Supplemental Digital Content is available in the text The presence of multiple primary tumors (MPT) in a single patient has been identified with an increasing frequency. A critical issue is to establish if the second tumor represents an independent primary cancer or a metastasis. Therefore, the assessment of MPT clonal origin might help understand the disease behavior and improve the management/prognosis of the patient.Herein, we report a 73-year-old male smoker who developed 2 primary lung cancers (adenocarcinoma and squamous cell carcinoma) and a malignant peritoneal mesothelioma (PM).Whole exome sequencing (WES) of the 3 tumors and of germline DNA was performed to determine the clonal origin and identify genetic cancer susceptibility.WT1 gene that might be involved in the carcinogenesis of nonasbestos-related mesothelioma. Finally, WES of the germinal DNA displayed several single nucleotide polymorphisms in DNA repair genes likely conferring higher cancer susceptibility.Both lung cancers were characterized by a high mutational rate with distinct mutational profiles and activation of tumor-specific pathways. Conversely, the PM harbored a relative low number of genetic variants and a novel mutation in the Overall, WES did not disclose any somatic genetic variant shared across the 3 tumors, suggesting their clonal independency; however, the carcinogenic effect of smoke combined with a deficiency in DNA repair genes and the patient advanced age might have been responsible for the MPT development. This case highlights the WES importance to define the clonal origin of MPT and susceptibility to cancer. Currently, the main criteria for defining the lineage of multiple unrelated intrapulmonary tumors compared with metastatic lesions are based on pathological and clinical assessments.,4 To date, several studies have described MPLC cases, but most of them have analyzed a limited number of genetic markers, resulting in a low accuracy and limited ability to establish cancers clonality.,6 Next generation sequencing (NGS) is a recent technology that can contribute to understanding the molecular mechanisms underlying tumor development by screening the whole DNA mutational profile.–10 Recently, Murphy et al applied the NGS approach to define the lineage of MPLC, demonstrating how genomic rearrangements were able to distinguish MPLC from metastatic lesions; however, the authors did not evaluate somatic and germinal mutational profiles. Once established that MPLC are primary and independent tumors, understanding the intrinsic genetic susceptibility to develop multiple cancers during the lifetime is crucial; indeed, those subjects with high predisposition might be enrolled in prevention programs and benefit from personalized follow-ups. Herein, we report an interesting case of a patient that developed 2 primary histologically distinct lung tumors and a malignant PM after 6 years. WES allowed us to deeply screen the 3 tumors, in order to identify a mutational signature specific for each malignancy and to establish the clonal origin of cancers. Concomitantly, the sequencing of normal genomic DNA (gDNA) allowed the identification of germline genetic variants potentially correlated with an individual risk of developing multiple cancers.The incidence of multiple primary tumors (MPT) during an individual's lifetime is increasing, mainly due to the advent of accurate cancer secondary prevention programs and the increase of life expectancy for cancer patients. The development of multiple primary lung cancers (MPLC) is an uncommon event, although the improvement in the diagnostic tests and novel therapies able to influence survival after the first diagnosis of cancer have led to an incidence peak that has grown up to 20% over the past 10 years.2We describe the case of a Caucasian male patient with a medical history of heavy smoking habit (100 pack-years), chronic obstructive pulmonary disease (COPD), and no exposure to asbestos. Before being referred to our unit, the patient was initially followed and treated in a different institution; hence, part of the patient's oncologic history was retrospectively retraced when he came to our attention Fig. A. The im2), which was discontinued after 2 cycles due to poor tolerance. Then, the patient experienced progressive worsening of clinical conditions and died in March 2015. Relevant images from CT-scans collected throughout the clinical history of the patients have been reported in Fig. The indeterminate nodule in the right upper lobe remained stable until September 2010, when an increase of its maximum diameters from 22 to 33 mm was reported; a positron emission tomography (PET) scan showed fluorodeoxyglucose (FDG) uptake limited to the right upper lobe lesion (SUV max: 6.2). Following this finding, the lesion was considered a metastasis of the original ADC and subsequently the patient received chemotherapy with carboplatin (AUC2), which was discontinued after 2 cycles for thrombocytopenia. During the subsequent assessments, the pulmonary lesion was substantially stable until November 2011, when a significant dimensional increase was observed. A subsequent PET-scan confirmed the right lung lesion as the only clearly detectable active site of disease , while no distant metastases were identified; therefore, surgery with potential curative intent for oligo-metastatic disease was proposed. Hence, in January 2012, the patient underwent right upper lobectomy and radical lymphadenectomy with postoperative diagnosis of keratinizing and moderately differentiated squamous cell carcinoma (SCC) of the lung with positivity for p63 at IHC  .–15 For this analysis, the ADC and the SCC samples were collected from stored surgical specimens , while the PM sample derived from the tissue collected during the abdominal biopsy.In order to understand whether ADC, SCC, and PM were unrelated cancers or shared a common clonal evolution, WES analysis was performed on the 3 tumors by HiSeq 2500 sequencer  as already described. This analysis displayed a predominance of C>A transversions in both lung cancers (ADC and SCC)  database and described in lung cancers: EHHADH (COSM5247826), KRAS (COSM512), OR4K2 (COSM1515038), and TP53 (COSM6549) in ADC; KIAA1324L (COSM396629), NFE2L2 (COSM396629), PEG3 (COSM5284477), POM121L12 (COSM393793), and WAC (COSM5311283) in SCC. Moreover, both histotypes carried mutations associated with potential therapeutic targets , or in a predictor of resistance to EGFR tyrosine kinase inhibitors (KRAS in ADC).We firstly extracted the somatic mutational signature from all the tumors according to base substitutions, as already described by Alexandrov et al.CC) Fig. A, corresnts Fig. A. Then, http://amp.pharm.mssm.edu/Enrichr/) also showed that different pathways were deregulated in ADC and SCC. Specifically, ADC was enriched with altered genes belonging to the MAPK pathway , whereas the mutations observed in SCC mostly affected genes involved in collagen modification, in extracellular matrix organization , and in the meiotic synapsis pathway . Conversely, the PM did not display COSMIC mutations or pathways associated with the carcinogenesis, probably due to the low number of somatic mutations (28); however, among these mutations, we identified 3 novel variants including 2 frameshift variants  and a missense variant (p.Ser71Phe WT1).The enrichment analysis using Reactome 2015 (http://snpeff.sourceforge.net).Germline analysis was also performed in order to discover genetic variants potentially linked to cancer predisposition. Germinal gDNA sequencing identified a total of 31,608 genetic variants of which 15,790 and 15,818 occurred in exons and nonexons regions, respectively Fig. B. In parEME1; rs26279, MSH3; rs8305, POLI; rs373572, RAD18; rs462779, REV3L; rs25487, XRCC1; rs1143634, IL1B)  involved in the etiology of the nicotine dependence  of 38 genes significantly mutated in a cohort of 660 lung ADC. Of note, mutation in PPP3CA co-occurred with an activating KRAS mutation (COSM512) as already described by Campbell et al. In addition, the mutation in the MGAM gene has been also observed in a comprehensive genome-wide characterization by Cancer Genome Atlas Research Network among 18 genes found significantly mutated in 230 lung ADC tumors. Across the 405 SCC-mutated genes, we found only 1 gene (NFE2L2) of 20 genes recurrent mutated in 484 lung SCC tumors; moreover, mutations in NFE3L2 gene have also been identified in 34% of 178 lung SCC tumors profiled by Cancer Genome Atlas Research Networt.Here, we describe an infrequent case of a patient who developed 2 histological distinct intrapulmonary tumors and a PM after 6 years. WES of the 3 tumors was performed to establish a clonal relationship. Although both lung ADC and SCC showed a similar mutational signature, characterized by a prominence of C>A substitutions, they did not share common somatic variants. Interestingly, the signature characterized by C>A mutations has been associated with smoke exposure in several cancers including lung ADC and SCCMAP3K4 and MAP2K1 genes, and a hotspot mutation in the KRAS codon 12; as it is known, the EGFR signaling pathway is one of the most frequently altered pathways in this histology. On the contrary, the SCC carried several mutations in genes involved in the extracellular matrix organization, a pathway often deregulated in cancer. In particular, we found a novel frameshift deletion  leading to a potential LOX protein destruction. LOX downmodulation has been found in SCC and its lack has been shown to induce the extracellular matrix disorganization leading to tumor development. Furthermore, in addition to being potentially involved in tumor development, some of the affected genes that were observed in this case might also play a relevant role in a targeted therapy approach in patients affected by lung cancer, possibly reducing sensitivity to currently registered agents or eventually representing potential targets for drugs that might become available for lung cancer in future. Although it is still unclear whether KRAS mutations are actually associated with resistance to EGFR inhibitors in lung cancer, aberrations of HGF signal are apparently involved in resistance to anti-EGFR and anti-VEGF targeted therapies. Contrarily, FLT-3 and mTOR might represent potentially actionable targets, as the former is sensitive to drugs such as dovitinib, while the latter is sensitive to everolimus.Furthermore, both lung tumors showed a specific gene signature linked to distinct pathways of activation. Specifically, the ADC harbored mutations in genes involved in EGFR signaling pathway, such as 2 novel genetic variants in the 3’UTR regions of BAP1 as the most altered gene. We also found an insertion in BAP1, potentially associated with a loss-of-function, and a deletion changing the reading frame in SETD2, a gene found altered in malignant pleural mesothelioma. In addition, we detected a novel mutation in the WT1 transactivation domain . Mutated WT1 has been already described in mesothelioma; interestingly, Park et al reported a patient with PM that harbored a point mutation within the transactivation domain of WT1 gene, demonstrating that this variant conferred an activation of its transcriptional role. However, the authors did not find any WT1 mutations in a further set of 32 asbestos-related mesothelioma patients, thus concluding that the WT1 pathway could be involved in the malignant transformation of nonasbestos-related mesothelioma. These data suggest that the p.Ser71Phe WT1 mutation might be implicated in the PM carcinogenesis process through the WT1 downstream pathway activation. Indeed, the mutation serine-71-phenylalanine (p.Ser71Phe) in WT1 gene is a nonconservative mutation that alters the properties of the protein by replacing the small and polar serine with the large and bulky side chain of a phenylalanine.Conversely, in PM, the distribution of base substitutions did not match any specific mutational signature, probably as a consequence of a relatively limited number of observed mutations (28 variants in PM vs >350 in the lung cancer lesions). Peritoneal mesothelioma is an extremely rare tumor and our sequencing data were in accordance with a previous study in which the authors performed WES on 7 PM finding a low mutational rate and EXO1 gene (rs1047840) with cigarette smoking has been described as conferring a significantly increased lung cancer risk, with a reported odds ratio equal to 1.72.According to the previous data and excluding a common lineage across the 3 tumors, we hypothesized that this patient could have an intrinsic predisposition to develop MPT. Indeed, the germinal gDNA sequencing showed that more than half of the variants were potentially associated with protein alterations. Notably, the analysis identified 21 genetic variants that were already described; of these, 62% were related to increased lung cancer risk. Among such variants, the association of the p.Glu589Lys in  Despite the only 2 cycles of carboplatin, considering the interval between treatment and SCC tumor collection (about 14 months), we cannot exclude the mutagenic effect induced by carboplatin.To the best of our knowledge, this is the first study that investigates the whole exome mutational profile of 3 MPT aimed at defining the clonal origin of the tumor lesions and also the germline assets in order to discover an individual genetic susceptibility to cancer predisposition. Our data support the hypothesis that the development of the 3 tumors was clonally independent, as they do not share a common mutational profile; however, we could not exclude the presence of mutations in regulatory regions, omitted by WES. The patient also carried several SNPs involved in nicotine dependence and DNA repair. The carcinogenic effects of tobacco smoke together with both a DNA repair deficiency and the advanced age of the patient may have led to a high mutation rate in the lung cancer lesions. It is also known that chemotherapy might affect the mutational status of eukaryote cells.BAP1 and SETD2) and that other mechanisms, such as microRNA deregulation, might be involved. In addition, the novel missense mutation in WT1 gene may also explain the PM development regardless of asbestos exposure.On the contrary, the low number of somatic mutations in PM suggests that its development is mainly caused by onset of mutations in driver genes (In conclusion, this study underlines how the germline assets could influence the cancer predisposition and how future WES studies on patients with MPT should be directed toward the genetic variants identification leading to cancer susceptibility. Our findings highlight the power of WES analysis in screening the mutational landscapes of patient with MPT in order to define the clonal feature and identify novel potential molecular targets for treatment.


ANTXR2, STK3, PDK4, CD163, MAL, GRAP, ID3, CTSZ, KIF1B and PLXDC2) whose coordinate pattern of expression was able to identify 98.4% of discovery cohort subjects correctly . In the validation cohort, the expression levels of the same 10 genes were able to identify 95.6% of subjects correctly when comparing AIS patients to asymptomatic controls , and 94.9% of subjects correctly when comparing AIS patients with stroke mimics . The transcriptional pattern identified in this study shows strong diagnostic potential, and warrants further evaluation to determine its true clinical efficacy.Early and accurate diagnosis of stroke improves the probability of positive outcome. The objective of this study was to identify a pattern of gene expression in peripheral blood that could potentially be optimised to expedite the diagnosis of acute ischaemic stroke (AIS). A discovery cohort was recruited consisting of 39 AIS patients and 24 neurologically asymptomatic controls. Peripheral blood was sampled at emergency department admission, and genome-wide expression profiling was performed via microarray. A machine-learning technique known as genetic algorithm k-nearest neighbours (GA/kNN) was then used to identify a pattern of gene expression that could optimally discriminate between groups. This pattern of expression was then assessed via qRT-PCR in an independent validation cohort, where it was evaluated for its ability to discriminate between an additional 39 AIS patients and 30 neurologically asymptomatic controls, as well as 20 acute stroke mimics. GA/kNN identified 10 genes ( Furthermore, such imaging techniques are often not immediately available in hospitals without dedicated stroke centres, such as smaller facilities and those which serve rural areas.5 As a result, crucial decisions regarding the triage of potential strokes by emergency department staff and emergency medical technicians are based on the assessment of overt patient symptoms using stroke recognition and severity scales such as the Cincinnati pre-hospital stroke scale (CPSS) and the National Institutes of Health stroke scale (NIHSS).4 In the hospital setting, the ability to identify stroke with such assessments is highly inconsistent, with an estimated sensitivity ranging from 44 to 85%, and specificity ranging from 64 and 98%.6 The sensitivity and specificity of these assessments are even lower in the pre-hospital setting,7 where the ability to quickly identify stroke facilitates the transfer of patients to stroke-ready hospitals, increasing the chances of appropriate treatment and positive outcome.8 Due to these current limitations, a rapidly measurable blood-based biomarker panel could be invaluable in informing pre-hospital and in-hospital decisions early in the acute phase of care, and could ultimately expedite access to interventional treatment.9Although neuroradiological imaging is the gold standard for diagnosis of stroke,9–13 More recently, the peripheral blood transcriptome has emerged as a potential source of stroke biomarkers, as preliminary reports have suggested that gene expression in the peripheral immune system is highly responsive to ischaemic brain injury.14–16 Most notably, Tang et al. identified a panel of 18 genes whose expression levels demonstrated the ability to discriminate between acute ischaemic stroke patients (AIS) and healthy controls with 93.5% sensitivity and 89.5% specificity using combined expression data generated from three blood draws obtained over the first 24 h of hospitalisation.17 While the necessity to obtain multiple blood samples limited this biomarker panel with regards to acute stroke triage, this work provided proof of principle that stroke-induced transcriptional changes in the peripheral immune system could be used to identify stroke with relatively high levels of accuracy. Thus, it is plausible that implementation of a robust biomarker discovery approach could identify transcriptional stroke markers with the potential to be diagnostically useful during the acute phase of care.As a result, there has been a substantial push for the identification of stroke-associated peripheral blood biomarkers. The earliest stroke biomarker studies focused on the peripheral blood proteome, and countless protein-based biomarker panels have been evaluated to date. While a handful of these protein-based panels have demonstrated a strong ability to differentiate between stroke patients and healthy controls lacking the presence of cardiovascular disease (CVD) risk factors, a majority have failed to achieve specificities and sensitivities approaching 90% when tested against clinically relevant control groups.18–20 The GA/kNN approach combines a powerful search heuristic, GA, with a non-parametric classification method, kNN. In GA/kNN analysis, a small combination of genes (referred to as a chromosome) is generated by random selection from the total pool of gene expression data  has been successfully used in a small number of cancer studies to identify diagnostically relevant biomarker panels with strong discriminatory ability.ion data . The abiion data . The abiion data . This enion data . The dision data , and theion data . This apWhile GA/kNN has proven robust in several applications in the field of cancer, it has yet to be utilised for biomarker discovery in the realm of cardiovascular disease (CVD). In this study, we applied the GA/kNN approach to analyse peripheral blood gene expression data generated via microarray to identify transcriptional patterns which could potentially be optimised for the detection of AIS in the acute phase of care.In order to identify potential transcriptional biomarkers for the identification of AIS, we first recruited a discovery cohort consisting of 39 AIS patients and 24 neurologically asymptomatic controls. In terms of demographic and clinical characteristics, AIS patients were older than controls, and displayed a higher prevalence of CVD risk factors such as hypertension and dyslipidaemia . FurtherANTXR2, STK3, PDK4, CD163, MAL, GRAP, ID3, CTSZ, KIF1B and PLXDC2) were able to classify 98.4% of subjects in the discovery cohort correctly with a sensitivity of 97.4% and specificity of 100%  method to estimate the relative contributions of each regressor to the total variance in composite RNA expression explained by the resultant regression model.he model , and washe model . In termhe model ; howeverhe model . Resultshe model . Taken aWe next used this composite RNA expression variable to examine the potential influence of stroke severity and time to blood draw on the pattern of gene expression observed across the top 10 genes. The composite RNA expression levels of the top 10 genes displayed a significant positive association with stroke severity as assessed by the NIHSS , suggestWe then tested the diagnostic ability of gene expression pattern identified in the discovery cohort in an independent validation cohort enroled via a second geographically and socioeconomically distinct clinical site . This validation cohort included an additional 39 AIS patients along with two different control groups, one consisting of 30 neurologically asymptomatic controls and the other consisting of 20 acute stroke mimics. Like in the discovery cohort, AIS patients were older than neurologically asymptomatic controls; however, AIS patients and asymptomatic controls were better matched in terms of the prevalence of comorbidities and CVD risk factors . AIS patPeripheral blood samples were once again obtained from patients at emergency department admission, and the expression levels of the top 10 genes identified by GA/kNN in the discovery cohort were measured via qRT-PCR. The overall pattern of differential expression between AIS patients and asymptomatic controls observed across the top 10 genes in the discovery cohort was also seen when comparing AIS patients and asymptomatic controls in the validation cohort . The strWhen comparing AIS patients to stroke mimics, the overall pattern of differential expression observed across the top 10 genes was identical to that observed when comparing AIS patients with asymptomatic controls; however, the magnitude of these expression differences was smaller in the case of several genes . DespiteThe primary objective of this study was to apply the GA/kNN approach to identify a pattern of gene expression in peripheral blood that could potentially be optimised to identify AIS in the acute phase of care. The 10 transcriptional markers identified by GA/kNN in our analysis proved robust in their combined ability to differentiate between AIS patients and controls in both the discovery cohort and the independent validation cohort; not only did these markers display levels of diagnostic accuracy that exceed those reported in a majority of previous stroke biomarker studies, they also demonstrated characteristics that suggest they have the potential to be clinically useful. Besides having diagnostic utility, some of the markers identified in this study may represent viable therapeutic targets in the context of stroke immunopathology.et al.N-methyl-D-aspartate receptor cleavage, could be used to differentiate between stroke patients and a combination of acute stroke mimics and neurologically asymptomatic controls with 92% sensitivity and 96% specificity.22 However, a majority of blood samples in this prior study were obtained between 24 and 72 h post-symptom onset, and it is currently unknown whether NR2 peptide would exhibit an equivalent level of diagnostic performance early in the acute phase of care. The 10-marker panel identified in our analysis was tested earlier in the progression of pathology, and thus exhibits an obvious advantage in that they has the potential to provide actionable diagnostic information at an early enough time point to influence critical triage decisions that has an impact on outcome.With regards to the countless number of peripheral blood biomarker explorations that have been performed to date, to our knowledge, only one prior investigation has reported similar levels of diagnostic accuracy to those which we observed in this study in terms discriminating between stroke patients and clinically relevant control populations. Dambinova 23 For this reason, a potential diagnostic for identification of acute stroke needs to be diagnostically robust across a wide time window with regards to the progression of stroke pathology. Another diagnostically beneficial characteristic we observed was that the stroke-associated pattern of expression across these 10 markers was positively correlated with the NIHSS. Thus, these markers may have utility in stratifying injury severity, information that is commonly considered when making decisions regarding the prescription of interventional treatment.4 These characteristics, along with the fact that we observed levels of sensitivity and specificity, which well exceed those achievable via the tools currently available to clinicians for the identification of stroke during acute triage, suggest that the 10-marker panel identified in our analysis has legitimate potential for future clinical implementation.The 10-marker panel identified in our analysis displayed several favourable characteristics that could make it well suited for identification of ischaemic stroke in the acute care setting. Most notably, the pattern of differential expression we observed between AIS patients and controls appeared to be relatively temporally stable. This is of clinical relevance from the standpoint that it is well established that acute stroke patients tend to arrive to the emergency department in two waves, the first within 4 h from symptom onset , and the second more than 8 h from symptom onset .CD163. It is well established that stroke induces a state of peripheral adaptive immune suppression characterised by a limited capacity of lymphoid cells to respond to antigen.25 This suppressed adaptive immune state leaves patients highly susceptible to post-stroke infection,26 which is the leading cause of death in the post-acute phase of care.27CD163 encodes for a protein known as cluster of differentiation 163 (CD163), a membrane-bound scavenger receptor for extracellular haemoglobin, which is predominantly expressed on immune populations of myeloid lineage.29 Mature CD163 is known to undergo ectodomain shedding to generate a soluble truncated peptide (sCD163), which has been shown in multiple studies to directly interact with lymphocytes and inhibit antigen-mediated activation.30–32 Interestingly, we observed elevated RNA expression levels of CD163 in the peripheral blood of AIS patients; it is possible that CD163 expression is increased in the innate peripheral immune system in response to stroke-induced increases in circulating free haemoglobin,33 subsequently driving an increase in levels of circulating sCD163, which act to suppress lymphocyte activation. In support of this hypothesis, unpublished preliminary data from our laboratory suggest that plasma levels of sCD163 are elevated in AIS patients during the acute phase of care, and are positively correlated with RNA expression levels of CD163 in whole blood. Ongoing work in our laboratory is aimed at characterising the relationship between peripheral-blood sCD163 levels and stroke-induced adaptive immune dysfunction, as CD163 may be therapeutically targetable as a means of rescuing adaptive immune responsiveness following stroke.Besides having diagnostic utility, some of the markers identified in this study may represent potential therapeutic targets in the context of stroke immunopathology. Perhaps, the most interesting of these markers from this standpoint is CD163, the markers identified in this study included several other genes that may be pathologically relevant within the context of the stroke-induced peripheral immune response. We observed downregulated expression levels of MAL and GRAP in the peripheral blood of AIS patients; both genes encode proteins that are critically involved in T-cell receptor activation and signal transduction.35 Furthermore, AIS patients exhibited elevated expression levels of STK3, a gene encoding a seine threonine kinase involved in pro-apoptotic signal transduction37 and suppression of lymphocyte proliferation.38 Taken as a whole, the differential regulation we observed across these genes is consistent with suppressed adaptive immune state induced in response to stroke, and may be mechanistically involved in blunting the responsiveness of the adaptive immune system following ischaemic brain injury. Conversely, two of the markers identified as being upregulated in the peripheral blood of AIS patients in this study, KIF1B and ANTXR2, may be mechanistically involved in the innate immune response to ischaemic insult. It is well established that stroke induces robust recruitment of myeloid-derived innate immune populations such as neutrophils and monocytes from the peripheral blood into the brain parenchyma;40 both genes encode proteins that have been shown to have a role in cellular adhesion and migration,41–44 and thus may be mechanistically involved in this process.In addition to Collectively, the findings reported here are exciting; however, it is important to note that this study was not without limitations. Perhaps, most notably was the fact that AIS patients and neurologically asymptomatic controls in our discovery cohort were not well matched with regards to several clinical and demographic characteristics; thus, intergroup differences in these factors had the potential to confound the selection of stroke-specific genes in our GA/kNN analysis. To account for this possible limitation, we utilised a relatively high termination cutoff for optimal solution selection; under these conditions, a confounding factor would have to be almost ubiquitously present in one group, and nearly ubiquitously absent in the other, for it to influence the selection of candidate genes. The results of our multiple regression analysis suggest that this strategy was largely successful; however, they did infer that medication status may influence the expression of the candidate genes. Despite this, the 10 candidate genes were still able to demonstrate high levels of diagnostic accuracy when discriminating between groups that were better matched in terms of these factors in the validation cohort.Taken as a whole, the results of this preliminary study demonstrate that a highly accurate RNA-based companion diagnostic for AIS is plausible using a relatively small number of markers, and also highlight the potential power of machine-learning approaches for biomarker discovery in the realm of CVD. The 10 transcriptional biomarkers identified in this study displayed levels of diagnostic performance that well exceed those reported in a majority of previous stroke biomarker investigations, as well as several characteristics that suggest that they may have true clinical utility for identification of ischaemic stroke during the acute phase of care. Furthermore, future exploration of these markers may reveal novel mechanisms that underlie the peripheral immune response to stroke, and lead to novel therapeutic targets in the context of stroke-induced immunopathology. Owing to the robust results of this preliminary analysis, the 10 transcriptional biomarkers identified in this study warrant further evaluation to determine their true clinical efficacy.45 The median time from symptom onset to blood draw was 5.3 h, as determined by the time the patient was last known to be free of AIS symptoms. In the case of patients who received thrombolytic therapy, blood samples were collected before the administration of recombinant tissue plasminogen activator. Injury severity was determined according to NIHSS at the time of blood draw. Control subjects were deemed neurologically normal by a trained neurologist at the time of enrolment. Demographic information was collected from either the subject or significant other by a trained clinician. All procedures were approved by the institutional review boards of the National Institute of Neurological Disorders/National Institute on Aging at the National Institutes of Health and Suburban Hospital. Written informed consent was obtained from all subjects or their authorised representatives before any study procedures.Acute ischaemic stroke patients and neurologically asymptomatic controls were recruited at Suburban Hospital, Bethesda, MD, USA, which serves an upper-class metro area bordering Washington DC. AIS cases were of mixed aetiology, and diagnosis was confirmed using magnetic resonance imaging according to the established criteria for diagnosis of acute ischaemic cerebrovascular syndrome.Peripheral whole-blood samples were collected via PAXgene RNA tubes  and stored at −80 °C until RNA extraction. Total RNA was extracted via the PreAnalytiX PAXgene blood RNA Kit (Qiagen) and automated using the QIAcube System (Qiagen). Quantity and purity of isolated RNA was determined via spectrophotometry . Quality of RNA was confirmed by chip capillary electrophoresis .RNA was amplified and biotinylated using the TotalPrep RNA Amplification Kit . Samples were hybridised to HumanRef-8 expression bead chips  containing 25,000 unique probes and scanned using the Illumina BeadStation. Raw probe intensities were background-subtracted, quantile-normalised and then summarised at the gene level using Illumina GenomeStudio. Sample labelling, hybridisation and scanning were performed per standard Illumina protocols. Raw data are assessable through the National Center for Biotechnology Information Gene Expression Omnibus via accession number GSE16561.z-transformed and GA/kNN analysis was performed using C source code developed by Li et al.20 compiled in Linux Mint. Two thousand near-optimal solutions were collected per sample using five nearest neighbours, majority rule, a chromosome length of five and a termination cutoff of 0.97. Leave-one-out cross-validation was performed using the top 50 ranked genes. The top 50 genes were tested against random gene combinations, which were selected using the R sample function .Normalised microarray data were filtered based on absolute fold difference between stroke and control; genes exhibiting a greater than 1.7 absolute fold difference in expression between AIS and control were retained for analysis. Filtered gene expression data were 1 As with the discovery cohort, AIS cases were of mixed aetiology, and diagnosis was confirmed via neuroradiological imaging. Patients admitted to the emergency department as suspected strokes based on the overt presentation of stroke-like symptoms, but receiving a negative diagnosis for stroke upon imaging according to the established acute ischaemic cerebrovascular syndrome diagnostic criteria,45 were identified as acute stroke mimics. Discharge diagnoses of stroke mimics included cases of seizures, complex migraines and other conditions, which induce neurological symptoms such as hypertensive encephalopathy. The median time from symptom onset to blood draw was 4.6 h and all blood was sampled before the administration of recombinant tissue plasminogen activator. Assessment of injury severity, screening of neurologically asymptomatic controls and collection of demographic information were performed in an identical manner. All procedures were approved by the institutional review boards of West Virginia University and Ruby Memorial Hospital. Written informed consent was obtained from all subjects or their authorised representatives before study procedures.AIS patients, acute stroke mimics and neurologically asymptomatic controls were recruited at Ruby Memorial Hospital, Morgantown, WV, USA, which serves an impoverished rural region of West Virginia that displays some of the highest CVD rates in the nation.B2M, PPIB and ACTB were amplified as references, and normalisation was performed using the NORMAgene data-driven normalisation algorithm.46Complementary DNA was generated from purified RNA using the Applied Biosystems high-capacity reverse transcription kit. For qPCR, target sequences were amplified from 10 ng of complementary DNA input using sequence-specific primers  and deteχ2-tests were used for comparison of dichotomous variables, whereas Student's t-tests were used for comparison of continuous variables. Spearman’s rho was used to assess the strength of correlational relationships. For multiple regression analysis, variance decomposition was performed using the relaimpo R package.21 Penalised logistic regression was performed using the logistf R package.47 The level of significance was established at 0.05 for all parametric statistical testing. In the cases of multiple comparisons, P-values were adjusted using Holm’s Bonferonni method.48Parametric statistical analysis was performed using SPSS  in combination with R 2.14 via the SPSS R integration plug-in.


In recent years mitochondrial DNA (mtDNA) has transitioned to greater prominence across diverse areas of biology and medicine. The recognition of mitochondria as a major biochemical hub, contributions of mitochondrial dysfunction to various diseases, and several high-profile attempts to prevent hereditary mtDNA disease through mitochondrial replacement therapy have roused interest in the organellar genome. Subsequently, attempts to manipulate mtDNA have been galvanized, although with few robust advances and much controversy. Re-engineered protein-only nucleases such as mtZFN and mitoTALEN function effectively in mammalian mitochondria, although efficient delivery of nucleic acids into the organelle remains elusive. Such an achievement, in concert with a mitochondria-adapted CRISPR/Cas9 platform, could prompt a revolution in mitochondrial genome engineering and biological understanding. However, the existence of an endogenous mechanism for nucleic acid import into mammalian mitochondria, a prerequisite for mitochondrial CRISPR/Cas9 gene editing, remains controversial. Engineering of mammalian mtDNA has been hampered by an inability to import nucleic acids into mitochondria.A limited toolkit exists for manipulation of mammalian mtDNA, relying on protein-only nucleolysis and heteroplasmy-shifting approaches.Although present in lower metazoans, the weight of evidence against an efficient endogenous RNA import mechanism in mammalian mitochondria is considerable.Controversially, the application of CRISPR/Cas9 for manipulation of mammalian mtDNA in human cells has been reported. Taken together, the respiratory chain and ATP synthase consist of ∼90 protein subunits, forming IMM-bound protein complexes. The vast majority of these proteins are encoded in and expressed from the nuclear genome; however, a subset is encoded within a spatially and heritably separate genome – the mitochondrial genome.From the initial alphaproteobacterial engulfment, that formed the first eukaryote through endosymbiosis, to the present-day organelle residing in mammalian cells, the relationship between mitochondria and their hosts has evolved substantially. Where once mitochondria-like symbionts were advantageous principally for their capacity to harness redox chemistries, the role of mitochondria in diverged eukaryotes, such as mammals, is much more intricately embedded in essential organismal function. Facilitation of these functions relies upon an electrochemical disequilibrium potential across the heavy-strand promoter (HSP) and the light-strand promoter (LSP), resulting in polycistronic transcripts that undergo substantial processing to yield the mature mRNA, tRNA, and rRNA molecules that are required for translation by mitochondrial ribosomes (mitoribosomes) bottleneckheteroplasmic state, where a substantial proportion of mtDNA molecules bear a pathogenic mutation that is partially rescued by the presence of wild-type molecules in the same cell Mammalian mitochondrial DNA (mtDNA) is a multi-copy, circular, double-stranded DNA molecule encoding 13 essential membrane-bound polypeptide subunits of the respiratory chain complexes I, III, IV, and ATP synthase, 22 tRNAs, and two ribosomal RNAs (rRNAs). At ∼16.5 kb, mammalian mtDNAs are relatively small and genetically compact, containing very little non-coding sequence and two overlapping genes It is well-established that 11 protein-coding mRNAs, encoding 13 polypeptides of respiratory chain complexes and ATP synthase, are transcribed from the mitochondrial genome and translated by mitoribosomes. In placental mammals a full complement of 22 functional tRNA species capable of recognizing 60 sense codons, and two rRNAs that are required for translation by mitoribosomes, are also encoded in mtDNA. Considering the substantial structural differences between mitochondrial and cytosolic tRNAs, the divergence and incompatibility of codon usage between mitochondrial and nuclear mRNAs, the lack of unassigned codons in mitochondrial open reading frames (ORFs) mitochondrial RNase P (mtRNase P). Both nuclear and mitochondrial RNases P liberate the 5′ ends of immature tRNA transcripts through structure-guided endonucleolytic processing. RNase P is an ancient enzyme, initially identified in bacteria, followed by eukaryotic nuclei and yeast mitochondria H1 RNA) that is necessary for catalytic function 1R methyltransferase, TRMT10C (MRPP1), a member of the short-chain dehydrogenase/reductase (SDR) family, SDR5C1 , and a protein with homology to PiIT N terminus (PIN) domain-like metallonucleases, PRORP (MRPP3) trans-acting RNA. This paradigm-shifting subclass of proteinaceous RNase P (PRORP) has since been identified in most eukaryal lineages . Similarly to nRNase P, RNase MRP possesses a RNA subunit (termed 7-2 RNA in the early literature) and several protein components, most of which are shared with nRNase P in vitro reconstitution experiments have suggested an RNase MRP-independent mechanism for primer processing in mtDNA replication, where the 3'-end of the RNA primer is generated by site-specific termination of transcription owing to G-quadruplex formation in nascent RNA, rather than cleavage by RNase MRP A further controversy concerning mitochondrial RNA processing by imported endogenous RNAs concerns the nRNase P-related endonuclease, the mitochondrial RNA processing ribonuclease , to the exclusion of any 5S rRNA molecule, and that would require substantial remodeling of the mitoribosomal central protuberance to accommodate 5S rRNA Val leads to destabilization of this tRNA and a switch in the structural RNA content of mt-LSU from mt-tRNAVal to mt-tRNAPheAnother area of debate concerning mitochondrial import of endogenous RNA in mammals focuses on the RNA content of mitoribosomes, specifically the existence of a minor structural rRNA species analogous to the polynucleotide phosphorylase (PNPase). PNPase is a homotrimeric 3′–5′ exoribonuclease which, together with mitochondrial RNA-specific helicase, hSUV3, forms the RNA degradasome in the mitochondrial matrix PNPT1), that were predicted to disrupt the homotrimer and therefore abolish any catalytic or transport function of PNPase, led to an accumulation of aberrantly processed mitochondrial RNA species within mitochondria, in line with the expectation of a role for PNPase in degradation of mitochondrial RNA A factor suggested to directly facilitate endogenous RNA import into mammalian mitochondria is a component of the mitochondrial RNA degradation machinery, In addition to the research concerning import of endogenous RNAs into mammalian mitochondria, discussed above, there also exists a less well interrogated literature suggesting both import and export of miRNAs bona fide mtDNA-encoded RNA species were enriched by many orders of magnitude. Such outer-membrane contaminants have been long discussed It is helpful to underscore the often-contradictory findings reported in the studies discussed above by reference to valuable data from a comprehensive, quantitative analysis of the human mitochondrial transcriptome biolistics in combination with endogenous mtDNA HR, which is highly active in these organisms. As the introduction of exogenous nucleic acids into mammalian mitochondria has been resoundingly unsuccessful thus far, alternative approaches to manipulating mtDNA in situ have emerged, relying on recognition and specific elimination of targeted mtDNA molecules present in a heteroplasmic population. Such developments hold significant potential for broad application in the treatment of currently incurable diseases arising from mutation of the mitochondrial genome.Despite innumerable innovative investigations that have yielded a substantial but mosaic literature on the subject, the vast majority of approaches to mitochondrial genome engineering have failed to be either efficient or robust, and progress has been glacial . Once imported into mitochondria, these REs (mtREs) bind to and cleave a DNA recognition site that is present in only one mtDNA haplotype. Because mammalian mitochondria do not possess any efficient DSB repair pathways, mtDNA molecules bearing DSBs are rapidly degraded, producing a shift in the heteroplasmic ratio copy number; sudden depletion of this copy number by DSB-mediated degradation results in replication of the remaining, intact mtDNA molecules, repopulating cells with the untargeted mtDNA haplotype. Further work with mtREs demonstrated their mtDNA heteroplasmy-shifting efficacy across multiple tissues in both transgenic and adeno-associated virus (AAV)-treated mice Early attempts to manipulate mtDNA heteroplasmy exploited bacterial restriction endonucleases (REs) directed to mitochondria by means of a N-terminal These platforms exploit developments in engineered nuclease technology for nuclear DNA manipulation Despite being a relatively new addition to the literature, the principles underlying mtZFNs and mitoTALENs have recently been applied by an independent laboratory to answer questions of basic mitochondrial molecular genetics Leishmania, to cultured human cells facilitated mitochondrial import of RNA from the cytosol Proceedings of the National Academy of SciencesLeishmania complex-mediated RNA import Over the past two decades a series of reports have suggested that RNA import into mammalian mitochondria could be facilitated by exogenous factors. One report described that addition of a multisubunit complex of approximately 500 kDa, isolated from mitochondria of Lys(CUU), which was demonstrated to partially localize to yeast mitochondria under stress conditions by the same investigators Other studies have claimed delivery of synthetic RNA to mammalian mitochondria through the use of two domains from yeast cytosolic tRNAGiven the controversial nature of mammalian mitochondrial RNA import, the publication of a study from Jo and colleagues, employing CRISPR/Cas9 to successfully manipulate mtDNA, was surprising Taken together, or separately, these data are extraordinary. The activity of CRISPR/Cas9 in mitochondria without any additional RNA import sequence implies that gRNAs were spontaneously imported into mitochondria from the cytosol. In addition, the data suggest that Cas9 protein has a previously undescribed, potent tropism for mitochondria because Cas9 bearing a NLS was localized to mitochondria. Further, the observation that targeting a single site in mtDNA for cleavage by CRISPR/Cas9 leads to depletion of only that locus, but not degradation of the entire molecule, is difficult to reconcile with the fact that mtDNA behaves as a unit Manipulation of mtDNA is an objective sought by many, from the laboratory to the bedside. The recent CRISPR/Cas9 revolution has transformed nuclear DNA manipulation, heralding the dawn of a new era in molecular biology and gene therapy. Although, given the many questions that remain regarding effective import of either endogenous or exogenous RNAs into mammalian mitochondria, it seems that the genome of this unusual organelle may be one of few genetic systems beyond the reach of CRISPR/Cas9 (see Outstanding Questions).Outstanding QuestionsCan RNA be imported into mammalian mitochondria? If so, what is the mechanism underlying this process?Given the dispensable/unknown function of RNAs proposed to be imported into mammalian mitochondria, what is the purpose of RNA import in such a system?How could gRNA be delivered to mammalian mitochondria in a manner compatible with efficient CRISPR/Cas9-mediated gene editing?Reliable import of nucleic acids into mammalian mitochondria, in concert with molecular tools currently in hand, would significantly advance the state of the art, and a functional CRISPR/Cas9 architecture in mitochondria could be revolutionary. However, given current prospects for efficient import of nucleic acids into mammalian mitochondria, a key prerequisite for genome editing by CRISPR/Cas9, the dream of a mitochondrial CRISPR/Cas9 panacea appears destined for a rude awakening.


The effects of metabolic approaches in ALS models encompass delays in onset of motor symptoms, protection of motor neurons and extension of survival, which signifies an important role of metabolism in the pathogenesis of the disease. There is now an urgent need to test metabolic approaches in controlled clinical trials. In addition, more detailed studies to better characterize the abnormalities in energy metabolism in patients with ALS and ALS models are necessary to develop metabolically targeted effective therapies that can slow the progression of the disease and prolong life for patients with ALS.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease primarily characterized by loss of motor neurons in brain and spinal cord. The death of motor neurons leads to denervation of muscle which in turn causes muscle weakness and paralysis, decreased respiratory function and eventually death. Growing evidence indicates disturbances in energy metabolism in patients with ALS and animal models of ALS, which are likely to contribute to disease progression. Particularly, defects in glucose metabolism and mitochondrial dysfunction limit the availability of ATP to CNS tissues and muscle. Several metabolic approaches improving mitochondrial function have been investigated  Amyotrophic lateral sclerosis (ALS) is a complex multi-pathogenic neurodegenerative disorder primarily characterized by the loss of upper motor neurons in the brain and lower motor neurons in the spinal cord and the brain stem  mice, the activities of the enzyme phosphofructokinase 1 (PFK1), the rate limiting enzyme in glycolysis, were reduced  were found to be reduced in the late symptomatic phases of the disease, indicating impairments in the TCA cycle pathway  butter fat and 0.15% (wt/wt) cholesterol. A 20% increase in the mean survival of these mice compared to mice fed with control diet was demonstrated and motor neuron loss was reduced. Moreover, SOD1G93A mice fed either a high fat diet  or with control diet  starting at 6 weeks of age also showed an increase in survival and the onset of motor symptoms and weight loss were delayed significantly. While mice treated with control diet died by 180 days, high fat died treated mice survived until 220 days and some of them survived more than 270 days , which thereby will improve oxidative phosphorylation of any fuel in addition to providing alternative fuel. This includes triheptanoin and α-ketoglutarate. Consistent with these theoretical notions, alternative fuels, metabolic supplements and mitochondrial function enhancers have been investigated with the purpose of improving ALS symptoms and delaying disease progression in mouse models of ALS, cell cultures and in patients with ALS. These metabolic potential treatments include triheptanoin, ketogenic diet, caprylic triglyceride, dichloroacetate, the Deanna protocol, pyruvate, lactate, creatine, coenzyme Q10, MitoQ, dexpramipexole, acetyl-L-carnitine and olesoxime, which we will discuss below. Their mechanisms and effects are also summarized in Figure G93A mice  containing the odd chain C7 fatty acid heptanoate. After hydrolysis in the gastro-intestinal tract, medium chain fatty acids diffuse into cells, the blood stream and into mitochondria. This enhances their availability to various organs including the CNS and muscles. In contrast, long chain fatty acids are bound to plasma proteins and require transporter proteins for uptake into cells and mitochondria  and at later symptomatic stages (25 weeks) of the disease was reduced. Downregulation of these metabolic enzymes in muscle even at early stages where grip strength was still intact suggests that metabolic defects in the energy producing pathways could contribute to disease progression in ALS  from P35 until P70 prevented downregulation of the enzymes such as succinate dehydrogenase, glutamic pyruvic transaminase 2 and the propionyl carboxylase β subunit compared to mice treated with control diet. Similarly, treatment with triheptanoin protected against motor neuron loss by 33% in the lumbar spinal cord  caprylic triglyceride improved motor performance in the rotarod test, protected against motor neuron loss and increased oxygen consumption in mitochondria isolated from the spinal cord , coenzyme Q10 (CoQ10) and ubiquinol in addition to MCTs if tolerated. It was claimed to have beneficial effects in anecdotal reports  is a pyruvate dehydrogenase (PDH) kinase inhibitor  and HG93A mouse model of ALS, pyruvate (1000 mg/kg/week i.p.) slowed disease progression and also improved motor symptoms in a rotarod test and survival by 10.5% when it was administered starting at the onset of disease  and UQ International (TT).KB applied for a patent for use of triheptanoin in ALS. The other author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bile acids, a structurally related group of molecules derived from cholesterol, have a long history as therapeutic agents in medicine, from treatment for primarily ocular diseases in ancient Chinese medicine to modern day use as approved drugs for certain liver diseases. Despite evidence supporting a neuroprotective role in a diverse spectrum of age-related neurodegenerative disorders, including several small pilot clinical trials, little is known about their molecular mechanisms or their physiological roles in the nervous system. We review the data reported for their use as treatments for neurodegenerative diseases and their underlying molecular basis. While data from cellular and animal models and clinical trials support potential efficacy to treat a variety of neurodegenerative disorders, the relevant bile acids, their origin, and the precise molecular mechanism(s) by which they confer neuroprotection are not known delaying translation to the clinical setting. Bile acids are a structurally related group of molecules derived from cholesterol that are widely known for their role as chemical detergents involved in the intestinal absorption and transport of fats and lipid-soluble nutrients  and chenodeoxycholic acid (CDCA). These primary bile acids can undergo conjugation with glycine or taurine prior to secretion in the bile to form glycocholic acid (GCA), taurocholic acid (TCA), glycochenodeoxycholic acid (GCDCA), and taurochenodeoxycholic acid (TCDCA). In the intestine, they can undergo dehydroxylation by gut bacteria to produce deoxycholic acid (DCA) and lithocholic acid (LCA). Further chemical modifications can also occur resulting in other minor species such as ursodeoxycholic acid (UDCA)  prior to exposure to Aβ peptide significantly decreased nuclear fragmentation and cytochrome c release that was dependent upon activation of the PI3K pathway  is characterized by dementia and memory loss that is associated with extracellular senile plaques of amyloid precursor protein derived amyloid-beta (Aβ) protein and intracellular neurofibrillary tangles consisting of the Tau protein  that express human amyloid precursor protein carrying the KM670/671NL Swedish double mutation and the human presenilin 1 L166P mutation under the regulation of a neuron-specific promoter, treatment via dietary supplementation of standard laboratory chow with 0.4% (wt/wt) of TUDCA significantly reduced amyloid plaque number in the frontal cortex and hippocampus, decreased injury to neurons measured by determining loss of, or damage to, neuronal fibers surrounding plaques, and improved memory retention measured via contextual, though not auditory, fear conditioning  that could predict the onset of either AD or amnestic mild cognitive impairment, considered an early precursor of AD, within 2–3 years with an accuracy of over 90% , which is characterized by the selective loss of dopaminergic neurons in the substantia nigra region of the brain and a resulting tremor, have been reported. Sodium nitroprusside (SNP)-induced cytotoxicity of human dopaminergic SH-SY5Y cells has been used as a model of PD. UDCA was found to dose-dependently (50–200 μM) decrease SNP-related cell death. UDCA reduced reactive oxygen species (ROS), reactive nitrogen species (peroxynitrite and nitric oxide), and helped to maintain intracellular glutathione (GSH) levels. Apoptosis markers including nuclear fragmentation, caspase activation, and cytochrome c release were correspondingly reduced. Inhibiting phosphatidylinositiol-3-kinase (PI3K) and Akt/PKB blocked the favorable effects of UDCA on SNP-induced cytotoxic cell death  data based on the genotypes of 2,525,705 SNPs in 4238 PD cases and 4239 non-PD controls, a total of 3 SNPs were found to be statistically associated with PD, including a non-synonymous missense variant in HSD3B7   gene mutation, a 2000 compound library was screened for significant improvement in mitochondrial membrane potential  neurotoxin is a widely used toxin model for PD, replicating most of the clinical and pathological features of PD in humans and animal models. TUDCA was found to play a role in ameliorating neurodegeneration in MPTP-induced degeneration of dopaminergic neurons in the nigrostriatal axis in C57BL/6 glutathione S-transferase pi (GSTP) null mice  have thus been used as a model for ALS. GUDCA was reported to reduce cell death in the NSC-34 hSOD1G93A cell model and to block caspase-9 activation  has prompted therapeutic strategies aimed at preventing neuronal cell death and promoting regeneration , activating LXR but not FXR in ventral midbrain dopaminergic neurons . A cluster of transcript-SNP pairs with the highest level of statistical significance and meeting correction for multiple testing was associated with HTT) gene , an autosomal dominantly inherited neurodegenerative disease, is caused by an expansion mutation in CAG triplet repeat number in exon 1 of the huntingtin . The disorder is characterized primarily by degeneration of Purkinje neurons of the cerebellum. TUDCA treatment was used to determine whether the survival of Purkinje cells and the onset and progression of ataxia was altered in a transgenic mouse model of SCA1, in which 30 copies of the human ATXN1 cDNA containing a CAG trinucleotide repeat of 82 repeats was under the control of the Pcp-2 Purkinje-cell specific promoter l  is another dominantly inherited neurodegenerative disorder characterized chiefly by progressive ataxia  is a normal cellular protein that when mutated becomes misfolded (PrPSc). PrPSc can then convert normally folded PrPC to the misfolded form. Accumulation of PrPSc causes loss of neurons, astrogliosis, and spongiform degeneration resulting in dementia, ataxia, and death. One approach to therapeutic development has been to block or interfere with the conversion of PrPC to PrPSc. TUDCA and UDCA were found to substantially reduce this conversion in cell-free aggregation assays as well as in both chronically and acutely infected mouse ScN2a neuroblastoma cells  is a very rare autosomal recessive disorder caused by mutations in the rd10 mouse model of retinitis pigmentosa, in which the mice carry a missense mutation in exon 7 of the Pde6b gene resulting in rod photoreceptor cell death within a month after birth, subcutaneous administration of TUDCA resulted in higher cone cell density in all 4 quadrants of the retina  or lecithin-retinol acyltransferase (LRAT) genes underlie Leber congenital amaurosis , another model for retinal degeneration  that can induce retinal degeneration in albino Balb/C mice  receptors, one of three ionotropic glutamate receptor subtypes that are expressed in inner retinal cells, by intravitreal injection of NMDA has frequently been used to model the cell death pathway that occurs in retinal diseases such as glaucoma  dose of TUDCA administered to rats 60 min after occlusion of the middle cerebral artery markedly reduced infarct size and apoptosis and preserved mitochondrial integrity , although open and shut times, slope conductance, and single channel current amplitude, were not significantly affected  that can modulate Trk activation, to activate signaling pathways that promote neuronal growth and survival (Rodrigues et al., Neuroprotective effects of several bile acids are well documented in a wide range of neurodegenerative diseases, including AD, PD, ALS, HD, and retinal degeneration, in cellular and animal models and in human clinical trials. Due to the historical precedent set by the use of bear bile in ancient Chinese medicine, the majority of the studies have focused on UDCA and its derivatives. However, bile acids are a relatively large group of structurally related molecules, thus little is known about the potential efficacy of other bile acid species or the roles of circulating vs. endogenous bile acids synthesized within the central nervous system. In addition, most of the mechanistic studies have been centered on apoptosis and related pathways Figure . HoweverGG and HA designed, wrote, revised, and edited the manuscript. Both authors approved the submitted version of the manuscript.This work was funded by the Lewis Katz School of Medicine at Temple University Department of Medical Genetics and Molecular Biochemistry and the Joseph & Rebecca Goodfriend Endowed Chair in Genetics.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.


Dynamic changes in protein phosphorylation govern the transitions between different phases of the cell division cycle. A “tug of war” between highly conserved protein kinases and the family of phosphoprotein phosphatases (PPP) establishes the phosphorylation state of proteins, which controls their function. More than three-quarters of all proteins are phosphorylated at one or more sites in human cells, with the highest occupancy of phosphorylation sites seen in mitosis. Spatial and temporal regulation of opposing kinase and PPP activities is crucial for accurate execution of the mitotic program. The role of mitotic kinases has been the focus of many studies, while the contribution of PPPs was for a long time underappreciated and is just emerging. Misconceptions regarding the specificity and activity of protein phosphatases led to the belief that protein kinases are the primary determinants of mitotic regulation, leaving PPPs out of the limelight. Recent studies have shown that protein phosphatases are specific and selective enzymes, and that their activity is tightly regulated. In this review, we discuss the emerging roles of PPPs in mitosis and their regulation of and by mitotic kinases, as well as mechanisms that determine PPP substrate recognition and specificity. Transitions between distinct phases of the cell cycle are governed by post-translational modifications of proteins. Protein phosphorylation is the most prevalent post-translational modification, with more than three-quarters of the human proteome being phosphorylated  activity, chromosome condensation, and microtubule aster formation , phosphotyrosine phosphatases (PTP), and aspartate-based protein phosphatases , Mg2+, Fe2+, and Zn2+ ions) in the catalytic center. The metal ions in the catalytic center of PPPs are crucial for the activation of a water molecule which triggers a nucleophilic attack on the phosphorous atom of the substrate phosphate group to hydrolyze the phosphate ester bond, thereby dephosphorylating the substrate , PP4, PP5, PP6, and PP7 Table . These a‴) , but differ in tissue expression and have specific functions by associating with isoform-specific partners  Table . Theoret‴) Table . PP4 andEntry into mitosis is often characterized as a dramatic increase in “bulk” phosphorylation that needs to be reversed by “bulk” dephosphorylation to allow cells to exit mitosis. These changes in protein phosphorylation are in general thought to be accompanied by a rise in kinase activity, most importantly Cdk1 activity, and a reduction in PP1 and PP2A phosphatase activities early in mitosis, followed by a reversal of the respective activities as cells start to exit mitosis. However, regulation of phosphorylation signaling in all phases of mitosis is highly dynamic requiring coordination of opposing kinase and PPP activities in a specific temporal and spatial manner to ensure orderly and accurate progression through mitosis to generate identical daughter cells , PP1 reverses AURKB activation loop phosphorylation (Thr-232 in human cells) at kinetochores  surrounded by linear sequence motifs containing either basic amino acids or acidic amino acids or proline  were more rapidly dephosphorylated than pSP sites and that this is due to the action of the phosphatase PP2A-B55  are 100% identical Shi, . How canBesides directly influencing the biochemical characteristics of the phosphorylation sites, short linear motifs (SLiMs) also contribute to the assembly of PPP holoenzymes and the recognition of substrates. SLiMs are often located in intrinsically disordered regions of proteins and the sequence composition of the SLiM regulates protein-protein interaction and binding affinity. SLiM-mediated interactions promote PPP holoenzyme assembly, substrate specificity, phosphatase activity, and subcellular targeting. The use of SLiMs as a primary mechanism for catalytic and regulatory subunit assembly was first discovered for PP1 and PP2B   through which they associate with the catalytic subunit  governing both classes of enzymes, it is becoming clear that protein kinases and phosphatases not only counteract each other on their shared substrates but also counteract each other directly as well as indirectly through elaborate feedforward and feedback loops. Identification of regulatory mechanisms governing the activity of distinct PPP holoenzymes including phospho-amino acid (Ser vs. Thr) and phosphorylation site motif (acidophilic vs. basophilic) preferences have greatly increased our knowledge of dephosphorylation signaling and demonstrate the importance of investigating PPP holoenzyme specific substrate preferences The identification of SLiM mediated subunit-subunit and subunit-substrate interaction mechanisms has greatly enhanced our ability to predict PPP regulatory subunits and substrates and build PPP-based networks. SLiMs provide an entry point for specifically targeting PPPs to discover their biological functions and substrates as well as roles in diseases. Phosphorylation signaling is frequently disrupted or deregulated in many types of cancer. Until now, the focus for therapeutic strategies has been on protein kinases. However, the inevitable emergence of kinase inhibitor resistance requires new therapeutic approaches and targeting regulatory PPP subunits and their interactions with catalytic subunits may prove to be a viable strategy. PPP inhibition activates the spindle assembly checkpoint and blocks mitotic exit, ultimately leading to mitotic catastrophe and cell death, a strategy often applied in cancer therapy. While global inhibition of PPP activity impacts all cells, targeting specific PPP holoenzymes via the SLiM motif could be tailored to signaling pathways frequently deregulated in cancer. For instance, selective inhibition of PP1 in mitosis has been shown to cause impaired tumor growth (Winkler et al., Another important area of investigation in PPP biology is the identification of substrates of specific PPP holoenzymes in order to connect kinases and PPPs on their shared substrates. Quantitative mass spectrometry-based proteomic and phosphoprotoemic approaches combined with small molecule kinase inhibitors, genetically-engineered ATP-analog sensitive kinases, substrate trapping mutants, proximity-labeling and crosslinking approaches have identified a plethora of kinases substrates. Using similar approaches when applicable and available, as well as inducible degron strategy and SLiM targeting peptides to identify the phosphatase substrateswill fill the gaps in our understanding of phosphorylation signaling networks. In addition, identification of upstream signaling inputs regulating PPP holoenzyme assembly, activity and substrate targeting will be important to dissect the complex regulatory behaviors.IN and AK wrote the manuscript and made a direct, intellectual contribution to the manuscript.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.


In contrast to Warburg’s idea of mitochondrial impairment, it is now evident that mitochondrial metabolism is largely intact, despite elevated glucose metabolism in cancer cells, and is even indispensable for tumor growth and survival3. Mitochondrial metabolic pathways, such as glutaminolysis, fatty acid oxidation, redox and one-carbon metabolism, are essential metabolic pathways for tumor growth and survival and thus are considered to be promising therapeutic targets for cancer therapy4. In addition, mitochondrial metabolism also confers resistance to conventional and targeted cancer therapy, implying that targeting mitochondrial metabolism could provide a promising combination strategy for cancer treatment5. Intriguingly, recent studies reveal that in addition to generating ATP, the mitochondrial electron transport chain is critical for tumor growth by providing an electron acceptor, such as NAD+, which is required for aspartate synthesis7. Importantly, it is now clear that the reprogramming of energy metabolism in cancer is directed by both intrinsic and extrinsic factors, such as oncogenic signaling and tumor microenvironment factors, which are actually known to cause cancer9.Since Otto Warburg first proposed that cancer cells have defective mitochondrial metabolism with enhanced aerobic glycolysis almost 100 years ago, uncovering the unique metabolic features of cancer has turned into one of the most vibrant and popular research areas in cancer research11. In particular, 13C metabolic flux analysis (13C-MFA) has provided a major driving force to extend our understanding of metabolic alterations in cancer12. Integration of these technologies has enabled the discovery of complicated networks in glucose, amino acid and fatty acid metabolism in cancer13. Beyond the area of macronutrients, current research in cancer metabolism is expanding to micronutrients, such as vitamins, which were underexplored in a cancer metabolism context but are being revisited as emerging key players in cancer metabolism and are drawing great attention as promising therapeutic targets17. Further, beyond the canonical function of metabolic enzymes catalyzing nutrients, moonlighting functions of metabolic enzymes are also emerging as functional sensing mechanisms linking metabolic status to tumor growth and survival. A growing body of evidence suggests that some metabolic enzymes, such as hexokinase 2 and pyruvate kinase M2 (PKM2), may play a critical role in tumor growth and survival by not only their canonical metabolic activities but also their primarily non-enzymatic functions mediated through protein–protein interactions that regulate signal transduction or transcription19.In recent years, metabolomics has joined genomics and proteomics to advance our understanding of the complicated nature of cancer metabolism in vitro and in vivo13C-MFA to a discussion of the emerging roles of new metabolic pathways involving vitamin D and non-metabolic functions of metabolic enzymes.In this special issue of cancer metabolism, we invited four review articles highlighting the expanding features of cancer metabolism research, which include coverage of the technological aspects of cancer metabolism research with a practical guide for using 20. They elegantly summarize how diverse technologies, including chromatography-coupled mass spectrometry, metabolite tracing, seahorse, redox coupling, genome editing and integrated multi-omics, have contributed to deciphering and identifying the complicated network of metabolic pathways in cancer. Furthermore, they also provide practical information about the advantages and limitations of each analytical tool for metabolism analysis. Finally, they provide a comprehensive overview of how major findings concerning altered metabolism or metabolic pathways in cancer have been achieved to date.Gina DeNicola’s group (Moffitt Cancer Center and Research Institute) reviews recent advances in cancer metabolism from a technological perspective13C-MFA21. This review provides a practical guide for using 13C-MFA with a discussion on its advantages and potential pitfalls, which will be helpful for cancer biologists interested in analyzing and interpreting 13C-MFA data to study metabolism. Importantly, this tool has been indispensable for uncovering alterations in the metabolism of macronutrients, such as glucose, amino acids and fatty acids, in cancer.Further, Maciek Antoniewicz (University of Delaware) provides an in-depth review of the technological aspects of cancer metabolism analysis, particularly focusing on 22. Recently, vitamin D has been recognized as a key player in diverse physiological and pathological processes beyond bone and calcium metabolism. Moreover, accumulating epidemiological and experimental evidence suggest that vitamin D metabolism and function are critical for the prevention and treatment of a broad spectrum of cancers. This review provides a comprehensive summary of the metabolism of vitamin D and its hormonal regulation in physiology and proposed antitumorigenic functions. Moreover, the authors summarize the diverse mechanisms that lead to the dysregulation of vitamin D metabolism and its role in cancer, which contributes to the development of cancer and resistance to vitamin D-based cancer therapy. Finally, they also discuss future directions to improve the efficiency of vitamin D-based cancer therapy while minimizing its serious side effect of hypercalcemia.As an example of a micronutrient important for cancer, Sang-Min Jeon’s group (Ajou University) reviews our current understanding of the metabolism and function of vitamin D in cancer23. Notably, they classify the non-canonical functions of metabolic enzymes into three categories with specific details: (1) enzymes with oncogenic non-canonical functions, such as protein kinases, transcription regulators or cytoskeleton modulators; (2) enzymes with tumor-suppressive non-canonical functions, such as counteracting hypoxia-inducible factor 1a and mitogen-activated protein kinase signaling or promoting p53 function; and (3) enzymes modulating non-canonical functions in a manner dependent on the metabolic state, such as integrating the metabolic state into the mitogenic signaling of mechanistic target of rapamycin complex 1, myelocytomatosis cellular oncogene or PKM2. They also discuss how future research is needed to better dissect the metabolic and non-metabolic functions of metabolic enzymes in cancer to design an effective cancer therapeutic strategy.Finally, Almut Schulze’s group (University of Wuerzburg) reviews the emerging non-canonical functions of key metabolic enzymes regulating signal transduction, transcription and the cytoskeleton25. Recently, the Food and Drug Administration approved a first-in-class cancer metabolism drug, enasidenib, which targets mutated isocitrate dehydrogenase 2 for acute myeloid leukemia, highlighting the promise of new drugs targeting metabolism26. In addition to enasidenib, multiple candidates targeting the Achilles’ heel of cancer metabolism are undergoing clinical trials27. Thus, it is likely that additional drugs targeting cancer metabolism will be developed for cancer therapy, either alone or in combination with conventional therapy, targeted therapy and immunotherapy.Beyond the role of aerobic glycolysis proposed by Warburg, our understanding of cancer metabolism has greatly evolved with the identification of new metabolic pathways using cutting-edge technologies. Now, cancer metabolism research is shifting into a new era with renewed hope for curing cancer and overcoming the limitations of conventional and targeted therapies


This paper focuses on the fabrication via simple melt blending of thermotropic liquid crystal polyester (TLCP) nanocomposites reinforced with a very small quantity of modified carbon nanotube (CNT) and the unique effects of the modified CNT on the physical properties of the nanocomposites. The thermal, mechanical, and rheological properties of modified CNT-reinforced TLCP nanocomposites are highly dependent on the uniform dispersion of CNT and the interactions between the CNT and TLCP, which can be enhanced by chemical modification of the CNT, providing a design guide of CNT-reinforced TLCP nanocomposites with great potential for industrial uses. Liquid-crystalline polymers (LCPs) have a phase of matter intermediate between the isotropic liquid and crystalline solid states termed as a mesophase or mesomorphic phase, and this LC state offers broad perspectives for various application materials that exhibit anisotropic but liquid properties . In geneThermotropic LCPs, that are a class of the materials used to produce anisotropic melts of relatively low viscosity, have been extensively investigated because of their excellent mechanical properties, superior chemical resistance, low gas and liquid permeability, low controllable coefficient of thermal expansion, excellent dimensional stability under temperature or in severe environment, and easy processability with high precision by extrusion and injection molding ,3,4,5. TCarbon nanotubes (CNTs) have attracted a great deal of scientific interest as advanced materials since they were first reported by Iijima . In partin situ polymerization, and melt blending. Among these processing techniques, melt blending process has been accepted as the simplest and the most effective method from an industrial perspective, because this process makes it possible to fabricate high performance polymer nanocomposites at low process costs, and facilitates commercial scale-up  versus θ as shown in Ea values of pure TLCP and TLCP/CNT nanocomposites were estimated to be 290.8, 293.7, 303.8, and 322.6 kJ/mol, respectively. As compared to pure TLCP, higher Ea value of TLCP nanocomposites suggested that they were more thermally stable than pure TLCP. In addition, TLCP nanocomposites containing modified CNT showed higher Ea values as compared to TLCP nanocomposites containing pristine CNT, suggesting that TLCP nanocomposites with more uniform dispersion of modified CNT were more thermally stable than those containing pristine CNT. This feature was also attributed to the interactions between modified CNT and TLCP matrix, which increase the activation of the thermal decomposition of TLCP matrix relative to pristine CNT. Similar observation has been reported that the enhanced thermal stability of CNT-reinforced poly (PEN) nanocomposites was associated with the increased interfacial interactions between modified CNT and PEN , where Gʹ and Gʺ are storage and loss moduli, respectively. The frequency ranges were varied between 0.05 and 450 rad/s, and the strain amplitude was applied to be within the linear viscoelastic ranges. The mechanical properties of TLCP nanocomposites were measured using an Instron 4465 testing machine, according to the procedures in the ASTM D 638 standard. Thermogravimertic analysis of TLCP nanocomposite were performed with a TA instrument SDF 2960 TGA over the temperature rage of 30~800 °C at a heating rate of 10 °C/min under N2.The chemical structure of CNT was characterized by means of room temperature FT-IR measurements in the range of 400~4,000 cmThis paper focused on the fabrication and characterization of thermotropic liquid crystal polymer (TLCP) nanocomposites reinforced with a very small quantity of modified carbon nanotube (CNT). The TLCP nanocomposites exhibited higher complex viscosity and more distinct shear thinning behavior than pure TLCP, resulting from strong interactions between CNT and TLCP matrix, which significantly influenced the relaxation behavior of polymer chains in the nanocomposites. The extent of the increase in the complex viscosity, storage modulus, and loss modulus of TLCP nanocomposites by the presence of modified CNT was more pronounced at low frequency as compared to those at high frequency. The decrease in the slopes of storage and loss moduli for TLCP/CNT nanocomposites with the introduction of modified CNT was attributed to the formation of the interconnected or network-like structures via the nanotube-nanotube and polymer-nanotube interactions, resulting in the pseudo solid-like behavior of TLCP nanocomposites. The incorporated CNT has a significant effect on the enhancement of thermal stability by acting as effective physical barriers against the thermal decomposition in TLCP nanocomposites. The improvement in the mechanical properties of TLCP nanocomposites resulted from the enhanced interfacial adhesion between modified CNT and TLCP as well as uniform dispersion of modified CNT in TLCP matrix, and their synergistic effect combining good interfacial interaction and uniform dispersion was more effective at lower CNT content relative to higher CNT content. This study demonstrates that the optimization of straight morphology and preferential alignment of CNT during melt processing as well as the enhanced interfacial adhesion between CNT and TLCP and uniform dispersion of CNT in TLCP matrix plays a key role in further improving the overall mechanical properties of TLCP nanocomposites.


Survivors of cerebral aneurysm rupture are at risk for significant morbidity and neurological deficits. Much of this is related to the effects of blood in the subarachnoid space which induces an inflammatory cascade with numerous downstream consequences. Recent clinical trials have not been able to reduce the toxic effects of free hemoglobin or improve clinical outcome. One reason for this may be the inability to identify patients at high risk for neurologic decline. Recently, haptoglobin genotype has been identified as a pertinent factor in diabetes, sickle cell, and cardiovascular disease, with the Hp 2-2 genotype contributing to increased complications. Haptoglobin is a protein synthesized by the liver that binds free hemoglobin following red blood cell lysis, and in doing so, prevents hemoglobin induced toxicity and facilitates clearance. Clinical studies in patients with subarachnoid hemorrhage indicate that Hp 2-2 patients may be a high-risk group for hemorrhage related complications and poor outcome. We review the relevance of haptoglobin in subarachnoid hemorrhage and discuss the effects of genotype and expression levels on the known mechanisms of early brain injury (EBI) and cerebral ischemia after aneurysm rupture. A better understanding of haptoglobin and its role in preventing hemoglobin related toxicity should lead to novel therapeutic avenues. Aneurysmal subarachnoid hemorrhage (aSAH) affects more than 30,000 people per year in the United States, with an estimated 30–50% mortality  and delayed cerebral infarcts (DCI) are the most studied delayed events and are known to contribute to poor outcomes after aSAH , and forming a clot. Within 24 h following aSAH, an intense polymorphonuclear leukocyte infiltration of the meninges is seen  and reactive oxygen species (ROS), primarily superoxide anion Shikama, . HoweverExtracellular Hb  will undergo a series of oxidative reactions generating metHb, which undergoes further oxidation producing free heme and free iron , Hb structure is often modified by the oxidation which reduces ability of CD163 to bind the complex  and heterozygous 2-1. In Western populations, it is estimated that the distribution of Hp 1-1 is ~16%, Hp 2-1 is ~48%, and Hp 2-2 is ~36%. Hp is cleaved into two subunits α and β chains, joined by a disulfide bond. Both alleles share the same β chain  in SAH inflammation have been an area of active research in recent years. TLRs belong to the pattern recognition receptors family and play a well-described role in innate immunity and inflammatory responses  is formed by brain microvascular endothelial cells (ECs) with tight junctions and astrocyte end feet encircling ECs and with the participation of pericytes. The BBB plays a fundamental role in brain homeostasis by regulating the entry of intravascular molecules from the blood into the brain Ueno, . StudiesThe EC has an active role following SAH and is critical to the BBB. Blood in the subarachnoid space rapidly stimulates the expression of cell adhesion molecules (CAMs) on the luminal surface of ECs , which activates coagulation by decreasing protein kinase C production (an antithrombotic), increasing endothelial production of tissue factor (procoagulant), and increasing platelet activation  may be higher for Hp1-1, although this is uncertain and requires further investigation . More reliable studies investigating bound CSF Hp  vs. free CSF Hp will help elucidate the mechanism of blood clearance and Hp protection after SAH.The glymphatic pathway  for clearance of CSF solutes has become an interesting topic of investigation. There is evidence that increasing solute size limits glymphatic/lymphatic system clearance, and furthermore, aSAH reduces its function (Iliff et al., Lastly, several limitations exist for experimental studies of Hp in mice. First, mice only have a single Hp genotype which corresponds to human Hp1, and therefore mice only express the Hp1-1 phenotype (Levy et al., Lysis of RBCs and release of free Hb/heme drives the majority of the pathological process involved in delayed brain injury following SAH. Haptoglobin and its polymorphism has been extensively studied in other disease states, so much so that a human plasma-derived Hp was developed and is being utilized in the treatment of hemolysis (Schaer D. J. et al., The conception of this article was by SB and JG. Both the writing and editing was performed by all authors. Authors SB, PP, DM, HZ contributed to the initial manuscript by writing individual portions of the manuscript. These were combined and edited by SB, and then submitted to HC, JG, PD, JC, and SD for expert editing and final preparation. Both JL and JA reviewed and critically edited the content of the manuscript.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.


Aquaporin 4 (AQP4) is the major water channel expressed in the central nervous system and is primarily expressed in astrocytes. Recently, accumulated evidence has pointed to AQP4 as a key molecule that could play a critical role in glioma development. Discoveries of the role of AQP4 in cell migration suggest that AQP4 could be a significant factor regarding glioma malignancies. However, the AQP4 expression levels in glioma have not been fully elucidated; furthermore, the correlation of AQP4 expression with glioma malignancy remains controversial. Here, we review the expression pattern and predictive significance of AQP4 in malignant glioma. The molecular mechanism of AQP4 as it pertains to the migration and invasion of human glioma cells has been summarized. In addition, the important roles of AQP4 in combating drug resistance as well as potential pharmacological blockers of AQP4 have been systematically discussed. More research should be conducted to elucidate the potential roles of AQP4 in malignant glioma for identifying the tumor type, progression stages and optimal treatment strategies. The observed experimental results strongly emphasize the importance of this topic for future investigations. To date, more than 13 water channel proteins (AQP1-13) have been identified in multiple mammalian species and appear to possess various regulatory functions in a wide variety of diseases, including brain edema , seizureGlioma is a frequently occurring malignant tumor in the central nervous system (CNS). Glioblastoma multiforme (GBM) is the most common and malignant type of brain tumors in adults. Most patients develop clinical symptoms over a short time span (approximately 3 months) and often die within 8-18 months of diagnosis . Fewer tIntriguingly, there are some discrepancies regarding the role of AQP4 in malignant gliomas. Using a pharmacological inhibitor and small-interfering RNA, Ding et al.  demonstrThe arrangement of AQP4 is morphologically spectacular, as it forms orthogonal arrays of particles (OAPs) that can be observed in freeze-fracture replicas. In brain tumors, these OAPs are redistributed to membrane domains apart from end-feet areas and are redistributed across the entire surface of the cell . WolburgWolburg et al.  also conin vivo situations. More efforts should be directed toward clarifying how in vitro culture conditions affect AQP4 expression. Previously, McCoy et al. [However, a study by Noell et al.  presentey et al.  confirmey et al.  also conRecent studies have called special attention to AQPs as potential diagnostic and therapeutic biomarkers . AQP4, wIn addition, a correlation between increased BBB permeability and elevated AQP4 levels has been observed , and AQPWarth et al.  investigThe authors additionally conducted an analysis regarding the comparison between AQP4 expression in glioblastoma centers and corresponding infiltration zones to identify whether different AQP4 levels between the tumor core and the infiltration zones are correlated to patient survival . Compare+ are released during cell migration [It has been postulated that the AQP4 expression levels are relevant to the migration of astrocytes , indicatigration . This prigration , 55, 56,The invasion of malignant glioma is a complex process most likely controlled by different genes and signaling pathways at multiple steps: reduced adhesion between tumor cells and other surrounding cells, increased tumor cell mobility, and degradation of the extracellular matrix . CurrentAs mentioned above, aside from water transport, other potential roles of AQP4 in the regulation, invasion and migration of gliomas have been slowly elucidated. These properties of AQP4 appear unrelated to the canonical and well-established role of AQP4 in edema formation and absorption, suggesting this protein as novel therapeutic target for treating brain tumors , 64.There are various controversies that should be discussed. Because disrupting the BBB is an important pathophysiological change during the progression of glioblastoma and could play a critical role in the development of drug resistance in the treatment process, the roles of AQP4 in the BBB is an especially contentious issue. Zhou et al.  found th+/K+-ATPase. It is known that decreasing the migratory ability of glioma cells is a potential means of overcoming resistance, and involvement of the Na+/K+-ATPase in the migration and proliferation of glioma cells has been previously confirmed [+/K+-ATPase activity could be used to combat apoptosis-resistant malignant gliomas. Furthermore, specific inhibition of Na+/K+-ATPase activity could disorganize the actin cytoskeleton (thus reducing motility and proliferation) and induce autophagy in malignant glioma models [+ uptake is suppressed in AQP4 knockout mice because of decreased oxygen delivery to tissue located farthest away from a vascular oxygen source. This finding could expedite research regarding Na+/K+-ATPase inhibition via AQP-4 mediated regulation of an ion imbalance. Thus, inhibiting oxygen delivery in AQP4-deficient cells may represent another important factor in regulating Na+/K+-ATPase activity and ultimately drug resistance.In addition, AQP4 might impact drug resistance through the ion transporter Naonfirmed . It has a models . A studya models  providedCollectively, the abovementioned findings suggest that the cancer-enhancing activity of AQP4 does more than simply create pathways for water flow and that additional distinguishing features could also be important. The role of AQP4 in human cancer has emerged as an area of intense research interest. Aquaporin inhibitors may therefore be a novel class of anti-tumor agents. Unfortunately, no such inhibitors are available to date, and attempts to produce small molecule inhibitors targeting aquaporins have been largely unsuccessful . Specifiin vivo [2-(Nicotinamide)-1,3,4-thiadiazole (TGN-020) is a low molecular weight compound that has recently been identified as an inhibitor of AQP4 . The autin vivo . Howeverin vivo .Propofol , an IV anesthetic commonly used in clinical practice, has been previously proven to influence neuronal apoptosis  and reduAn article published by Gunnarson et al.  has showTetraethylammonium (TEA) has been indicated to inhibit water permeability of AQP4 in transfected oocytes  and has Arylsulfonamides are a class of compounds that have shown promise for the development of pharmacological agents targeting AQPs . SulfonaXenopus oocytes [The carbonic anhydrase inhibitor acetazolamide and anti-epileptic drugs such as topiramate and zonisamide  have bee oocytes  as well  oocytes ; however oocytes .Xenopus oocytes.Bumetanide is known as a loop diuretic drug that inhibits the NKCC cotransporter in the ascending limb of the loop of Henle in the kidneys  and was As stated above, there are several reports of AQP4 inhibitors ; unfortunately, follow-up studies of these compounds by other investigators using different assays have failed to confirm AQP4 inhibition. For a detailed discussion on the issues related to the discovery of AQP inhibitors, please refer to the recent review by Verkman et al. . One intIt is believed that identifying specific AQP4-selective inhibitors is currently a high priority in ongoing research. With an increasing number of novel compounds being tested, it is possible to begin elucidating a role of AQP blockers in mitigating pathologies such as brain oedema, glioblastoma, and breast and colon cancers as well as many other clinically important disorders . HopefulAQP4 is dissociated from the OAPs and redistributes across the entire surface of glioma cells under tumor conditions. AQP4 expression levels could correlate to the tumor grade, as AQP4 expression levels increase with higher glioma grades. More importantly, the redistribution of AQP4 and OAPs could be one of the earliest indicators of glioma transformation. However, the facilitating role of AQP4 in the infiltration and invasion of malignant glioblastoma cells requires additional research. Furthermore, the important roles of AQP4 in preventing drug resistance during glioma chemotherapy and of potential novel pharmacological blockers of AQP4 have been elucidated. We should be aware of the various unresolved questions regarding AQP4 in human glioblastoma. Further discovery of the dynamics of OAPs and AQP4 molecules in tumor may be critical to gain insight into the potential role of AQP4 in malignant glioma regarding the prevention, treatment, and classification of the tumor type and progression stages. Although the observations summarized in this review should be confirmed with more studies, we believe that they could provide critical information for the design of more focused research that will allow for systematic and definitive evaluation of the role of AQP4 in glioma treatments. More effort should be directed toward clarifying the newly discovered functions and molecular mechanisms of AQP4 in malignant gliomas.