia, whereas NGFR knockdown alone inhibits mechanical allodynia. Collectively the results highlight the ability of OSCC to co-opt different components of the neurotrophin pathway in metastasis, PNI, and pain., (© 2022 The Authors. Advanced Biology published by Wiley-VCH GmbH.)

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Background: Mutations in the postsynaptic transmembrane protein neuroligin-3 are highly correlative with autism spectrum disorders (ASDs) and intellectual disabilities (IDs). Fear learning is well studied in models of these disorders, however differences in fear response behaviours are often overlooked. We aim to examine fear behaviour and its cellular underpinnings in a rat model of ASD/ID lacking Nlgn3., Methods: This study uses a range of behavioural tests to understand differences in fear response behaviour in Nlgn3 -/y  rats. Following this, we examined the physiological underpinnings of this in neurons of the periaqueductal grey (PAG), a midbrain area involved in flight-or-freeze responses. We used whole-cell patch-clamp recordings from ex vivo PAG slices, in addition to in vivo local-field potential recordings and electrical stimulation of the PAG in wildtype and Nlgn3 -/y  rats. We analysed behavioural data with two- and three-way ANOVAS and electrophysiological data with generalised linear mixed modelling (GLMM)., Results: We observed that, unlike the wildtype, Nlgn3 -/y  rats are more likely to response with flight rather than freezing in threatening situations. Electrophysiological findings were in agreement with these behavioural outcomes. We found in ex vivo slices from Nlgn3 -/y  rats that neurons in dorsal PAG (dPAG) showed intrinsic hyperexcitability compared to wildtype. Similarly, stimulating dPAG in vivo revealed that lower magnitudes sufficed to evoke flight behaviour in Nlgn3 -/y  than wildtype rats, indicating the functional impact of the increased cellular excitability., Limitations: Our findings do not examine what specific cell type in the PAG is likely responsible for these phenotypes. Furthermore, we have focussed on phenotypes in young adult animals, whilst the human condition associated with NLGN3 mutations appears during the first few years of life., Conclusions: We describe altered fear responses in Nlgn3 -/y rats and provide evidence that this is the result of a circuit bias that predisposes flight over freeze responses. Additionally, we demonstrate the first link between PAG dysfunction and ASD/ID. This study provides new insight into potential pathophysiologies leading to anxiety disorders and changes to fear responses in individuals with ASD., (© 2022. The Author(s).)

Stress response pathways protect the lung from the damaging effects of environmental toxicants. Here we investigate the role of the fragile X mental retardation protein (FMRP), a multifunctional protein implicated in stress responses, in the lung. We report that FMRP is expressed in murine and human lungs, in the airways and more broadly. Analysis of airway stress responses in mice and in a murine cell line ex vivo, using the well-established naphthalene injury model, reveals that FMRP-deficient cells exhibit increased expression of markers of oxidative and genotoxic stress and increased cell death. Further inquiry shows that FMRP-deficient cells fail to actuate the integrated stress response pathway (ISR) and upregulate the transcription factor ATF4. Knockdown of ATF4 expression phenocopies the loss of FMRP. We extend our analysis of the role of FMRP to human bronchial BEAS-2B cells, using a 9,10-phenanthrenequinone air pollutant model, to find that FMRP-deficient BEAS-2B cells also fail to actuate the ISR and exhibit greater susceptibility. Taken together, our data suggest that FMRP has a conserved role in protecting the airways by facilitating the ISR. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Grain boundary velocity has been believed to be correlated to curvature, and this is an important relationship for modeling how polycrystalline materials coarsen during annealing. We determined the velocities and curvatures of approximately 52,000 grain boundaries in a nickel polycrystal using three-dimensional orientation maps measured by high-energy diffraction microscopy before and after annealing at 800°C. Unexpectedly, the grain boundary velocities and curvatures were uncorrelated. Instead, we found strong correlations between the boundary velocity and the five macroscopic parameters that specify grain boundary crystallography. The sensitivity of the velocity to grain boundary crystallography might be the result of defect-mediated grain boundary migration or the anisotropy of the grain boundary energy. The absence of a correlation between velocity and curvature likely results from the constraints imposed by the grain boundary network and implies the need for a new model for grain boundary migration.

Background: Methotrexate (MTX) is widely used in chemotherapy but its associated hepatotoxicity is a major complication, limiting its use. This study evaluates possible therapeutic effect of oral alpha-ketoglutarate (AKG) supplementation against MTX-induced hepatotoxicity., Methods: HepG2 cells were used to evaluate in-vitro cyto-protection conferred by AKG against MTX induced cytotoxicity. For in-vivo animal study, rats were divided into three groups. Group-I served as control. Group-II animals were administered single intraperitoneal injection of MTX (20 mg/kg/body weight), while Group-III received MTX as in group-II followed by oral AKG (2 gm/kg body weight) for 5 days. 99m Tc-Mebrofenin hepatobiliary study was performed under a gamma camera to determine real time functional status of rats' livers. Multiple parameters concerning hepatic mebrofenin uptake and excretion, including T peak and T 1/2 peak in control and treated animals were determined. Biochemical analysis of the liver homogenate in terms of hepatic enzyme activities in serum, antioxidant status, tissue factor activity, tissue collagen content and histological analysis of the liver tissue were also done., Results: AKG supplementation significantly reversed MTX induced derangement in activities of serum liver enzymes [ALT and ALP (p = 0.003); AST (p = 0.005)], antioxidant status [LPO and GSH (p = 0.005); CAT (p = 0.004)], tissue factor activity (p = 0.005) and tissue collagen content (p = 0.005). Functional imaging confirmed that hepatic retention and fractional biliary excretion were significantly abnormal in MTX treated group (T peak : 234 s ± 40 s; T 1/2 peak : 846sec ± 32sec) as compared to AKG supplemented group (T peak : 144 s ± 35sec; T 1/2peak : 468sec ± 27sec). Hepatic extraction fraction (HEF) was 92.2 ± 1.8%, 48.7 ± 2.6% and 69.8 ± 4.3% in control, MTX and AKG supplemented rats respectively., Conclusion: 99m Tc-mebrofenin imaging strongly suggests therapeutic action of AKG in protecting liver damage by MTX in rats. Functional imaging parameters correlated well with biochemical and histopathological findings., (Copyright © 2020 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

Background: Colon cancer cells can migrate and metastasize by undergoing epithelial-to-mesenchymal transition (EMT). Mesenchymal stem cells (MSCs) are non-cancerous, multipotent adult stem cells, which can also migrate. In this study, we wanted to compare the biological, physical, and functional properties of these migratory cells., Materials and Methods: HT-29 and HCT-116, two human colon carcinoma cell lines, represent less aggressive and more aggressive cancer cells, respectively. MSCs were isolated from human bone marrow. After confirming the identity of all the cell types, they were evaluated for E-cadherin, β1-integrin, Vimentin, ZEB-1, β-catenin, and 18S rRNA using Q-PCR. MMP-2 and MMP-9 activity were evaluated using gelatin zymography. Functional tests like wound healing assay, migration assay, and invasion assay were also done. Biomechanical properties like cell stiffness and non-specific adhesion (between indenter probe and cell membrane) were evaluated through nanoindentation using atomic force microscopy (AFM)., Results: Expression of EMT and stem cell markers showed typical expression patterns for HT-29, HCT-116, and MSCs. Functional tests showed that MSCs migrated faster than malignant cells. MMP-2 and MMP-9 activity reinforced this behavior. Interestingly, the migration/invasion capacity of MSCs was comparable to aggressive HCT-116, and more than HT-29. MSCs also showed the maximum cell stiffness and non-specific cell-probe adhesions, followed by HCT116 and HT29 cells., Conclusions: Our findings indicate that the migratory properties of MSCs is comparable or even greater than that of cancer cells and despite their high migration potential, they also have the maximum stiffness., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

In this report, we discuss the design of a novel collagen/pectin (CP) hybrid composite hydrogel (CPBG) containing in-situ mineralized bioactive glass (BG) particles to simulate an integrative 3D cell environment. Systematic analysis of the CP sol revealed collagen and pectin molecules interacted regardless of both possessing similar net negative charge through the mechanism of surface patch binding interaction. Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) confirmed this associative interaction which resulted in the formation of a hybrid crosslinked network with the BG nanoparticles acting as pseudo crosslink junctions. Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray Analysis (EDAX) and Transmission Electron Microscopy (TEM) results confirmed uniform mineralization of BG particles, and their synergetic interaction with the network. The in-vitro bioactivity tests on CPBG indicated the formation of bone-like hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ) microcrystals on its surface after interaction with simulated body fluid. This hydrogel was loaded with a model antifungal drug amphotericin-B (AmB) and tested against Candida albicans. The AmB release kinetics from the hydrogel followed the Fickian mechanism and showed direct proportionality to gel swelling behavior. Rheological analysis revealed the viscoelastic compatibility of CPBG for the mechanical load bearing applications. Cell viability tests indicated appreciable compatibility of the hydrogel against U2OS and HaCaT cell lines. FDA/PI on the hydrogel portrayed preferential U2OS cell adhesion on hydrophobic hydroxyapatite layer compared to hydrophilic surfaces, thereby promising the regeneration of both soft and hard tissues., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cancer invading into nerves, termed perineural invasion (PNI), is associated with pain. Here, we show that oral cancer patients with PNI report greater spontaneous pain and mechanical allodynia compared with patients without PNI, suggesting that unique mechanisms drive PNI-induced pain. We studied the impact of PNI on peripheral nerve physiology and anatomy using a murine sciatic nerve PNI model. Mice with PNI exhibited spontaneous nociception and mechanical allodynia. Perineural invasion induced afterdischarge in A high-threshold mechanoreceptors (HTMRs), mechanical sensitization (ie, decreased mechanical thresholds) in both A and C HTMRs, and mechanical desensitization in low-threshold mechanoreceptors. Perineural invasion resulted in nerve damage, including axon loss, myelin damage, and axon degeneration. Electrophysiological evidence of nerve injury included decreased conduction velocity, and increased percentage of both mechanically insensitive and electrically unexcitable neurons. We conclude that PNI-induced pain is driven by nerve injury and peripheral sensitization in HTMRs.

Oral cancer patients experience pain at the site of the primary cancer. Patients with metastatic oral cancers report greater pain. Lack of pain identifies patients at low risk of metastasis with sensitivity = 0.94 and negative predictive value = 0.89. In the same cohort, sensitivity and negative predictive value of depth of invasion, currently the best predictor, were 0.95 and 0.92, respectively. Cancer pain is attributed to cancer-derived mediators that sensitize neurons and is associated with increased neuronal density. We hypothesized that pain mediators would be overexpressed in metastatic cancers from patients reporting high pain. We identified 40 genes overexpressed in metastatic cancers from patients reporting high pain (n = 5) compared to N0 cancers (n = 10) and normal tissue (n = 5). The genes are enriched for functions in extracellular matrix organization and angiogenesis. They have oncogenic and neuronal functions and are reported in exosomes. Hierarchical clustering according to expression of neurotrophic and axon guidance genes also separated cancers according to pain and nodal status. Depletion of exosomes from cancer cell line supernatant reduced nociceptive behavior in a paw withdrawal assay, supporting a role for exosomes in cancer pain. The identified genes and exosomes are potential therapeutic targets for stopping cancer and attenuating pain.

Aberrant protein synthesis and protein expression are a hallmark of many conditions ranging from cancer to Alzheimer's. Blood-based biomarkers indicative of changes in proteomes have long been held to be potentially useful with respect to disease prognosis and treatment. However, most biomarker efforts have focused on unlabeled plasma proteomics that include nonmyeloid origin proteins with no attempt to dynamically tag acute changes in proteomes. Herein we report a method for evaluating de novo protein synthesis in whole blood liquid biopsies. Using a modification of the "bioorthogonal noncanonical amino acid tagging" (BONCAT) protocol, rodent whole blood samples were incubated with l-azidohomoalanine (AHA) to allow incorporation of this selectively reactive non-natural amino acid within nascent polypeptides. Notably, failure to incubate the blood samples with EDTA prior to implementation of azide-alkyne "click" reactions resulted in the inability to detect probe incorporation. This live-labeling assay was sensitive to inhibition with anisomycin and nascent, tagged polypeptides were localized to a variety of blood cells using FUNCAT. Using labeled rodent blood, these tagged peptides could be consistently identified through standard LC/MS-MS detection of known blood proteins across a variety of experimental conditions. Furthermore, this assay could be expanded to measure de novo protein synthesis in human blood samples. Overall, we present a rapid and convenient de novo protein synthesis assay that can be used with whole blood biopsies that can quantify translational change as well as identify differentially expressed proteins that may be useful for clinical applications.

Serotonin is an important neurotransmitter and neuromodulator. Disruption of the serotonergic system has been implicated in various psychiatric disorders such as schizophrenia and bipolar disorder. Most of the drugs targeting these neurotransmitter systems are classified primarily as agonists or inverse agonists/antagonists, with their described function being limited to activating the canonical signaling pathway(s), or inhibiting the pathway(s) respectively. Previous work with the human 5-HT 2A has shown the receptor to be activated by dopamine, also an endogenous ligand. Dopamine is the cognate ligand of the dopaminergic system, which significantly overlaps with the serotonergic system in the brain. The two systems innervate many of the same brain areas, and the central serotonergic systems also regulate dopamine functions. Our aim was to investigate the downstream signaling set up by the receptor on being activated by dopamine. We show that dopamine is a functionally selective ligand at 5-HT 2A and have examined dopamine as a ligand with respect to some receptor-dependent phenotypes. Our results show that dopamine acts as an agonist at the human serotonin 2A receptor and brings about its activation and internalization. Using in vitro assays, we have established differences in the signaling pathways set up by dopamine as compared to serotonin. Using site-specific mutagenesis we have identified residues important for this functional selectivity, shown by dopamine at this receptor. Our identification of specific residues important in the functional selectivity of dopamine at 5-HT 2A could have far reaching implications for the field of GPCR signaling and drug-design. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries., Competing Interests: Declarations of interest None., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Recent advances in neurobiology have provided several molecular entrees for targeted treatments for Fragile X syndrome (FXS). However, the efficacy of these treatments has been demonstrated mainly in animal models and has not been consistently predictive of targeted drugs' response in the preponderance of human clinical trials. Because of the heterogeneity of FXS at various levels, including the molecular level, phenotypic manifestation, and drug response, it is critically important to identify biomarkers that can help in patient stratification and prediction of therapeutic efficacy. The primary objective of this study was to assess the ability of molecular biomarkers to predict phenotypic subgroups, symptom severity, and treatment response to metformin in clinically treated patients with FXS. We specifically tested a triplex protein array comprising of hexokinase 1 (HK1), RAS (all isoforms), and Matrix Metalloproteinase 9 (MMP9) that we previously demonstrated were dysregulated in the FXS mouse model and in blood samples from patient with FXS. Seventeen participants with FXS, 12 males and 5 females, treated clinically with metformin were included in this study. The disruption in expression abundance of these proteins was normalized and associated with significant self-reported improvement in clinical phenotypes (CGI-I in addition to BMI) in a subset of participants with FXS. Our preliminary findings suggest that these proteins are of strong molecular relevance to the FXS pathology that could make them useful molecular biomarkers for this syndrome.

Neuronal activity is responsible for the high energy consumption in the brain. However, the cellular mechanisms draining ATP upon the arrival of a stimulus are yet to be explored systematically at the post-synapse. Here, we provide evidence that a significant fraction of ATP is consumed upon glutamate stimulation to energize mGluR-induced protein synthesis. We find that both mGluR and NMDAR alter protein synthesis and ATP consumption with distinct kinetics at the synaptic-dendritic compartments. While mGluR activation leads to a rapid and sustained reduction in neuronal ATP levels, NMDAR activation has no immediate impact on the same. ATP consumption correlates inversely with the kinetics of protein synthesis for both receptors. We observe a persistent elevation in protein synthesis within 5 minutes of mGluR activation and a robust inhibition of the same within 2 minutes of NMDAR activation, assessed by the phosphorylation status of eEF2 and metabolic labeling. However, a delayed protein synthesis-dependent ATP expenditure ensues after 15 minutes of NMDAR stimulation. We identify a central role for AMPK in the correlation between protein synthesis and ATP consumption. AMPK is dephosphorylated and inhibited upon mGluR activation, while it is phosphorylated upon NMDAR activation. Perturbing AMPK activity disrupts receptor-specific modulations of eEF2 phosphorylation and protein synthesis. Our observations, therefore, demonstrate that the regulation of the AMPK-eEF2 signaling axis by glutamate receptors alters neuronal protein synthesis and bioenergetics., (© 2020 The Authors.)

The ribosomal p70 S6 Kinase 1 (S6K1) has been implicated in the etiology of complex neurological diseases including autism, depression and dementia. Though no major gene disruption has been reported in humans in RPS6KB1, single nucleotide variants (SNVs) causing missense mutations have been identified, which have not been assessed for their impact on protein function. These S6K1 mutations have the potential to influence disease progression and treatment response. We mined the Simon Simplex Collection (SSC) and SPARK autism database to find inherited SNVs in S6K1 and characterized the effect of two missense SNVs, Asp14Asn (allele frequency = 0.03282%) and Glu44Gln (allele frequency = 0.0008244%), on S6K1 function in HEK293, human ES cells and primary neurons. Expressing Asp14Asn in HEK293 cells resulted in increased basal phosphorylation of downstream targets of S6K1 and increased de novo translation. This variant also showed blunted response to the specific S6K1 inhibitor, FS-115. In human embryonic cell line Shef4, Asp14Asn enhanced spontaneous neural fate specification in the absence of differentiating growth factors. In addition to enhanced translation, neurons expressing Asp14Asn exhibited impaired dendritic arborization and increased levels of phosphorylated ERK 1/2. Finally, in the SSC families tracked, Asp14Asn segregated with lower IQ scores when found in the autistic individual rather than the unaffected sibling. The Glu44Gln mutation showed a milder, but opposite phenotype in HEK cells as compared to Asp14Asn. Although the Glu44Gln mutation displayed increased neuronal translation, it had no impact on neuronal morphology. Our results provide the first characterization of naturally occurring human S6K1 variants on cognitive phenotype, neuronal morphology and maturation, underscoring again the importance of translation control in neural development and plasticity., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Oral squamous cell carcinoma (OSCC) is one of the leading cancers in the world. OSCC patients are managed with surgery and/or chemoradiation. Prognoses and survival rates are dismal, however, and have not improved for more than 20 years. Recently, the concept of precision medicine was introduced, and the introduction of targeted therapeutics demonstrated promising outcomes. This article reviews the current understanding of initiation, progression, and metastasis of OSCC from both genetic and epigenetic perspectives. In addition, the applications and integration of omics technologies in biomarker discovery and drug development for treating OSCC are reviewed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Whether fragile X mental retardation protein (FMRP) target mRNAs and neuronal activity contributing to elevated basal neuronal protein synthesis in fragile X syndrome (FXS) is unclear. Our proteomic experiments reveal that the de novo translational profile in FXS model mice is altered at steady state and in response to metabotropic glutamate receptor (mGluR) stimulation, but the proteins expressed differ under these conditions. Several altered proteins, including Hexokinase 1 and Ras, also are expressed in the blood of FXS model mice and pharmacological treatments previously reported to ameliorate phenotypes modify their abundance in blood. In addition, plasma levels of Hexokinase 1 and Ras differ between FXS patients and healthy volunteers. Our data suggest that brain-based de novo proteomics in FXS model mice can be used to find altered expression of proteins in blood that could serve as disease-state biomarkers in individuals with FXS.

The most conserved molecular phenotype of Fragile X Syndrome (FXS) is aberrant protein synthesis. This has been validated in a variety of experimental model systems from zebrafish to rats, patient-derived lymphoblasts and fibroblasts. With the advent of personalized medicine paradigms, patient-derived cells and their derivatives are gaining more translational importance, not only to model disease in a dish, but also for biomarker discovery. Here we review past and current practices of measuring protein synthesis in FXS, studies in patient derived cells and the inherent challenges in measuring protein synthesis in them to offer usable avenues of modeling this important metabolic metric for further biomarker development.

Objective: Autologous cultured explants of human oral mucosal epithelial cells (OMEC) are a potential therapeutic modality in patients of bilateral ocular surface disease (OSD) with incapacitating dry eye. Mycophenolate mofetil (MMF) has been found to upregulate the mucin production in conjunctival goblet cells in vitro. The aim of this study was to evaluate the effects of MMF on mucin expression in primary cultures of OMEC., Methods: With informed consent, oral mucosal epithelial tissue samples were obtained from patients undergoing oral surgery for non-malignant conditions. OMEC were cultured on human amniotic membrane (HAM) scaffold for 2 weeks. Mucin expression was quantified using RT-PCR and qPCR before and after treating cultured OMEC with MMF., Results: Morphological studies revealed a confluent sheet of proliferating, stratified oral mucosal epithelial cells. Mucin mRNAs were elucidated by RT-PCR. Compared to untreated controls, MUC1, MUC15 and MUC16 mRNAs and MUC1 protein expression were found to be upregulated in MMF treated primary cultures of OMEC, as assessed by qPCR and immunocytochemistry respectively., Conclusion: Our findings demonstrate that MMF can act as a novel enhancer of mucin production in OMEC in vitro. It has the potential to improve dry eye in patients undergoing OMEC transplantation for bilateral OSD.

The incidence of oral cancer in the United States is increasing, especially in young people and women. Patients with oral cancer report severe functional pain. Using a patient cohort accrued through the New York University Oral Cancer Center and immune-competent mouse models, we identify a sex difference in the prevalence and severity of oral cancer pain. A neutrophil-mediated endogenous analgesic mechanism is present in male mice with oral cancer. Local naloxone treatment potentiates cancer mediator-induced orofacial nociceptive behavior in male mice only. Tongues from male mice with oral cancer have significantly more infiltrating neutrophils compared to female mice with oral cancer. Neutrophils isolated from the cancer-induced inflammatory microenvironment express beta-endorphin and met-enkephalin. Furthermore, neutrophil depletion results in nociceptive behavior in male mice. These data suggest a role for sex-specific, immune cell-mediated endogenous analgesia in the treatment of oral cancer pain.

Once activated at the surface of cells, G protein-coupled receptors (GPCRs) redistribute to endosomes, where they can continue to signal. Whether GPCRs in endosomes generate signals that contribute to human disease is unknown. We evaluated endosomal signaling of protease-activated receptor-2 (PAR 2 ), which has been proposed to mediate pain in patients with irritable bowel syndrome (IBS). Trypsin, elastase, and cathepsin S, which are activated in the colonic mucosa of patients with IBS and in experimental animals with colitis, caused persistent PAR 2 -dependent hyperexcitability of nociceptors, sensitization of colonic afferent neurons to mechanical stimuli, and somatic mechanical allodynia. Inhibitors of clathrin- and dynamin-dependent endocytosis and of mitogen-activated protein kinase kinase-1 prevented trypsin-induced hyperexcitability, sensitization, and allodynia. However, they did not affect elastase- or cathepsin S-induced hyperexcitability, sensitization, or allodynia. Trypsin stimulated endocytosis of PAR 2 , which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR 2 , which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR 2 -dependent hyperexcitability of nociceptors, and PAR 2 association with β-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR 2 A cholestanol-conjugated PAR 2 antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR 2 signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR 2 antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases., Competing Interests: Conflict of interest statement: P.M. and G.A.H. work for Takeda Pharmaceuticals, Inc. Research in N.W.B.’s laboratory is supported in part by Takeda Pharmaceuticals, Inc. N.W.B. and G.A.H. are founding scientists of Endosome Therapeutics, Inc.

Stress is known to cause contrasting patterns of morphological and physiological plasticity in the hippocampus and amygdala. An obligatory cellular process underlying such neural changes is de novo translation and alterations in protein expression. Yet the nature of the translational response to stress in neurons remains largely unexplored. Even less is known about how glia are affected. Using a click-chemistry-based method to label the de novo proteome in live brain slices, we monitored translation in neurons and astrocytes of the basolateral amygdala (BLA) and dorsal hippocampal area CA3 (dCA3) in rats at different time-points after a single 2-hr exposure to immobilization stress. We observed enhancements in neuronal translation in both brain regions 1 hour after stress. This initial increase persisted in the BLA up to 10 days afterwards. In contrast, dCA3 neuronal translation gradually decreased to below control levels 10 days later. Translation profiles of dCA3 astrocytes followed timelines similar to neurons, but in BLA astrocytes translation peaked 1 day later and remained elevated 10 days later. Together our results demonstrate that stress causes an immediate upregulation of protein synthesis in both amygdalar and hippocampal neurons and astrocytes. However, these two areas eventually exhibit opposite temporal profiles of protein expression well after the end of stress. These findings identify new metrics of stress-induced plasticity at the level of cell-type specific proteomic landscape that may provide important insights into the molecular basis of the divergent temporal effects of stress across brain regions and biological scales., (© 2018 Wiley Periodicals, Inc.)

Oral squamous cell carcinoma (OSCC) is one of the leading cancers in the world. OSCC patients are managed with surgery and/or chemoradiation. Prognoses and survival rates are dismal, however, and have not improved for more than 20 years. Recently, the concept of precision medicine was introduced, and the introduction of targeted therapeutics demonstrated promising outcomes. This article reviews the current understanding of initiation, progression, and metastasis of OSCC from both genetic and epigenetic perspectives. In addition, the applications and integration of omics technologies in biomarker discovery and drug development for treating OSCC are reviewed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Patients with oral cancer report severe pain during function. Inflammation plays a role in the oral cancer microenvironment; however, the role of immune cells and associated secretion of inflammatory mediators in oral cancer pain has not been well defined. In this study, we used 2 oral cancer mouse models: a cell line supernatant injection model and the 4-nitroquinoline-1-oxide (4NQO) chemical carcinogenesis model. We used the 2 models to study changes in immune cell infiltrate and orofacial nociception associated with oral squamous cell carcinoma (oSCC). Oral cancer cell line supernatant inoculation and 4NQO-induced oSCC resulted in functional allodynia and neuronal sensitization of trigeminal tongue afferent neurons. Although the infiltration of immune cells is a prominent component of both oral cancer models, our use of immune-deficient mice demonstrated that oral cancer-induced nociception was not dependent on the inflammatory component. Furthermore, the inflammatory cytokine, tumor necrosis factor alpha (TNFα), was identified in high concentration in oral cancer cell line supernatant and in the tongue tissue of 4NQO-treated mice with oSCC. Inhibition of TNFα signaling abolished oral cancer cell line supernatant-evoked functional allodynia and disrupted T-cell infiltration. With these data, we identified TNFα as a prominent mediator in oral cancer-induced nociception and inflammation, highlighting the need for further investigation in neural-immune communication in cancer pain.

Aberrant neuronal translation is implicated in the etiology of numerous brain disorders. Although mTORC1-p70 ribosomal S6 kinase 1 (S6K1) signaling is critical for translational control, pharmacological manipulation in vivo has targeted exclusively mTORC1 due to the paucity of specific inhibitors to S6K1. However, small molecule inhibitors of S6K1 could potentially ameliorate pathological phenotypes of diseases, which are based on aberrant translation and protein expression. One such condition is fragile X syndrome (FXS), which is considered to be caused by exaggerated neuronal translation and is the most frequent heritable cause of autism spectrum disorder (ASD). To date, potential therapeutic interventions in FXS have focused largely on targets upstream of translational control to normalize FXS-related phenotypes. Here we test the ability of two S6K1 inhibitors, PF-4708671 and FS-115, to normalize translational homeostasis and other phenotypes exhibited by FXS model mice. We found that although the pharmacokinetic profiles of the two S6K1 inhibitors differed, they overlapped in reversing multiple disease-associated phenotypes in FXS model mice including exaggerated protein synthesis, inappropriate social behavior, behavioral inflexibility, altered dendritic spine morphology, and macroorchidism. In contrast, the two inhibitors differed in their ability to rescue stereotypic marble-burying behavior and weight gain. These findings provide an initial pharmacological characterization of the impact of S6K1 inhibitors in vivo for FXS, and have therapeutic implications for other neuropsychiatric conditions involving aberrant mTORC1-S6K1 signaling.

Brain-derived neurotrophic factor (BDNF) plays an important role in neurodevelopment, synaptic plasticity, learning and memory, and in preventing neurodegeneration. Despite decades of investigations into downstream signaling cascades and changes in cellular processes, the mechanisms of how BDNF reshapes circuits in vivo remain unclear. This informational gap partly arises from the fact that the bulk of studies into the molecular actions of BDNF have been performed in dissociated neuronal cultures, while the majority of studies on synaptic plasticity, learning and memory were performed in acute brain slices or in vivo. A recent study by Bowling-Bhattacharya et al., measured the proteomic changes in acute adult hippocampal slices following treatment and reported changes in proteins of neuronal and non-neuronal origin that may in concert modulate synaptic release and secretion in the slice. In this paper, we place these findings into the context of existing literature and discuss how they impact our understanding of how BDNF can reshape the brain.

Stimulus-triggered protein synthesis is critical for brain health and function. However, due to technical hurdles, de novo neuronal translation is predominantly studied in cultured cells, whereas electrophysiological and circuit analyses often are performed in brain slices. The different properties of these two experimental systems create an information gap about stimulus-induced alterations in the expression of new proteins in mature circuits. To address this, we adapted two existing techniques, BONCAT and SILAC, to a combined proteomic technique, BONLAC, for use in acute adult hippocampal slices. Using BDNF-induced protein synthesis as a proof of concept, we found alterations in expression of proteins involved in neurotransmission, trafficking, and cation binding that differed from those found in a similar screen in cultured neurons. Our results indicate important differences between cultured neurons and slices, and suggest that BONLAC could be used to dissect proteomic changes underlying synaptic events in adult circuits. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The PI3K enhancer PIKE links PI3K catalytic subunits to group 1 metabotropic glutamate receptors (mGlu1/5) and activates PI3K signaling. The roles of PIKE in synaptic plasticity and the etiology of mental disorders are unknown. Here, we show that increased PIKE expression is a key mediator of impaired mGlu1/5-dependent neuronal plasticity in mouse and fly models of the inherited intellectual disability fragile X syndrome (FXS). Normalizing elevated PIKE protein levels in FXS mice reversed deficits in molecular and cellular plasticity and improved behavior. Notably, PIKE reduction rescued PI3K-dependent and -independent neuronal defects in FXS. We further show that PI3K signaling is increased in a fly model of FXS and that genetic reduction of the Drosophila ortholog of PIKE, CenG1A rescued excessive PI3K signaling, mushroom body defects, and impaired short-term memory in these flies. Our results demonstrate a crucial role of increased PIKE expression in exaggerated mGlu1/5 signaling causing neuronal defects in FXS., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Fragile X syndrome (FXS) is the most frequent cause of inherited intellectual disability and autism. It is caused by the absence of the fragile X mental retardation 1 (FMR1) gene product, fragile X mental retardation protein (FMRP), an RNA-binding protein involved in the regulation of translation of a subset of brain mRNAs. In Fmr1 knockout mice, the absence of FMRP results in elevated protein synthesis in the brain as well as increased signaling of many translational regulators. Whether protein synthesis is also dysregulated in FXS patients is not firmly established. Here, we demonstrate that fibroblasts from FXS patients have significantly elevated rates of basal protein synthesis along with increased levels of phosphorylated mechanistic target of rapamycin (p-mTOR), phosphorylated extracellular signal regulated kinase 1/2, and phosphorylated p70 ribosomal S6 kinase 1 (p-S6K1). The treatment with small molecules that inhibit S6K1 and a known FMRP target, phosphoinositide 3-kinase (PI3K) catalytic subunit p110β, lowered the rates of protein synthesis in both control and patient fibroblasts. Our data thus demonstrate that fibroblasts from FXS patients may be a useful in vitro model to test the efficacy and toxicity of potential therapeutics prior to clinical trials, as well as for drug screening and designing personalized treatment approaches., (© 2014 WILEY PERIODICALS, INC.)

Individual bacteria and shifts in the composition of the microbiome have been associated with human diseases including cancer. To investigate changes in the microbiome associated with oral cancers, we profiled cancers and anatomically matched contralateral normal tissue from the same patient by sequencing 16S rDNA hypervariable region amplicons. In cancer samples from both a discovery and a subsequent confirmation cohort, abundance of Firmicutes (especially Streptococcus) and Actinobacteria (especially Rothia) was significantly decreased relative to contralateral normal samples from the same patient. Significant decreases in abundance of these phyla were observed for pre-cancers, but not when comparing samples from contralateral sites (tongue and floor of mouth) from healthy individuals. Weighted UniFrac principal coordinates analysis based on 12 taxa separated most cancers from other samples with greatest separation of node positive cases. These studies begin to develop a framework for exploiting the oral microbiome for monitoring oral cancer development, progression and recurrence.

Background: Metastasis to the cervical (neck) lymph nodes is one of the most significant clinical factors responsible for death from oral squamous cell carcinoma (SCC). Therefore, the lymph nodes are frequently removed when the tumor is excised (neck dissection), even though the majority of patients will not benefit from the extra surgery. Two subtypes of oral SCC distinguished by the presence of tumor genomic aberrations +3q, -8p, +8q and/or +20 differ in risk for metastasis - high for the 3q8pq20 subtype, harboring one or more of the aberrations and low for the non-3q8pq20 subtype, lacking these alterations. A prior analysis of the literature suggested genes differentially methylated in the two subtypes. Therefore, the goal of this study was to further investigate the methylation status of candidate biomarkers of the non-3q8pq20 subtype, and evaluate their utility for identifying patients at low risk for metastasis., Methods: Methylation status of genes in a cohort of 52 oral SCC patients with at least five year follow up was determined by pyrosequencing. Gene expression levels were determined by quantitative RT-PCR. Growth following re-expression of HOXA9 in cultured oral SCC cells was assessed by proliferation and colony formation assays., Results: A pilot study evaluating methylation levels of HOXA9, MT1A and HOXA11 promoters in DNA from 12 tumors (six each of the 3q8pq20 and non-3q8pq20 subtypes) revealed that only HOXA9 was differentially methylated. Significant differences in methylation levels of HOXA9 were observed amongst the 52 oral SCCs with respect to genomic subtype and nodal status (p = 0.014, and p = 0.024, respectively, Wilcoxon rank sum test). High levels of HOXA9 methylation and low levels of expression in oral SCC cell lines were observed compared to HaCaT, a non-tumorigenic keratinocyte cell line. Re-expression of HOXA9 in the SCC4 oral cancer cell line resulted in diminished proliferation and colony formation., Conclusions: HOXA9 methylation is frequent in oral cancers and levels are higher in tumors with greater risk of metastasis. Expression of HOXA9 is low in cells with high levels of methylation and reduced expression appears to confer a growth advantage.

The proper regulation of translation is required for the expression of long-lasting synaptic plasticity. A major site of translational control involves the phosphorylation of eukaryotic initiation factor 2 α (eIF2α) by PKR-like endoplasmic reticulum (ER) kinase (PERK). To determine the role of PERK in hippocampal synaptic plasticity, we used the Cre-lox expression system to selectively disrupt PERK expression in the adult mouse forebrain. Here, we demonstrate that in hippocampal area CA1, metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) is associated with increased eIF2α phosphorylation, whereas stimulation of early- and late-phase long-term potentiation (E-LTP and L-LTP, respectively) is associated with decreased eIF2α phosphorylation. Interesting, although PERK-deficient mice exhibit exaggerated mGluR-LTD, both E-LTP and L-LTP remained intact. We also found that mGluR-LTD is associated with a PERK-dependent increase in eIF2α phosphorylation. Our findings are consistent with the notion that eIF2α phosphorylation is a key site for the bidirectional control of persistent forms of synaptic LTP and LTD and suggest a distinct role for PERK in mGluR-LTD.

Although antipsychotic drugs can reduce psychotic behavior within a few hours, full efficacy is not achieved for several weeks, implying that there may be rapid, short-term changes in neuronal function, which are consolidated into long-lasting changes. We showed that the antipsychotic drug haloperidol, a dopamine receptor type 2 (D₂R) antagonist, stimulated the kinase Akt to activate the mRNA translation pathway mediated by the mammalian target of rapamycin complex 1 (mTORC1). In primary striatal D₂R-positive neurons, haloperidol-mediated activation of mTORC1 resulted in increased phosphorylation of ribosomal protein S6 (S6) and eukaryotic translation initiation factor 4E-binding protein (4E-BP). Proteomic mass spectrometry revealed marked changes in the pattern of protein synthesis after acute exposure of cultured striatal neurons to haloperidol, including increased abundance of cytoskeletal proteins and proteins associated with translation machinery. These proteomic changes coincided with increased morphological complexity of neurons that was diminished by inhibition of downstream effectors of mTORC1, suggesting that mTORC1-dependent translation enhances neuronal complexity in response to haloperidol. In vivo, we observed rapid morphological changes with a concomitant increase in the abundance of cytoskeletal proteins in cortical neurons of haloperidol-injected mice. These results suggest a mechanism for both the acute and long-term actions of antipsychotics.

Introduction: Wellbutrin (bupropion hydrochloride; WB), an anti-depressant of the aminoketone class is new highly selective norepinephrine and dopamine reuptake inhibitor; it is effective in the treatment of patients with major depression., Materials and Methods: To investigate the in vitro effects of WB in human cultured peripheral blood lymphocytes and human cortical neural (HCN2) cell lines, micronucleus, sister chromatid exchange analysis, cellular viability, and comet assays were employed. The present study is to our knowledge, the first report on WB genotoxicity in cultured human peripheral blood lymphocytes and its cytotoxicity in the HCN2 cell line. We have also investigated the genotoxic potential of WB to induce chromosomal aberrations., Results: WB-induced cytotoxicity (measured as reduction of the nuclear division index) possibly prevented the division of damaged cells., Conclusion: We conclude that although, WB exerts potential genotoxic effects in cultured lymphocytes, its cytogenetic effects are very unlikely to occur in blood cultures of WB-administered subjects.

Fragile X syndrome (FXS) is the leading inherited cause of autism and intellectual disability. Aberrant synaptic translation has been implicated in the etiology of FXS, but most lines of research on therapeutic strategies have targeted protein synthesis indirectly, far upstream of the translation machinery. We sought to perturb p70 ribosomal S6 kinase 1 (S6K1), a key translation initiation and elongation regulator, in FXS model mice. We found that genetic reduction of S6K1 prevented elevated phosphorylation of translational control molecules, exaggerated protein synthesis, enhanced mGluR-dependent long-term depression (LTD), weight gain, and macro-orchidism in FXS model mice. In addition, S6K1 deletion prevented immature dendritic spine morphology and multiple behavioral phenotypes, including social interaction deficits, impaired novel object recognition, and behavioral inflexibility. Our results support the model that dysregulated protein synthesis is the key causal factor in FXS and that restoration of normal translation can stabilize peripheral and neurological function in FXS., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Persons with pervasive developmental disorders (PDD) exhibit a range of cognitive deficits that hamper their quality of life, including difficulties involving communication, sociability, and perspective-taking. In recent years, a variety of studies in mice that model genetic syndromes with a high risk of PDD have provided insights into the underlying molecular mechanisms associated with these disorders. What is less appreciated is how the molecular anomalies affect neuronal and circuit function to give rise to the cognitive deficits associated with PDD. In this review, we describe genetic mutations that cause PDD and discuss how they alter fundamental social and cognitive processes. We then describe efforts to correct cognitive impairments associated with these disorders and identify areas of further inquiry in the search for molecular targets for therapeutics for PDD.

Treatment for fragile X syndrome and related autism spectrum disorders has long been thought to be effective only during a narrow window early in development. In this issue of Neuron, Michalon et al. (2012) dispel this myth., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Tuberous sclerosis complex (TSC) and fragile X syndrome (FXS) are caused by mutations in negative regulators of translation. FXS model mice exhibit enhanced metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD). Therefore, we hypothesized that a mouse model of TSC, ΔRG transgenic mice, also would exhibit enhanced mGluR-LTD. We measured the impact of TSC2-GAP mutations on the mTORC1 and ERK signaling pathways and protein synthesis-dependent hippocampal synaptic plasticity in ΔRG transgenic mice. These mice express a dominant/negative TSC2 that binds to TSC1, but has a deletion and substitution mutation in its GAP-domain, resulting in inactivation of the complex. Consistent with previous studies of several other lines of TSC model mice, we observed elevated S6 phosphorylation in the brains of ΔRG mice, suggesting upregulated translation. Surprisingly, mGluR-LTD was not enhanced, but rather was impaired in the ΔRG transgenic mice, indicating that TSC and FXS have divergent synaptic plasticity phenotypes. Similar to patients with TSC, the ΔRG transgenic mice exhibit elevated ERK signaling. Moreover, the mGluR-LTD impairment displayed by the ΔRG transgenic mice was rescued with the MEK-ERK inhibitor U0126. Our results suggest that the mGluR-LTD impairment observed in ΔRG mice involves aberrant TSC1/2-ERK signaling., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Till today, there has been some hesitation to accept the role of fine needle aspiration cytology (FNAC) in pelvic mass. We have tried to study the role of ultrasonography (USG) and computed tomography (CT) guided FNAC as diagnostic and supportive investigation for ovarian tumors., Aim: To evaluate the current status of image-directed percutaneous aspiration of ovarian neoplasm for the purpose of early detection of malignancy., Materials and Methods: Seventy-four fine needle aspirations of ovarian neoplasms were performed between January 2007 and December 2008 by transabdominal approach under USG and CT guidance and correlated with histopathological findings and tumor markers., Results: A total of 47 (63.5%) cases were assessed as malignant and 21 (28.3%) as benign and 6 (8.1%) as inconclusive. The neoplastic lesions were categorized as per World Health Organization (WHO) classification., Conclusion: With the availability of modern techniques, USG and CT guided FNAC can be an optimum modality for the diagnosis of primary and metastatic ovarian neoplasms and evaluation of recurrent malignant tumors, which has great impact on patient management consequently.

Purpose: Problems in management of oral cancers or precancers include identification of patients at risk for metastasis, tumor recurrence, and second primary tumors or risk for progression of precancers (dysplasia) to cancer. Thus, the objective of this study was to clarify the role of genomic aberrations in oral cancer progression and metastasis., Experimental Design: The spectrum of copy number alterations in oral dysplasia and squamous cell carcinomas (SCC) was determined by array comparative genomic hybridization. Associations with clinical characteristics were studied and results confirmed in an independent cohort., Results: The presence of one or more of the chromosomal aberrations +3q24-qter, -8pter-p23.1, +8q12-q24.2, and +20 distinguishes a major subgroup (70%-80% of lesions, termed 3q8pq20 subtype) from the remainder (20%-30% of lesions, non-3q8pq20). The 3q8pq20 subtype is associated with chromosomal instability and differential methylation in the most chromosomally unstable tumors. The two subtypes differ significantly in clinical outcome with risk for cervical (neck) lymph node metastasis almost exclusively associated with the 3q8pq20 subtype in two independent oral SCC cohorts., Conclusions: Two subtypes of oral lesions indicative of at least two pathways for oral cancer development were distinguished that differ in chromosomal instability and risk for metastasis, suggesting that +3q,-8p, +8q, and +20 constitute a biomarker with clinical utility for identifying patients at risk for metastasis. Moreover, although increased numbers of genomic alterations can be harbingers of progression to cancer, dysplastic lesions lacking copy number changes cannot be considered benign as they are potential precursors to non-3q8pq20 locally invasive, yet not metastatic oral SCC.

Internalization and recycling of G-protein coupled receptors are important cellular processes regulating receptor function. These are receptor-subtype and cell type-specific. Although important, trafficking variations between receptor isoforms of different species has received limited attention. We report here, differences in internalization and recycling between rat and human serotonin 2A receptor (5-HT(2A)R) isoforms expressed in human embryonic kidney 293 cells in response to serotonin. Although the human and rat 5-HT(2A)Rs differ by only a few amino acids, the human receptor takes longer to recycle to the cell surface after internalization, with the additional involvement of beta arrestin-2 and G-protein receptor kinase 2. The interaction of beta arrestin-2 with the human receptor causes the delay in recycling and is dependent on a primate-specific ASK motif present in the C-terminus of the receptor. Conversion of this motif to NCT, the corresponding sequence present in the rat isoform, results in the human isoform trafficking like the rat receptor. Replacing the serine 457 with alanine in the ASK motif of human isoform resulted in faster recycling, although with continued arrestin-dependent internalization. This study establishes significant differences between the two isoforms with important implications in our understanding of the human 5-HT(2A)R functions; and indicates that extrapolating results from non-human receptor isoforms to human subtypes is not without caveats.

The serotonergic system has been implicated in emotional and cognitive function. In particular, 5-HT(2A) (5-hydroxytrytamine receptor 2A) is attributed to a number of disorders like schizophrenia, depression, eating disorders and anxiety. 5-HT(2A), being a GPCR (G-protein coupled receptor), is important in the pharmaceutical industry as a proven target for these disorders. Despite their extensive clinical importance, the structural studies of this protein is lacking due to difficulties in determining its crystal structure. We have performed sequence analysis and molecular modeling of 5-HT(2A) that has revealed a set of conserved residues and motifs considered to play an important role in maintaining structural integrity and function of the receptor. The analysis also revealed a set of residues specific to the receptor which distinguishes them from other members of the subclass and their orthologs. Further, starting from the model structure of human 5-HT(2A) receptor, docking studies were attempted to envisage how it might interact with eight of its ligands (such as serotonin, dopamine, DOI, LSD, haloperidol, ketanserin, risperidone and clozapine). The binding studies of dopamine to 5-HT(2A) receptor can bring up better understanding in the etiology of a number of neurological disorders involving both these two receptors. Our sequence analysis and study of interactions of this receptor with other ligands reveal additional residue hotspots such as Asn 363 and Tyr 370. The function of these residues can be further analyzed by rational design of site-directed mutagenesis. Two distinct binding sites are identified which could play important roles in ligand binding and signaling.

Serotonergic and dopaminergic systems, and their functional interactions, have been implicated in the pathophysiology of various CNS disorders. Here, we use recombinant serotonin (5-HT) 2A (5-HT2A) receptors to further investigate direct interactions between dopamine and 5-HT receptors. Previous studies in Xenopus oocytes showed that dopamine, although not the cognate ligand for the 5-HT2A receptor, acts as a partial-efficacy agonist. At micromolar concentrations, dopamine also acts as a partial-efficacy agonist on 5-HT2A receptors in HEK293 cells. Like 5-HT, dopamine also induces receptor-internalization in these cells, although at significantly higher concentrations than 5-HT. Interestingly, if the receptors are first sensitized or "primed" by subthreshold concentrations of 5-HT, then dopamine-induced internalization occurs at concentrations approximately 10-fold lower than when dopamine is used alone. Furthermore, unlike 5-HT-mediated internalization, dopamine-mediated receptor internalization, alone, or after sensitization by 5-HT, does not depend on PKC. Dopamine-internalized receptors recycle to the surface at rates similar to those of 5-HT-internalized receptors. Our results suggest a previously uncharacterized role for dopamine in the direct activation and internalization of 5-HT2A receptors that may have clinical relevance to the function of serotonergic systems in anxiety, depression, and schizophrenia and also to the treatment of these disorders.