Influence of MMR, MGMT Promotor Methylation and Protein Expression on Overall and Progression-Free Survival in Primary Glioblastoma Patients Treated with Temozolomide.

Glioblastoma is the most common malignant brain tumor in adults. Standard treatment includes tumor resection, radio-chemotherapy and adjuvant chemotherapy with temozolomide (TMZ). TMZ methylates DNA, whereas O6-methylguanine DNA methyltransferase (MGMT) counteracts TMZ effects by removing the intended proteasomal degradation signal. Non-functional MGMT mediates the mismatch repair (MMR) system, leading to apoptosis after futile repair attempts. This study investigated the associations between MGMT promoter methylation, MGMT and MMR protein expression, and their effect on overall survival (OS) and progression-free survival (PFS) in patients with glioblastoma. MGMT promoter methylation was assessed in 42 treatment-naïve patients with glioblastoma WHO grade IV by pyrosequencing. MGMT and MMR protein expression was analyzed using immunohistochemistry. MGMT promoter methylation was present in 52%, whereas patients <70 years of age revealed a significantly longer OS using a log-rank test and a significance threshold of p ≤ 0.05. MGMT protein expression and methylation status showed no correlation. MMR protein expression was present in all patients independent of MGMT status and did not influence OS and PFS. Overall, MGMT promoter methylation implicates an improved OS in patients with glioblastoma aged <70 years. In the elderly, the extent of surgery has an impact on OS rather than the MGMT promoter methylation or protein expression.

Acute Infectious Proctitis in Adolescents.

The diagnosis of acute proctitis requires understanding who is at risk, being aware of symptoms, and leveraging a thorough sexual history with appropriate risk stratification to make the diagnosis. Cases have been concentrated in adolescents (ages 15-19 years), young adults (ages 20-24 years), men and transgender women who have sex with men, and those with a history of human immunodeficiency virus infection. Black adolescents experience a disproportionately high number of cases of proctitis due to an intersection of concentrated cases in sexual networks and delayed screening/diagnosis due to health care access barriers. Signs and symptoms include purulent discharge, bleeding, pain, tenesmus, pruritus, diarrhea or constipation, weight loss, or fever. Multisite sexually transmitted infection testing should be offered based on risk stratification (eg, history of condomless anal sex, oral intercourse, number of sex partners). Further management includes promotion of barrier protection and preexposure prophylaxis, routine surveillance, partner notification, and routine access to preventive immunizations.

Co-incidental C9orf72 expansion mutation-related frontotemporal lobar degeneration pathology and sporadic Creutzfeldt-Jakob disease.

BACKGROUND: The C9orf72 hexanucleotide expansion mutation is the most common cause of genetic frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and combined FTD-ALS. Its underlying neuropathology combines TDP-43 pathology and dipeptide repeat protein (DPR) deposits and may also associate with other neurodegeneration-associated protein aggregates. Herein we present a unique combination of C9orf72 mutation with sporadic Creutzfeldt-Jakob disease (CJD) in a 74-year-old patient with rapidly progressive dementia. METHODS: Detailed neuropathological examination including immunohistochemistry for several proteinopathies. Genetic analysis was conducted by repeat primed polymerase chain reaction (PCR). Furthermore, we analyzed additional C9orf72 mutation carriers for prion-protein (PrP) deposits in brain tissue and screened the cerebellar cortex of other CJD cases for p62/DPR neuronal inclusions to assess the frequency of combined pathologies. RESULTS: Postmortem brain examination of a patient with a rapidly progressive neurological deterioration of 8 months' duration confirmed the diagnosis of CJD. She harbored valine homozygosity at PRNP codon 129. In addition, a frontotemporal lobar degeneration (FTLD)-pattern with TDP-43 protein aggregates and p62+/C9RANT+ positive inclusions along with a high degree of Alzheimer-related pathology (A3B3C3) were identified. The suspected C9orf72 expansion mutation was confirmed by repeat-primed PCR. Screening of 13 C9orf72 cases showed no pathological PrP aggregates and screening of 100 CJD cases revealed no other C9orf72 expansion mutation carriers. CONCLUSION: A combination of a C9orf72 expansion mutation-related FTLD with sporadic CJD in the same patient is rare. While the rarity of both diseases makes this concurrence most likely to be coincidental, questions regarding a potential link between these two neurodegenerative pathologies deserve further studies.

Quantitative Measurement of the Improvement Derived From a 10-Mo Progressive Exercise Program to Improve Balance and Function in Women at Increased Risk for Fragility Fractures.

INTRODUCTION/BACKGROUND: Osteoporosis is a common disorder and is associated with an increased risk of bone fracture. Falls are a proximate cause of a high proportion of medical costs and mortality. Improving balance can reduce the risk of falls and improve health outcomes, especially for the at-risk population of people with osteoporosis and osteopenia. The FrameWorksTM exercise program is a formal, standardized, informational and interventional 10-month exercise program. The purpose of this study was to quantitatively assess the improvement in standing balance, functional reach, and overall confidence in balance after participating in the 10-month program. METHODOLOGY: This study is a prospectively designed study with a pre and post study measurement of balance metrics. Sixty-two female participants, 45 years of age or older and at increased risk for fragility fractures, completed the 10-month program as well the pre and post program testing. Confidence was measured with the Activities-specific Balance Confidence Scale, a self-reported survey. Balance was measured digitally by means of testing with a NeuroCom® Basic Balance Master® system. Measurements were made of the Limits of Stability (LOS) Test and Modified Clinical Test of Sensory Interaction on Balance (mCTSIB). Balance was clinically assessed with the Functional Reach Test (FRT). RESULTS: Participation in the 10 months FrameWorksTM program resulted in improvement in quantitative measures of balance (Composite Sway Velocity, -12%, p < 0.001; End point excursion, 17.1%, p < 0.000001). A clinical measure of balance, the Functional Reach Test, improved, (2.9 cm, p < 0.0001). Participation also resulted in improvement in balance confidence (9.4 %, p < 0.00001). A height increase was observed (0.6 cm, p < 0.000001). CONCLUSIONS: The 10-month FrameWorksTM program improves balance and confidence in women at risk for fragility fractures. By improving balance and confidence, people are less likely to fall and therefore sustain fewer fractures and associated injuries.

Neuropathology-driven Whole-genome Sequencing Study Points to Novel Candidate Genes for Healthy Brain Aging.

PURPOSE: Understanding the healthy brain aging process is key to uncover the mechanisms that lead to pathologic age-related neurodegeneration, including progression to Alzheimer disease (AD). We aimed to address the issue of pathologic heterogeneity that often underlies a clinical AD diagnosis. METHODS: We performed a deep whole-genome sequencing study aiming to identify variants that are associated specifically with healthy brain aging. PATIENTS: We examined samples from the community-based longitudinal Vienna Transdanubian Aging study comparing neuropathologically "healthy" aging in individuals above 80 years of age with pure AD patients of the same age. RESULTS: Focusing on potentially functional variants, we discovered a single variant (rs10149146) that lies on the autophagy-associated TECPR2 gene and was carried by 53.6% of the "healthy" brain elderly individuals (15/28). An additional nonsynonymous variant on the CINP gene (encoding a cell cycle checkpoint protein) was also found in 46% of healthy controls. Both variants are absent from all AD cases. TECPR2 and CINP appear to be "partner" genes in terms of regulation and their associated transcription factors have been previously implicated in AD and neurodegeneration. CONCLUSIONS: Our study underlines the strength of neuropathology-driven definitions in genetic association studies and points to a potentially neuroprotective effect of key molecules of autophagy and cell cycle control.

Molecular diagnostic testing of diffuse gliomas in the real-life setting: A practical approach.

Typing of diffuse gliomas according to the WHO 2016 Classification of Tumors of the Central Nervous System is based on the integration of histology with molecular biomarkers. However, the choice of appropriate methods for molecular analysis and criteria for interpretation of test results is left to each diagnostic laboratory. In the present study, we tested the applicability of combined immunohistochemistry, direct sequencing, and multiplex ligation-dependent probe amplification (MLPA) for diagnostic assessment of IDH1/2 mutation status, chromosome 1p/19q status, and TERT promoter mutations. To this end, we analyzed a consecutive series of 165 patients with diffuse low- and high-grade gliomas (WHO grade II and III) from three Austrian centers in which tissue specimens were routinely processed. We could reliably detect IDH1/2 mutations by combining immunohistochemistry, direct sequencing, and MLPA analysis. MLPA analysis also allowed reliable detection of combined whole chromosomal arm 1p/19q codeletion when using carefully selected criteria providing an optimal balance between sensitivity and specificity. Direct sequencing proved to be suitable for identification of TERT promoter mutations, although its analytical performance remains to be assessed. To conclude, we propose a practicable combination of methods and criteria which allow reliable molecular diagnostic testing of diffuse gliomas in the real-life setting.
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Overexpression of minichromosome maintenance protein 10 in medulloblastoma and its clinical implications.

BACKGROUND: Overexpression of minichromosome maintenance (MCM) proteins 2, 3, and 7 is associated with migration and invasion in medulloblastoma (MB). However, expression profiling of all prereplication complex (pre-RC) has not been addressed in MBs. PROCEDURE: We performed mRNA expression profiling of a large set of pre-RC elements in cell lines and tumor tissues of MB. RNAi technology was employed for functional studies in MB cell lines. RESULTS: Our data showed that most of the pre-RC components are significantly overexpressed in MB. Among all pre-RC mRNAs, MCM10 showed the highest level of expression (∼500- to 1,000-fold) in MB cell lines and tissues compared to the levels detected in cerebellum. In addition, RNAi silencing of MCM10 caused reduced cell proliferation and cell viability in MB cells. CONCLUSIONS: Taken together, our study reveals that the pre-RC is dysregulated in MB. In addition, MCM10, a member of this complex, is significantly overexpressed in MB and is required for tumor cell proliferation.

Dura mater is a potential source of Aß seeds.

Deposition of amyloid-ß (Aß) in the brain parenchyma and vessels is one of the hallmarks of Alzheimer disease (AD). Recent observations of Aß deposition in iatrogenic Creutzfeldt-Jakob disease (iCJD) after dural grafting or treatment with pituitary extracts raised concerns whether Aß is capable of transmitting disease as seen in prion diseases by the disease-associated prion protein. To address this issue, we re-sampled and re-evaluated archival material, including the grafted dura mater of two cases with iCJD (28 and 33-years-old) without mutations in the AßPP, PSEN1 and PSEN2 genes, and carrying ε3/ε3 alleles of the APOE gene. In addition, we evaluated 84 dura mater samples obtained at autopsy (mean age 84.9 ± 0.3) in the community-based VITA study for the presence of Aß deposition. We show that the dura mater may harbor Aß deposits (13 %) in the form of cerebral amyloid angiopathy or amorphous aggregates. In both iCJD cases, the grafted dura mater had accumulated Aß. The morphology and distribution pattern of cerebral Aß deposition together with the lack of tau pathology distinguishes the Aß proteinopathy in iCJD from AD, from that seen in young individuals without cognitive decline carrying one or two APOE4 alleles, and from that related to traumatic brain injury. Our novel findings of Aß deposits in the dura mater, including the grafted dura, and the distinct cerebral Aß distribution in iCJD support the seeding properties of Aß. However, in contrast to prion diseases, our study suggests that such Aß seeding is unable to reproduce the full clinicopathological phenotype of AD.

KINFix--A formalin-free non-commercial fixative optimized for histological, immunohistochemical and molecular analyses of neurosurgical tissue specimens.

An optimal fixative should ideally combine the advantages of formalin fixation and freezing, allowing for good preservation of histology and molecular components, easy handling and storage, lack of toxicity, and low costs. Most of these criteria are fulfilled by ethanol-based solutions, and due to our good experience with the commercial RCL2 fixative, reflected by our published single-center trial, we initiated a multicenter ring trial. However, during its course, RCL2 was discontinued on the market. Therefore, we created our own agent, KINFix, composed of the same main constituents as RCL2, and employed it in our laboratory with similar results. Here we present our evaluation of the three fixatives formalin, RCL2, and KINFix from the perspective of histopathology as well as nucleic acid and protein analyses in comparison to fresh frozen tissues together with the multicenter ring trial data for RCL2. We observe that RCL2 and KINFix offer comparable histomorphology and superior template for molecular analyses than formalin. Moreover, KINFix as freely available fixative might overcome some of the difficulties related to the commercial agents. Therefore, we conclude that KINFix might be an attractive complement to formalin in tissue processing and advocate its use in neuropathological practice.

Clinicopathological description of two cases with SQSTM1 gene mutation associated with frontotemporal dementia.

There is a strong genetic influence on the clinicopathological phenotypes associated with frontotemporal lobar degeneration (FTLD) and frontotemporal dementia (FTD). Intracellular deposition of TDP-43 is the phenotypical hallmark of a frequent subgroup of cases. Mutations in the sequestosome 1 (SQSTM1) gene have rarely been found in individuals with FTD. Here we provide a comprehensive clinicopathological description of two cases with a SQSTM1 mutation. The clinical phenotype of patient 1 (mutation p.Glu396*) was compatible with the behavioural variant (bv) of FTD. TDP-43 pathology was consistent with the features of type B of FTLD-TDP pathology. However, prominent neuronal granular cytoplasmic TDP-43 immunoreactivity and abundant oligodendroglial inclusions, proven by colocalization with the oligodendroglial-marker TPPP/p25, were also seen. The clinical phenotype of patient 2 was compatible with bvFTD associated with parkinsonism and bulbar symptoms in the later stage. Genetic testing of patient 2 identified a C9orf72 repeat expansion mutation together with a missense mutation (p.Arg212Cys) in SQSTM1. TDP-43 pathology was characterized by neuritic profiles compatible mostly with type A. In contrast to patient 1, p62 pathology was seen to a greater extent as TDP-43 immunoreactivity in neurons. Using an antibody that detects poly(GP) peptides produced via repeat associated non-ATG translation associated with expanded hexanucleotide repeat in the C9orf72 gene, we confirmed the presence of pathognomonic inclusions. The present study supports previous observations on amyotrophic lateral sclerosis (ALS) that SQSTM1 mutations consistently associate with TDP-43 pathology. The co-presence of C9orf72 mutation may influence the phenotype, thus finding one FTLD (or ALS) related mutation does not exclude the presence of further influential genetic alterations. Oligodendroglial TDP-43 pathology is considerable in some forms of FTLD-TDP, thus their evaluation might be considered to be included in classification systems.

Essential role for mammalian apurinic/apyrimidinic (AP) endonuclease Ape1/Ref-1 in telomere maintenance.

The major mammalian apurinic/apyrimidinic endonuclease Ape1 is a multifunctional protein operating in protection of cells from oxidative stress via its DNA repair, redox, and transcription regulatory activities. The importance of Ape1 has been marked by previous work demonstrating its requirement for viability in mammalian cells. However, beyond a requirement for Ape1-dependent DNA repair activity, deeper molecular mechanisms of the fundamental role of Ape1 in cell survival have not been defined. Here, we report that Ape1 is an essential factor stabilizing telomeric DNA, and its deficiency is associated with telomere dysfunction and segregation defects in immortalized cells maintaining telomeres by either the alternative lengthening of telomeres pathway (U2OS) or telomerase expression (BJ-hTERT), or in normal human fibroblasts (IMR90). Through the expression of Ape1 derivatives with site-specific changes, we found that the DNA repair and N-terminal acetylation domains are required for the Ape1 function at telomeres. Ape1 associates with telomere proteins in U2OS cells, and Ape1 depletion causes dissociation of TRF2 protein from telomeres. Consistent with this effect, we also observed that Ape1 depletion caused telomere shortening in both BJ-hTERT and in HeLa cells. Thus, our study describes a unique and unpredicted role for Ape1 in telomere protection, providing a direct link between base excision DNA repair activities and telomere metabolism.

Atypical sporadic CJD-MM phenotype with white matter kuru plaques associated with intranuclear inclusion body and argyrophilic grain disease.

We describe an atypical neuropathological phenotype of sporadic Creutzfeldt-Jakob disease in a 76-year-old man. The clinical symptoms were characterized by progressive dementia, gait ataxia, rigidity and urinary incontinence. The disease duration was 6 weeks. MRI did not show prominent atrophy or hyperintensities in cortical areas, striatum or thalamus. Biomarker examination of the cerebrospinal fluid deviated from that seen in pure Alzheimer's disease. Triphasic waves in the EEG were detected only later in the disease course, while 14-3-3 assay was positive. PRNP genotyping revealed methionine homozygosity (MM) at codon 129. Neuropathology showed classical CJD changes corresponding to the MM type 1 cases. However, a striking feature was the presence of abundant kuru-type plaques in the white matter. This rare morphology was associated with neuropathological signs of intranuclear inclusion body disease and advanced stage of argyrophilic grain disease. These alterations did not show correlation with each other, thus seemed to develop independently. This case further highlights the complexity of neuropathological alterations in the ageing brain.

Genetically engineered microvesicles carrying suicide mRNA/protein inhibit schwannoma tumor growth.

Microvesicles (MVs) play an important role in intercellular communication by carrying mRNAs, microRNAs (miRNAs), non-coding RNAs, proteins, and DNA from cell to cell. To our knowledge, this is the first report of delivery of a therapeutic mRNA/protein via MVs for treatment of cancer. We first generated genetically engineered MVs by expressing high levels of the suicide gene mRNA and protein-cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT) in MV donor cells. MVs were isolated from these cells and used to treat pre-established nerve sheath tumors (schwannomas) in an orthotopic mouse model. We demonstrated that MV-mediated delivery of CD-UPRT mRNA/protein by direct injection into schwannomas led to regression of these tumors upon systemic treatment with the prodrug (5-fluorocytosine (5-FC)), which is converted within tumor cells to 5-fluorouracil (5-FU)-an anticancer agent. Taken together, these studies suggest that MVs can serve as novel cell-derived "liposomes" to effectively deliver therapeutic mRNA/proteins to treatment of diseases.

A pan-European study of the C9orf72 repeat associated with FTLD: geographic prevalence, genomic instability, and intermediate repeats.

We assessed the geographical distribution of C9orf72 G(4) C(2) expansions in a pan-European frontotemporal lobar degeneration (FTLD) cohort (n = 1,205), ascertained by the European Early-Onset Dementia (EOD) consortium. Next, we performed a meta-analysis of our data and that of other European studies, together 2,668 patients from 15 Western European countries. The frequency of the C9orf72 expansions in Western Europe was 9.98% in overall FTLD, with 18.52% in familial, and 6.26% in sporadic FTLD patients. Outliers were Finland and Sweden with overall frequencies of respectively 29.33% and 20.73%, but also Spain with 25.49%. In contrast, prevalence in Germany was limited to 4.82%. In addition, we studied the role of intermediate repeats (7-24 repeat units), which are strongly correlated with the risk haplotype, on disease and C9orf72 expression. In vitro reporter gene expression studies demonstrated significantly decreased transcriptional activity of C9orf72 with increasing number of normal repeat units, indicating that intermediate repeats might act as predisposing alleles and in favor of the loss-of-function disease mechanism. Further, we observed a significantly increased frequency of short indels in the GC-rich low complexity sequence adjacent to the G(4) C(2) repeat in C9orf72 expansion carriers (P < 0.001) with the most common indel creating one long contiguous imperfect G(4) C(2) repeat, which is likely more prone to replication slippage and pathological expansion.

Interlaboratory comparison of IDH mutation detection.

Isocitrate dehydrogenase (IDH) mutational testing is becoming increasingly important. For this, robust and reliable assays are needed. We tested the variation of results between six laboratories of testing for IDH mutations. Each laboratory received five unstained slides from 31 formalin-fixed paraffin-embedded (FFPE) glioma samples, and followed its own standard IDH diagnostic routine. All laboratories used immunohistochemistry (IHC) with an antibody against the most frequent IDH1 mutation (R132H) as a first step. Three laboratories then sequenced only IHC negative cases while the others sequenced all cases. Based on the overall analysis, 13 samples from 11 tumors had an R132H mutation and one tumor showed an R132G mutation. Results of IHC for IDH1 R132H mutations in all six laboratories were completely in agreement, and identified all R132H mutations. Upon sequencing the results of two laboratories deviated from those of the others. After a review of the entire diagnostic process, on repeat (blinded) testing one laboratory was completely in agreement with the overall result. A change in technique did only partially improve the results in the other laboratory. IHC for the IDH1 R132H mutation is very reliable and consistent across laboratories. IDH sequencing procedures yielded inconsistent results in 2 out of 6 laboratories. Quality assurance is pivotal before IDH testing is made part of clinical management of patients.

Opioid Legislation in Patients With Inflammatory Bowel Disease: A State-wide Retrospective Cohort Study.

BACKGROUND: Indiana State opioid prescription legislation has been shown to decrease overall opioid prescriptions. However, this effect has not been studied in specific diseases associated with chronic pain such as inflammatory bowel disease (IBD). We aimed to determine the effect of state opioid prescription legislation on opioid prescribing patterns in IBD. METHODS: A retrospective cohort analysis using an interrupted time-series from December 15, 2010 to July 1, 2018, with 2 time periods separated by Title 844 of the Indiana Administrative Code, in a statewide health care system capturing the majority of the state's population including all adult patients with IBD. The primary outcome was opioid prescription rate per person-year. RESULTS: In total, 9436 patients met inclusion criteria. After legislation, the total number of opioid orders per patient-year continued to increase (0.543, 95% CI, 0.528-0.558, to 0.663, 95% CI, 0.654-0.672), with fewer scripts from the emergency department (0.113, 95% CI, 0.106-0.120, to 0.092, 95% CI, 0.088-0.095) and more from outpatient providers (0.303, 95% CI, 0.292-0.314 to 0.432, 95% CI, 0.424-0.439). There were increases in biologic (0.206, 95% CI, 0.197-0.215 to 0.517, 95% CI, 0.509-0.525) and steroid (0.182, 95% CI, 0.173-0.190 to 0.237, 95% CI, 0.232-0.243) prescriptions per person-year following legislation. Factors associated with heavy opioid use included chronic steroids (odds ratio, 5.030; 95% CI, 4.176-6.054), history of IBD-related surgery (odds ratio, 2.807; 95% CI, 2.367-3.323) and current smoking (odds ratio, 2.650; 95% CI, 2.223-3.158). CONCLUSIONS: Despite legislation and the increased use of disease-modifying drugs, statewide opioid prescriptions continued to increase. The increase in opiate use, high steroid use, and significant health care utilization suggests poor underlying disease control.

State legislation, which has decreased opioid prescribing in the general population, has not had similar effects in patients with inflammatory bowel disease. This is the first study assessing opioid legislation and its effects in a chronic pain condition.

Extracellular-Vesicle-Based Cancer Panels Diagnose Glioblastomas with High Sensitivity and Specificity.

Glioblastoma is one of the most devastating neoplasms of the central nervous system. This study focused on the development of serum extracellular vesicle (EV)-based glioblastoma tumor marker panels that can be used in a clinic to diagnose glioblastomas and to monitor tumor burden, progression, and regression in response to treatment. RNA sequencing studies were performed using RNA isolated from serum EVs from both patients (n = 85) and control donors (n = 31). RNA sequencing results for preoperative glioblastoma EVs compared to control EVs revealed 569 differentially expressed genes (DEGs, 2XFC, FDR < 0.05). By using these DEGs, we developed serum-EV-based biomarker panels for the following glioblastomas: wild-type IDH1 (96% sensitivity/80% specificity), MGMT promoter methylation (91% sensitivity/73% specificity), p53 gene mutation (100% sensitivity/89% specificity), and TERT promoter mutation (89% sensitivity/100% specificity). This is the first study showing that serum-EV-based biomarker panels can be used to diagnose glioblastomas with a high sensitivity and specificity.

Identification of c-myc-dependent proteins in the medulloblastoma cell line D425Med.

High c-myc levels are linked to poor prognosis in medulloblastoma (MB), and it was the aim of the current study to search for c-myc-dependent proteins in the MB cell line D425Med. For this purpose D425Med cells and cells with knocked-down c-myc (by siRNA) were analysed by a gel-based differential proteomics study using mass spectrometry. Heterogeneous nuclear ribonucleoproteins C1/C2, heterogeneous nuclear ribonucleoprotein A/B, stathmin, endoplasmic reticulum protein ERp29 precursor and guanidinoacetate N-methyltransferase were c-myc dependently expressed. Signalling, the protein machinery, metabolism and endoplasmic reticulum function may be affected and these results enable studying tumour tissue for these proteins as potential dignity markers or pharmacological targets.

SRPX Emerges as a Potential Tumor Marker in the Extracellular Vesicles of Glioblastoma.

Extracellular vesicles (EVs) may be used as a non-invasive screening platform to discover markers associated with early diagnosis, prognosis, and treatment response. Such an approach is invaluable for diseases such as glioblastoma, for which only a few non-invasive diagnostic or prognostic markers are available. We used mass spectrometry to analyze proteomics profiles of EVs derived from four glioblastoma cell lines and human primary astrocytes (HPAs) and found that SRPX is the only protein enriched in the majority of glioblastoma EVs that was absent in the HPA-derived EVs. Then, we evaluated the relationship between SRPX protein expression and tumor grade using immunohistochemical staining (IHC) and performed colony formation and viability assays to analyze the possible function of SRPX in glioblastoma. SRPX mRNA and protein expression were associated with tumor grade. Moreover, temozolomide (TMZ)-resistant tumor tissues showed highly positive SRPX staining, compared to all other tumor grades. Additionally, glioblastoma cells displayed enhanced SRPX gene expression when exposed to TMZ. Knockdown of SRPX gene expression via siRNA inhibited cell viability. Taken together, the results of this study suggest that SRPX can be used as a novel tumor marker for diagnostic and prognostic purposes and can also be a therapeutic target for glioblastomas.

Functional Medicine Approach to Patient Care Improves Sleep, Fatigue, and Quality of Life in Patients With Inflammatory Bowel Disease.

Background: A Functional Medicine program was developed at an inflammatory bowel disease (IBD) center with the goal of integrating strategies to address modifiable lifestyle factors and to complete a 6-week elimination diet under the direction of a trained Functional Medicine dietitian and Functional Medicine providers. Methods: From January 2019 to November 2019, patients with controlled, but persistent, symptoms from IBD were offered enrollment. Each of the 5 visits incorporated an educational session focused on nutrition followed by a session focusing on modifiable lifestyle factors. The patients were placed on a supervised 6-week elimination diet. At each visit, patients completed the SIBDQ (Short Inflammatory Bowel Disease Questionnaire), FSS (Fatigue Severity Scale), PSQI (The Pittsburgh Sleep Quality Index), and MSQ (Medical Symptoms Questionnaire). Statistical analysis was performed using the Wilcoxon matched pairs signed-rank test. Results: Nineteen patients enrolled (2 men: 1 ulcerative colitis [UC], 1 Crohn's disease [CD]; 17 women: 3 UC, 14 CD). 15 patients completed all modules. There was improvement in all patient-reported outcomes (PROs) (FSS, P < .001; PSQI, P < .001; SIBDQ, P < .001; MSQ, P < .001). Every patient who completed the last session demonstrated weight loss. Conclusions: The psychoemotional roots to immune disease states, particularly IBD, are complicated and often not addressed in traditional care. We are just beginning to understand the impact of nutrition, sleep, stress, movement, and relationships on IBD. In this cohort, utilizing Functional Medicine as an adjunct to traditional care resulted in improvement in all PROs.