Recent studies have suggested that macrophages have a critical role in the development of alcohol-induced inflammation in the liver. To define the precise pathogenic function of these cells during alcoholic liver disease (ALD), it is extremely important to conduct extensive studies in clinical settings that further elucidate the phenotypic diversity of macrophages in the context of ALD. Research to date already has identified several characteristics of macrophages that underlie the cells’ actions, including macrophage polarization and their phenotypic diversity. Other analyses have focused on the contributions of resident versus infiltrating macrophages/monocytes, as well as on the roles of macrophage mediators, in the development of ALD. Findings point to the potential of macrophages as a therapeutic target in alcoholic liver injury. Future studies directed toward understanding how alcohol affects macrophage phenotypic switch in the liver and other tissues, whether the liver microenvironment determines macrophage function in ALD, and if targeting of macrophages alleviates alcoholic liver injury, will provide promising strategies to manage patients with alcoholic hepatitis. Alcoholic liver disease (ALD) is a complex disease that affects millions of people worldwide and eventually can lead to liver cirrhosis and liver cancer . Aside from the direct cytotoxic and the oxidative-stress–mediated effects that alcohol and its metabolite, acetaldehyde, exert on hepatocytes, alcohol ingestion activates both the innate and adaptive immune responses in the liver. These responses involve multiple hepatic cell types, including resident macrophages, natural killer cells, natural killer T cells, lymphocytes, and neutrophils. In particular, resident macrophages in the liver, also known as Kupffer cells, are important for clearing pathogens, including bacteria, viruses, immune complexes, bacterial products called endotoxin or lipopolysaccharide (LPS), and tumor cells, from the liver . ResearcThe activation of circulating monocytes and accumulation of macrophages in the liver are important pathophysiological features in patients with ALD. However, the role of hepatic macrophages in the pathogenesis of ALD has not been fully elucidated. This review will discuss some of the new findings in monocyte/macrophage biology, provide an update of the current studies on the involvement of liver macrophages in ALD, and identify remaining questions to be addressed in order to develop macrophage-targeted therapy for ALD.Macrophages, which play an important role in the initial innate immune response to infection with pathogens or other insults, fall into two main categories—infiltrating macrophages and tissue-resident macrophages. Infiltrating macrophages are derived from precursor cells called monocytes that circulate throughout the body and are recruited into the tissues when an inflammatory reaction occurs. Tissue-resident macrophages, in contrast, always remain localized within one tissue, serving as sentries and first line of defense against any infection or injury in that tissue.+) are divided into the two major subsets based on their CD14 and CD16 expression, as well as on their expression of markers called CCR2 and CX3/CR1:Monocytes are circulating innate immune cells formed from progenitor cells in the bone marrow; the monocytes then differentiate into numerous subsets of macrophages . In bothhiCD16− and CCR2+/CX3CR1lo.The predominant subset, representing 90 percent of circulating monocytes, is the classical subset characterized by the marker combination CD14dimCD16+ and CCR2−/CX3CR1hi/CCR5hi  or CD14hiCD16+ and CCR2−/CX3CR1hi/CCR5hi (intermediate monocytes).The less abundant nonclassical monocyte subset further can be divided into two groups characterized by the marker combinations CD14hi monocytes, which are similar to the human CD14hiCD16− monocytes, and nonclassical Ly6Clo monocytes, which are similar to human CD14dimCD16+ monocytes. These human and murine cells patrol in the blood vessels until they are recruited to the organs in case of an injury or insult. Although the gene-expression profiles related to activation and trafficking have been well conserved between murine and human monocytes, the ratios of various subsets can differ between mice and humans. Therefore, it is important to be careful and take these differences into consideration when extending experimental murine monocyte studies to human disease.1Analogous to their human counterparts, murine subsets include classical Ly6CResident macrophages can be found in a variety of tissues, such as the brain, skin, lungs, liver, and spleen. Tissue-resident macrophages exhibit a large diversity of phenotypes and functions, based on their tissue of residence, raising the question of the origin of these cells . Recent The Kupffer cells in the liver are the largest population of tissue-resident macrophages and largely contribute to inflammatory reactions in the liver. The innate immune function of Kupffer cells not only is critical in the body’s response to liver injury but also is crucial in tolerogenic responses to antigens in the liver. Kupffer cells are located in the hepatic sinusoids and fall into two major subsets :Radiosensitive macrophages that are replaced rapidly by hematopoietic precursors and are important in inflammatory reactions; andRadioresistant, long-lived Kupffer cells that do not participate in inflammatory foci.Mouse models frequently are used to investigate various aspects of macrophage function. However, as with the monocyte precursors, differences in the characteristics of murine and human macrophages exist that must be taken into account when using mice as preclinical models of disease . For exaMacrophages have a unique ability to alter their phenotypes and, thus, their functions, depending on tissue microenvironmental cues, such as the presence of cytokines, growth factors, pathogen-associated molecular pattern molecules (PAMPs), and damage-associated molecular pattern molecules (DAMPs). This process is known as polarization and results in the emergence of two macrophage phenotypes labeled M1 and M2 macrophages. M1 macrophages primarily have proinflammatory effects. For example, classically activated M1 macrophages help mediate the initial defense against intracellular bacteria and viruses; in addition, they are important for the response to a tissue injury. The M1 macrophages produce proinflammatory and stress mediators and cytokines, such as IL-1, tumor necrosis factor alpha (TNFα), interferon γ, IL-12, IL-18, nitric oxide, and reactive oxygen species (ROS), and can activate adaptive immune responses . Once thThe functional heterogeneity of macrophages is reflected in their differential, sometimes opposing, roles in various diseases . For exaAlthough it is convenient to divide macrophages into M1 and M2 cells, it is important to note that this division is oversimplified. The M1 and M2 macrophages only represent the two extremes of a full spectrum of phenotypes, and within either category there are subpopulations with different phenotypes and functions. For example, the M2 cells can be classified into at least two subtypes, wound-healing and immune-regulatory macrophages . The wouThe plasticity of macrophage phenotypes is controlled by various intracellular molecular mechanisms, including signaling proteins, transcription factors, and epigenetic events. For example, activation of macrophages via TLRs and interferon receptors, which induces a signaling mechanism involving a molecule called STAT1,In ALD, macrophage imprinting and polarization to M1 or M2 phenotypes is influenced by cytokine mediators in the liver. The detailed investigation of pathways activated by cytokines and stress proteins in the liver during ALD will provide insights into the polarization of resident versus infiltrating liver macrophages.Macrophages seem to play a central role in ALD. In fact, recent findings suggest the coexistence and complex inter-actions of different types of macrophages in ALD . Thus, iIncreased macrophage numbers have been reported in both early  and late  stages of ALD , althougThe levels of chemokines involved in monocyte recruitment, particularly MCP-1, MIP-1α, and MIP-1β, were increased in the liver of patients with ALD .In analyses of gene-expression profiles, the expression of inflammatory genes was higher in macrophages from patients with alcohol-related cirrhosis than in macrophages from patients with Hepatitis C virus–related cirrhosis .Factors that imply monocyte activation, such as neopterin and leukocyte-function–associated antigen 3, were elevated in ALD patients .Circulating monocytes from ALD patients express TNFα receptors and spontaneously produce TNFα. When stimulated by LPS, they release even higher levels of TNFα . Highly Patients with alcoholic hepatitis and/or cirrhosis exhibit elevated levels of other cytokines  and chemokines produced by circulating monocytes and liver macrophages . These iGlobal gene-expression profiling of liver samples from patients with alcohol-related cirrhosis demonstrated unique gene-expression patterns that differed between early and late stages of cirrhosis. Genes expressed at much higher levels in early than late stage of cirrhosis included those related to macrophage activation, proliferation, and migration , emphasiAdditional clinical studies evaluating macrophages and circulating monocytes from human patients at different stages of ALD are needed to understand the precise functional contributions of monocytes/macrophages to disease progression.Kupffer cells are liver-resident macrophages that are activated through the CD14/TLR4 receptor complex in response to increased intestinal translocation of LPS during prolonged alcohol consumption and which may contribute to alcohol-induced liver injury. Animal studies have revealed that acute and chronic ethanol administration are associated with signs of CD14/TLR4 activation of macrophages in the liver, including upregulation of CD14 as well as increased production of TNFα, MCP-1, and ROS . FurtherResearchers have investigated how alcohol consumption may trigger Kupffer-cell activation. ROS production may be one of the mechanisms contributing to increased sensitization of Kupffer cells to LPS in the alcoholic liver . During Another essential component in alcohol-mediated Kupffer-cell activation is the CD14/TLR4 receptor complex. LPS-induced activation of this receptor complex on Kupffer cells triggers downstream signaling kinases , ultimately leading to the induction of the proinflammatory cytokines TNFα, IL-6, and MCP-1. Consistent with this model, Kupffer cells from alcohol-fed mice are sensitized to LPS and exhibit increased LPS responses, leading to higher levels of TNFα  and MCP-Inhibition of Kupffer-cell activation and reduction of proinflammatory cytokines—particularly inhibition of proinflammatory cytokine production by Kupffer cells—has been a major focus of efforts to alleviate ALD. For example, it may be possible to reverse Kupffer-cell sensitization by treating alcohol-exposed Kupffer-cell primary cultures with adiponectin, an anti-inflammatory adipokine . TreatmeTissue-resident macrophages, such as Kupffer cells in the liver, not only protect against pathogens but also help nourish and maintain the cells  and ensure tissue homeostasis. However, under stress conditions caused by infection or by inflammation in the absence of infection , additional monocytes infiltrate the damaged tissue and differentiate into macrophages that help clear the pathogens, remove dead cells and cell debris, and restore tissue homeostasis. In fact, in many disease models  the tissue macrophages that have been described actually are derived from such infiltrating monocytes .4), which causes liver damage, results in an influx of infiltrating macrophages that can increase the total number of hepatic macrophages tenfold  and activation of heart muscle cells   .+CD16+ monocytes, which correspond to the Ly-6Clow infiltrating macrophages, occurs in a variety of inflammatory diseases, including rheumatoid arthritis, atherosclerosis, asthma, atopic eczema, pancreatitis, and alveolar proteinosis. Nonclassical CD14+CD16+ monocytes also expand in the circulation and liver of patients with chronic liver disease, suggesting their involvement in the progression of liver inflammation and fibrogenesis  of the nonclassical CD14ogenesis .The heterogeneous populations of both resident and infiltrating macrophages present in the liver have multiple functions that are relevant to ALD see :They can serve as antigen-presenting cells that display foreign molecules on their surface, thereby triggering adaptive immune responses.They may exhibit liver proteins that have been modified by malondialdehyde-acetaldehyde  . This moThey normally produce antimicrobial peptides and mediators and have microbial killing activities; however, these functions may be compromised during ALD.Through activation of TLR-mediated signaling, they may lead to increased expression of immunoinhibitory receptors called PD-1 and TIM-3 on T cells, thereby impairing antimicrobial activity in patients with alcoholic hepatitis .hi infiltrating macrophages) produce a variety of proinflammatory mediators, including ROS, reactive nitrogen species, proinflammatory cytokines, and chemokines, thereby causing tissue damage.Certain subpopulations  have yielded a reduction in alcohol-induced inflammatory responses in murine liver .Studies using globular adiponectin to induce IL-10 production in Kupffer cells via the enzyme heme oxygenase-1 alleviated murine ALD . InductiEfforts centering on the MCP-1 and MIF produced by Kupffer cells and infiltrating macrophages in the mouse alcoholic liver identified these chemokines as effective targets .Strategies targeting stress-induced heat-shock protein 90 with specific inhibitors—an approach currently assessed in clinical trials for cancer—helped ameliorate ALD by inhibiting macrophage inflammatory responses in murine liver .These studies collectively support clinical evaluations of macrophage-targeting therapies in alcoholic-hepatitis patients. Clinical research combining biologics, small-molecule drugs, and antioxidant therapies targeting macrophage function and phenotype may provide lasting therapeutic efficacy in alcoholic hepatitis and cirrhosis.As in many chronic inflammatory diseases, macrophages have emerged as critical players and perhaps a therapeutic target in ALD. However, depleting all hepatic macrophages will not be an effective approach because of the heterogeneity and phenotype diversity of these cells; the specific populations to be targeted for maximum benefit remain to be determined.AdipokineA bioactive factor produced and secreted by fat (adipose) tissue that can modulate the function of other tissuesAlveolar proteinosisalveoli with a protein-like material that prevents ventilation of the affected area; results in shortness of breath, coughing, chest pain, weight loss, and spitting up of bloodA chronic lung disease characterized by the filling of the AlveoliSac-like structures in the lungs where the gas exchange between the inhaled air and blood takes placeAutoantibodyAn immune molecule  formed in response to, and acting against, one of the individual’s own normal tissue constituentsChemokinecytokines that induce the movement of leukocytes Any of a family of small Complement systemphagocytosis)A complex system of about 20 distinct proteins, their receptors, and related regulatory proteins that induce the destruction  of cells during an immune response as well as regulate various other biologic functions Damage-associated molecular pattern molecules (DAMPs)Molecules that can initiate an immune response in response to cell or tissue damage EndotoxinToxic molecule associated with the membranes of certain bacteria that are released when the cells are disrupted and have numerous biologic effects ; endotoxins are composed of lipopolysaccharides (LPS)EpigeneticPertaining to mechanisms that alter the activity of genes without changing their DNA sequences Fate mappingAn experimental approach to determine the origins of various tissues in the adult organism from the embryonic structures and to track the development of specific cells through several developmental stagesLeukocytesAny of variety of white blood cells, such as monocytes or lymphocytesMacrophage polarizationcytokines); the two main types of polarized macrophages are M1  and M2  macrophages, each of which produces specific cytokines and induces specific immune responsesProcess during which macrophages acquire specific characteristics and functions in response to external signals ; malondialdehyde can interact with certain DNA building blocks, forming DNA adducts, which can induce mutations in the DNAPathogen-associated molecular pattern molecules (PAMPs)Molecules that can initiate an immune response in response to infection with a pathogen PhagocytosisProcess by which a cell  takes up microorganisms or cell fragments in membrane-enclosed vesicles in which the engulfed material is killed and digestedSteatohepatitisCondition in which fat droplets accumulate in the liver  with simultaneous inflammation of the liverStellate cellCell type found in the liver with a characteristic star-like shape that is mainly responsible for fat storage in the liver as well as for collagen production; source of excess collagen produced during hepatitisTolerogenicCapable of inducing immunologic tolerance low infiltrating macrophages in tissue repair and wound healing can be utilized to prevent chronic liver inflammation during the early phase of ALD. The conversion of the proinflammatory tissue-damaging Ly-6Chi infiltrating macrophages to anti-inflammatory tissue restorative Ly-6Clow cells can serve as a target for treatment of advanced stages of ALD, such as alcoholic steatohepatitis, and has been suggested in human and mouse studies (There are many questions that still need to be addressed with regard to the role of hepatic macrophages in ALD development. For example, how do infiltrating monocytes differentiate within the liver during ALD? What are the tissue-environmental cues and molecular-signaling pathways that drive the reprogramming of infiltrating macrophages in the alcoholic liver? Another important question is how the number of infiltrating macrophages is controlled after tissue homeostasis is reestablished. Do excess cells undergo apoptosis or do they emigrate? Moreover, in-depth knowledge of the molecules and pathways that control and regulate the phenotype and functions of hepatic macrophages is critical for developing therapeutic strategies to treat ALD. For example, the functions of Ly-6C studies . Inhibit studies . These t


Exposure to toxic metals poses a serious human health hazard based on ubiquitous environmental presence, the extent of exposure, and the toxicity and disease states associated with exposure. This global health issue warrants accurate and reliable models derived from the risk assessment process to predict disease risk in populations. There has been considerable interest recently in the impact of environmental toxicants such as toxic metals on the epigenome. Epigenetic modifications are alterations to an individual's genome without a change in the DNA sequence, and include, but are not limited to, three commonly studied alterations: DNA methylation, histone modification, and non-coding RNA expression. Given the role of epigenetic alterations in regulating gene and thus protein expression, there is the potential for the integration of toxic metal-induced epigenetic alterations as informative factors in the risk assessment process. In the present review, epigenetic alterations induced by five high priority toxic metals/metalloids are prioritized for analysis and their possible inclusion into the risk assessment process is discussed. The US Environmental Protection Agency (EPA) conducts human health risk assessments for toxic chemicals using a four tier process as part of the Integrated Risk Information System (IRIS) Program EPA, . This foin vivo and in vitro models, the mode of action for a particular agent is investigated . Epigenetic alterations can be induced by environmental stimuli, and much attention has been given to the role of the epigenome in human disease Skinner, ; a role The transcriptome, which is the total transcribed RNA of a cell at a given point in time, is regulated primarily through transcription and mRNA stability and degradation. DNA methylation, histone modifications, and miRNA all regulate the transcriptome through transcriptional processes. The proteome, the total protein of the cell, is the functional mediator between the genome and the cell and is regulated primarily through post-transcriptional regulation of mRNA transcripts Figure . EpigeneDNA methylation is the most well-studied epigenetic alteration. The transfer of a methyl group to cytosine forms 5-methyl cytosine (5-MeC) which occurs preferentially, though not exclusively, on cytosines in CpG dinucleotides. The DNA methyltransferase (DNMT) family mediates the methylation of cytosines through the transfer of a single methyl group from S-adenosine methionine (SAM) to cytosine. At present, there has been no identification of a single enzyme which exclusively demethylates DNA  gene was correlated with prostate cancer  aids in decisions for therapeutic strategies for glioma  proteins. MBD proteins may directly block transcription factor binding or recruit chromatin modifiers that alter the chromatin structure into a more transcriptionally repressive environment  of mRNA. miRNA target the transcripts for degradation or to prevent translation, resulting in decreased target protein expression, though it has been reported that miRNAs may activate translation  contaminating drinking water around the globe. Chronic exposure through contaminated drinking water constitutes a major health concern, and it is estimated that hundreds of millions of individuals are exposed, a large percentage of which are estimated to exceed the World Health Organization (WHO) drinking water limit of 10 ppb  and pentavalent arsenate (iAsV) to trivalent and pentavalent methylated forms. The primary enzyme involved in the required reduction and methylation reactions is arsenic methyltransferase (AS3MT), which utilizes SAM as a methyl donor  promoter has been positively associated with arsenic exposure in individuals from West Bengal, India  and Ras association  domain family member 1 (RASSF1A)        , deleted in bladder cancer 1 (DBC1), G0/G1switch 2 (G0S2), family with sequence similarity 83, member A (FAM83A), keratin 7 (KRT7), zinc finger and SCAN domain containing 12 (ZSCAN12), epiregulin (EREG), and thioesterase superfamily member 4 (THEM4)  and H3 arginine 17 (H3R17) in a dose-dependent manner in human keratinocytes , and the proto-oncogenes C-Jun and C-Fos  gene was analyzed; there was a positive correlation between mRNA levels and H3 acetylation  Substance Priority List . Although exposure differs dramatically based on location, occupation, and diet, the 2009 National Health and Nutrition Examination Study (NHANES), estimates that ~2% of American women of reproductive age exceed the oral RfD for mercury EPA, . This isGSTM1) in women with elevated blood mercury levels (above 2.9 μg/L). However, no statistical correlation was found and the study did not ascertain GSTM1 expression levels  promoter and increasing mercury levels   decreased total histone protein (THP) and was associated with decreased H3K27 mono-methylation  along with decreased histone H3 acetylation in the promoter region of brain derived neurotrophic factor  was associated with increased expression of miR-302b, miR-367, miR-372, miR-196b, and miR-141  , there was an inverse association between lead exposure and methylation of the promoter region of the gene Collagen Type I, Alpha 2 (COL1A2)  , excision repair cross-complementation group 1 (ERCC1), X-ray repair complementing defective repair 1 (XRCC1), and 8-oxoguanine DNA glycosylase (OGG1) , which regulates the steroid hormone cortisol. This increase in expression was associated with DNA hypomethylation of the HSD11B2 gene, suggesting epigenetic mechanisms in cadmium-mediated reproductive toxicity  dehydrogenase 2  promoter were increased compared to untransformed cells, suggesting chronic cadmium exposure may alter transcriptional responses through histone modification  in human bronchial epithelial cells  promoters and the silencing of their expression , which are associated with varying health outcomes. Such biomarkers associated with disease may be used as an additional tool that could inform the risk assessment process.There is the potential that epigenetic alterations can be used as biomarkers of adverse health outcomes, representing a new tool in predicting metal-associated diseases (Mulero-Navarro and Esteller, In the clinical settings, epigenetic biomarkers may be useful as early detection of changes associated with chronic diseases or cancers. For example, epigenetic alterations associated with tumor suppressors or oncogenes may inform an individual's cancer susceptibility (Verma et al., Clearly, additional studies are needed to ascertain metal-associated epigenetic alterations not only at different exposure levels, but also at varied developmental time points. Such information is critical not only to refine dose-response predictions, but to differentiate between epigenetic alterations that naturally differ with respect to age (Bocklandt et al., Epigenetic alternations may be a useful tool in predicting differential gene expression in response to toxic-metal exposure. As mentioned previously, epigenetic alterations can affect gene expression in a number of ways, including histone modifications which influence transcription, expression of miRNAs which regulate mRNA stability and subsequent translation, and modification of CpG methylation patterns leading to altered transcription. Of the three, alteration of DNA methylation patterns is supported by the largest body of evidence. Methylation of CpG sites in the promoter regions of genes is often associated with silencing the expression of that gene (Weber et al., The potential for heritability associated with forms of epigenetic alterations provides novel possibilities in the risk assessment process. Epigenetic modifications can be mitotically inherited, and as a result may be passed on to future generations (Rakyan and Whitelaw, in utero exposure to several toxic metals such as arsenic and cadmium are associated with epigenetic alterations. Epigenetic alterations not only provide the possibility of informing later life health outcomes, but may be useful in predicting adverse health outcomes from a specific early life metal exposure. Inter-individual differences in metabolism may actually be mediated in part by the epigenetic modifications. In certain cases, metal-specific epigenetic alterations may have the possibility of informing researchers of an individual's previous exposure and their specific associated changes in gene expression or health effects.As discussed in the review, In addition, epigenetic alterations to the germ line may provide insight into trans-generational differences in disease patterns or susceptibility (Sasaki and Matsui, While the number of publications on metals-induced epigenetic changes continues to rise, there are gaps in research that need to be filled prior to their incorporation into the risk assessment process. Where data are derived from humans, sample sizes need to be large and data confirmed and replicated cross-cohorts. The data should be collected at different time points during development and/or growth to determine the temporal stability of the changes and establish potential developmental windows of susceptibility. Moreover, additional studies are required to examine the associations between epigenetic alterations, functional cellular consequences and adverse health outcomes through the use of epidemiologic as well as traditional toxicological studies. Such studies require an examination across multiple tissues, at multiple doses, for multiple durations, and at different time periods during development or life stage.The epigenome serves as a link between an individual's genome and their response to environmental cues. As such, the changes to the epigenome can act as both a snapshot of an individual's response to various environmental stressors, as well as a putative predictor of an individual's susceptibility to future stressors and disease outcome. In order to fully understand these relationships it will be necessary to conduct studies that compare both epigenetic data with functional measures such as gene and protein expression within the same study and controlled exposure scenarios. Taken together, the epigenetic data may provide novel contributions to the risk assessment process, but currently much remains unknown about the association between these modifications and functional changes in gene and protein expression.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, we expect other proteins involved in Ca2+ signaling in the mitochondria are affected with CaMKII inhibition. Several outstanding questions remain for CaMKII signaling in heart mitochondria. Most importantly, how does CaMKII, without the recognized N-terminal mitochondrial targeting sequence transfer to mitochondria?CaMKII is a newly discovered resident of mitochondria in the heart. Mitochondrial CaMKII promotes poor outcomes after heart injury from a number of pathological conditions, including myocardial infarction (MI), ischemia reperfusion (IR), and stress from catecholamine stimulation. A study using the inhibitor of CaMKII, CaMKIIN, with expression delimited to myocardial mitochondria, indicates that an underlying cause of heart disease results from the opening of the mitochondrial permeability transition pore (mPTP). Evidence from electrophysiological and other experiments show that CaMKII inhibition likely suppresses mPTP opening by reducing Ca However, a number of other channels have been described for these processes  through induction of both apoptosis and necrosis ,  to measure ion currents, such as the MCU current, across the inner mitochondrial membrane. In order for the patch pipette to access the mitochondrial inner membrane, the mitochondria must be swollen to rupture the outer mitochondrial membrane. These manipulations raise the issue of whether, or how closely, the channel activity observed with patch-clamp corresponds to the 2+ sources . However, specificity of the inhibitor CaMKIIN occurs even in overexpression systems, where for example both CaMKII and PKC could phosphorylate the same consensus site. When each kinase was co-expressed with CaMKIIN, only phosphorylation by CaMKII was inhibited  for CaMKII phosphorylation is shared by a number of other kinases, for example, PKCdelta (2+ homeostasis. CaMKII activity elevates diastolic sarcoplasmic reticulum (SR) Ca2+ leak (Curran et al., 2+ overload (Zhang et al., 2+ homeostasis promotes mitochondrial Ca2+ overload (Lemasters et al., + homeostasis in heart failure (Wagner et al., + accumulation in heart failure was shown to influence mitochondrial Ca2+ load via enhanced NCLX-mediated Ca2+ removal (Maack et al., 2+ levels.CaMKII activity outside of mitochondria contributes to mitochondrial Ca2+ (Miller and Kennedy, The role of CaMKII functioning in the mitochondria in physiology and disease is in the early stages of research and discovery. CaMKII may be central to regulating mitochondrial homeostasis as its activity is regulated by both CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The main input to the basal ganglia, the corticostriatal pathway, shows some of the earliest signs of neuropathology in Huntington’s disease (HD), an inherited neurodegenerative condition that typically strikes in mid-life with progressively deteriorating cognitive, emotional, and motor symptoms. Although an effective treatment remains elusive, research on transgenic animal models has implicated dysregulation of glutamate (Glu), the excitatory amino acid released by corticostriatal neurons, in HD onset. Abnormalities in the control of Glu transmission at the level of postsynaptic receptors and Glu transport proteins play a critical role in the loss of information flow through downstream circuits that set the stage for the HD behavioral phenotype. Parallel but less-well characterized changes in dopamine (DA), a key modulator of Glu activation, ensure further deficits in neuronal communication throughout the basal ganglia. Continued analysis of corticostriatal Glu transmission and its modulation by DA, including analysis at the neurobehavioral level in transgenic models, is likely to be an effective strategy in the pursuit of HD therapeutics. Aberrant function of basal ganglia circuitry plays an important role in multiple neuropathological conditions Reiner, . Here weHD afflicts approximately 1 in 10,000 persons of European descent. Other populations are also affected but at a slightly lower incidence Harper, . The larhuntingtin (htt) gene located on exon 1 of chromosome 4  repeat in the Following clinical diagnosis, usually triggered by motor symptom onset, HD is fatal within 15–20 years. During the initial stages of the disorder, HD patients experience cognitive deficits in mood, attention, procedural memory and executive functions  results mainly from loss of medium spiny neurons (MSNs), which account for >90% of the striatal neuronal population. Striatal neuronal loss is usually accompanied by loss of cortical pyramidal neurons (CPNs), primarily in motor and premotor areas  models. Truncated models solely express the first exon of the The truncated R6 line is the oldest and one of the most studied transgenic mouse models. R6/2 mice, with a CAG repeat length of ~150, have an aggressive phenotype. R6/2 mice have a shortened lifespan of 3–5 months with symptom onset beginning as early as 1 month of age  and bacteria artificial chromosome (BAC). Several lines of the YAC mouse model have been generated based on the number of CAG repeats: YAC18 (control), YAC46, YAC72, and YAC128. Compared to truncated models, YAC and BAC mice have a slower disease progression. As is the case with humans, the YAC128 mouse model displays striatal followed by cortical atrophy as well as hyperkinetic followed by hypokinetic activity in an age-dependent manner  pyramidal tract (PT)-type neurons; and (2) intra-telencephalically projecting (IT)-type neurons. PT-type neurons project to ipsilateral striatum, thalamus, and subthalamic nucleus (STN) as well as the regions of the spinal cord and brain stem. IT-type neurons project to the ipsilateral and contralateral striatum and other cortical layers  play a modulatory role. Evidence suggests D1-like receptor signaling facilitates glutamatergic signaling in direct pathway, striatonigral MSNs. In contrast, D2-like receptor activation inhibits MSNs in the indirect pathway Figure . TherefoMotor function is in part shaped by CPNs projecting to striatal MSNs in the so-called corticostriatal pathway. The cerebral cortex and striatum are the most affected brain areas in HD, with massive MSN loss in the striatum. Not all MSNs however are equally vulnerable . The dysfunction of the direct pathway in the later stage of the disease would result in an inability to facilitate movement; resulting in a rigidity and bradykinesia (see above). Thus changes in output from striatal MSNs, which receives Glu input from the cortex, correlates with the behavioral phenotype of HD.Particular focus has been placed on the neuronal activity of the cortex and striatum. Regardless of genetic construct and phenotypic onset, changes ranging from membrane properties to impaired neuronal processing in the corticostriatal pathway have been identified in multiple mouse models of HD  in HOM mice . Spectral analysis of LFPs recorded from the striatum of freely behaving R6/2 mice revealed an increase in power in theta (7–14 Hz) and gamma (35–45 Hz) bands , and excitatory amino-acid carrier 1 (EAAC1). Note GLT1 is also known as in EAAT2 in humans and is primarily located on glial cells. Glu transporters play a vital role in maintaining the level of extracellular Glu by removing Glu from the synapse; dysregulation of this Glu uptake can result in slow Glu clearance prompting Glu spillover and increased receptor activation  mediated by Glu. The evoked EPSCs are decreased in Q175 mice, indicating dysregulation of Glu transmission in this model  administration  in either D1 or D2 receptors have been studied. Using electrophysiology with these mice a DA receptor-specific modulation of EPSC and NMDA and AMPA currents was found. D1 receptor agonist increased EPSC in D1 MSNs projecting to the direct pathway whereas D2 receptor agonist decreased EPSCs in D2 cells of the indirect pathway. Changes in NMDA and AMPA current were also DA receptor-specific. A D1 agonist resulted in increased current in direct pathway MSNs, and a D2 receptor agonist resulted in decreased NMDA and AMPA current in the indirect pathway MSNs . Electrophysiological data from mice expressing GFP now suggest the direct pathway is affected in the early stages of HD via D1 receptor activation. More than likely it is a combination of D1 receptor activation in the direct pathway in conjunction with decreased function of the indirect pathway. Since both theories would result in increased GABAergic output to the direct pathway, the ultimate result is an exacerbated HD phenotype.HD is a multifaceted disorder displaying cognitive, psychiatric, and motor deficits. The dysregulation in the corticostriatal pathway contributes to these associated characteristics of HD and potentially underlie the HD phenotype. Dysregulation of Glu and DA have been documented in HD patients as well as rodent models, and the abnormal electrophysiology, due to these neurotransmitters, further indicates that disrupted communication in the corticostriatal pathway in HD.Both authors contributed to this review article. KDB: prepared a draft, which was updated and edited by GVR.We gratefully acknowledge support from the CHDI Foundation (Grants A-7449).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AbstractIn the treatment of intracanal and periodontal infections, the local application of antibiotics and other therapeutic agents in the root canal or in periodontal pockets may be a promising approach to achieve sustained/controlled drug release, high antimicrobial activity and low systemic side effects. The conventional method for the elimination of subgingival microbial infection includes mechanical debridement, irrigation with antimicrobial agents or surgical access. But, the effectiveness of conventional nonsurgical treatment is limited by lack of accessibility to bacteria in deeper periodontal pockets, and/or does not completely eliminate intracanal microorganisms. Surgical intervention may be beneficial but cannot be done in all cases, medically compromised cases and also in patients not willing to be subjected to surgical therapy. Development of local drug delivery systems provides an answer to all such difficulties. This comprehensive review tries to cover the detailed information about the latest advances in the various local drug delivery systems, their indications, contraindications and their advantages over systemic drug therapy. Periodontal diseases are bacterial infections characterized by inflammation and destruction of the attachment apparatus, often leading to tooth loss. The most common forms of periodontal diseases, gingivitis and periodontitis are caused by bacteria adjacent to or associated with periodontal structures. These bacteria, along with calculus and other local factors, are the principal components that perpetuate the disease process . It is important to keep the pathogenic microflora of the pocket suppressed in order to maintain health of the periodontal tissues . Modalities of treatment may be a non surgical approach or a surgical approach [4].Plaque constitutes highly organized bacterial populations. Elevated proportions of some subgingival microbial species have been associated with destructive periodontal disease activity. Potential periodontal pathogens include Aggregtibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythus, Peptostreptococcous micros, Campylobacter rectus, Eiknella corrodens, Fusobacterium nucleatum, Eubacterium species, Treponema denticola, Beta-hemolytic streptococci, a variety of enteric rods and pseudomonas, Enterococci, Staphylococci and possibly yeasts [6].Debridement of the root surface by scaling and root planing came into a relatively common use in the first half of the past century and has become the central feature held in common by all currently used forms of periodontal therapy . Endodontic biofilms represent a common cause of persistent infections in the root canal because of their resistance to antimicrobial compounds [10]. It is of particular concern that resistant microbial species such as Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Streptococcus species, Actinomyces species and Candida species, among others may remain in root canals .Scaling and root planing has been effective in immediately decreasing the microbial load but recolonization of the same can occur as early as 60 days after scaling and root planing. Hence, chemical therapy has been advocated to inhibit pathogenic microflora and encourage inhibition of non pathogenic microflora [13].Periodontal and endodontic therapies can be grouped into three broad categories: mechanical debridement and cleaning to eliminate bacteria; treatment aimed at killing or affecting the metabolism of the infectious organism, such as antiseptics and antibiotics, and treatment that affect the environment of the infectious organism [14].Although the antimicrobial efficacy of conventional irrigants is generally recognized, they may produce some complications such as tissue irritation or hypersensitivity .Systemic administration has been useful in treating periodontal pockets, but it involves a relatively high dose with repeated intake over a prolonged period of time to achieve the required inhibitory concentrations in the sulcular fluid. This increases the chances of development of resistance, of alteration of commensal flora and of increased potential for adverse effects like allergic/ anaphylactic reaction, gastric disturbances, superinfection, nausea, vomiting, etc. Based on duration of action (Greenstein & Tonetti 2000) [ A) sustained release devices−Drug delivery for less than 24 hrs−require multiple applications−follow first order kineticsB) controlled delivery devices−duration of drug release exceeds 24 hrs−administered once−follow zero order kineticsCharacteristics of locally delivered antimicrobials17].Before any antimicrobial agent can be recommended for periodontal or intracanal a number of basic and important conditions have to be fulfilled  .• Hypersensitivity to conventional intracanal irrigants like sodium hypochlorite .• Delivery of antimicrobials using ultrasonic devices is contraindicated in asthmatics, infective conditions such as AIDS, tuberculosis and those with cardiac pacemakers [Recent local drug delivery agents and periodontal diseases20]. A multidisciplinary approach to the delivery of therapeutics to target tissues is needed for a precise control of pharmacokinetics and pharmacodynamics of the drug to ensure adequate concentration at the targeted site.Antimicrobial or antiseptic agents are applied locally for the treatment of periodontitis in combination with mechanical root debridement. Subgingival irrigation with drug solutions provides high concentrations in the periodontal pocket for only short periods. In consequence, repeated oral irrigation is required to exert bactericidal or bacteriostatic effects. The mode by which a drug is delivered to a specific site of action has a significant role on its efficacy. The use of controlled drug release devices may well improve the antimicrobial efficacy in the periodontal pocket, with consequent clinical benefits. The efficacy of drug delivery systems is mainly affected by the biological environment and the properties of the polymer and the drug [21]. The mode of delivery primarily controls the drug’s success and failure, as the choice of a drug is often influenced by the way it is administered. Various drug delivery devices for intrapocket therapy include:Potential release mechanisms involved for controlled drug release can be: (i) desorption of surface-bound/adsorbed drugs; (ii) diffusion through the carrier matrix; (iii) diffusion (in the case of nanocapsules) through the carrier wall; (iv) carrier matrix erosion; and (v) a combined erosion /diffusion process [22]. Whereas, to decrease the speed of releasing the drug, the monolithic fibers were developed by impregnating drug into the molten polymers, spinning it on high temperature followed by a quick cooling [23]. Aimetti et al [24] evaluated the clinical, radiological and microbiological response to the local delivery of tetracycline fibers along with scaling and root planing at sites with persistent periodontal lesions and obtained the greatest advantage in the treatment of up to 12 months following treatment.1) Fibers- Fibers are thread – like devices placed in periodontal pocket and secured with periodontal dressing, ensuring a sustained release of drug at the site. Fibers used in periodontal disease are of two types – hollow and monolithic fibers. Hollow fibers contain a reservoir of drug, which is released by a simple diffusion through reservoir walls [25]. Periochip is the controlled subgingival delivery of chlorhexidine, and was developed by Perio Products Ltd, Jerusalem, Israel. This is an orange brown rectangular chip, rounded at one end. It measures 5 mm x 4 mm x 0.3 mm, weighs about 7.4 mg and contains 2.5 mg of chlorhexidine gluconate, which is incorporated in a biodegradable matrix of hydrolyzed gelatin cross linked with glutaraldehyde. Paolantonio et al [26] conducted a study to evaluate clinical and microbiologic effects of chlorhexidine chips when used as an adjunct to scaling and root planing. The adjunctive use of chlorhexidine chip resulted in significant probing depth reduction and a clinical attachment level gain compared to scaling and root planing alone. The results were concomitant with a significant benefit of scaling and root planing plus chlorhexidine treatment on subgingival microflora. In an in vitro study, Perugini et al [27] showed that the composite micro material films made of three layers of polymers (chitosan/ PLGA /chitosan), compared to the monolayer films, represent a suitable dosage form to prolong ipriflavone release for 20 days. Jeffcoat et al [29] showed a significant change in probing depth and clinical attachment levels after the placement of a biodegradable chlorhexidine gelatin chip in the treatment of adult periodontitis. Natural polymer like gelatin, [28] chitosan,  collagen [26] and synthetic polymers like polyvinyl alcohol (PVA), poly  (PLGA), [30] are commonly used to synthesize films.2) Films- Films are implantable matrix delivery devices with encapsulation of drug, in a manner that they are distributed throughout the polymer and control release occurs by drug diffusion and/or matrix dissolution or erosion. Films of various polymers have been made for the controlled release of therapeutic agents. The release action depends on the type of polymer used to manufacture the chip. The ease of insertion with minimal pain, control on dosage, dimension and shape of the films make them an ideal device to be used in periodontal pocket. The thickness of the film should not exceed 400 μm as well as it should have sufficient adhesiveness to ensure no interference in maintaining oral hygiene habits. Films that release drugs by diffusion alone are prepared by using water-insoluble non-degradable polymers, whereas those that are released by diffusion and matrix erosion or dissolution use soluble or biodegradable polymers [18] demonstrated a significant reduction in the clinical and microbiological parameters after using elyzol (metronidazole gel) and atridox (doxycycline gel). Perinetti et al [32] compared the clinical healing and the microbiological findings following repeated intrasulcular applications of 1% metronidazole or 1% chlorhexidine gels in persistent periodontal pockets previously treated by scaling and root planing and obtained a similar reduction with both gels at 1% concentration.3) Gels- Gels are injectable semi solid devices containing an adequate concentration of drug and delivered at specific site. They are easy to prepare and administer. They further possess a property of bioadhesivity that enhances the retention time in the periodontal pocket. Salvi et al [33]. Alendronate gel has been found to increase bone formation on local delivery into the periodontal pocket. In patients with type 2 diabetes mellitus and chronic periodontitis, local delivery of 1% alendronate gel into periodontal pockets resulted in a significant increase in the probing depth reduction, clinical attachment level gain, and improved bone fill compared to placebo gel as an adjunct to scaling and root planning [34].Novel local drug delivery agents used for the treatment of periodontal diseases are alendronate and simvastatin. Alendronate (4-amino 1-hydroxybutylidine bisphosphonate), a novel bisphosphonate is a very potent inhibitor of bone resorption. The net effect of alendronate on bone formation might be explained by its inhibition of osteoclasts, thus affecting bone maturation and remodeling. Once taken up by bone, alendronate has a prolonged skeletal retention  and significant amounts can be released in the resorptive process which may in turn provide protection to the alveolar bone [35] showed a greater decrease in gingival index and probing depth and a clinical attachment level gain with significant defect fill at sites treated with scaling and root planing plus locally delivered SMV gel in patients with chronic periodontitis.Simvastatin (SMV) is a specific competitive inhibitor of 3-hydroxy-2-methyl-glutaryl coenzyme-A reductase. Pardeep et al [36] compared and evaluated the clinical effects of topical subgingival application following scaling and root planing of a new biodegradable xanthan based chlorhexidine gel as compared with a gel containing chlorhexidine and metronidazole and scaling and root planing alone. Xanthan based chlorhexidine gel showed the greatest improvements in clinical parameters and a maximum reduction in occurrence of P. gingivalis and an almost similar reduction for Aggregatibacter actinomycetemcomitans and fusobacterium species.Bhardawaj et al [37] conducted a study to evaluate the effect of a controlled release device containing minocycline microspheres (Arestin TM) on the treatment of chronic periodontitis. The results of this study concluded that treatment with scaling and root planing plus minocycline microspheres (Arestin™) is more effective and safer than scaling and root planing alone in reducing the signs of chronic periodontitis.4) Microparticulate system- This system consists of an encapsulation of the drug into a polymer, which dissolves gradually releasing the drug at the target site. It is a highly stable system for the delivery of an optimum concentration of the drug in the pocket. Gopinath et al [22]. Nanosizing of drugs can lead to a dramatic increase in their absorption and bioavailability leading to a subsequent reduction in drug dose [38]. Owing to its small size, it acquires a high dispersibility in an aqueous medium and controlled release rate. The polymer-based nanoparticles were prepared via micellar polymerization, resulting in powder material with particle size in the range of 50–180 nm [20]. In an attempt to obtain a novel delivery system adequate for the treatment of periodontal disease, triclosan-loaded polymeric  nanoparticles were prepared by emulsification–diffusion process. A preliminary in vivo study in dogs with induced periodontal defects suggested that triclosan-loaded nanoparticles penetrate through the junctional epithelium [30]. These systems reduce the frequency of administration and further provide a uniform distribution of the active agent over an extended period of time [22].5) Nanoparticulate system- Modern drug delivery systems are designed for targeted controlled slow drug release. Up to now, polymer or microparticle-based hydrogels have been applied in dentistry, which can affect the rate of release because of their structure. Recently, intensive research has been performed all over the world to improve the effectiveness of delivery systems. The nanoparticulate system provides several advantages as compared with microspheres, microparticles and emulsion-based delivery systems, including high dispersibility in an aqueous medium, controlled release rate and increased stability. Due to their small size, nanoparticles penetrate regions that may be inaccessible to other delivery systems, such as the deep periodontal pockets. These systems reduce the frequency of administration and further provide a uniform distribution of the active agent over an extended period of time [Recent Intracanal local drug delivery systems39] conducted a study in which chlorhexidine-loaded devices were prepared with ethyl cellulose, to form a needle-like device suitable to be inserted in the root canal. Chitosan, PLGA and polymethylmethacrylate have been used as coatings for absorbent paper to examine the controlled release of chlorhexidine digluconate loaded-paper points [40].Intracanal local drug delivery agents usually consist of antibiotics to eliminate bacterial infection and non steroidal anti inflammatory drugs to reduce/eliminate post operative pain, after mechanical cleaning has been done i.e. post bio-mechanical preparation (BMP). Many systems of different shapes  have been proposed for placement in the root canal as a temporary dressing for local drug delivery and healing. Huang et al [41] developed amoxicillin-loaded microparticles (5–38 μm) by spray-drying. The antimicrobial activity of amoxicillin was preserved when it was encapsulated, and in vitro antimicrobial activity studies demonstrated that the MIC 90 against E. faecalis was achieved over 6 days.1) Microparticulate systems- Novel formulations based on microparticles, which appear promising for the accessibility to the conventionally inaccessible parts of the root canal which have been developed. Sousa et al [42] used a combination of light and PLGA nanoparticles (150–200 nm) loaded with the photoactive drug methylene blue to treat infections caused by E. faecalis. Chitosan nanoparticles were also found to display a high degree of antibacterial activity against E. faecalis in in-vitro culture, and good compatibility with the endodontic sealer [43].2) Nanoparticulate systems- Use of nanoparticles as local intracanal drug delivery system is also gaining popularity nowadays. Pagonis et al [The management of periodontal diseases and endodontic infections has traditionally focused on the use of anti-infective irrigating solutions and the use of mechanical procedures to eliminate infectious tissue and to hinder disease progression. Several sustained and controlled-release antimicrobials have been developed and tested. Novel drug-loaded microspheres or nanoparticulate systems can be considered as good carriers for the controlled release of several active substances/ antimicrobial agents suited to such specific sites as periodontal pockets or dental cavities. Most have shown positive clinical and microbiological effects, but increased the cost of therapy. The benefits like improved patient compliance and pharmacokinetic response, easy access to the diseased site, and lower drug dosages may overcome the cost factor. Site specific administration of simple injectable formulations of micro/nano particles in the periodontal pocket or in the root canal cavity would reduce the number of treatment sessions for patients, and may serve as an adjuvant to surgical protocols offering a means of saving teeth which is the ultimate goal of any dental treatment. Sources of Funding- NoneDisclosures- None


CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF  as well as variola virus and vaccinia virus . The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested . Filoviridae [Ebolavirus and Marburgvirus], Arenaviridae [Lassa fever virus (LASV)], Bunyaviridae [Rift Valley fever virus (RVFV) and Crimean Congo hemorrhagic fever virus (CCHFV)], and Flaviviridae . Most are zoonotic, vector-borne, and may cause sporadic, unanticipated, and devastating outbreaks in endemic areas  (n = 4), MARV (n = 3), CCHFV (n = 3), RVFV (n = 1) and LASV (n = 1) were obtained from the United States Army Medical Research Institute of Infectious Diseases, Ft. Detrick, Maryland. Genomic DNA from VACV virus (n = 6) was obtained from NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH. The two target amplicons of VARV  representing all VARV sequence variants for these genetic regions  were obt2 and YFVAs previously described, culture supernatants from standard isolates of the four serotypes of DENV were employed . The folFiloviridae, Flaviviridae and Poxviridae) so as to achieve maximum strain coverage with the least number of primers. Primer sets were designed to simultaneously amplify two different regions in each virus or viral group: NP and L genes of ebolaviruses  and MARV, S segment of CCHFV, M and S segments of RVFV, L segment of LASV, NS5 regions of DEN and YF, and the RNA pol (RAP94 and RPO147) genes of VACV and VARV; a total of 57 primers. The amplicons were ~500 bp in length (range 399–685 bp). The primer sequences contained no more than three degenerate positions and had a melting temperature of around 72–75°C  were introduced, where required, to account for sequence variations. A complete list of all LDR primers is provided in Orthopoxvirus species (cowpox and monkeypox viruses). The flavivirus primers were designed to identify and distinguish DENV and YFV without cross-reactivity with a panel of flaviruses.The PCR and LDR primers for each of the target regions for all the virus groups were evaluated in separate assays individually. This was performed to evaluate satisfactory signal detection from each of the regions selected. Primers that failed to produce either PCR amplicon or generated less than two LDR products were replaced, and new primers were designed. The LDR primers were designed such that they were able to differentiate between the three species of ebolavirus tested . The poxvirus primers were designed such that they were able to discriminate between VARV and VACV and would not cross-react or produce false positive signals with other Nucleic acid extraction and one-step RT-PCR amplification , ligase detection reaction (LDR), and universal array were performed as described previously , 38, witSudan ebolavirus for which there is a single experiment. We included the following geographic and genotypic variants: four different strains of three species of ebolavirus ; standard strains of DENV serotypes 1–4; VARV ; and CCHFV .The analyses were repeated in at least 2 different experiments with the exception of in vitro transcribed using the mMessage in vitro transcription kit . Following DNAse treatment, the synthetic RNA was purified using an RNeasy column . The quantity of RNA generated for each transcript was determined using the Ribogreen® RNA Quantitation Kit  following the manufacturer’s instructions.Synthetic RNA fragments of the viruses were prepared for LASV, RVFV, and YFV, MARV and CCHFV . The tar10 to 1x100 copies/ml) were used in the PCR reaction to determine the limit of detection. Second, the quantified VACV virus DNA stocks obtained from BEI Resources were serially diluted in nuclease-free water and 5 μl of each of the DNA dilutions was used in the PCR reaction to determine limit of detection. Third, serial dilutions (ten-fold) of viral culture stocks were prepared for EBOV (Zaire ebolavirus ‘95) from inactivated standard stock cultures. Dilutions were prepared in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum . RNA was extracted from 140 μl of each dilution, and RT-PCR-LDR/universal array was used to determine the analytical sensitivity of the assay.The limit of detection (LOD) of the different viruses was determined in the following manner. First, 10-fold serial dilutions of the synthetically prepared, previously quantified, RNA transcripts of LASV, RVFV, YFV, MARV and CCHFV .4 genome equivalents/reaction.The limits of detection using either synthetically transcribed RNA or dilutions of whole virus are shown in We describe a multiplex detection assay that can simultaneously detect and differentiate 7 VHF viruses, VARV and VACV, in a single assay and is amenable to adaptation to a high-throughput format. The PCR/LDR/universal array was validated on 53 different samples including viral culture filtrates, vaccine strain of viruses, serum specimens from patients with DENV infection, nucleic acid from VACV viruses and PCR amplicons <500bp in size of VARV. Since the viruses that cause the VHF syndrome include variant viral species with diverse geographical distributions, we attempted to include viruses from several geographic areas and viral species when applicable. The assay has some built-in redundancy, by including 6 possible signals for each virus (only 2 of which are required for identification), to account for the genetic diversity often encountered in RNA viruses. We encountered signal drop-out in some of the viral species but the assay was able to detect all viruses included in the panel. Additionally, the number of signals detected, or the fluorescent intensity of the signals generated, was robust for all samples tested.Early stages of infection for most of these viruses are relatively non-specific. For some viruses, such as RFV and Lassa, the classical hemorrhagic manifestations may be absent, making accurate and timely diagnosis challenging. A multiplexed detection assay such as the PCR/LDR has a potential benefit in being able to screen for several pathogens in an endemic area or in symptomatic returning travelers. In fact, there are several multiplex assays that have been developed in the recent years to address this issue . The anaSudan ebolavirus and Zaire ebolavirus, have been consistently recognized as causing large outbreaks since 1976 with the most recent in 2014 [Tai Forest ebolavirus (formerly Cote d’Ivoire), Reston and Bundibugyo ebolaviruses have occurred less frequently [Tai Forest ebolavirus has been implicated in a single human infection acquired during the autopsy of a wild chimpanzee [Reston ebolavirus causes VHF and death in primates and illness in pigs [Reston ebolavirus is thought to be non-pathogenic in humans, the WHO has cautioned that the effect of the infection in the immunosuppressed, pregnant women, and children is unknown [Bundibugyo ebolavirus is known to be pathogenic. The outbreak in Uganda (2007–8) resulted in 100 cases with a fatality rate of ~40% [Zaire ebolavirus as well as the isolates of Sudan and Reston ebolaviruses tested  and Sierra Leone (KM233116), and isolates of Bundibugyo ebolavirus (NC_014373), and Tai Forest ebolavirus (FJ217162). Zaire ebolavirus 1976 (KC242801) is included to show the original alignment used for assay design. Nucleotides in red font indicate mismatches. Only relevant areas of the DNA sequence with primer binding sites are shown. Numbers indicate nucleotide positions in GenBank.Amplicon 1 and 2 PCR primer alignments of the NP and L genes of 2014 (PDF)Click here for additional data file.S2 FigZaire ebolavirus isolates from Guinea (KJ660348) and Sierra Leone (KM233116). Zaire ebolavirus 1976 (KC242801) is included to show the original alignment used for assay design. Nucleotides in red font indicate mismatches. Yellow indicates the single base variant at the junction ligation site. The terminal nucleotide “T” at the 3’end highlighted in green indicates an addition to prevent quenching of fluorescence of Cy-3 dye by the adjacent guanine bases. Only relevant areas of the DNA sequence with primer binding sites are shown. Numbers indicate nucleotide positions in GenBank.Amplicon 1 and 2 LDR ligation primer alignments and ligation sites of the NP and L genes of 2014 (PDF)Click here for additional data file.S1 TableMultiple primers were designed for each amplicon for the different viruses and are numbered; multiple primers for the same virus were required to accommodate sequence variations and these are then differentiated by the letters a, b, c and so forth. The underlined nucleotides indicate the sequence of the universal tails.(PDF)Click here for additional data file.S2 TablePrimer names indicate the virus detected as well as the amplicon on which the LDR primer pairs were designed. For example, the first primer was designed to detect RESTV and was designed on a nucleotide position on amplicon 1. When more than one primer pair was designed for a particular virus, they were indicated with different numbers 1,2,3 etc. When multiple primers were designed at any LDR position an alphabetical suffix is included . An amino blocking group (Blk) was used at the 5’end of the upstream primers and downstream primers were labeled with Cy-3 at the 3’ end. The first 20 nucleotides of the upstream primer (underlined) are the zip-code complements.(PDF)Click here for additional data file.


New insights into the natural roles of these transporters are described and discussed, focusing on the knowledge obtained or suggested by post-genomic research. The new information reviewed here provides clues into the unexpectedly complex roles of these transporters, including a proposed indirect regulation of the stress response machinery and control of membrane potential and/or internal pH, with a special emphasis on a genome-wide view of the regulation and evolution of MDR/MXR-MFS transporters.Multidrug/Multixenobiotic resistance (MDR/MXR) is a widespread phenomenon with clinical, agricultural and biotechnological implications, where MDR/MXR transporters that are presumably able to catalyze the efflux of multiple cytotoxic compounds play a key role in the acquisition of resistance. However, although these proteins have been traditionally considered drug exporters, the physiological function of MDR/MXR transporters and the exact mechanism of their involvement in resistance to cytotoxic compounds are still open to debate. In fact, the wide range of structurally and functionally unrelated substrates that these transporters are presumably able to export has puzzled researchers for years. The discussion has now shifted toward the possibility of at least some MDR/MXR transporters exerting their effect as the result of a natural physiological role in the cell, rather than through the direct export of cytotoxic compounds, while the hypothesis that MDR/MXR transporters may have evolved in nature for other purposes than conferring chemoprotection has been gaining momentum in recent years. This review focuses on the drug transporters of the Major Facilitator Superfamily (MFS; drug:H All living cells exhibit elaborate molecular mechanisms to protect themselves from external assault, such as the exposure to drugs and other cytotoxic compounds. The simultaneous acquisition of resistance to a wide range of structurally and functionally unrelated cytotoxic chemicals has become a widespread phenomenon in nature known as Multidrug/Multixenobiotic resistance (MDR/MXR)  and confer MDR/MXR resistance in bacteria, fungi and more complex eukaryotes.Although drug resistance might be mediated by various mechanisms, membrane transporters that presumably catalyze the efflux of a broad range of cytotoxic substrates play an important role in MDR/MXR processes  to guide the development of new prophylactic, diagnosis, and therapeutic approaches to the rising number of drug resistant fungal infections  approaches has gained considerable momentum in recent years. Remarkably, the identification of alterations occurring in gene or protein expression following exposure to a drug or xenobiotic compound can contribute to identify the cellular pathways that are most relevant to the stress response, including MDR/MXR transporters and how their complex regulation and putative physiological roles may also influence drug resistance. Genomic/proteomic variations can be identified through transcriptomics and quantitative proteomics. Expression proteomic approaches are also important to identify alterations at the level of post-translational modifications associated with exposure to xenobiotic and cytotoxic compounds. Another very important tool in yeast research are gene knockout collections. Chemogenomic analyses to identify determinants of resistance to a particular compound in yeast can be performed using homozygous , haploinsufficiency  and multicopy- and overexpression (gene dosage > 100%) collections. Many MDR/MXR transporters are directly linked to the ability of cells to grow in the presence of a particular stressing condition, and most of the compounds to which these transporters provide protection have been identified through the assessment of the fitness phenotypes of the corresponding deletion mutants under chemical stress. In fact, under control conditions these growth phenotypes are not detectable and would not have been identified. Furthermore, in several cases the presence of the gene is required for growth under a given stress, and yet there are no alterations at the expression or protein content level, meaning that many of these conditions would not have been discovered using expression-based methodologies such as DNA microarrays or proteomics. However, the identification of an MDR/MXR transporter-encoding gene as a determinant of resistance does not necessarily mean that the gene product is involved directly in the efflux of that compound from the cell. As discussed in Section The MDR/MXR Phenomenon, some of these transporters might have natural physiological roles that indirectly enable chemoprotection, for example through the alteration of plasma membrane potential and/or lipid composition. For this reason, lipidomic analyses can also be of particular interest, given the role of sphingolipids and ceramides as signaling mediators in a growing number of pathways and in lipid-based signaling in the yeast response to stress   and the 14-spanner drug:H+ antiporter family 2 (DHA2)  might also provide an explanation for the involvement of Qdr2 in the oxidative stress response  and mono- and divalent cations , ARN1, ARN2, SIT, and ENB1 (ARN subfamily), GEX1 and GEX2 (GEX subfamily)  Table . Unlike y) Table . Among ty) Table . ConversVBA5 was associated with MDR, its overexpression leading to increased susceptibility to 4-NQO and quinidine  have been obtained, based on the increasing abundance of available genome sequences, which may help to understand several of the issues raised regarding the action of such transporters , with the identification of 10 main gene lineages  was also reconstructed  further hints at a more complex involvement of these MDR/MXR-MFS transporters in chemoprotection.S. cerevisiae, mostly coming from genome-wide experiments. This information, gathered in the YEASTRACT database (www.yeastract.com)  whose expression and/or localization was found to change in response to MMS treatment leading to replicative stress , Msn2 (71%), Msn4 (64%), and Yap1 (61%) regulating more than 60% of the DHA1/DAG genes Figure . Msn2 anAll but one of the DHA1/DAG genes are also regulated by nutrient availability transcription factors. Thirteen transcription factors involved in the response to amino acid availability have been found to regulate 70% of the DHA1/DAG genes. Among these transcription factors, Gcn4 and Bas1 stand out as the two that regulate the majority (more than 60%) of these genes Figure . Gcn4 isqdr3 null mutants under glucose deprivation , Hap2 (36%), and Tye7 (32%). Mig3 plays a major role in carbon catabolite repression and ethanol response, but is also involved in the response to toxic agents, while Hap2 is one of the key regulators of respiration and Tye7 is an activator of glycolytic gene expression. Among the 27 DHA genes regulated by carbon source related transcription factors, eight are regulated by five or more of these transcription factors, including Atr1 (5 TFs), Vba4 (5 TFs), Arn3 (6 TFs), Arn4 (5 TFs), Ymr279c (7 TFs), Hol1 (5 TFs), Tpo1 (5 TFs), and Aqr1 (12 TFs) Figure . Very liARN2, TPO1, and TPO4 was registered in cells growing on oleate as carbon source  or direct  evidences, and the number of independent studies supporting each regulatory interaction can vary greatly. Here we have opted to show all interactions described for each TF-target gene considered, although we distinguish between expression evidence and DNA-binding evidence  show that it is at least partially similar to that in S. cerevisiae .Further characterization of drug resistance MFS proteins in pathogenic yeasts or phytopathogenic fungi will benefit from studies in S. cerevisiae to higher eukaryotes, Tpo1 homologs encoding putative plasma membrane MFS transporters from the plant model Arabidopsis thaliana were analyzed to uncover new roles for these transporters in plant abiotic stress tolerance (Cabrito et al., ZIFL1 transcription levels were found to be elevated in plants treated with the herbicide 2,4-D, and heterologous expression in yeast complemented the sensitivity phenotype in tpo1 null mutant cells stressed with 2,4-D (Cabrito et al., S. cerevisiae and A. thaliana, to uncover novel roles for plant MFS transporters, which may open new avenues for the development of efficient strategies to improve crop productivity or decontaminate polluted soils.The knowledge gathered in yeast MDR/MXR-MFS transporters can also be extrapolated to other systems and less accessible higher eukaryotes such as plants. Abiotic stress is an important contributor to loss of plant productivity worldwide under agricultural conditions. The clarification of the mechanisms underlying MDR/MXR in plants will be instrumental in paving the way for the development of efficient strategies to improve crop tolerance to abiotic stress, in particular regarding the potential role of MDR/MXR transporters in the plant response to heavy-metal contamination, high salinity, pesticides, and other xenobiotic compounds. Considerable progress has been made on the characterization of plant ABC transporters, which have been shown to play a pivotal role in numerous physiological processes (van den Brule and Smart, S. cerevisiae has significantly increased our knowledge on the evolution, physiology and regulation of MDR/MXR-MFS transporters, contributing to elucidate the mystery of the redundancy and promiscuity of these transporters and their physiological relevance. In the near future, more specialized global approaches such as metabolomics and lipidomics are expected to expand on the recent findings here reviewed, in particular regarding the possible indirect role of these transporters in the regulation of the stress response, either through metabolic regulation, alteration of the plasma membrane composition, or other mechanisms. The exploitation of high-throughput technologies and the knowledge gathered in yeast can prove invaluable to guide studies in pathogenic fungi, to develop more robust yeast strains for biotechnological applications, and to improve crop protection, with promising repercussions from medicine to biotechnology and agriculture.In recent years, the use of genome-wide approaches in the model yeast The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Central neural circuits orchestrate the behavioral and autonomic repertoire that maintains body temperature during environmental temperature challenges and alters body temperature during the inflammatory response and behavioral states and in response to declining energy homeostasis. This review summarizes the central nervous system circuit mechanisms controlling the principal thermoeffectors for body temperature regulation: cutaneous vasoconstriction regulating heat loss and shivering and brown adipose tissue for thermogenesis. The activation of these thermoeffectors is regulated by parallel but distinct efferent pathways within the central nervous system that share a common peripheral thermal sensory input. The model for the neural circuit mechanism underlying central thermoregulatory control provides a useful platform for further understanding of the functional organization of central thermoregulation, for elucidating the hypothalamic circuitry and neurotransmitters involved in body temperature regulation, and for the discovery of novel therapeutic approaches to modulating body temperature and energy homeostasis. Life-threatening protein denaturation looms as Tcore increases, and reductions in membrane fluidity, ion fluxes, and enzyme performance accompany significant reductions in Tcore. The fundamental central neural circuits for thermoregulation orchestrate behavioral and autonomic repertoires that maintain Tcore during thermal challenges, sensed by thermal receptors, that arise from either the ambient or the internal  environment. A variety of other neural circuits and neurochemical modulators impinge on the fundamental thermoregulatory pathways to produce the alterations in Tcore that occur with circadian and ultradian periodicities2 and those that accompany many behavioral states, such as fever during sickness, increased Tcore during psychological stress and at ovulation, and reductions in Tcore during sleep, sepsis, or exposure to metabolic distress . Recent research in the central nervous system (CNS) control of Tcore focuses principally on: (a) elaborating the CNS pathways and neurotransmitter systems involved in the fundamental central thermoregulatory network; (b) the modulation of activity within the fundamental central thermoregulatory network by non-thermal factors and behavioral states; and (c) pharmacological manipulation of the CNS thermoregulatory network for a variety of therapeutic goals .Body temperature  and central  thermoreceptors that provides a consolidated assessment of Tcore and, importantly, of imminent threats to Tcore. The primary thermoeffector mechanisms recruited for both cold defense and centrally driven hyperthermias  include: (a) thermoregulatory behaviors to reduce the loss of heat produced during basal metabolism; (b) cutaneous vasoconstriction (CVC) to conserve heat in the body core and limit heat loss to the environment; and (c) heat production (thermogenesis). The principal sources of metabolic heat production, beyond those contributing to basal metabolic rate , are brown adipose tissue (BAT), whose sympathetic neural input fuels mitochondria that shunt proton fluxes into heat production, and shivering behavior in skeletal muscle, dependent on the inefficiency of ATP utilization to generate heat. Effector mechanisms for heat defense include: (a) thermoregulatory behavior to increase heat loss; (b) cutaneous vasodilation, which in humans includes a sympathetic vasodilator outflow6, combined with increased cardiac output7 and visceral vasoconstriction10 to facilitate core heat loss from the body surface; and (c) evaporative cooling .In its simplest form, the fundamental thermoregulatory network can be modeled as a reflex15. Along these same lines, most research subjects are furry mammals that are strongly dependent on non-sweating mechanisms for evaporative cooling. Although this has limited the available detail on the CNS pathways regulating human sweating17, considerable insight has been gained on the CNS regulation of other mechanisms of evaporative cooling, such as panting20. Additionally, since most of the basic neuroscience of CNS thermoregulatory pathways has been derived from experiments in rodents, including the exclusivelyin vitro studies of neurons with intrinsic thermosensitivity, the translation of the conclusions, including the circuit model inApart from important “first responders”, the CNS pathways controlling and mediating thermoregulatory behaviors remain incompletely defined27 and of CVC-promoting neurons in the median preoptic nucleus (MnPO)29.The CNS thermoregulatory control of the sympathetic outflows mediating CVC and BAT thermogenesis and of the somatic motoneurons producing shivering is effected through parallel but distinct, effector-specific, integrative/efferent circuits –25 that31 and a glutamatergic excitation32; the latter inputs, potentially arising from neurons in the MnPO that project to the DMH, are synaptically connected to BAT33 and express the leptin receptor34. These glutamatergic inputs to DMH32 could provide the excitation required to drive the BAT sympathoexcitatory neurons and the shivering-promoting neurons in DMH when their POA inhibitory input is reduced during skin cooling or fever35. Although intrinsically warm-sensitive (W-S) 39, are postulated to play a key role in central thermoregulation by providing a prominent core temperature-modulated, GABAergic38 regulation of thermogenesis-promoting neurons in DMH  and BAT thermogenesis mediated by the action of prostaglandin E2 (PGE2) on its EP3 receptors45. Similarly, the fundamental thermoregulatory network mediates stress-induced hyperthermia48. Unraveling the complexity of the thermoregulatory circuitry in the hypothalamus52, including the phenotypic characterization of the projection neurons34 and their synaptic interactions that mediate the circadian13 and many behavioral54 modulations in Tcore, continues to pose significant research challenges for understanding the “heart” of the CNS thermoregulatory network. The downstream targets of the hypothalamic projection neurons for thermoregulation are the sympathetic and somatic premotor neurons in the rostral ventromedial medulla, centered on the rostral raphe pallidus (rRPa). Midbrain  and medullary 60) pathways to these premotor neurons exert important modulatory influences on the thermoregulatory activation of thermoeffector organs  , POA neu in DMH , the concore. These are integrated with the brain temperature information potentially derived from the discharge of W-S, GABAergic preoptic neurons of the central thermoregulatory network to regulate thermoeffector activities. The membranes of thermoreceptor afferent neurons contain transient receptor potential (TRP) cation channels whose temperature-dependent conductances transduce skin temperature into primary thermoreceptor afferent neuronal activity. The TRPM8 channel, activated by menthol and cooling, is the strongest candidate for the cutaneous cold receptor  channel67. Primary thermoreceptor dorsal root ganglion neurons synapse on thermoreceptive-specific, lamina I spinal  dorsal horn cells68, and these, in turn, collateralize to innervate the thalamus, providing the neural substrate for cutaneous thermosensory perception and localization69, and the pontine lateral parabrachial nucleus (LPB)71, which is responsible for triggering involuntary  thermoregulatory responses. Spinal lamina I skin cooling-responsive neurons provide a glutamatergic excitation to neurons in the external lateral subdivision of the lateral parabrachial nucleus (LPBel), which, in turn, project principally to the MnPO of the POA73, while a parallel, spinoparabrachial glutamatergic pathway excites POA-projecting neurons in the dorsal subnucleus of the LPB (LPBd)74 in response to skin warming74. Thus, activations of POA-projecting LPBd and LPBel neurons, driven respectively by cutaneous, and likely visceral, warm and cold thermoreceptor stimuli, initiate heat defense and cold defense inhibitions and excitations, respectively, in CVC sympathetic outflow and cutaneous blood flow, in BAT sympathetic outflow and BAT thermogenesis, and in shivering EMGs and shivering thermogenesis to maintain a homeostatic Tcore.Cold and warm thermoreceptors in the skin and viscera provide the extracranial thermal signals relating to skin temperature and Tcore  pH, inorganic cations, or endovanilloids, thereby providing a basis for such factors to influence thermoeffector activation and thus Tcore. For instance, TRPV1 channels, potentially on the terminals of afferents in the peritoneum, are stimulated by an endogenous ligand to tonically inhibit BAT thermogenesis, which results in a hyperthermic response to TRPV1 antagonist administration90. Although these afferents were not directly tested for their thermal responsiveness, the hyperthermic response to TRPV1 antagonism was not altered by changes in Tcore. The relative influences and the interactions of thermal and non-thermal stimuli on the conductance of the relevant TRPV1 channels could play a role in determining their effect on the level of thermoeffector activation to thermoreceptor stimuli.Providing an additional layer of complexity in the CNS regulation of Tcore; and (b) on augmenting thermogenesis to elevate energy expenditure with the goal of weight loss through consumption of the high-energy lipid stores in white adipose tissue. Novel approaches to reducing Tcore would have immediate benefits in treating intractable fevers that are unresponsive to cyclooxygenase inhibitors. Therapeutic hypothermia can have beneficial effects on survival and on reducing brain and tissue damage in ischemic insults such as cardiac arrest, stroke, and neonatal encephalopathy94. Extended space travel  also may require pharmacological induction of a hypothermic, hibernation-like state to reduce energy consumption and psychological stress.Interest in pharmacological modulation of the central thermoregulatory network has focused: (a) on reducing CVC and thermogenesis to lower Tcore must arise from changes in the level of activation of thermoeffector tissues. Although modulating neuronal discharge at any site within the central thermoregulatory network would be expected to alter Tcore, centrally generated hyperthermias, such as fever, that arise from altered activity in hypothalamic thermoregulatory neurons could be most effectively reduced by manipulating thermoeffector efferent pathways96. Indeed, directly inhibiting the discharge of neurons in the rRPa area, including the functionally significant premotor neurons that control the principal thermoeffectors, produced a fall in brain temperature of approximately 14°C in an ambient environment of 15°C97, and stimulation of α2 adrenergic receptors in the rRPa could completely block or prevent a lipopolysaccharide-evoked fever95. Similarly, central administration of an adenosine A1 receptor (A1AR) agonist reduced rat Tcore by 10°C in an ambient temperature of 15°C60 and mouse Tcore by 5°C in an ambient temperature of 4°C98. Of particular interest, in rats, this hypothermia, which was produced by a blockade of the cold-evoked activation of BAT and shivering thermogenesis, was long-lasting and paralleled by marked reductions in heart and respiratory rate, in EEG, and in behavior60—all reminiscent of those occurring in hibernation and torpor, which require central stimulation of the A1AR101. The discovery of a role for TRP channels in thermal sensation and in thermoeffector activation has stimulated research into the pharmacological modulation of TRP channels in the central thermoregulatory network to abrogate the normal cold defense mechanisms and allow Tcore to fall in a cool ambient environment. By reducing the activation of cooling-responsive skin thermoreceptors, a TRPM8 antagonist reduced rat Tcore by approximately 1°C when the rats were in an ambient temperature of 19°C63. Stimulating TRPV1 reduced mouse Tcore by approximately 12°C when mice were exposed to a 10°C ambient temperature102, and this effect was potentiated by the addition of a TRPM8 antagonist103. The location of the relevant, hypothermic TRPV1 channels remains unknown.Under basal metabolic and movement conditions, changes in T107, but through its consumption of lipid and glucose energy stores and oxygen, thermogenic metabolism in BAT is a neurally regulated contributor to energy homeostasis. Thus, particularly in the face of an elevated consumption of energy-rich food , a chronic reduction in cooling-evoked BAT thermogenesis would contribute to the augmented adipose energy stores that characterize obesity. Indeed, mice without BAT exhibit a propensity for obesity and diabetes109; conversely, overexpression of uncoupling protein-1 (UCP-1), principally responsible for thermogenesis in BAT, mitigates obesity induced by a high-fat diet110. Several anti-obesity therapies currently being explored are based on increased activation of BAT thermogenesis, through either activation of the central thermoregulatory network to increase the sympathetic outflow to BAT111 or an alteration in the cellular biochemical pathways in brown adipocytes or a hyperplasia of BAT to augment thermogenesis. The consistent findings that obese humans have significantly reduced cooling-activated BAT112, and that the basal  sympathetic activation of BAT is reduced in rats fed a high-fat diet114, and that a vagal afferent input to the NTS mediates the reduced cooling-evoked BAT activity in rats fed a high-fat diet115 not only support a role for reduced BAT activity in the excess adipose accumulation of obesity but also highlight the significance of non-thermal inputs to the central thermoregulatory network83 in influencing even the most basic thermoregulatory responses.Not only is BAT a thermogenic thermoeffector, including in adult humanscore that accompany a wide range of behaviors and in response to many hormones and drugs arise through altered non-thermal inputs to, or neurochemical modulation of, the neural activity within the fundamental thermoregulatory network. The latter, as well as thermoreceptor-based strategies, are being researched as therapeutic approaches in which the central thermoregulatory networks are recruited to alter Tcore and metabolism.Considerable progress has been achieved in revealing the functional organization of the dedicated thermoregulatory network within the CNS that provides the fundamental neural control of the thermoregulatory effectors: thermoregulatory behavior, CVC, and BAT and shivering thermogenesis, although many of the details of the neurophysiology and neuroanatomy of the central thermoregulatory network remain active areas of investigation. The changes in T