Morphologic Features of Invasion in Lung Adenocarcinoma: Diagnostic Pitfalls.

Reproducibility of pulmonary invasive adenocarcinoma diagnosis is poor when applying the World Health Organization (WHO) classification. In this article, we aimed first to explain by 3-dimensional morphology why simple pattern recognition induces pitfalls for the assessment of invasion as applied in the current WHO classification of pulmonary adenocarcinomas. The underlying iatrogenic-induced morphologic alterations in collapsed adenocarcinoma in situ overlap with criteria for invasive adenocarcinoma. Pitfalls in seemingly acinar and papillary carcinoma are addressed with additional cytokeratin 7 and elastin stains. In addition, we provide more stringent criteria for a better reproducible and likely generalizable classification.

Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer.

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis.

Circulating autoantibodies to alpha-enolase (ENO1) and far upstream element-binding protein 1 (FUBP1) are negative prognostic factors for pancreatic cancer patient survival.

Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis due to a lack of early diagnostic markers and effective therapy. In PDA patients, the glycolytic enzyme and plasminogen receptor alpha-enolase (ENO1) and the transcription factor far upstream element-binding protein 1 (FUBP1) are upregulated and elicit the production of autoantibodies (aAb) that discriminate healthy subjects from PDA patients, with the latter mostly directed to post-translational phosphorylated isoforms. Here, the correlation of prognosis with circulating ENO1 and FUBP1aAb, and their protein tissue expression was analyzed in PDA patients. Circulating ENO1 and FUBP1 aAb was analyzed in two cohorts of PDA patients by ELISA (n = 470), while tissues expression was observed by immunohistochemistry (n = 45). Overall survival (OS) was estimated using the Kaplan-Meier method, while the Cox model was used to estimate the hazard ratios (HR) adjusted for the main prognostic factors. Logistic models were applied to assess associations between death and its risk indicators. All statistical analyses were performed with Stata version 15. Unlike ENO1 aAb, there was a significant correlation between FUBP1 aAb and FUBP1 expression in tumors (p = 0.0268). In addition, we found that high ENO1 (p = 0.016) and intermediate FUBP1 aAb levels (p = 0.013) were unfavorable prognostic factors. Notably, it was found that high anti-FUBP1 aAb level is a good prognostic marker for tail-body PDA (p = 0.016). Our results suggest that different levels of circulating aAb to ENO1 and FUBP1 predict a poor outcome in PDA patients and can be used to improve therapeutic strategies.

Cohort profile: the Turin prostate cancer prognostication (TPCP) cohort.

Introduction: Prostate cancer (PCa) is the most frequent tumor among men in Europe and has both indolent and aggressive forms. There are several treatment options, the choice of which depends on multiple factors. To further improve current prognostication models, we established the Turin Prostate Cancer Prognostication (TPCP) cohort, an Italian retrospective biopsy cohort of patients with PCa and long-term follow-up. This work presents this new cohort with its main characteristics and the distributions of some of its core variables, along with its potential contributions to PCa research. Methods: The TPCP cohort includes consecutive non-metastatic patients with first positive biopsy for PCa performed between 2008 and 2013 at the main hospital in Turin, Italy. The follow-up ended on December 31st 2021. The primary outcome is the occurrence of metastasis; death from PCa and overall mortality are the secondary outcomes. In addition to numerous clinical variables, the study's prognostic variables include histopathologic information assigned by a centralized uropathology review using a digital pathology software system specialized for the study of PCa, tumor DNA methylation in candidate genes, and features extracted from digitized slide images via Deep Neural Networks. Results: The cohort includes 891 patients followed-up for a median time of 10 years. During this period, 97 patients had progression to metastatic disease and 301 died; of these, 56 died from PCa. In total, 65.3% of the cohort has a Gleason score less than or equal to 3 + 4, and 44.5% has a clinical stage cT1. Consistent with previous studies, age and clinical stage at diagnosis are important prognostic factors: the crude cumulative incidence of metastatic disease during the 14-years of follow-up increases from 9.1% among patients younger than 64 to 16.2% for patients in the age group of 75-84, and from 6.1% for cT1 stage to 27.9% in cT3 stage. Discussion: This study stands to be an important resource for updating existing prognostic models for PCa on an Italian cohort. In addition, the integrated collection of multi-modal data will allow development and/or validation of new models including new histopathological, digital, and molecular markers, with the goal of better directing clinical decisions to manage patients with PCa.

Full cost of diagnostic pathology for lung carcinoma in Italy: results from four Pathology Units.

Objective: To calculate the full cost of diagnostic pathology tests for Non-Small Cell Lung Cancer (NSCLC) across four Italian Pathology Units. Methods: Pathology Units were located in private (2) and public (2) hospitals distributed across the Italian territory (North: 2; Centre: 1; South: 1). Pathologists provided via questionnaire data on tests on NSCLC samples along with the identification and quantification of the necessary healthcare resources (diagnostic technologies, laboratory instruments and personnel). Resources were valued according to hospital-specific unit, yearly and hourly costs (disposables; technologies; professional clusters). Results: The full cost per NSCLC tissue sample included histopathological immunophenotypic and required molecular analysis. Overall, it reached € 659.77 and it was mainly composed of direct costs (77.69%). The processing of a NSCLC tissue sample was labour intensive, as a relevant share of the full cost (44.98%) was actually due to personnel costs, with laboratory technicians, biologists and pathologist driving this finding (17.09%,12.43% and 10.81%, respectively). Conclusions: The results of this research can facilitate the negotiation of new dedicated tariffs for NSCLC sample processing with the national or local third party-payers.

Validation of a Simple, Rapid, and Cost-Effective Method for Acute Rejection Monitoring in Lung Transplant Recipients.

Despite advances in immunosuppression therapy, acute rejection remains the leading cause of graft dysfunction in lung transplant recipients. Donor-derived cell-free DNA is increasingly being considered as a valuable biomarker of acute rejection in several solid organ transplants. We present a technically improved molecular method based on digital PCR that targets the mismatch between the recipient and donor at the HLA-DRB1 locus. Blood samples collected sequentially post-transplantation from a cohort of lung recipients were used to obtain proof-of-principle for the validity of the assay, correlating results with transbronchial biopsies and lung capacity tests. The results revealed an increase in dd-cfDNA during the first 2 weeks after transplantation related to ischemia-reperfusion injury (6.36 ± 5.36%, p < 0.0001). In the absence of complications, donor DNA levels stabilized, while increasing again during acute rejection episodes (7.81 ± 12.7%, p < 0.0001). Respiratory tract infections were also involved in the release of dd-cfDNA (9.14 ± 15.59%, p = 0.0004), with a positive correlation with C-reactive protein levels. Overall, the dd-cfDNA percentages were inversely correlated with the lung function values measured by spirometry. These results confirm the value of dd-cfDNA determination during post-transplant follow-up to monitor acute rejection in lung recipients, achieved using a rapid and inexpensive approach based on the HLA mismatch between donor and recipient.

mEPE-score: a comprehensive grading system for predicting pathologic extraprostatic extension of prostate cancer at multiparametric magnetic resonance imaging.

OBJECTIVE: To investigate the diagnostic accuracy of the PI-RADS v2.1 multiparametric magnetic resonance imaging (mpMRI) features in predicting extraprostatic extension (mEPE) of prostate cancer (PCa), as well as to develop and validate a comprehensive mpMRI-derived score (mEPE-score). METHODS: We retrospectively reviewed all consecutive patients admitted to two institutions for radical prostatectomy for PCa with available records of mpMRI performed between January 2015 and December 2020. Data from one institution was used for investigating diagnostic performance of each mEPE feature using radical prostatectomy specimens as benchmark. The results were implemented in a mEPE-score as follows: no mEPE features: 1; capsular abutment: 2; irregular or spiculated margin: 3; bulging prostatic contour, or asymmetry of the neurovascular bundles, or tumor-capsule interface > 1.0 cm: 4; ≥ 2 of the previous three parameters or measurable extraprostatic disease: 5. The performance of mEPE features was evaluated using the five diagnostic parameters and ROC curve analysis. RESULTS: Two-hundred patients were enrolled at site 1 and 76 at site 2. mEPE features had poor sensitivities ranging from 0.08 (0.00-0.15) to 0.71 (0.59-0.83), whereas specificity ranged from 0.68 (0.58-0.79) to 1.00. mEPE-score showed excellent discriminating ability (AUC > 0.8) and sensitivity = 0.82 and specificity = 0.77 with a threshold of 3. mEPE-score had AUC comparable to ESUR-score (p = 0.59 internal validation; p = 0.82 external validation), higher than or comparable to mEPE-grade (p = 0.04 internal validation; p = 0.58 external validation), and higher than early-and-late-EPE (p < 0.0001 internal and external validation). There were no significant differences between readers having different expertise with EPE-score (p = 0.32) or mEPE-grade (p = 0.45), but there were significant differences for ESUR-score (p = 0.02) and early-versus-late-EPE (p = 0.03). CONCLUSIONS: The individual mEPE features have low sensitivity and high specificity. The use of mEPE-score allows for consistent and reliable assessment for pathologic EPE. KEY POINTS: • Individual PI-RADS v2.1 mpMRI features had poor sensitivities ranging from 0.08 (0.00-0.15) to 0.71 (0.59-0.83), whereas Sp ranged from 0.68 (0.58-0.79) to 1.00. • mEPE-score is an all-inclusive score for the assessment of pEPE with excellent discriminating ability (i.e., AUC > 0.8) and Se = 0.82, Sp = 0.77, PPV = 0.74, and NPV = 0.84 with a threshold of 3. • The diagnostic performance of the expert reader and beginner reader with pEPE-score was comparable (p = 0.32).

SKP2 drives the sensitivity to neddylation inhibitors and cisplatin in malignant pleural mesothelioma.

BACKGROUND: The combination of pemetrexed and cisplatin remains the reference first-line systemic therapy for malignant pleural mesothelioma (MPM). Its activity is moderate because of tumor aggressiveness, immune-suppressive environment and resistance to chemotherapy-induced immunogenic cell death (ICD). Preliminary and limited findings suggest that MPM cells have deregulated ubiquitination and proteasome activities, although proteasome inhibitors achieved disappointing clinical results. METHODS: Here, we investigated the role of the E3-ubiquitin ligase SKP/Cullin/F-box (SCF) complex in cell cycle progression, endoplasmic reticulum (ER)/proteostatic stress and ICD in MPM, and the therapeutic potential of the neddylation/SCF complex inhibitor MLN4924/Pevonedistat. RESULTS: In patient-derived MPM cultures and syngenic murine models, MLN4924 and cisplatin showed anti-tumor effects, regardless of MPM histotype and BAP1 mutational status, increasing DNA damage, inducing S- and G2/M-cell cycle arrest, and apoptosis. Mechanistically, by interfering with the neddylation of cullin-1 and ubiquitin-conjugating enzyme UBE2M, MLN4924 blocks the SCF complex activity and triggers an ER stress-dependent ICD, which activated anti-MPM CD8+T-lymphocytes. The SKP2 component of SCF complex was identified as the main driver of sensitivity to MLN4924 and resistance to cisplatin. These findings were confirmed in a retrospective MPM patient series, where SKP2 high levels were associated with a worse response to platinum-based therapy and inferior survival. CONCLUSIONS: We suggest that the combination of neddylation inhibitors and cisplatin could be worth of further investigation in the clinical setting for MPM unresponsive to cisplatin. We also propose SKP2 as a new stratification marker to determine the sensitivity to cisplatin and drugs interfering with ubiquitination/proteasome systems in MPM.

HLA-DRB1 mismatch-based identification of donor-derived cell free DNA (dd-cfDNA) as a marker of rejection in heart transplant recipients: A single-institution pilot study.

BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) is considered a reliable marker of organ damage with potential applications in the follow-up of transplant recipients. METHODS: In this work we present an assay based on the donor-recipient HLA-mismatch (human leukocyte antigen) at the HLA-DRB1 locus to monitor rejection by quantifying the percentage of dd-cfDNA using a droplet digital PCR (polymerase chain reaction) technique. A panel of probes targeting the HLA-DRB1 locus and covering >85% genetic variability was validated and used to assess dd-cfDNA levels in a prospective cohort of 19 adult heart transplant recipients (mean age 50.9±14.8 years). The assay was carried out on a total of 232 liquid biopsies collected at the same time as endomyocardial biopsy (EMB) during routine post-transplant follow-up. RESULTS: Results show a significant increase of dd-cfDNA related to ischemia-reperfusion injury (2.22±2.09%) and to acute cellular rejection (1.71±3.10%) compared to stable conditions (0.43±1.04%, p < 0.0001). On the contrary, no increase was observed during infections or vascular complications, underlining the potential role of this biomarker for rejection monitoring. With a cut-off of 0.11%, the test showed 70.8% specificity (95% CI, 58.17% - 81.40%) and 64.2% sensitivity (95% CI, 49.80% - 76.86%) in discriminating acute rejection from no rejection. CONCLUSIONS: These data demonstrate that this HLA mismatch-based droplet digital PCR method is effective for monitoring rejection in heart transplant recipients. Compared to next generation sequencing approaches, it is far more flexible, less expensive and provides faster results.

Predictor Analysis in Radiofrequency Ablation of Benign Thyroid Nodules: A Single Center Experience.

Purpose: To confirm the efficacy of ultrasound (US) guided radiofrequency ablation (RFA) in the treatment of benign thyroid nodules, we evaluated as primary outcome the technical efficacy and clinical success in a single center dataset. The secondary outcome was to find a correlation between nodules' pre-treatment features and volume reduction rate (VRR) ≥75% at 12 months after RFA and during follow-up period. Methods: This retrospective study included 119 consecutive patients (99 females, 20 males, 51.5 ± 14.4 years) with benign thyroid nodules treated in our hospital between October 2014 and December 2018 with a mean follow-up of 26.8 months (range 3-48). Clinical and US features before and after RFA were evaluated by a US examination at 1, 3, 6, 12 months and annually thereafter up to 48 months. Results: The median pre-treatment volume was 22.4 ml; after RFA we observed a statistically significant volume reduction from the first month (11.7 ml) to the last follow-up (p < 0.001 for all follow-up times). The median VRR was 47.1, 55.3, 61.2, 67.6, 72.8, 71.3, and 62.9% at 1, 3, 6, 12, 24, 36, and 48 months of follow-up respectively, showing a progressive significant improvement up to 24 months (VRRs 1 vs 3 months, 3 vs 6 months and 6 vs 12 months p < 0.001, 12 vs 24 months p = 0.05) while no differences at 24 vs 36 and 36 vs 48 months were observed. Symptoms improved significantly (complete resolution 64.35%, partial resolution 35.65%), and neck circumference was reduced as compared to pre-treatment (p < 0.001). Lower pre-treatment neck circumference (37.5 vs 36.0 cm, p = 0.01) was a positive predictor of VRR ≥75% at 12 months. Macrocystic echostructure (HR 2.48, p 0.046) and pre-treatment volume >22.4 ml (HR 0.54, p 0.036) were found to be independent positive and negative predictors of VRR ≥75% respectively. One-month post RFA VRR ≥50% represented the best positive predictor of technical success. Conclusions: This study confirmed the efficacy of RFA in the treatment of benign thyroid nodules. In particular we show that by selecting macrocystic nodules smaller than 22.4 ml better long-term response can be achieved, which is predicted by an early shrinkage of the nodule.

Intron 4-5 hTERT DNA Hypermethylation in Merkel Cell Carcinoma: Frequency, Association with Other Clinico-pathological Features and Prognostic Relevance.

Merkel cell carcinoma (MCC) is an aggressive skin tumor with neuroendocrine differentiation, mainly affecting elderly population or immunocompromised individuals. As methylation of the human telomerase reverse transcriptase (mhTERT) has been shown to be a prognostic factor in different tumors, we investigated its role in MCC, in particular in intron 4-5 where rs10069690 has been mapped and recognized as a cancer susceptibility locus. DNA methylation analysis of hTERT gene was assessed retrospectively in a cohort of 69 MCC patients from the University of Bologna, University of Turin and University of Insubria. Overall mortality was evaluated with Kaplan-Meier curves and multivariable Royston-Parmar models. High levels of mhTERT (mhTERThigh) (HR = 2.500, p = 0.015) and p63 (HR = 2.659, p = 0.016) were the only two clinico-pathological features significantly associated with a higher overall mortality at the multivariate analysis. We did not find different levels of mhTERT between MCPyV (+) and (-) cases (21 vs 14, p = 0.554); furthermore, mhTERThigh was strongly associated with older age (80.5 vs 72 years, p = 0.026), no angioinvasion (40.7% vs 71.0%, p = 0.015), lower Ki67 (50 vs 70%, p = 0.005), and PD-L1 expressions in both tumor (0 vs 3%, p = 0.021) and immune cells (0 vs 10%, p = 0.002). mhTERT is a frequently involved epigenetic mechanism and a relevant prognostic factor in MCC. In addition, it belongs to the shared oncogenic pathways of MCC (MCPyV and UV-radiations) and it could be crucial, together with other epigenetic and genetic mechanisms as gene amplification, in determining the final levels of hTERT mRNA and telomerase activity in these patients.

Protective Role of the M-Sec-Tunneling Nanotube System in Podocytes.

BACKGROUND: Podocyte dysfunction and loss are major determinants in the development of proteinuria. FSGS is one of the most common causes of proteinuria, but the mechanisms leading to podocyte injury or conferring protection against FSGS remain poorly understood. The cytosolic protein M-Sec has been involved in the formation of tunneling nanotubes (TNTs), membrane channels that transiently connect cells and allow intercellular organelle transfer. Whether podocytes express M-Sec is unknown and the potential relevance of the M-Sec-TNT system in FSGS has not been explored. METHODS: We studied the role of the M-Sec-TNT system in cultured podocytes exposed to Adriamycin and in BALB/c M-Sec knockout mice. We also assessed M-Sec expression in both kidney biopsies from patients with FSGS and in experimental FSGS (Adriamycin-induced nephropathy). RESULTS: Podocytes can form TNTs in a M-Sec-dependent manner. Consistent with the notion that the M-Sec-TNT system is cytoprotective, podocytes overexpressed M-Sec in both human and experimental FSGS. Moreover, M-Sec deletion resulted in podocyte injury, with mitochondrial abnormalities and development of progressive FSGS. In vitro, M-Sec deletion abolished TNT-mediated mitochondria transfer between podocytes and altered mitochondrial bioenergetics. Re-expression of M-Sec reestablishes TNT formation and mitochondria exchange, rescued mitochondrial function, and partially reverted podocyte injury. CONCLUSIONS: These findings indicate that the M-Sec-TNT system plays an important protective role in the glomeruli by rescuing podocytes via mitochondrial horizontal transfer. M-Sec may represent a promising therapeutic target in FSGS, and evidence that podocytes can be rescued via TNT-mediated horizontal transfer may open new avenues of research.

Retrospective analysis of the ultrasound features of resected thyroid nodules.

PURPOSE: Several ultrasound (US) risk stratification systems (US-RSSs) have been proposed to stratify the risk of malignancy (ROM) of thyroid nodules. This risk might be overestimated due to selection bias and comparison with the cytological report alone. Our study aimed to compare ROM and diagnostic performance of three guidelines (ATA, AACE/ACE/AME, EUTIRADS) and evaluate the changes in unnecessary biopsy according to the nodule size cutoff for biopsy, using histology as gold standard. METHODS: This retrospective observational study included 146 consecutive patients who underwent surgery after US and cytological characterization. We analyzed the effectiveness and accuracy of three US-RSSs. RESULTS: 46.6% of nodules were diagnosed as malignant. Applying US-RSS, the percentage of nodules that should have been analyzed by biopsy was 84.25% with ATA, 69.86% with EUTIRADS and 64.38% with AACE/ACE/AME systems. The ROM was 94.9%, 86.0%, 87.0% for high-risk category, 36.4%, 32.0%, 35.4% for intermediate-risk category and 22.9%, 0.0%, 22.9% for low-risk category by ATA, AACE/ACE/AME and EUTIRADS systems, respectively. EUTIRADS and AACE/ACE/AME systems were more accurate in differentiating malignant from benign cases. ATA score was the more sensitive US-RSS to identify malignant tumors within the high-risk category. About the unnecessary biopsies, in the intermediate-risk category, the application of the size criterion helps to increase specificity in all systems. CONCLUSIONS: The US categorization of low and high-risk thyroid nodules using current US-RSSs helps alone to determine the optimal treatment option. Nodule size remains relevant to recommend biopsy for the intermediate-risk category.

PD-1 (PDCD1) promoter methylation in Merkel cell carcinoma: prognostic relevance and relationship with clinico-pathological parameters.

Merkel cell carcinoma is an aggressive neuroendocrine skin tumor, for which several non-conclusive prognostic factors of adverse clinical behavior have been reported. As promoter methylation of the immune checkpoint receptor CD279/PD-1/PDCD1(mPDCD1) has been shown to be a prognostic factor in different cancers, we investigated its role in Merkel cell carcinoma. mPDCD1was assessed retrospectively in a cohort of 69 Merkel cell carcinoma patients from the University of Bologna, University of Turin and University of Insubria. Kaplan-Meier curves and log-rank tests were calculated for all variables. To assess the influence of mPDCD1, the Cox proportional hazards model and different Royston-Parmar models were evaluated. High PDCD1 methylation (mPDCD1high) was associated with a higher overall mortality at both the univariate analysis (log rank test: χ2 = 5.17, p = 0.023; permutation test: p = 0.023) and the multivariate analysis (HR = 2.111, p = 0.042). The other variables associated with a higher overall mortality at the multivariate analysis were clinical stage III-IV (HR = 2.357, p = 0.008), size > 2 cm (HR = 2.248, p = 0.031) and Merkel cell polyomavirus (HR = 0.397, p = 0.015). Further, mPDCD1high was strongly associated with older age (81 vs 76 years, p = 0.042), absence of immune cells (92.6%, p < 0.001), no expression of PD-L1 by immune cells (70.4%, p = 0.041) and by both immune and tumor cells (70.4%, p = 0.001). mPDCD1 is a valid prognostic parameter in patients affected by Merkel cell carcinoma. In addition, it could provide an estimate of the global PD-1/PD-L1 expression with potentially relevant implications from a therapeutic point of view.

ELOVL5 mutations cause spinocerebellar ataxia 38.

Spinocerebellar ataxias (SCAs) are a heterogeneous group of autosomal-dominant neurodegenerative disorders involving the cerebellum and 23 different genes. We mapped SCA38 to a 56 Mb region on chromosome 6p in a SCA-affected Italian family by whole-genome linkage analysis. Targeted resequencing identified a single missense mutation (c.689G>T [p.Gly230Val]) in ELOVL5. Mutation screening of 456 independent SCA-affected individuals identified the same mutation in two further unrelated Italian families. Haplotyping showed that at least two of the three families shared a common ancestor. One further missense variant (c.214C>G [p.Leu72Val]) was found in a French family. Both missense changes affect conserved amino acids, are predicted to be damaging by multiple bioinformatics tools, and were not identified in ethnically matched controls or within variant databases. ELOVL5 encodes an elongase involved in the synthesis of polyunsaturated fatty acids of the ω3 and ω6 series. Arachidonic acid and docosahexaenoic acid, two final products of the enzyme, were reduced in the serum of affected individuals. Immunohistochemistry on control mice and human brain demonstrated high levels in Purkinje cells. In transfection experiments, subcellular localization of altered ELOVL5 showed a perinuclear distribution with a signal increase in the Golgi compartment, whereas the wild-type showed a widespread signal in the endoplasmic reticulum. SCA38 and SCA34 are examples of SCAs due to mutations in elongase-encoding genes, emphasizing the importance of fatty-acid metabolism in neurological diseases.