Oral squamous cell carcinoma (OSCC) has a remarkably high incidence worldwide, and a fairly serious prognosis, encouraging further research into advanced technologies for noninvasive methods of making early diagnoses, ideally in primary care settings.Our purpose was to examine the validity of using advanced noninvasive technologies in diagnosis of OSCC by identifying and evaluating relevant published reports.MEDLINE, EMBASE, and CINAHL were searched to identify clinical trials and other information published between 1990 and 10 June 2014; the searches of MEDLINE and EMBASE were updated to November 2014. Study selection: Studies of noninvasive methods of diagnosing OSCC, including oral brush biopsy, optical biopsy, saliva-based oral cancer diagnosis, and others were included.Data were abstracted and evaluated in duplicate for possible relevance on two occasions at an interval of 2 months before being included or excluded.This study identified 163 studies of noninvasive methods for diagnosing OSCC that met the inclusion criteria. These included six studies of oral brush biopsy, 42 of saliva-based oral diagnosis, and 115 of optical biopsy. Sixty nine of these studies were assessed by the modified version of the QUADAS instrument. Saliva-based oral cancer diagnosis and optical biopsy were found to be promising noninvasive methods for diagnosing OSCC.The strength of evidence was rated low for accuracy outcomes because the studies did not report important details required to assess the risk for bias.It is clear that screening for and early detection of cancer and pre-cancerous lesions have the potential to reduce the morbidity and mortality of this disease. Advances in technologies for saliva-based oral diagnosis and optical biopsy are promising pathways for the future development of more effective noninvasive methods for diagnosing OSCC that are easy to perform clinically in primary care settings. Oral cancer is the eighth most common cancer worldwide and represents a significant disease burden. If detected at an early stage, survival from oral cancer is better than 90% at 5 years, whereas survival of patients presenting with late stage disease is only 30%. The 5-year survival rate for oral cancer has remained less than 50% over the last 50 years for the following reasons ,2: (i) mAlthough the precise sequence and number of events required for tumourigenesis remains unknown, understanding of tumourigenesis may help in development of more effective methods for diagnosis and treatment. A recent series of experiments performed by Hahn et al. ,4 and HaAccounting for 96% of all oral cancers, squamous cell carcinoma (SCC) is usually preceded by dysplasia presenting as white epithelial lesions on the oral mucosa (leukoplakia). Leukoplakias develop in 1–4% of the population . These bThe peak incidence of oral squamous cell carcinoma (OSCC) is between the ages of 45 and 75 years. The increasing number of older persons worldwide, together with concomitant increases in the incidence of malignancies, are creating a pressure on healthcare systems . The perMinimally invasive intervention is considered one of the most important developments in the healthcare industry. The global market for minimally invasive intervention is steadily growing, its annual growth rate being approximately 10% . As patiIn 1986, Bouquot noted that as many as 10% of US adults have some form of oral abnormality that requires histopathological assessment . HoweverOptical biopsy involves light–tissue interactions and different types of spectroscopy depending on the nature of the tissues and type of excitation light used -18. The List 1 Summary of the most common non-invasive methods for diagnosing oral squamous cell carcinomaToluidine blueConventional oral brush biopsyOral brush biopsy coupled with computer-assisted analysisOral brush biopsyGenomic substancesTranscriptomc substancesProteomic substancesSaliva-Based Oral Cancer DiagnosisChemiluminescence Tissue fluorescence imaging (VELscope)Light-based detection systems:Tissue fluorescence spectroscopyRaman spectroscopyElastic scattering SpectroscopyDifferential path-length spectroscopyNuclear magnetic resonance spectroscopyConfocal reflectance microscopy (CRM)Optical Coherence TomographyAngle-resolved low coherence interferometry (A/LCI)OthersOptical Biopsy:DNA-analysisBiomarkers:Laser capture microdissectionThis systematic review studied noninvasive methods for diagnosing OSCC.Why is TB staining unreliable?What is the sensitivity and specificity of oral brush biopsy?How does the accuracy of different types of optical biopsy compare with that of surgical biopsy for diagnosis of OSCC?What changes may develop in oral saliva in OSCC patients and those who are at high risk of OSCC and are these changes diagnostic for OSCC?This study was designed to answer the following four key questions.Exception for one study from 1986, MEDLINE, EMBASE, the Cochrane Library, and CINAHL were searched to identify clinical trials and other information published between 1990 and 10 June 2014; the searches of MEDLINE and EMBASE were updated to November 2014. The major terms and concepts searched included (but were not limited to) the following: surgical biopsy, optical biopsy, saliva-based oral cancer diagnosis, and saliva changes in people with cancer. Table The fundamental a priori criteria for inclusion of studies were that they involved direct comparison of a noninvasive method with surgical biopsy; 10 or more patients were enrolled for the purpose of making a primary diagnosis of an oral mucosal abnormality; and the findings were published as an English-language, full-length, peer-reviewed article. List 2 shows all of the study inclusion criteria.List 2 Inclusion criteriaStudies addressing Key Questions 1 and/or 2Studies prospective in designEnrollment of 10 or more patients for the purpose of diagnosisThe patients assessed by the gold standard Toluidine blue staining,Oral brush biopsy,Optical BiopsyNon-invasive methods in diagnosis of OSCCFluorescence spectroscopy,Raman spectroscopy,Elastic scattering spectroscopy,Differential path-length spectroscopy,Optical TomographyNuclear magnetic resonance spectroscopyOptical Biopsy:Enhanced dye fluorescence,Ratio imagingAuto-fluorescence spectroscopy,Saliva-based oral cancer diagnosisSaliva:Genomic substance,Trans-criptomic mRNA,Proteomic substancesOral Saliva changes in cancer patients:English-languageFull-length articlePeer-reviewed articleAbstracts of articles identified by the investigator were evaluated in duplicate for possible relevance on two occasions at intervals of 2 months; 313 abstracts were thus identified. When exclusion and inclusion criteria were applied at the abstract level, 62 abstracts were excluded. The 286 full-length articles of studies that seemed relevant at the abstract level were then obtained and the full articles examined to ascertain whether they met the inclusion criteria; 194 of these articles met the inclusion criteria  instrument developed by Whiting et al. was used to assess the internal validity of each of the included studies because we considered the original form of QUADAS adequate (List 3) . The modList 3 (Modified Diagnostic Accuracy Studies (QUADAS) instrument developed by Whiting et al.)Category 1: Spectrum compositionWas the spectrum of patients described in the paper and was it chosen adequately?Were selection criteria described clearly?Was the method of population recruitment consecutive?Was the setting of the study relevant?Was disease prevalence and severity reported? (not included in QUADAS)Category 2a: Index test and reference standard: Selection and executionIn light of current technology, was the reference standard chosen appropriate to verify test results?Is it possible that a change in the technology of the index test has occurred since this paper was published? (not included in QUADAS)Was there an abnormally long time period between the performance of the test under evaluation and the confirmation of the diagnosis with the reference standard?Was the execution of the index test described in sufficient detail to permit replication of the test?Was the execution of the reference standard described in sufficient detail to permit replication of the test?Did the whole sample, or a random selection of the sample, receive verification using a reference standard of diagnosis?Did all patients receive the same reference standard regardless of the index test result?Were the results of the index test incorporated in the results of the reference standard?Was the cut-off value pre-specified or acceptable in light of previous research? (not included in QUADAS)Was treatment started based on the knowledge of the index test results before the reference standard was applied? (not included in QUADAS)Category 2b: Index test and reference standard: InterpretationWere the index test results interpreted blind to the results of the reference standard?Were the reference standard results interpreted blind to the results of the index test?Was clinical data available when test results were interpreted?Is data presented on observer or instrument variation that could have affected the estimates of test performance? (not included in QUADAS)Category 3: AnalysisWere appropriate results presented  and were these calculated appropriately? (not included in QUADAS)Was a measure of precision of the results presented ? (not included in QUADAS)Were uninterpretable/indeterminate/ intermediate results reported and included in the results?Was the threshold value specified retrospectively based on analysis of the results? (not included in QUADAS)Were reasons for drop-out from the study reported?Were subgroup analyses pre-specified and clinically relevant? (not included in QUADAS)Were results presented in a 2 × 2 data table? (not included in QUADAS)Was any indication of the utility of the test provided? (not included in QUADAS)Category 4: Research PlanningWas an appropriate sample size calculation performed and were sufficient patients included in the study? (not included in QUADAS)Were study objectives clearly reported? (not included in QUADAS)Was there any evidence that a study protocol had been developed before the study was started? (not included in QUADAS)List 4 Quality assessment instrument (the full list of items modified)Was the study prospective in design?Were the patients assessed by the gold standard Were patients assessed by a reference standard regardless of the biopsy resultsWas funding for this study provided by a source that doesn’t have an obvious financial interest in the findings of the studyDid the study account for inter-reader/scorer differences?Were the reader(s) of the investigated procedure blinded to the results of the reference standard?Were readers of the reference standard blinded to the results of the study?Were the readers of the investigated procedure blinded to all other clinical information?Were readers of the reference standard blinded to all other clinical information?The experience of the investigation’s team in the relevant fieldThe strength of evidence supporting each major conclusion was graded as high, moderate, low, or insufficient. According to the modified version of the QUADAS instrument, the proportion of tools covering each item were classified as follows: Grade I, 75–100%; Grade II, 50–74%; Grade III, 25–49%; and Grade IV, 0–24% . StudiesStatistical analysis was performed using Microsoft office XL version 2007, which was used to calculate the 30 variables in the modified version of QUADAS . The iteThis study identified 163 studies of noninvasive methods for diagnosing OSCC that met the inclusion criteria. These included six studies of oral brush biopsy, 42 of saliva-based oral diagnosis, and 115 of optical biopsy. Sixty nine of these studies were assessed by the modified version of the QUADAS instrument   and supravital staining outside the body, which usually involves preparation of slides of detached cells ,21. TB iThe goal of the highly sensitive and specific technique of oral brush biopsy is to provide a sample by a less painful and simpler means than scalpel or punch biopsy. The accuracy of brush tests has been the subject of many published studies. In every study in which oral lesions have been simultaneously assessed by both a brush biopsy and surgical biopsy, this test has been shown to have both sensitivity and specificity well over 90% ,25. OralSaliva can be considered a mirror of bodily health. The multifarious components of saliva not only protect the integrity of oral tissues, but also provide clues to various local and systemic conditions and diseases. The components of saliva are continually being explored as markers of various diseases and for monitoring general health . In the Tumour cells may inhibit or produce biochemical substances referred to as tumour markers. These can be normal endogenous products that are produced at a greater rate in cancer cells or the products of newly switched on genes that are quiescent in normal cells -35. Tumo), extracellular matrix-degrading proteinases , hypoxia markers , epithelial-mesenchymal transition markers , epithelial tumour factors (Cyfra 21–1), cytokeratins , microRNA molecules, and hypermethylation of cancer-related genes (p16 and death-associated protein kinase) [Relevant markers include oncogenes , anti-oncogenes , cytokines -8, and -1β), growth factors  -43.. [. [. [Markers in the form of changes in the host DNA of dysplastic or cancer cells include point mutation, deletion, translocation, amplification, and methylation Table . Loss of. ,44 found. , and hav. ,46. The . ,48. Loss. ,48. Usin. ,50. Rosa. [. [.  have rep. [. [. -54.TableIt has been speculated that salivary mRNA is contained in apoptotic bodies ,56 or ac. [. [. [. [Carbonylation signifies oxidative damage to proteins: there is reportedly a substantial increase in salivary carbonyls (246%) in OSCC patients, indicating that their epithelial cells are being exposed to significant free radical attack . The sen.  reported. [.  and Rajk. [. [.  found si. [. [. . Arellan[. [. [.  used LumLight-based systems depend on the assumption that absorption and reflection of light differs between normal tissues and those with metabolic or structural changes. Vizilite Plus with TBlue system , LED Dental  Microlux/DL  and Orascoptic DK  are light-based oral cancer screening aids that have been developed with the aim of assisting identification of early stage precancerous and cancerous lesions. LED Dental, a handheld device with an illuminated chemiluminescent stick, emits visible light in the 430 nm wavelength that causes fluorescent excitation of certain compounds in tissues. After the patients have rinsed their mouths with acetic acid, the oral cavity is examined. With Microlux (AdDent) and ViziLite , the oral cavity is examined with a battery-powered fibreoptic visible light source rather than a chemiluminescent stick; again, prior rinsing with acetic acid is required. These devices are not sensitive or specific for diagnosing any type of oral lesion. Only pathologic examination of tissue can definitively determine the biologic nature of a lesion -70. The It has already been established that autofluorescence optical biopsy can produce diagnostically useful information about human oral cavity tissues ,76. BothIt is well known that all tissues fluoresce and that malignant tissues fluoresce less than normal tissues: thus, these tissues have different spectral characteristics. Studies have shown that when an ultraviolet or near ultraviolet light source is used, normal oral mucosa emits more green fluorescence than neoplastic lesions ,101. MalAutofluorescence imaging has recently been shown to improve detection of premalignant and malignant oral lesions ,101. ThiFluorescence can be slightly enhanced by exogenously applying fluorescent drugs such as 5-aminolevulinic acid (5-ALA), which induces protoporphyrin IX  -107. RecThis technique compares a photochemical or end metabolic product that is known to be increased in disease states with another product that is known to be depleted. For example, as described above, 5-ALA enhances PPIX, which fluoresces red after excitation with blue light. The same excitation results in green fluorescence of molecules such as NAD and FADH, which are depleted in malignant tissues with a high metabolic rate -114. Shi6 to 1 in 108) photons undergo a process called Raman or inelastic scattering, in which photons are discharged from the material or ‘scattered’ at a differing wavelength than the incident photon; it is this wavelength shift that is recorded in Raman spectroscopy [The Raman effect was first discovered by Professor Raman of Calcutta University, for which he was awarded the Nobel prize in 1930 . This eftroscopy ,118.The Raman effect occurs when light impinges on a molecule and interacts with the electron cloud and bonds of that molecule. In the spontaneous Raman effect, which is a form of light scattering, a photon excites a molecule from the ground state to a virtual energy state. When the molecule relaxes, it emits a photon and goes into a different rotational or vibrational state. The difference in energy between the original state and this new state leads to a shift in the emitted photon’s frequency away from the excitation wavelength ,109,110.Raman spectroscopy is being investigated as a diagnostic tool for characterising cancer cells and early malignant changes and distinguishing these cells from normal cells. It has a distinct advantage over other optical techniques: it provides information on molecular composition and structure of living tissue ,119,120.In 2009, Harris et al. ,126 examESS makes diagnoses by objective statistical and analytical methods rather than by subjective interpretation of images. It provides optical geometrical information that is based on white light reflectance. In ESS, photons hit tissue and are backscattered without changes in wavelength. The relative intensity of this backscattering is influenced by the composition of the interrogated tissue, specifically the relative concentration of scatterers  and absorbers . A scattering event carries with it all the characteristics of the cellular components, which are called “scattering centres”. Pathological scattering centres may originate from disorganised epithelial orientation and architecture, changes in morphology of epithelial surface thickness and texture, cell crowding, increased distance from subepithelial collagen layer, enlargement and hyperchromicity of cell nucleus, increased concentration of metabolic organelles, and presence of abnormal protein packages or particles ,107,136.Müller et al. used three spectroscopic techniques to assess 91 tissue sites from 15 patients with varying degrees of oral malignancy  and eight healthy volunteers. By fitting a linear combination of collagen and the reduced form of NADH fluorescence spectra to intrinsic tissue fluorescence spectra that had been excited by 337 nm and 358 nm laser light, these researchers obtained direct biochemical information regarding oral tissue native fluorophores with autofluorescence spectroscopy. They measured wavelength-dependent absorption and scattering coefficients by diffuse reflectance spectroscopy to provide information regarding tissue absorption and structure, such as haemoglobin concentration and stroma density. They then obtained light ESS information resulting from single backscattering from epithelial cell nuclei by subtracting the diffusely reflected component from the measured reflectance; this provided information concerning the size distribution of cell nuclei. They described this method as trimodal spectroscopy and reported a sensitivity and specificity of 96% and 96%, respectively, in distinguishing cancerous/dysplastic  from normal tissue. In addition, the authors were able to distinguish dysplastic from cancerous tissue with a sensitivity of 64% and a specificity of 90% .Lovat et al. studied ESS measurements collected in vivo and matched them with pathologic findings of histological specimens taken from identical sites within Barrett oesophagus. They examined 181 matched biopsy sites from 81 patients. ESS detected high risk sites with 92% sensitivity and 60% specificity and differentiated high risk sites from inflammation with sensitivity and specificity of 79% . SharwanDifferential path-length spectroscopy, a recently developed fibreoptic point measurement technique, measures scattered photons that have travelled in predetermined path lengths. Differential path-length spectroscopy is considered to be a form of ESS that has fixed photon path length, fixed photon visitation depth, and absolute measurement of absorbers. This technology was developed at the Erasmus Medical Centre, Rotterdam, the Netherlands ,150. TheThis technology uses light scattering either to construct an image, as in OCT, or to measure the average size of different cell structures, thus providing objective information about degree of dysplasia, as in angle-resolved low coherence interferometry (a/LCI).This is analogous to ultrasound imaging except that it uses light rather than sound. The high spatial resolution of OCT enables noninvasive in vivo “optical biopsy” and provides immediate and localised diagnostic information. The recent development of a Fourier domain mode lock swept source-based OCT system has helped to simultaneously achieve a high speed (>100 kHz A-scan rate) and good spatial resolution (<4 μm). In addition, the development of various miniature scanning probes that provide high-speed three-dimensional OCT pictures has been reported ,153-161.A/LCI, an emerging biomedical imaging technology that uses the properties of scattered light to measure the average size of different cell structures, including cell nuclei, directly measures diagnostically relevant subcellular features of epithelial tissues up to 500 μm below the surface. Unlike OCT, which is a subjective method because it requires image interpretation, a/LCI performs an objective analysis of tissue and delivers direct confirmation of precancerous disease to the physician -106. WaxNMR exploits the magnetic properties of certain atomic nuclei to determine the physical and chemical properties of atoms or the molecules in which they are contained. It relies on the phenomenon of nuclear magnetic resonance and can provide detailed information about the structure, dynamics, reaction state, and chemical environment of molecules. This technology allows three-dimensional study of atoms in molecules; the larger the magnet, the more sensitive the device. Using NMR, it is possible to view how protein links with DNA -106. NMRThis distinguishes different biomolecules by probing chemical bond vibrations and using these molecular and submolecular patterns to define and differentiate pathological from normal tissues ,185. OptPatients with potentially malignant oral lesions referred to specialist centres are faced with long waiting times, leading to significant diagnostic delays . In the TB is one of the oldest noninvasive methods for diagnosing OSCC. Being highly sensitive and moderately specific for malignant lesions, it can be used for screening and to rule out suspicious areas. However, it is an unreliable technique because it is highly subjective and depends on the experience of the investigator. Unlike sampling of uterine cervix cells, analysis of surface epithelial cells of the oral cavity and oropharynx by standard exfoliative cytology (brush biopsy) has proven to be unreliable, identifying as few as 31% of dysplastic tissues . ComputeExfoliative cytology based on a nanobiochip sensor platform for oral cancer detection has been described. The diagnosis of oral carcinoma by oral brush biopsy with exfoliative cytology based on nanobiochip sensor platform has 97–100% sensitivity and 86% specificity ,186,187.A point of care device for testing saliva to detect oral cancer that is not yet commercially available has been developed by the University of California, Los Angeles (UCLA) Collaborative Oral Fluid Diagnostic Research Laboratory, led by Dr David Wong ,189. TheMost optical biopsy technologies are still in experimental studies and clinical trials. The FDA, Health Canada, and the World Health Organization have approved VELscope®  for direct visualisation of autofluorescence of oral cavity and diagnosis of oral carcinoma ,191. TheA/LCI technology is unique in being able to evaluate microstructure in epithelial tissues up to 500 μm below the surface. The technique uses real-time analysis of light scattered from tissues to detect enlargement of nuclei and other organelle-related changes indicative of early cancer progression. This is an alternative to OCT, which aims to construct a high-resolution image that is analysed either by a trained expert or computer algorithm to provide diagnostic information. In contrast, a/LCI measures diagnostically relevant variables directly and provide them to the physician to enable faster, more direct confirmation of precancerous disease. To date, the technique has been shown to accurately detect pre-cancer in the oesophagus, colon, oral cavity and cervix. Oncoscope  is now developing a/LCI commercially. In the United States, FDA approval for this application of Oncoscope’s system is expected in 2016 ,191.Saliva-based oral cancer diagnosis and optical biopsy are promising noninvasive methods for diagnosing OSCC with high sensitivity and reliable specificity that are easy for primary care practitioners to perform clinically. These technologies provide objective information and do not require special experience for interpretation of the information obtained. They could be widely used in the near future as reliable routine modalities for oral cancer diagnosis and evaluation of the degree of dysplasia of pre-cancerous lesions. It is clearly evident that screening and early detection of cancer and its precursors have the potential to reduce the morbidity and mortality of this disease. These technologies may change the consequences of this disease in the near future.It is clear that screening for and early detection of cancer and pre-cancerous lesions have the potential to reduce the morbidity and mortality of this disease. Advances in technologies for saliva-based oral diagnosis and optical biopsy are promising pathways for the future development of more effective noninvasive methods for diagnosing OSCC that are easy to perform clinically in primary care settings.


Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid , glycodeoxycholic acid , and glycochenodeoxycholic acid . Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury. N-acetyl-p-benzoquinone imine (NAPQI), which, along with the depletion of hepatic glutathione, is recognized to be a critical initiating step in the development of hepatotoxicity  mg/kg APAP) compared to the initial 48 h (20.5 [3.1–57.5] mg/kg APAP), indicating a decline in the median daily dose between study day 1 and day 2. Daily doses were within the recommended dose range for APAP use in children .APAP overdose patients may be relatively asymptomatic during the initial 24 hours following APAP overdose, as the development of overt toxicity may not be apparent until 24 to 48 hours after the time of ingestion. Thus, patients are evaluated for the potential risk of hepatotoxicity through the use of a nomogram  based oAll children in Group C received treatment with NAC. Since the efficacy of NAC in preventing hepatic injury is a function of the time it is administered relative to the time of the drug overdose , the datSummary data, presented as peak values, for bile acids by subject group are presented in Due to the limited subject sampling scheme of this pediatric study, relative comparison of temporal changes in bile acids, APAP protein adducts and ALT in the early hours after the APAP overdose was not possible. As an alternative, the time to reach peak measurement for individual bile acids was examined and compared as a function of APAP protein adduct concentrations. APAP protein adduct values ≥ 1.0 nmol/mL have been shown to have high sensitivity and specificity  in patients with APAP liver injury  ,24. As sIn subsequent analysis, ROC analysis was conducted to identify bile acid cut points that maximized the sum of sensitivity and specificity for discriminating patients with peak adducts < 1.0 nmol/mL or ≥ 1.0 nmol/mL. TDCA values > 0.56 uM had the highest area under the curve value for discriminating subjects at the adduct toxicity cut point of 1.0 nmol/mL , followeIn previous work, APAP protein adducts and ALT were shown to be higher in patients with greater delays in receiving treatment with NAC . Thus, tThus, overall, the data suggest that several of the conjugated bile acids associate with toxicity severity in APAP overdose, as defined by both ALT values and APAP protein adduct concentrations.Bile acids are formed during the biotransformation of cholesterol through two metabolic pathways known as the “classical” or neutral pathway and the “alternative” or acidic pathway. The classical pathway produces primary bile acids and involves microsomal cholesterol 7α-hydroxylase (CYP7A1), while the alternative pathway involves biotransformation of cholesterol through mitochondrial sterol 27-hydroxylase (CYP27A1) followed by oxysterol 7α-hydroxylase (CYP7B1) . More thThe increased availability of metabolomics technology has enabled investigations of the relationship between small molecules of endogenous metabolism and the development of liver injury in experimental models of APAP toxicity ,13,25,34primary bile acids showed that CA was lower in the APAP overdose group (compared to Group B), while CDCA was only reduced in Group A when compared to Group B. The lower levels of CA in the APAP overdose subjects could be consistent with a protective mechanism of the liver and a shift from the classical to the alternative pathway of metabolism, as has been shown for non-alcoholic fatty liver disease [Examination of the  disease .Cyp7A1 and Cyp8B1) and cholesterol transport (Abcd1) were down-regulated, while genes that increased bile acid transport  were up-regulated [in vitro [The mechanistic significance of elevated bile acids in children with APAP overdose and liver injury is unclear. Previous studies in rodent models of APAP toxicity ,37 foundegulated . During egulated . Treatmeegulated , was proegulated . Taurinein vitro . The tauin vitro . Thus, tin vitro . Bile acAPAP protein adducts reflect the contribution of oxidative metabolism to APAP toxicity  and are It has also possible that perturbations of bile acid profiles may be useful in the future to examine the homeostatic integrity of the liver in the recovery and regeneration stages of liver injury. This concept has been tested in a previous clinical study in which bile acid profiles in biliary fluid were examined pre- and post- liver transplant as a functional indicator of metabolic capacity . The limSeveral additional limitations of the present study merit consideration. The three subject groups differed somewhat by age and by distribution of gender . In addiThe data provided herein have application to other studies of drug induced liver injury and suggests a potential role for bile acid profiles as sensitive determinants of liver injury in drug development. Additional research to further understand the mechanistic significance of bile acid elevations in APAP exposure and toxicity is of interest, especially in light of recent studies that have illustrated the role of bile acids as signaling molecules that are important in lipid and glucose metabolism. Finally, this study illustrates how metabolomic approaches can be used as a tool for biomarker discovery and hypothesis generation in the clinical setting of drug toxicity and liver injury.


Ischemic stroke is the third leading cause of death and disability. Up until now, all clinical trials testing potential stroke neuroprotectants failed. For this reason attention of researchers has been focusing on the identification of brain endogenous neuroprotective mechanisms activated after cerebral ischemia. In this context, ischemic preconditioning and ischemic post-conditioning represent two neuroprotecive strategies to investigate in order to identify new molecular target to reduce the ischemic damage.Most of the current focus on developing neuroprotective therapies is aimed at preventing neuronal death. However, these approaches have not been successful despite many years of clinical trials mainly because the numerous side effects observed in humans and absent in animals used at preclinical level. Recently, the research in this field aims to overcome this problem by developing strategies which induce, mimic, or boost endogenous protective responses and thus do not interfere with physiological neurotransmission. Preconditioning is a protective strategy in which a subliminal stimulus is applied before a subsequent harmful stimulus, thus inducing a state of tolerance in which the injury inflicted by the challenge is mitigated. Tolerance may be observed in ischemia, seizure, and infection. Since it requires protein synthesis, it confers delayed and temporary neuroprotection, taking hours to develop, with a pick at 1–3 days. A new promising approach for neuroprotection derives from post-conditioning, in which neuroprotection is achieved by a modified reperfusion subsequent to a prolonged ischemic episode. Many pathways have been proposed as plausible mechanisms to explain the neuroprotection offered by preconditioning and post-conditioning. Although the mechanisms through which these two endogenous protective strategies exert their effects are not yet fully understood, recent evidence highlights that the maintenance of ionic homeostasis plays a key role in propagating these neuroprotective phenomena. The present article will review the role of protein transporters and ionic channels involved in the control of ionic homeostasis in the neuroprotective effect of ischemic preconditioning and post-conditioning in adult brain, with particular regards to the Na The blood–brain barrier was preserved thanks to the reduced activity of matrix metalloproteinases and cell adhesion molecules Gidday, .+-ATPase, acid-sensing ion channels, Na+/H+ exchange, Na+/K+/2Cl− cotransport and Na+/Ca2+ exchanger) and transporters for metabolites.Improved capacity to preserve cellular ionic and pH homeostasis: preconditioning regulates expression and activity of ion transport systems , the plasma membrane Ca2+-ATPase (PMCA), the Na+/H+ exchange (NHE), the Na+/K+/2Cl− cotransport (NKCC) and the acid-sensing cation channels (ASIC).Ischemic brain conditioning activates intracellular biological responses to a potential lethal insult; therefore it is predictable that organs reinforce their tolerance when exposed to a sublethal insult prior or after a harmful episode of ischemia by increasing energy metabolism or delaying anoxic depolarization after the onset of the subsequent ischemic insult  is a protein that aids in the active transport of sodium, potassium, and chloride into and out of cells; it is widely distributed throughout the body and it has important functions in organs that secrete fluids. NKCC2 is found specifically in the kidney, where it serves to extract sodium, potassium, and chloride from the urine so that they can be reabsorbed into the blood  mice the lack of NHE1 attenuated the damage induced by conditions mimicking cerebral ischemia in vitro, and this effect was confirmed also in vivo  is protective in adult myocardium in a model of ischemia followed by reperfusion; however, the effect is unclear in immature myocardium  during the first 2 min of reperfusion  during brain post-conditioning few information are available. In a recent work it has been demonstrated that in hippocampal neurons exposed to hypoxia conditioning mimiking an in vitro post-conditioning, NKCC1 is strongly activated through binding with STE20/SPS1-related proline/alanine-rich kinase (SPAK)  superfamily, which are Na+- selective cation channels sensitive to amiloride  are neuronal voltage-insensitive cationic channels activated by extracellular protons. ASIC proteins are a subfamily of the ENaC/Deg superfamily of ion channels. ASICs are potential drug targets for treating a wide variety of conditions linked to both the CNS and PNS. Acid-sensing cation channels (ASIC) have been extensively studied both in physiological and in pathological conditions for their wide distribution in the nervous system and since they represent key targets in many different aspects of acidosis, a condition that occurs during ischemic stroke, as previously analyzed  diseases. Indeed, ischemic brain pH falls to 6.0 due to the accumulation of lactic acid and to the protons produced by ATP hydrolysis , including Akt  is an antiporter membrane protein that removes calcium from cells. It consists of nine transmembrane segments that can mediate Ca2+ and Na+ fluxes across the plasma membrane. Depending on the intracellular concentrations of Ca2+, [Ca2+]i, and Na+, [Na+]i, NCX can operate either in the forward mode, coupling the uphill extrusion of Ca2+ to the influx of Na+ ions, or in the reverse mode, mediating the extrusion of Na+ and the influx of the Ca2+ ions  increase this exchanger mediates the entrance of sodium ions and the extrusion of calcium ions in tight dependence of their electrochemical gradient. The way in which the antiporter works (forward or reverse) depends on several factors such as membrane potential, Na+ and Ca2+ gradient. Therefore, NCX represents an important actor in the regulation of pathophysiological and physiological responses to increases of [Ca2+]i and [Na+]i  is a transport protein in the plasma membrane of cells that functions removing calcium (Ca2+) from the cell; so it is also an important regulator of the calcium concentration in the extracellular space. This protein is the only plasmamembrane high affinity Ca2+ transporting system. It removes Ca2+ from the cytosol in all eukaryotic cells, and for most of them it is the only Ca2+ exporting system together with Na+/Ca2+ exchanger which represent the principal Ca2+ exchanger in heart cells and neurons (Jensen et al., 2+ affinity of the pump, however renders it the only system capable of fine-tuning its exchanges with the external ambient (Brini et al., The plasma membrane Ca22+ -ATPase may export intracellular Ca2+ even with relatively lower transport capacity during prolonged membrane hyperpolarization and during conditions of increased intracellular Na+ concentrations, as occurred under ischemic conditions. Ohta et al. (2+ (Kato et al., Conversely to other plasma membrane buffering systems, such as NCX and ATPases, plasma membrane Caa et al.  suggesteMoreover, in gerbils, ischemic preconditioning by pre-exposure to 2-min ischemia is extremely effective in protecting against ischemic cell death since there is a modification of the electrophysiological properties of CA1 neurons (Kawai et al., The identification of the players involved in the control of ionic homeostasis during pre and post-conditioning should provide more direct opportunities for translational neuroprotection trials. Indeed, the present review, summarizing the specific effect of the plasmamembrane proteins involved in the maintenance of cellular ionic concentration, would provide information of which transporters needed to be activated or inhibited in order to protect the brain by the ischemic injury.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.


Neurofibromatosis type 1, tuberous sclerosis complex, and Von Hippel-Lindau disease, historically classified as the phakomatoses, are hereditary multisystem disorders characterized by the presence of hamartoma, which carry the risk of malignant transformation. The alteration of tumor suppressor genes seems to be at the basis of their pathophysiogenetic mechanism. Lisch and choroidal nodules in neurofibromatosis type 1, retinal astrocytomas in tuberous sclerosis complex, and retinal capillary hemangioma in Von Hippel-Lindau disease are the principal ophthalmic hamartomatous manifestations. The advent of novel imaging techniques such as near infrared reflectance and optical coherence tomography has provided unprecedented insight on the choroidal and retinal features of these diseases. These methods have improved early diagnosis and the ongoing surveillance in these conditions. Among an array of treatment modalities, antivascular endothelial growth factor therapy has been used in the management of retinal hamartomas but results have been varied. This review is an update on the pathophysiogenetic mechanisms, ophthalmic manifestations, and novel treatment strategies in the phakomatoses with emphasis on the role of imaging techniques. Van der Hoeve in 1932 included neurofibromatosis type 1 (NF1), tuberous sclerosis complex (TSC), and Von Hippel-Lindau disease (VHL) in a group named “phakomatoses.” The conditions were defined as hereditary multisystem disorders, which share the common characteristics of the presence of “spots, tumefactions, and cysts” which were described as “phakomata” (from the Greek term “phakos” for mother-spot), which proliferate and carry the risk of malignant transformation , but advRecent progress in multimodal imaging techniques, like near infrared reflectance (NIR) and optical coherence tomography (OCT), has enhanced our knowledge on the retinal manifestations of these diseases, providing unprecedented information on the morphological aspects of hamartomas. These methods have improved early diagnosis and the ongoing surveillance in these conditions. Indeed, facilitated and noninvasive imaging with NIR has led to propose choroidal nodules as an additional diagnostic criterion in NF1, which aids diagnosis in children and in uncertain cases . PeripapThe present paper is a detailed review of the ophthalmological features in the phakomatoses with particular attention to novel imaging findings and ongoing management methods.NF1 is an autosomal dominant disease due to deletions or mutations of the neurofibromin gene located on chromosome 17p11.2 . The preOphthalmological features included in the NIH diagnostic criteria of NF1 are Lisch nodules and optic pathway gliomas (OPG) . Other oLisch nodules of the iris represent the main ocular manifestation of NF1. They usually appear from the age of 2 years onwards and approximately 50% of children present nodules; by adulthood this number increases to over 90% –29. The Ragge et al. reported that they were more frequently localized in the inferior hemifield due to the sunlight-shielding effects of the upper eyelid ; howeverGlaucoma onset is rarely seen in NF1, although multiple pathogenic mechanisms related to angle closure have been proposed. The most commonly reported mechanisms are infiltration of the anterior chamber by neurofibromas that obstruct the angle, secondary angle closure by neurofibromatous cysts or increased thickness of the ciliary body and choroid, neovascular glaucoma, and developmental angle abnormalities –36. OptiChoroidal abnormalities of NF1 are often difficult to detect via fundus biomicroscopic examination and fluorescein angiography .Indocyanine green angiography in NF1 patients shows delayed perfusion of the choriocapillaris in areas corresponding to choroidal nodules , 46. YasThe enhanced depth imaging function of spectral domain OCT allows the visualization of deeper ocular structures than traditional OCT as the peak sensitivity is placed behind the retinal pigment epithelium . Ayata eMoreover, through manual segmentation measurements of retinal layer and choroidal thickness using OCT software, we demonstrated generalized choroidal and retinal thinning in patients with NF1 . We showRetinal microvascular anomalies consisting in small (second or third order venule) tributaries of the inferior or superior-temporal vein denominated “corkscrew retinal vessels,” “hemangioma-like,” or “ball of thread” retinal microvessels have been reported and these seem to be overlying areas corresponding to choroidal nodules as shown by NIR imaging  51, 52], 5251, 5Other retinal alterations such as multiple retinal capillary hemangiomas, astrocytic hamartomas, and, rarely, combined hamartomas of the retina and the retinal pigment epithelium (CHRRPE) were described . OCT feaOptic pathway gliomas (OPG) have the most dramatic impact on visual acuity in NF1 patients. They are present in 15%–20% of children with NF1 before the age of eight . These tSeveral studies demonstrated a correlation between OPGs in NF1 affected children and OCT evidenced thinning of the retinal nerve fiber layer (RNFL) , 62, 63.We speculated that this could be due to the age related, chronic nature of NF1 or that choroidal and retinal thinning could also involve the macular and peripapillary RNFL and the GCL-IPL , 66. FurHigh levels of VEGF have been demonstrated in plexiform neurofibromas and malignant peripheral nerve sheath tumors . Interes TSC1 and TSC2 located on chromosomes 9q34 and 16p13, which encode for hamartin and tuberin, respectively [Tuberous sclerosis complex is a multisystemic disease outlined by hamartomas involving the central nervous system, eye, skin, kidneys, heart, lung, and liver. This condition was first described by Bourneville in 1880 who documented typical tuber-like cerebral lesions in the human brain at postmortem examination . In 1932ectively . The incectively .The typical retinal lesions are astrocytic hamartomas, which can be the first manifestation of disease and are among the major criteria for diagnosis. They have been reported in 44% to 48% of patients in two large case series , 73. RetThe “younger tuberous bodies” are not easy to identify with ophthalmoscopy due to their translucent or blurry aspect. At times the only sign that can help to identify these lesions is a perilesional circular reflex. They are often localized near a vessel, which gives it an interrupted aspect. These vessels are usually frail and can lead to vitreous hemorrhage. The “older tuberous bodies” are usually peri- or epipapillary and can be confused with optic disc drusen. Their size can range between one-half and four optic disk diameters. Their structure has a high acoustic density at B-scan ultrasonography due to calcifications in the lesions. The transitional lesions have a smooth margin appearance and can be multinodular in the center. Fluorescein angiography can help to recognize the vascularization pattern and to distinguish younger from older lesions. In most cases visual acuity is not affected due to extramacular localization of lesions. Visual field defects and a progressive loss of visual acuity can be associated with optic disc hamartomas for their mechanical pushing effect . Rowley Other features of the retina found in TSC are retinal pigmentary changes including both hyperpigmented areas  and areas of hypopigmentation with a “punched out” appearance that can be found both at the posterior pole and in the midperiphery . Rowley Shields et al. studied retinal hamartomas with time domain OCT in 15 patients and found typical characteristics such as hyperreflectivity and “round, confluent, moth-eaten empty spaces with posterior shadowing” on the surface of the tumor and disorganization of the internal retinal layers. Moreover, the authors observed a gradual progression from normal to tumorous retina in all cases, inner retinal disorganization in 3 cases (20%), outer retina disorganization in 0 cases (0%), and disorganization of the entire retina in 5 patients (33%). Furthermore, OCT showed moderate retinal traction over the tumor in 4 cases, optically empty spaces in 10 cases, and shadowing posterior to the tumor in 14 cases (93%). Shallow elevation or edema of the retina adjacent to the tumor was found in 13% and 27% of cases, respectively, and macular edema was found in 20% of cases . SolimanOther ocular findings reported by Rowley et al. included palpebral angiofibromas in 39% of patients, nonparalytic strabismus in 5% (4 exotropia and 1 esotropia), iris and choroidal colobomas in 3%, and areas of iris depigmentation in 2%. Myopia was present in 27%, hyperopia in 22%, and astigmatism (>0.75 D) in 27% of patients; such distribution of refractive errors is fairly similar to the distribution among normal individuals, as assessed by population studies . Lopez eIn a review by Mennel et al., flat lesions evolved to more prominent forms without symptoms in about 10% of patients but spontaneous regression was also reported . TherefoRetinal toxicity has been associated with the drug vigabatrin, which is often used as first-choice therapy in the treatment of refractory seizures typical of TSC . RetinalVon Hippel-Lindau (VHL) disease has an autosomal dominant transmission and is caused by a germline variation of the VHL tumor suppressor gene positioned on the short arm of chromosome 3p25-26 , 93. AppRCH are either benign vascular neoplastic processes or congenital lesions . They deOn ophthalmoscopic examination, RCHs are confined, globular, red-orange vascular tumors, typically observed in the juxtapapillary or the peripheral retina. They vary in size from 10 to 3000 microns or more. Peripheral lesions are most commonly found in the superior-temporal quadrant . In 15% Small peripheral RCHs are sometimes difficult to detect with funduscopy and are better seen with fluorescein angiography where lesions are characterized by hyperfluorescence. The feeder vessel will hyperfluoresce and is well evidenced in the arterial phase whereas the draining vein is clearly seen in the venous phase. In the late phases the tumor shows hyperfluorescence and leakage .Vessels in HB are fenestrated leading to exudation/leakage in contrast to normal retinal vessels. The differential diagnosis of RCH is with Wyburn-Mason disease (racemose hemangioma), Coats' disease, retinal macroaneurysm, vasoproliferative retinal tumor, and retinal cavernous hemangioma .Shields et al. described OCT characteristics of RCH, which showed thickening and structural alterations of the retina with mild shadowing of the underlying structures. These authors highlighted the importance of OCT in evaluating intra- and subretinal fluid; longstanding retinal edema in these lesions had features similar to cystoid macular edema whereas the chronic persistence of subretinal edema seemed to have caused a reduction of thickness of the layer corresponding to photoreceptors . Rarely Treatment of RCH usually includes laser photocoagulation, photodynamic therapy, cryotherapy, radiation, and surgical removal. In the literature various authors recommend early treatment of RCH when lesions tend to enlarge and become more difficult to treat with time . NeverthSingh et al. describe their experience in the management of RCH in 77 eyes of patients with VHL. Eighty-two percent of RCH were initially observed for a period of 84 months and the authors suggested only close observation in lesions smaller than 1500 microns, which are not visually threatening , 108. PhPhotodynamic therapy (PDT) is aimed at vascular endothelial cells and has also been employed in the management of RCH , 110. HoThe phakomatoses require a multidisciplinary approach and the ophthalmologist plays a fundamental role in diagnosis and management. Imaging techniques in ophthalmology have made groundbreaking progress in the past decade and NIR may lead to the inclusion of choroidal nodules as the 8th diagnostic criteria in NF1. OCT provides novel information on retinal hamartomas and enhanced depth imaging technology enables visualization of the choroid in vivo providing further information on the morphology of the choroid and choroidal nodules in NF1. Peripapillary RNFL analysis with OCT is a noninvasive tool and is an adjunct in monitoring optic pathway gliomas. Furthermore, hand-held OCT enables faster examination of the RNFL in children who are unable to collaborate due to their young age and neurological conditions. An enormous bulk of research has led to the establishment of optimal doses and protocols for intravitreal anti-VEGF treatment in retinal pathology, but little information is available for this treatment in the phakomatoses and reports on the use of anti-VEGF agents are contrasting and incomplete. The future might hold new drug delivery systems for the intravitreal treatment of symptomatic retinal hamartomas.


In light of literature evidence that embryonic stem cells (ESCs) are tolerant of DNMT mutations and advancement in the technology of gene targeting, it is now possible to introduce desired mutations in DNMT loci to generate suitable ESC lines that can help understand the underlying mechanisms by which abnormal levels of DNMTs or their specific mutations/alleles result in abnormal neurogenesis. In the future, these models can facilitate development of suitable drugs for treatment of neuropsychiatric disorders.Epigenetic mechanisms underlie differentiation of pluripotent stem cells into different lineages that contain identical genomes but express different sets of cell type-specific genes. Because of high discordance rates in monozygotic twins, epigenetic mechanisms are also implicated in development of neuropsychiatric disorders such as schizophrenia and autism. In support of this notion, increased levels of DNA methyltransferases (DNMTs), DNMT polymorphisms, and dysregulation of DNA methylation network were reported among schizophrenia patients. These results point to the importance of development of DNA methylation machinery-based models for studying the mechanism of abnormal neurogenesis due to certain DNMT alleles or dysregulated DNMTs. Achieving this goal is strongly confronted by embryonic lethality associated with altered levels of epigenetic machinery such as DNMT1 and expensive approaches in developing  During postblastocyst stage of mammalian development, the embryo undergoes implantation and the cells of the inner cell mass undergo lineage specification. As a result, cells of different lineages, despite being genetically identical, express unique set of genes that are lineage-specific while keeping the nonspecific genes silent. This difference in expression patterns is achieved due to the developmental cues in the embryo but is maintained in form of distinctive epigenetic patterns of the genomes. These epigenetic marks are heritable such that they are propagated in all the daughter cells within the lineage. de novo methyltransferases DNMT3A and DNMT3B can impart new methylation marks on unmethylated DNA, DNMT1 is responsible for maintenance of DNA methylation throughout life [ de novo and maintenance DNMTs are reflected in their expression patterns: DNMT3A and DNMT3B are involved in epigenetic reprogramming and expressed mainly in the germline [DNA methylation, histone modifications, and regulatory noncoding RNAs constitute the main epigenetic marks of mammalian genomes. Of these three, DNA methylation or covalent addition of methyl group at the fifth carbon of cytosines in genomic DNA is the earliest to be reported and most well studied in terms of its establishment, maintenance, and erasure . Most cout life . These cgermline , 7, plurgermline  whereas Because of their central role in creation and maintenance of DNA methylation patterns and the inverse correlation between DNA methylation and gene expression, DNMTs have been the subjects of intense research. In particular, the role of DNMTs and the influence of their dysregulation on the process of differentiation or maintenance of differentiated state are becoming unraveled in the recent past. Recent studies also indicate that dysregulation of DNMTs may be a disease-causing mechanism among patients with schizophrenia and bipolar disorders. Epigenetic mechanisms have been proposed to be involved in pathophysiology of schizophrenia and autism because of high discordance rates between monozygotic twins . Althoug in vitro and animal models developed by different investigators that have helped in gaining insights into the critical role of DNMTs in pluripotency and differentiation  basedntiation . I will  de novo methyltransferases whereas DNMT1 is a maintenance methyltransferase. In addition to these enzymes, DNMT3L is another DNMT family member which is catalytically inactive. DNMT3L interacts with DNMT3A and DNMT3B and is important for establishment of certain methylation marks in the germline. Once DNA methylation marks are established on unmethylated DNA, they are maintained by DNMT1 in a postreplicative manner (As mentioned above, DNMT3A and DNMT3B are thee manner . Dnmt1 were disrupted showed reduction in DNA methylation levels and normal growth rates and morphology, but embryos derived from them showed stunted growth and developmental delay and did not survive after midgestation [ Dnmt3Bnull/null embryos fail to develop to term due to developmental defects, Dnmt3Anull/null embryos survive to term but fail to survive beyond four weeks [ Dnm3Lnull/null mice survive but their conceptuses have DNA methylation defects. Dnmt3Lnull/null females show stochastic imprinting patterns in their oocytes [ Dnmt3Lnull/null males show loss of spermatogonia with wide-spread DNA methylation defects that reduce the chances of survival of their progeny [Indications that DNMTs are absolutely essential for development came from mouse transgenic studies. ESCs in which both alleles ofestation . Howeverestation . In subsestation . Similarur weeks . In cont oocytes  resultin progeny . Taken t Dnmt1c/c mouse ESCs that do not produce DNMT1 showed that in absence of DNMT1 ESCs do not lose their self-renewal capacity and their ability to initiate differentiation but the mutant cells do not survive differentiation [ Dnmt1c/c-derived neurons to survive [ Dnmt1 promoter [ Dnmt1 promoter and enable the expression of DNMT1 in ESCs [The functional roles of DNMTs in the differentiation program were only established recently. Transgenic studies in which a catalytically inactive DNMT1 mutant was expressed inntiation . For ins survive . In anotpromoter . These mpromoter . This st in ESCs . It is t in ESCs . Taken tExperiments using transgenic mice lacking DNMT3A and/or DNMT3B suggest that in addition to DNMT1, DNMT3A and DNMT3B also influence differentiation. For instance, in absence of DNMT3A and DNMT3B, ESCs retain their proliferative capacity and self-renewal but progressively lose DNA methylation . Interes de novo DNMTs in the brain samples with schizophrenia. Consistent with this expectation, overexpression of DNMT3A was also observed in GABAergic neurons of schizophrenia patients [ BDNF and GABAergic genes. This finding is in support of the hypothesis that overexpression of DNMT1 downregulates both specific GABAergic and glutamatergic genes [ REELIN, BDNF, GAD67, and the repressed GABAergic and glutamatergic genes is unknown. In addition to the reports that related overexpression of DNMT1 in brain samples from schizophrenia patients, certain alleles of Dnmt1, Dnmt3A, and Dnmt3L have been found to be associated with schizophrenia [Because of their central role in the properties of pluripotent and adult stem cells, and their differentiation potential, abnormal regulation of DNMTs or their mutations are expected to cause a broad spectrum of human disease conditions. However, their essential requirement during embryogenesis makes many of the mutations in DNMTs unrecoverable due to embryonic lethality . Mutatiopatients . Mechaniic genes . It is iophrenia . In summ in vivo models available to investigate the mechanistic basis by which overexpression of DNMTs or specific alleles of DNMTs cause phenotypes associated with schizophrenia. Animal models to investigate the mechanistic role of DNMT defects are very expensive as they require gene targeting experiments using conditional alleles that affect DNMTs in neuronal lineages. In addition, materials such as neuronal progenitors and neurons in early stages of differentiation are limited in their availability per animal and require multiple animals to be used in each study. In vitro models help address these problems because it is possible to genetically modify ESCs or neuronal progenitor cells, scale them up to required quantities, and induce differentiation into specific types of neurons, which in turn can be obtained in sufficient amounts. To this date, Dnmttet/tet ESCs constitute the only cell line in which DNMT1 overexpression in ESCs results in abnormal neuronal differentiation. This transgenic cell line was obtained by inserting tet-off cassettes into the endogenous Dnmt1 promoters of wild-type ESCs. As a result of transactivation mediated by the tet-off cassettes, the levels of DNMT1 in Dnmttet/tet ESCs are five times higher than the wild-type cells [ Dnmttet/tet neurons appear to “remember” that DNMT1 was overexpressed in ESC stage and this memory results in abnormal neurogenesis. While there is an ESC model to investigate the role of DNMT1 overexpression in abnormal neurogenesis, the Dnmt1tet/tet neurons do not have elevated levels of DNMT1 in neurons and therefore do not represent the neurons of schizophrenia patients with psychosis.At present, there are no suitablepe cells . These Epe cells . InteresFrom the description above, it is clear that there is an absolute need for development of suitable cell-based models to study the mechanisms by which elevated levels of DNMT1 and DNMT3A, or other DNMT family members, specific disease-associated DNMT alleles cause development of abnormal neurons. These models would then give us an opportunity to test whether the resultant neurons share any molecular/phenotypic features of neurons from schizophrenia patients or from patients with other psychiatric disorders. The task of cell-based models to generate neurons that overexpress DNMTs or express specific schizophrenia-associated DNMT variants can be accomplished with relative ease because ESCs are tolerant to DNMT levels and even loss of methylation . ESCs ar In vitro models offer a simple platform to investigate the disease-causing mechanisms because genetically altered ESC lines have well-defined mutations and, barring these specific changes, the remainder of the genome in each of these mutant cell lines is identical to the wild-type cells. As a result, the genotype-phenotype correlation would be clear and unequivocal. Following determination of the exact molecular defect, the in vitro models also become attractive tools for large-scale screening using potential drug targets to correct the abnormal neuronal phenotypes. Such high-throughput screens are difficult to perform with animal models. It is important to note that the details of affected cell types in the brain of schizophrenia patients are not well-established. In this context, in vitro differentiation of genetically altered ESCs might result in neuronal cell types that may not be representative of the cell types affected in schizophrenia. Although there is no complete picture, literature evidence suggests dysfunction of both GABAergic and Glutamatergic neurons in schizophrenia [The possibility of development of ESC models offers certain advantages and disadvantages in their utility to understand the mechanistic basis by which dysregulation of DNMTs is associated with disorders such as schizophrenia.ophrenia , 40. In ophrenia , 42. In Research in the recent past by different groups has uncovered the important role of DNMTs in schizophrenia. It is also possible that, in the future, more DNMT alleles will be associated with other neuropsychiatric disorders. However, investigation of the mechanisms by which the dysregulation of DNMTs results in these disorders using animal models can be highly expensive and laborious. The availability of suitable knockout cell lines, advancements in the technology for targeted gene modifications, and constitutive expression of cDNAs in stem cells together provide a viable alternative option to animal models. Using this approach, elucidation of the mechanistic basis by which altered DNMT levels or specific DNMT alleles cause abnormal neuronal differentiation can pave the way in the future towards possible therapeutic interventions to neuropsychiatric disorders.


PTPRJ) gene was identified as a common event in the lymphomagenesis of these B-cell lymphomas. The present study aimed to determine the cellular pathways affected by the inactivation of these TSGs including PTPRJ in FL and DLBCL tumourigenesis.The analysis of cellular networks and pathways involved in oncogenesis has increased our knowledge about the pathogenic mechanisms that underlie tumour biology and has unmasked new molecular targets that may lead to the design of better anti-cancer therapies. Recently, using a high resolution loss of heterozygosity (LOH) analysis, we identified a number of potential tumour suppressor genes (TSGs) within common LOH regions across cases suffering from two of the most common forms of Non-Hodgkin’s lymphoma (NHL), Follicular Lymphoma (FL) and Diffuse Large B-cell Lymphoma (DLBCL). From these studies LOH of the protein tyrosine phosphatase receptor type J . Moreover, upregulation of the VEGF, MAPK and ERBB signalling pathways was also observed in NHL cases with LOH of PTPRJ, indicating that LOH-driving events causing inactivation of PTPRJ, apart from possibly inducing a constitutive activation of these pathways by reduction or abrogation of its dephosphorylation activity, may also induce upregulation of these pathways when inactivated. This finding implicates these pathways in the lymphomagenesis and progression of FL and DLBCL.Pathway analytical approaches identified that candidate TSGs located within common LOH regions participate within cellular pathways, which may play a crucial role in FL and DLBCL lymphomagenesis . These analyses also identified genes within the interactome of PTPRJ (i.e. PTPN11 and B2M) that when inactivated in NHL may play an important role in tumourigenesis. We also detected genes that are differentially expressed in cases with and without LOH of The evidence obtained in this research supports findings suggesting that FL and DLBCL share common pathogenic mechanisms. Also, it indicates that PTPRJ can play a crucial role in the pathogenesis of these B-cell tumours and suggests that activation of PTPRJ might be an interesting novel chemotherapeutic target for the treatment of these B-cell tumours.The online version of this article (doi: 10.1186/1471-2164-15-390) contains supplementary material, which is available to authorized users. The identification of altered pathways in tumour cells has provided a more holistic understanding of the pathogenic mechanisms that underlie the genesis, progression and chemoresponse of cancer. As a consequence, malfunction of a gene or a group of genes must be analysed as part of a complex network of components that are highly related to each other. However, this analysis is limited by factors such as the lack of analytic tools to convert and integrate, in a feasible and reliable manner, large amounts of data derived from different high-throughput genomic approaches into outputs with more biological meaning.Non-Hodgkin’s lymphoma (NHL) represents a highly biological and clinical heterogeneous group of blood cancers , 2. Two In a previous study, the integration of copy number (CNV) and gene expression profiling (GEP) data from DLBCL and FL cases, allowed us to identify common and disease-specific genetic alterations targeting known oncogenic pathways, such as the mitogen activated protein kinase (MAPK) and apoptosis signalling pathways, unmasking common pathogenic mechanisms underlying the malignant phenotype of these biologically and genetically distinct NHL subtypes . LikewisPTPRJ (protein tyrosine phosphatase receptor type J) as a novel TSG in the tumourigenesis of FL and DLBCL, with LOH of PTPRJ identified as a common event in FL and DLBCL. LOH of PTPRJ was also confirmed by a decrease of heterozygosity of a microsatellite targeting PTPRJ loci in these NHL cases. In addition, FL cases with LOH exhibited a significant downregulation of PTPRJ [PTPRJ, as this protein tyrosine phosphatase has been implicated in the oncogenesis of breast, lung, colorectal, thyroid and meningioma cancers [In vitro experiments, using agonist peptides of PTPRJ and the oncogenic silencing of PTPRJ expression by microRNA-328 expression [As part of our recent LOH studies, we also implicated of PTPRJ . Several cancers –12. Furt cancers –19. None cancers , 21. Thipression –24.PTPRJ in order to provide a better understanding of the role of PTPRJ in the lymphomagenesis of FL and DLBCL. These analyses identified metabolic pathways as one of the most enriched and affected pathways resulting from the inactivation of candidate TSGs, indicating that these cellular pathways might play an important role in FL and DLBCL tumourigenesis. Additionally, inactivation of PTPRJ was shown to affect the expression of a number of genes and pathways that are regulated by PTPRJ through protein-protein interactions.Since the role of PTPRJ in normal and malignant B-cell differentiation is poorly understood, we used pathway and genomic analyses to identify cellular pathways that may be altered by the inactivation of MAPK6 (mitogen-activated protein kinase 6), PTPN11  and ANXA7 (annexin A7) showed the highest number of links with other genes . The high number of protein links of these proteins suggests that a possible inactivation of these genes may cause a major deregulation of patwhays by altering the function of a high number of genes. Thus, these genes are shown as the most interesting candidate TSGs in NHL lymphomagenesis.In order to determine whether the candidate tumour suppressor genes (TSGs) located within common LOH regions across DLBCL and FL cases interact and participate within common cellular networks, a global interactome of a total of 262 genes affected by LOH events , was creFASLG was classified into the cytokines and growth factor family and together with PTPRJ into the cell differentiation markers family. EP300 was found in the translocated cancer gene, transcription factor and tumour suppressor gene families. Additionally, MAPK6, PTPN11 and NFATC3 were identified as members of the protein kinases, oncogenes and transcription factors families, respectively.In addition to the global interactome study, a gene set enrichment analysis (GSEA) was performed to classify the 262 candidate TSGs targeted by LOH events, thenceforth also referred in this study as LOH genes, into curated gene families and also to determine the pathways that are mostly enriched with these genes. This approach categorized these LOH genes into gene families . Several cellular pathways were identified to be enriched with genes located within LOH regions , indicating that these candidate TSGs have been previously implicated in multiple forms of cancer  gene was implicated as a novel TSG in the lymphomagenesis of DLBCL and FL [PTPRJ might have an effect on mechanisms regulating the expression of PTPRJ [. This significant reduction in PTPRJ transcript abundance may result in haploinsufficiency of this TSG and thus, play a crucial role in the FL and DLBCL tumourigenesis. Based on this, we performed in silico pathway and gene expression analyses in order to provide more information about the unclear role and function of PTPRJ in normal and malignant B-cell development and to understand the effect of LOH of PTPRJ in the lymphomagenesis of FL and DLBCL from a more cellular perspective.In a recent study, the protein tyrosine phosphatase receptor type J , GRB2 (growth factor receptor-bound protein 2), ERBB2 , MET (met proto-oncogene (hepatocyte growth factor receptor), PDGFRB , CTNNB1 (catenin (cadherin-associated protein), beta 1, 88 kDa), PLCG1 , CBL (Cas-Br-M (murine) ecotropic retroviral transforming sequence), KIT , NTRK1 , KDR (kinase insert domain receptor (a type III receptor tyrosine kinase)), PXN (paxillin), GAB1 (GRB2-associated binding protein 1), LEPR (leptin receptor), EPOR (erythropoietin receptor), GHR (growth hormone receptor), CXCR4 (chemokine (C-X-C motif) receptor 4), CSK (c-src tyrosine kinase), INSR (insulin receptor), JAK2 (Janus kinase 2), LCK (lymphocyte-specific protein tyrosine kinase), GRIN2B , TEK  and TIE1 (tyrosine kinase with immunoglobulin-like and EGF-like domains 1). Interestingly, these 24 genes were found to significantly enrich important signalling pathways in lymphocyte biology, which suggests that a double inactivation of PTPRJ and PTPN11 might have an aberrant effect on the function of these pathways  data from NHL patients with known LOH status for PTPRJ , to detes Figure A, suggess Figure B and 3C.PTPRJ, we compared 262 candidate TSGs located within LOH regions against the 400 genes with differential expression between cases with retention and LOH of PTPRJ. This approach identified only the NFATC3  gene, located at 16q22, to be commonly inactivated and differentially expressed in NHL cases with LOH of PTPRJ . Furthermore, It is important to mention that LOH of NFATC3 was found in 58% of NHL, 48% of FL and 67% of DLBCL cases [In order to identify genes, previously found to be inactivated in our high resolution LOH study , and thaThe implementation of pathway analyses on candidate tumour suppressor genes (TSGs) found to be targeted by loss of heterozygosity in NHL, provided a holistic perspective of the shared cellular mechanisms underlying the lymphomagenesis of the commonest forms of NHL, FL and DLBCL. This analytical approach unmasked the pathways that are mostly altered by the genetic inactivation of TSGs potentially caused by LOH events in NHL and revealed common pathogenic mechanisms between FL and DLBCL. The analysis of candidate tumour suppressor genes, previously identified to be located within common LOH regions across FL and DLBCL cases , via intDisruption of metabolic pathways such as energy production or biosynthesis of amino acids, nucleosides, etc. are one of the hallmarks in cancer, as the malignant cells demand a higher activity in energy metabolism and biosynthesis in order to sustain their constitutive and accelerated proliferation and their malignant performance , 26. TheB2M was found to be inactivated in NHLs and its inactivation has been associated as a mechanism to evade the immune surveillance in DLBCL and other types of cancer [Using an annotated network database enriched with protein-protein interactions and associations, we were able to visualize the interactome of PTPRJ in relation to candidate TSGs located within LOH regions. This analysis determined that PTPRJ does not interact directly with any other possible LOH genes; however, this TSG indirectly interacts with 22 possible inactivated TSGs by LOH in NHL cases. This finding suggests that the malignant phenotype of tumoural B-cells is orchestrated by an uncontrolled activation of oncogenic signaling pathways, which may be caused by the inactivation of the dephosphorylation activity of PTPRJ together with the inactivation of other TSGs that might regulate direct targets of PTPRJ within these pathways. For instance, PTPRJ interacts directly with GRB2, a pivotal protein in signal transduction that activates the RAS-MAPK pathway, which is inactivated by the dephosphorylation activity of PTPRJ. GRB2 was also found to interact directly with B2M in an unclear manner . Interesf cancer , 31. In PTPN11, highly targeted by LOH events in NHL, was also identified in the PTPRJ interactome. PTPN11 encodes a protein tyrosine phosphatase (PTP) with two SH2 domains which acts as an intracellular signalling transducer of growth factors and cytokines receptors by regulation of the MAPK pathway [PTPN11 block the T-cell and B-cell development [PTPN11 gene have been detected in low frequencies in leukemia cases; however, alterations in this gene predispose patients with Noolan syndrome to several types of leukemia (mainly juvenile myelomonocytic leukemia JMML) and other types of cancer. [PTPRJ and PTPN11 may induce a constitutive activation of oncogenic pathways regulated by these PTPs. pathway . Furtherelopment . Mutatio cancer. –36. InteNFATC3 was identified as being significantly upregulated in NHL cases with LOH of PTPRJ. Interestingly, LOH of NFATC3 was identified to be very common across FL and DLBCL cases and copy-neutral events were found to be the driving cause of LOH of NFATC3 in these cases, based on the previously described high resolution LOH approach in these NHL cases [++ -dependent protein that regulates T-cell activation and migration, proliferation and angiogenesis by regulation of VEGF [+CD25+ T-cells (Treg cells) interact with activated CD4+ T-cells to suppress the anti-tumour activity of infiltrated CD4+ T-cells in B-cell NHL tumours and thus, induce immune tolerance to these tumours [reg cells also suppress the cytotoxic activity of CD8+ T-cells [B2M, the malignant B-cell-mediated recruitment of Treg cells and regulation of infiltrating CD4+, possibly through NFATC3, and CD8+ T-cells. Further studies are required to determine the role of NFATC3 and its inactivation in B-cell lymphomagenesis, the copy-neutral events driving LOH of NFATC3 and its relation with the inactivation of PTPRJ, which is known to inhibit the TCR-mediated T-cell activation by dephosphorylation of LAT and PLCG1 pathways [Additionally, the gene HL cases . NFATC3  of VEGF . Moreove of VEGF . In addi tumours . Interes T-cells . These fpathways , 41.PTPRJ, which suggests that the inactivation of PTPRJ might affect pathways whose ultimate goals are to switch on/off transcription factors and as a result of this, to induce an aberrant expression of these genes. In addition, using a Gene Set Enrichment Analysis (GSEA) of the differentially expressed genes between cases with LOH and retention; several pathways related to cancer and lymphocyte function were identified to be significantly upregulated in cases with LOH. This upregulation may be due to an indirect effect of the LOH of PTPRJ, which may cause a constitutive activation of pathways and may result in an abnormal expression of genes downstream of these pathways. Nonetheless, further functional studies  are required to validate these results and confirm the effect of LOH of PTPRJ in the expression of these oncogenic pathways.Although gene expression data was used to infer pathways of a protein tyrosine phosphatase, which lacks a DNA-binding regulatory motif, we were able to identify genes with different patterns of expression between cases with retention and LOH of ++ concentration [++ signalling pathways. Unexpectedly, signalling mediated by the Fc epsilon receptor I was found to be also upregulated in LOH cases; however, despite that this receptor for IgE is expressed in early stages of B-cells [For instance, the VEGF signalling pathway was found as the most significant upregulated pathway in cases with LOH of PTPRJ. This finding was expected as PTPRJ dephosphorylates VEGFR-2 and inhibits the VEGF-mediated cell proliferation, migration, angiogenesis and anti-apoptosis signalling . Malignantration , 47, it  B-cells , its relKi) by activation of MAPK pathway [PTPRJ transcript abundance between FL and DLBCL cases. This discrepancy may indicate differences in the LOH-driving events targeting this TSG in FLs and DLBCLs, which is supported by the significant downregulation of PTPRJ in FL cases, but not in DLBCL with LOH [On the other hand, the most enriched pathways in cases with retention of PTPRJ were DNA polymerase, TP53 signaling and proteasome pathways. It is likely that the upregulation of these pathways in cells with functional PTPRJ are the consequence of the activation of tumour suppressor activities, such as controlling cell proliferation and proapoptotic signalling through dephosphorylation of VEGFR, MAPK and PI3K pathways –18. The  pathway . Further pathway , 51, detPTPRJ. Furthermore, these findings suggest that PTPRJ plays a crucial role in the lymphomagenesis of FL and DLBCL as this TSG was found to induce aberrant expression of genes and PTPRJ-regulated pathways in NHL cases with LOH. Finally, it is important to highlight that the role of the most promising TSGs and pathways affected by LOH in FL and DLBCL tumourigenesis will need to be validated in further in vitro modeling experiments.In conclusion, the use of pathway analytical approaches has provided more evidence supporting the presence of common pathogenic mechanisms underlying the lymphomagenesis of FL and DLBCL. Specifically, this work identified genes and pathways affected by LOH of A total of 42 NHL cases, 21 DLBCL and 21 FL, were analysed using a high resolution LOH approach, which identified 46 common LOH regions across FL and DLBCL cases, harbouring 262 candidate tumour suppressor genes . The gloPTPRJ as a novel TSG in the lymphomagenesis of FL and DLBCL [PTPRJ on genes and pathways. Thus, to identify candidate TSGs affected by LOH events that interact directly or indirectly with PTPRJ and play a role together with PTPRJ, within common cellular networks, we extracted and analyzed the interactome of PTPRJ from the global interactome enriched with the 262 candidate TSGs [In order to extend on recently published findings implicating nd DLBCL  and to uate TSGs , using tate TSGs , 8.PTPRJ, we used our previously described linear gene expression profiling (GEP) data from cases (19 FLs and 20 DLBCLs) with a known LOH status (15 RET and 14 LOH) [PTPRJ and the 262 candidate TSGs identified within common LOH regions in NHL cases [NFATC3  between cases with retention and LOH of PTPRJ was carried out using an independent t-test (α < 0.05).To determine differentially expressed genes between cases with retention and LOH of  14 LOH) , 8. The  14 LOH)  was emplPTPRJ was performed using the above mentioned GEP data from NHL cases with a known LOH status (15 RET and 14 LOH) for PTPRJ in the GSEA v 2.0 software. The collection of curated pathways from the KEGG database and the default settings (without collapsing GEP data) were used for this analysis. Significance level was set at an α < 0.05.The identification of gene sets that were upregulated in cases with RET and LOH of Classification of LOH genes into curated gene families. (DOC 32 KB)Additional file 1: List of gene sets with chemical and genetic perturbations (CGP) enriched with genes within LOH regions. (DOC 46 KB)Additional file 2: Gene sets upregulated in cases with retention (RET) ofPTPRJ. (DOC 128 KB)Additional file 3: Gene sets upregulated in cases with LOH ofPTPRJ. (DOC 151 KB)Additional file 4:


In this review, we discuss molecular tools that enable the selective manipulation of astrocytic signaling, including the tools to selectively activate and inactivate astrocyte signaling in vivo. Lastly, we highlight a few tools in development that present strong potential for advancing our understanding of the role of astrocytes in physiology, behavior, and pathology.Astrocytes are the predominant glial type in the central nervous system and play important roles in assisting neuronal function and network activity. Astrocytes exhibit complex signaling systems that are essential for their normal function and the homeostasis of the neural network. Altered signaling in astrocytes is closely associated with neurological and psychiatric diseases, suggesting tremendous therapeutic potential of these cells. To further understand astrocyte function in health and disease, it is important to study astrocytic signaling  There are four major families of GPCRs that are distinguished by their composition, ability to activate intracellular signaling cascades, and the functional consequences associated with their activation. The importance of GPCRs is underscored by the fact that these molecules are the most common targeted class of proteins of therapeutic agents. Astrocytes express each of the major classes of GPCRs    , has also been used in cultured astrocytes, with again varying results  have been expressed in astrocytes, primarily to elicit Caes Table . In vivo2+ when using these optogenetic actuators in astrocytes. Extracellular Ca2+ entry through ChR2 variants seems the most logical mechanism, but a recent comparative study found that intracellular stores are the primary source  transgenic mouse line  was engineered through directed molecular evolution  were developed .The optogenetic and pharmacogenetic approaches for activating astrocyte loxp mice, was developed to study cell-specific disease mechanisms. In this model, the endogenous Mecp2 gene is silenced by insertion of a loxP-Stop cassette, but can be activated when combined with Cre- or Cre-ER system  in the brain  and lentiviral vectors , a GPCR predominately coupled to Gi signaling  conditional knockout mouse was generated to study the role of astrocytic GPCR signaling in aging-related neuroinflammation  regulated intracellular stores in the endoplasmic reticulum. Astrocytes express only one of three subtypes of this receptor, IP3R type 2 (IP3R2)  in the IP3R2 KO model , significantly reducing the number of astrocytes responding to Gq-GPCR activation or neuronal activity in multiple brain regions , which binds cytosolic IP3 molecules, was used to suppress astrocytic Ca2+ signaling in vitro and ATP-induced astrocytic Ca2+ responses in vivo D  and sequential release of IP3 are the key steps in Gq-GPCR mediated intracellular Ca2+ release in astrocytes in vivo . In this system, the expression of TeNT required Cre expression to remove the floxed stop cassette and tetracycline transactivator (tTA) to activate the TRE. TRE-loxP-STOP-loxP-TeNTΔ1-GFP mice were crossed with GFAP-tTA and GFAP-CreERt2 lines to create a triple transgenic mouse line. In the triple transgenic mice TeNT can be expressed in astrocytes following tamoxifen treatment; the expression of TeNT is suppressed in the presence of doxycycline. In this model, astrocyte expression of TeNT requires that two different transgenes driven by the GFAP promoter be expressed in the same astrocyte markedly increasing the probability of astrocyte specific expression. Further, as TeNT was directly tagged with eGFP, it is possible to identify all cells expressing TeNT. In contrast to the impaired sleep pattern in dnSNARE mice, astrocytic TeNT-expressing mice showed unaltered sleep patterns compared to triple transgenic mice without tamoxifen or double transgenic mice with tamoxifin  are known to interfere with synaptic vesicular release as well as other processes dependent on vesicular protein trafficking  knockout model has become an attractive model for studying the role of Glt-1 in vivo. The Glt-1 Het model does not suffer from the more obvious adverse effects of the full Glt-1 knockout, and displays several interesting behavioral phenotypes and has allowed for the study of Glt-1 in several brain pathologies  gap junctions. Astrocytes express two major connexin proteins (Cx43 and Cx30) with germline and conditional knockout mouse models existing , it is very important to consider the consequences of expression during development.At this time, there is not a single gene delivery system that can be used to exclusively express transgenes or recombine naïve genes in astrocytes. The common astrocyte marker proteins  are either expressed in alternate subsets of mature CNS cells  and opto-beta-1 (Gs linked)  is conditionally inactivated in Gα11 KO (gna11–/–) mice, which do not have obvious behavior defect  to generate forebrain specific and neuronal Gαq/Gα11 double KO mice  available for expressing molecules in astrocytes. Currently, all available transcriptional units drive transgene expression in cells other than astrocytes. Further, during development, most astrocyte promoters drive gene expression in progenitor cells that give rise to neurons, oligodendrocytes, and astrocytes  may yield different results. These conflicts require additional studies using multiple tools  to refine our understanding of astrocyte function. Currently, there are several important limitations in the genetic tools available for perturbing astrocyte function In summary, while genetic manipulation of astrocytes is in its early stages of development, genetic models have already provided important insight into the role of astrocytes in physiology, behavior, and neurological diseases. It seems very likely that future advances in this field will depend largely on genetic approaches currently available as well as those under development.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.