Metabolic, Mitochondrial, and Inflammatory Effects of Efavirenz, Emtricitabine, and Tenofovir Disoproxil Fumarate in Asymptomatic Antiretroviral-Naïve People with HIV.

This study aimed to comprehensively assess the metabolic, mitochondrial, and inflammatory effects of first-line efavirenz, emtricitabine, and tenofovir disoproxil fumarate (EFV/FTC/TDF) single-tablet regimen (STR) relative to untreated asymptomatic HIV infection. To this end, we analyzed 29 people with HIV (PWH) treated for at least one year with this regimen vs. 33 antiretroviral-naïve PWH. Excellent therapeutic activity was accompanied by significant alterations in metabolic parameters. The treatment group showed increased plasmatic levels of glucose, total cholesterol and its fractions (LDL and HDL), triglycerides, and hepatic enzymes (GGT, ALP); conversely, bilirubin levels (total and indirect fraction) decreased in the treated cohort. Mitochondrial performance was preserved overall and treatment administration even promoted the recovery of mitochondrial DNA (mtDNA) content depleted by the virus, although this was not accompanied by the recovery in some of their encoded proteins (since cytochrome c oxidase II was significantly decreased). Inflammatory profile (TNFα, IL-6), ameliorated after treatment in accordance with viral reduction and the recovery of TNFα levels correlated to mtDNA cell restoration. Thus, although this regimen causes subclinical metabolic alterations, its antiviral and anti-inflammatory properties may be associated with partial improvement in mitochondrial function.

Extranodal natural killer/T-cell lymphoma nasal type in a western population: Molecular profiling identifies new therapeutic targets.

(A) Correlation matrix of unsupervised co-regulated genes, based on the 208 genes included in the NanoString platform. Some of the clusters of co-regulated genes corresponded to the following: Inflammatory cells; Epstein-Barr virus; B-cells; Cytotoxic T-cells; T-cells; and Proliferation. (B) Analysis of genomic alterations by targeted sequencing. Distribution of mutations in the 62 analyzed genes. Rows correspond to sequenced genes, columns represent individual patients. Color coding: green, missense; blue, synonymous; pink, frameshift; violet, Indel; red, stop gained; yellow, UTR.

Variants affecting diverse domains of MEPE are associated with two distinct bone disorders, a craniofacial bone defect and otosclerosis.

PURPOSE: To characterize new molecular factors implicated in a hereditary congenital facial paresis (HCFP) family and otosclerosis. METHODS: We performed exome sequencing in a four-generation family presenting nonprogressive HCFP and mixed hearing loss (HL). MEPE was analyzed using either Sanger sequencing or molecular inversion probes combined with massive parallel sequencing in 89 otosclerosis families, 1604 unrelated affected subjects, and 1538 unscreened controls. RESULTS: Exome sequencing in the HCFP family led to the identification of a rare segregating heterozygous frameshift variant p.(Gln425Lysfs*38) in MEPE. As the HL phenotype in this family resembled otosclerosis, we performed variant burden and variance components analyses in a large otosclerosis cohort and demonstrated that nonsense and frameshift MEPE variants were significantly enriched in affected subjects (p = 0.0006-0.0060). CONCLUSION: MEPE exerts its function in bone homeostasis by two domains, an RGD and an acidic serine aspartate-rich MEPE-associated (ASARM) motif inhibiting respectively bone resorption and mineralization. All variants associated with otosclerosis are predicted to result in nonsense mediated decay or an ASARM-and-RGD-truncated MEPE. The HCFP variant is predicted to produce an ASARM-truncated MEPE with an intact RGD motif. This difference in effect on the protein corresponds with the presumed pathophysiology of both diseases, and provides a plausible molecular explanation for the distinct phenotypic outcome.

Homozygous and heterozygous disruptions of ANK3: at the crossroads of neurodevelopmental and psychiatric disorders.

AnkyrinG, encoded by the ANK3 gene, is involved in neuronal development and signaling. It has previously been implicated in bipolar disorder and schizophrenia by association studies. Most recently, de novo missense mutations in this gene were identified in autistic patients. However, the causative nature of these mutations remained controversial. Here, we report inactivating mutations in the Ankyrin 3 (ANK3) gene in patients with severe cognitive deficits. In a patient with a borderline intelligence, severe attention deficit hyperactivity disorder (ADHD), autism and sleeping problems, all isoforms of the ANK3 gene, were disrupted by a balanced translocation. Furthermore, in a consanguineous family with moderate intellectual disability (ID), an ADHD-like phenotype and behavioral problems, we identified a homozygous truncating frameshift mutation in the longest isoform of the same gene, which represents the first reported familial mutation in the ANK3 gene. The causality of ANK3 mutations in the two families and the role of the gene in cognitive function were supported by memory defects in a Drosophila knockdown model. Thus we demonstrated that ANK3 plays a role in intellectual functioning. In addition, our findings support the suggested association of ANK3 with various neuropsychiatric disorders and illustrate the genetic and molecular relation between a wide range of neurodevelopmental disorders.

Aerobic capacity and mitochondrial function in bipolar disorder: a longitudinal study during acute phases and after clinical remission.

Background: Aerobic capacity has shown to predict physical and mental health-related quality of life in bipolar disorder (BD). However, the correlation between exercise respiratory capacity and mitochondrial function remains understudied. We aimed to assess longitudinally intra-individual differences in these factors during mood episodes and remission in BD. Methods: This study included eight BD patients admitted to an acute psychiatric unit. Incremental cardiopulmonary exercise test (CPET) was conducted during acute episodes (T0), followed by constant work rate cycle ergometry (CWRCE) to evaluate endurance time, oxygen uptake at peak exercise (VO2peak) and at the anaerobic threshold. The second test was repeated during remission (T1). Mitochondrial respiration rates were assessed at T0 and T1 in peripheral blood mononuclear cells. Results: Endurance time, VO2peak, and anaerobic threshold oxygen consumption showed no significant variations between T0 and T1. Basal oxygen consumption at T1 tended to inversely correlate with maximal mitochondrial respiratory capacity (r=-0.690, p=0.058), and VO2peak during exercise at T1 inversely correlated with basal and minimum mitochondrial respiration (r=-0.810, p=0.015; r=-0.786, p=0.021, respectively). Conclusions: Our preliminary data showed that lower basal oxygen consumption may be linked to greater mitochondrial respiratory capacity, and maximum oxygen uptake during the exercise task was associated with lower basal mitochondrial respiration, suggesting that lower oxygen requirements could be associated with greater mitochondrial capacity. These findings should be replicated in larger samples stratified for manic and depressive states.

Impact of Cervical Cancer on Quality of Life and Sexuality in Female Survivors.

Cervical cancer is the fourth most frequent cancer in women worldwide, and the 11th most frequent neoplasm in Spain. Despite the optimization of treatments and a 5-year survival rate of 70%, side effects and sequelae are described after treatment. The treatments have physical, psychological and sociocultural consequences that deteriorate the quality of life of patients. One of the sequelae that worries patients is the impairment of sexual function and satisfaction, considered a fundamental dimension of the human being. The aim of this study was to examine quality of life and sexual function and satisfaction among Spanish cervical cancer survivors. A retrospective case-control study was conducted between 2019 and 2022. The sample consisted of 66 patients who completed the Female Sexual Function Index, the Golombok Rust Sexual Satisfaction Inventory and European Organization for Research and Treatment of Cancer quality of life questionnaire. The control group consisted of women without cervical cancer and gynecological pathologies obtained using the so-called online virtual sampling method. The patient group consisted of women with cervical cancer who completed treatment. Cervical cancer survivors reported sexual dysfunction and impaired sexual satisfaction in almost half of the domains. Quality of life was also affected, with pain and fatigue being the most frequent symptoms in these patients. Our results indicate that there is dysfunction, sexual dissatisfaction and a lower level of quality of life in cervical cancer survivors than in healthy women without pathology.

Integrated Multi-Omics Analysis for Inferring Molecular Players in Inclusion Body Myositis.

Inclusion body myositis (IBM) is an acquired inflammatory myopathy affecting proximal and distal muscles that leads to weakness in patients over 50. It is diagnosed based on clinical and histological findings in muscle related to inflammation, degeneration, and mitochondria. In relation to IBM, a shortage of validated disease models and a lack of biomarkers and effective treatments constitute an unmet medical need. To overcome these hurdles, we performed an omics analysis of multiple samples from IBM patients (saliva, fibroblasts, urine, plasma, and muscle) to gain insight into the pathophysiology of IBM. Degeneration was evident due to the presence of amyloid ß peptide 1-42 (Aß1-42) in the saliva of the analyzed IBM patients. The presence of metabolic disarrangements in IBM was indicated by an imbalanced organic acid profile in fibroblasts and urine. Specifically, abnormal levels of L-pyroglutamic and orotic acid were supported by the abnormal expression of related metabolites in plasma and urine (glutathione and pyrimidines) and the aberrant expression of upstream gene regulators (L2HGDH, IDH2, OPLAH, and ASL) in muscle. Combined levels of L-pyroglutamic and orotic acid displayed an outstanding biomarker signature in urine with 100% sensitivity and specificity. The confirmation of systemic metabolic disarrangements in IBM and the identification of novel biomarkers reported herein unveil novel insights that require validation in larger cohorts.

Coordinated local RNA overexpression of complement induced by interferon gamma in myositis.

Complement proteins are deposited in the muscles of patients with myositis. However, the local expression and regulation of complement genes within myositis muscle have not been well characterized. In this study, bulk RNA sequencing (RNAseq) analyses of muscle biopsy specimens revealed that complement genes are locally overexpressed and correlate with markers of myositis disease activity, including the expression of interferon-gamma (IFNγ)-induced genes. Single cell and single nuclei RNAseq analyses showed that most local expression of complement genes occurs in macrophages, fibroblasts, and satellite cells, with each cell type expressing different sets of complement genes. Biopsies from immune-mediated necrotizing myopathy patients, who have the lowest levels of IFNγ-induced genes, also had the lowest complement gene expression levels. Furthermore, data from cultured human cells showed that IFNγ upregulates complement expression in macrophages, fibroblasts, and muscle cells. Taken together, our results suggest that in myositis muscle, IFNγ coordinates the local overexpression of complement genes that occurs in several cell types.

Unravelling inclusion body myositis using a patient-derived fibroblast model.

BACKGROUND: Inclusion body myositis (IBM) is an inflammatory myopathy clinically characterized by proximal and distal muscle weakness, with inflammatory infiltrates, rimmed vacuoles and mitochondrial changes in muscle histopathology. There is scarce knowledge on IBM aetiology, and non-established biomarkers or effective treatments are available, partly due to the lack of validated disease models. METHODS: We have performed transcriptomics and functional validation of IBM muscle pathological hallmarks in fibroblasts from IBM patients (n = 14) and healthy controls (n = 12), paired by age and sex. The results comprise an mRNA-seq, together with functional inflammatory, autophagy, mitochondrial and metabolic changes between patients and controls. RESULTS: Gene expression profile of IBM vs control fibroblasts revealed 778 differentially expressed genes (P-value adj < 0.05) related to inflammation, mitochondria, cell cycle regulation and metabolism. Functionally, an increased inflammatory profile was observed in IBM fibroblasts with higher supernatant cytokine secretion (three-fold increase). Autophagy was reduced considering basal protein mediators (18.4% reduced), time-course autophagosome formation (LC3BII 39% reduced, P-value < 0.05), and autophagosome microscopic evaluation. Mitochondria displayed reduced genetic content (by 33.9%, P-value < 0.05) and function (30.2%-decrease in respiration, 45.6%-decline in enzymatic activity (P-value < 0.001), 14.3%-higher oxidative stress, 135.2%-increased antioxidant defence (P-value < 0.05), 11.6%-reduced mitochondrial membrane potential (P-value < 0.05) and 42.8%-reduced mitochondrial elongation (P-value < 0.05)). In accordance, at the metabolite level, organic acid showed a 1.8-fold change increase, with conserved amino acid profile. Correlating to disease evolution, oxidative stress and inflammation emerge as potential markers of prognosis. CONCLUSIONS: These findings confirm the presence of molecular disturbances in peripheral tissues from IBM patients and prompt patients' derived fibroblasts as a promising disease model, which may eventually be exported to other neuromuscular disorders. We additionally identify new molecular players in IBM associated with disease progression, setting the path to deepen in disease aetiology, in the identification of novel biomarkers or in the standardization of biomimetic platforms to assay new therapeutic strategies for preclinical studies.

Birth-weight differences at term are explained by placental dysfunction and not by maternal ethnicity. Study in newborns of first generation immigrants.

OBJECTIVE: The aim of the study was to investigate the influence of ethnicity and cerebroplacental ratio (CPR) on the birth weight (BW) of first generation Indo-Pakistan immigrants' newborns. METHODS: This was a retrospective study in a mixed population of 620 term Caucasian and Indo-Pakistan pregnancies, evaluated in two reference hospitals of Spain and Italy. All fetuses underwent a scan and Doppler examination within two weeks of delivery. The influence of fetal gender, ethnicity, GA at delivery, CPR, maternal age, height, weight and parity on BW was evaluated by multivariable regression analysis. RESULTS: Newborns of first generation Indo-Pakistan immigrants were smaller than local Caucasian newborns (mean BW mean= 3048 ± 435 g versus 3269 ± 437 g, p < .001). Multivariable regression analysis demonstrated that all studied parameters, but maternal age and ethnicity, were significantly associated with BW. The most important were GA at delivery (partial R2 = 0.175, p < .001), CPR (partial R2 = 0.032, p < .001), and fetal gender (partial R2 = 0,029, p < .001). CONCLUSIONS: The propensity to a lower BW, explained by placental dysfunction but not by maternal ethnicity is transmitted to newborns of first generation immigrants. Whatever are the factors implied they persist in the new residential setting.

Premenstrual and menstrual changes reported after COVID-19 vaccination: The EVA project.

BACKGROUND: Menstrual disorders were not reported as a possible secondary effect in any of the clinical trials for the SARS-CoV-2 vaccines. AIM: To describe the prevalence of perceived premenstrual and menstrual changes after COVID-19 vaccine administration. DESIGN: Cross-sectional study. METHODS: A total of 14,153 women (mean age 31.5 ± 9.3 years old) who had received the full course of vaccination at least three months earlier were included in this cross-sectional study. Data including the type of vaccine administered, perceived changes in the amount and duration of menstrual bleeding, presence of clots, cycle length, and premenstrual symptoms were collected through a retrospective online survey from June to September 2021. RESULTS: Of the women who participated in this study, 3136 reported no menstrual changes and 11,017 (78% of the study sample) reported experiencing menstrual cycle changes after vaccination. In summary, women who reported menstrual changes after vaccination were older (overall p < 0.001) and slightly more smokers (p = 0.05) than women who did not report any changes. The most prevalent changes in relation to premenstrual symptoms were increased fatigue (43%), abdominal bloating (37%), irritability (29%), sadness (28%), and headaches (28%). The most predominant menstrual changes were more menstrual bleeding (43%), more menstrual pain (41%), delayed menstruation (38%), fewer days of menstrual bleeding (34.5%), and shorter cycle length (32%). CONCLUSION: Women vaccinated against COVID-19 usually perceive mild menstrual and premenstrual changes. Future studies are warranted to clarify the physiological mechanisms behind these widely reported changes.

Developmental disruption and restoration of brain synaptome architecture in the murine Pax6 neurodevelopmental disease model.

Neurodevelopmental disorders of genetic origin delay the acquisition of normal abilities and cause disabling phenotypes. Nevertheless, spontaneous attenuation and even complete amelioration of symptoms in early childhood and adolescence can occur in many disorders, suggesting that brain circuits possess an intrinsic capacity to overcome the deficits arising from some germline mutations. We examined the molecular composition of almost a trillion excitatory synapses on a brain-wide scale between birth and adulthood in mice carrying a mutation in the homeobox transcription factor Pax6, a neurodevelopmental disorder model. Pax6 haploinsufficiency had no impact on total synapse number at any age. By contrast, the molecular composition of excitatory synapses, the postnatal expansion of synapse diversity and the acquisition of normal synaptome architecture were delayed in all brain regions, interfering with networks and electrophysiological simulations of cognitive functions. Specific excitatory synapse types and subtypes were affected in two key developmental age-windows. These phenotypes were reversed within 2-3 weeks of onset, restoring synapse diversity and synaptome architecture to the normal developmental trajectory. Synapse subtypes with rapid protein turnover mediated the synaptome remodeling. This brain-wide capacity for remodeling of synapse molecular composition to recover and maintain the developmental trajectory of synaptome architecture may help confer resilience to neurodevelopmental genetic disorders.

Neuronal induction and bioenergetics characterization of human forearm adipose stem cells from Parkinson's disease patients and healthy controls.

Neurodegenerative diseases, such as Parkinson's disease, are heterogeneous disorders with a multifactorial nature involving impaired bioenergetics. Stem-regenerative medicine and bioenergetics have been proposed as promising therapeutic targets in the neurologic field. The rationale of the present study was to assess the potential of human-derived adipose stem cells (hASCs) to transdifferentiate into neuronal-like cells (NhASCs and neurospheres) and explore the hASC bioenergetic profile. hASC neuronal transdifferentiation was performed through neurobasal media and differentiation factor exposure. High resolution respirometry was assessed. Increased MAP-2 neuronal marker protein expression upon neuronal induction (p<0.05 undifferentiated hASCs vs. 28-36 days of differentiation) and increased bIII-tubulin neuronal marker protein expression upon neuronal induction (p<0.05 undifferentiated hASCs vs. 6-28-36 days of differentiation) were found. The bioenergetic profile was detectable through high-resolution respirometry approaches in hASCs but did not lead to differential oxidative capacity rates in healthy or clinically diagnosed PD-hASCs. We confirmed the capability of transdifferentiation to the neuronal-like profile of hASCs derived from the forearms of human subjects and characterized the bioenergetic profile. Suboptimal maximal respiratory capacity trends in PD were found. Neuronal induction leading to positive neuronal protein expression markers is a relevant issue that encourages the suitability of NhASC models in neurodegeneration.

Assessment of mitochondrial toxicity in newborns and infants with congenital cytomegalovirus infection treated with valganciclovir.

BACKGROUND: Ganciclovir/valganciclovir is currently indicated during the first 6 months of life in symptomatic children with congenital cytomegalovirus (CMV) infection. However, this treatment may have the potential to induce mitochondrial toxicity due to off-target inhibition of DNA-polymerases. Similar anti-HIV drugs have been associated with mitochondrial toxicity but this has never been explored in CMV. OBJECTIVE: To determine the potential mitochondrial toxicity profile at the genetic, functional and biogenesis level in peripheral blood mononuclear cells from a cohort of newborns and infants with symptomatic congenital CMV infection (treated with valganciclovir, untreated and uninfected controls). DESIGN: Longitudinal, observational and controlled study. SETTING AND PATIENTS: Subjects were recruited at the tertiary referral Hospital Sant Joan de Déu and experiments were conducted at IDIBAPS-Hospital Clínic of Barcelona, Spain. CMV-infected newborns underwent comprehensive monthly clinical follow-up. METHODS: Mitochondrial parameters, audiometry and neurological assessment were measured at baseline, 3-6 and 12 months after inclusion in the study. The Kruskal-Wallis test for k-independent samples and Friedman tests for repeated measurements were applied. RESULTS: Complex IV, citrate synthase enzymatic activities and mtDNA remained preserved in congenital CMV-infected infants treated with valganciclovir compared with controls (p>0.05 in all cases). CONCLUSIONS: No evidence of mitochondrial toxicity was found in infants treated with valganciclovir for congenital CMV.

An integrated prognostic model for diffuse large B-cell lymphoma treated with immunochemotherapy.

Diffuse large B-cell lymphoma (DLBCL), the most frequent non-Hodgkin's lymphoma subtype, is characterized by strong biological, morphological, and clinical heterogeneity, but patients are treated with immunochemotherapy in a relatively homogeneous way. Here, we have used a customized NanoString platform to analyze a series of 197 homogeneously treated DLBCL cases. The platform includes the most relevant genes or signatures known to be useful for predicting response to R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone) in DLBCL cases. We generated a risk score that combines the International Prognostic Index with cell of origin and double expression of MYC/BCL2, and stratified the series into three groups, yielding hazard ratios from 0.15 to 5.49 for overall survival, and from 0.17 to 5.04 for progression-free survival. Group differences were highly significant (p < 0.0001), and the scoring system was applicable to younger patients (<60 years of age) and patients with advanced or localized stages of the disease. Results were validated in an independent dataset from 166 DLBCL patients treated in two distinct clinical trials. This risk score combines clinical and biological data in a model that can be used to integrate biological variables into the prognostic models for DLBCL cases.

Peripheral T-cell lymphoma: molecular profiling recognizes subclasses and identifies prognostic markers.

Peripheral T-cell lymphoma (PTCL) is a clinically aggressive disease, with a poor response to therapy and a low overall survival rate of approximately 30% after 5 years. We have analyzed a series of 105 cases with a diagnosis of PTCL using a customized NanoString platform (NanoString Technologies, Seattle, WA) that includes 208 genes associated with T-cell differentiation, oncogenes and tumor suppressor genes, deregulated pathways, and stromal cell subpopulations. A comparative analysis of the various histological types of PTCL (angioimmunoblastic T-cell lymphoma [AITL]; PTCL with T follicular helper [TFH] phenotype; PTCL not otherwise specified [NOS]) showed that specific sets of genes were associated with each of the diagnoses. These included TFH markers, cytotoxic markers, and genes whose expression was a surrogate for specific cellular subpopulations, including follicular dendritic cells, mast cells, and genes belonging to precise survival (NF-κB) and other pathways. Furthermore, the mutational profile was analyzed using a custom panel that targeted 62 genes in 76 cases distributed in AITL, PTCL-TFH, and PTCL-NOS. The main differences among the 3 nodal PTCL classes involved the RHOAG17V mutations (P < .0001), which were approximately twice as frequent in AITL (34.09%) as in PTCL-TFH (16.66%) cases but were not detected in PTCL-NOS. A multivariate analysis identified gene sets that allowed the series of cases to be stratified into different risk groups. This study supports and validates the current division of PTCL into these 3 categories, identifies sets of markers that can be used for a more precise diagnosis, and recognizes the expression of B-cell genes as an IPI-independent prognostic factor for AITL.

Estimation of the number of synapses in the hippocampus and brain-wide by volume electron microscopy and genetic labeling.

Determining the number of synapses that are present in different brain regions is crucial to understand brain connectivity as a whole. Membrane-associated guanylate kinases (MAGUKs) are a family of scaffolding proteins that are expressed in excitatory glutamatergic synapses. We used genetic labeling of two of these proteins (PSD95 and SAP102), and Spinning Disc confocal Microscopy (SDM), to estimate the number of fluorescent puncta in the CA1 area of the hippocampus. We also used FIB-SEM, a three-dimensional electron microscopy technique, to calculate the actual numbers of synapses in the same area. We then estimated the ratio between the three-dimensional densities obtained with FIB-SEM (synapses/µm3) and the bi-dimensional densities obtained with SDM (puncta/100 µm2). Given that it is impractical to use FIB-SEM brain-wide, we used previously available SDM data from other brain regions and we applied this ratio as a conversion factor to estimate the minimum density of synapses in those regions. We found the highest densities of synapses in the isocortex, olfactory areas, hippocampal formation and cortical subplate. Low densities were found in the pallidum, hypothalamus, brainstem and cerebellum. Finally, the striatum and thalamus showed a wide range of synapse densities.

Mapping arginine methylation in the human body and cardiac disease.

PURPOSE: Arginine methylation (ArgMe) is one of the most ubiquitous PTMs, and hundreds of proteins undergo ArgMe in, for example, brain. However, the scope of ArgMe in many tissues, including the heart, is currently underexplored. Here, we aimed to (i) identify proteins undergoing ArgMe in human organs, and (ii) expose the relevance of ArgMe in cardiac disease. EXPERIMENTAL DESIGN: The publicly available proteomic data is used to search for ArgMe in 13 human tissues. To induce H9c2 cardiac-like cell hypertrophy glucose is used. RESULTS: The results show that ArgMe is mainly tissue-specific; nevertheless, the authors suggest an embryonic origin of core ArgMe events. In the heart, 103 mostly novel ArgMe sites in 58 nonhistone proteins are found. The authors provide compelling evidence that cardiac protein ArgMe is relevant to cardiomyocyte ontology, and important for proper cardiac function. This is highlighted by the fact that genetic mutations affecting methylated arginine positions are often associated with cardiac disease, including hypertrophic cardiomyopathy. The pilot experimental data suggesting significant changes in ArgMe profiles of H9c2 cells upon induction of cell hypertrophy using glucose is provided. CONCLUSIONS AND CLINICAL RELEVANCE: The work calls for in-depth investigation of ArgMe in normal and diseased tissues using methods including clinical proteomics.