Partial purification and MALDI-TOF MS analysis of UN1, a tumor antigen membrane glycoprotein.

UN1 is a membrane glycoprotein that is expressed in immature human thymocytes, a subpopulation of peripheral T lymphocytes, the HPB acute lymphoblastic leukemia (ALL) T-cell line and fetal thymus. We previously reported the isolation of a monoclonal antibody (UN1 mAb) recognizing the UN1 protein that was classified as "unclustered" at the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. UN1 was highly expressed in breast cancer tissues and was undetected in non-proliferative lesions and in normal breast tissues, indicating a role for UN1 in the development of a tumorigenic phenotype of breast cancer cells. In this study, we report a partial purification of the UN1 protein from HPB-ALL T cells by anion-exchange chromatography followed by immunoprecipitation with the UN1 mAb and MALDI-TOF MS analysis. This analysis should assist in identifying the amino acid sequence of UN

Mass Spectrometry-Based Metabolomic and Proteomic Strategies in Organic Acidemias

Organic acidemias (OAs) are inherited metabolic disorders caused by deficiency of enzymatic activities in the catabolism of amino acids, carbohydrates, or lipids. These disorders result in the accumulation of mono-, di-, or tricarboxylic acids, generally referred to as organic acids. The OA outcomes can involve different organs and/or systems. Some OA disorders are easily managed if promptly diagnosed and treated, whereas, in others cases, such as propionate metabolism-related OAs (propionic acidemia, PA; methylmalonic acidemia, MMA), neither diet, vitamin therapy, nor liver transplantation appears to prevent multiorgan impairment. Here, we review the recent developments in dissecting molecular bases of OAs by using integration of mass spectrometry-(MS-) based metabolomic and proteomic strategies. MS-based techniques have facilitated the rapid and economical evaluation of a broad spectrum of metabolites in various body fluids, also collected in small samples, like dried blood spots. This approach has enabled the timely diagnosis of OAs, thereby facilitating early therapeutic intervention. Besides providing an overview of MS-based approaches most frequently used to study the molecular mechanisms underlying OA pathophysiology, we discuss the principal challenges of metabolomic and proteomic applications to OAs

Metabolomic fingerprinting of renal disease progression in Bardet-Biedl syndrome reveals mitochondrial dysfunction in kidney tubular cells.

Chronic kidney disease (CKD) is a major clinical sign of patients with Bardet-Biedl syndrome (BBS), especially in those carrying BBS10 mutations. Twenty-nine patients with BBS and 30 controls underwent a serum-targeted metabolomic analysis. In vitro studies were conducted in two kidney-derived epithelial cell lines, where Bbs10 was stably deleted (IMCD3-Bbs10-/-cells) and over-expressed. The CKD status affected plasmatic metabolite fingerprinting in both patients with BBS and controls. Specific phosphatidylcholine and acylcarnitines discriminated eGFR decline only in patients with BBS. IMCD3-Bbs10-/ cells displayed intracellular lipidaccumulation, reduced mitochondrial potential membrane and citrate synthase staining. Mass-Spectrometry-based analysis revealed that human BBS10 interacted with six mitochondrial proteins, in vitro. In conclusion, renal dysfunction correlated with abnormal phosphatidylcholine and acylcarnitines plasma levels in patients with BBS; in vitro, Bbs10 depletion caused mitochondrial defects while human BBS10 interacted with several mitochondria-related proteins, suggesting an unexplored role of this protein

The first knock-in rat model for glutaric aciduria type I allows further insights into pathophysiology in brain and periphery.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra-and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new ther-apeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery

Minimally Invasive Surgery for the Management of Lung Cancer

Lung cancer is the leading cause of cancer-related death and the most diagnosed cancer. The treatment of Non-Small Cell Lung Cancer (NSCLC) depends on clinical staging. Surgical radical resection is recommended for patients with stage 1 or 2 of disease and represents the treatment of choice. In the last decades, the surgical approach for lung cancer changed moving from an open approach to a minimally invasive approach, represented by Video Assisted Thoracic Surgery (VATS) and Robot-Assisted Thoracic Surgery (RATS). In this chapter, we illustrate the characteristics of lung cancer, the diagnosis, the classification, the staging and the preoperative evaluation. Then we focus on the surgical treatment of lung cancer and on how it has changed during the years. We explain the open approach represented by the traditional posterolateral thoracotomy and by the muscle-sparing thoracotomy. We illustrate VATS approach and evolution: from the hybrid approach to the pure VATS that can be triportal, biportal or even uniportal. Then, we focus on RATS approach, characterized by the use of multiple ports in the same intercostal space and how it evolved toward the uniportal approach. The objective is to combine the advantage of uniportal VATS (lower postoperative pain, enhanced recovery) and RATS (better visualization, more degrees of movements)

Exercise with food withdrawal at thermoneutrality impacts fuel use, the microbiome, AMPK phosphorylation, muscle fibers, and thyroid hormone levels in rats

Exercise under fasting conditions induces a switch to lipid metabolism, eliciting beneficial metabolic effects. Knowledge of signaling responses underlying metabolic adjustments in such conditions may help to identify therapeutic strategies. Therefore,

Beyond N staging in colorectal cancer: Current approaches and future perspectives

Traditionally, lymph node metastases (LNM) evaluation is essential to the staging of colon cancer patients according to the TNM (tumor–node–metastasis) system. However, in recent years evidence has accumulated regarding the role of emerging pathological features, which could significantly impact the prognosis of colorectal cancer patients. Lymph Node Ratio (LNR) and Log Odds of Positive Lymph Nodes (LODDS) have been shown to predict patients’ prognosis more accurately than traditional nodal staging and it has been suggested that their implementation in existing classification could help stratify further patients with overlapping TNM stage. Tumor deposits (TD) are currently factored within the N1c category of the TNM classification in the absence of lymph node metastases. However, studies have shown that presence of TDs can affect patients’ survival regardless of LNM. Moreover, evidence suggest that presence of TDs should not be evaluated as dichotomic but rather as a quantitative variable. Extranodal extension (ENE) has been shown to correlate with presence of other adverse prognostic features and to impact survival of colorectal cancer patients. In this review we will describe current staging systems and prognostic/predictive factors in colorectal cancer and elaborate on available evidence supporting the implementation of LNR/LODDS, TDs and ENE evaluation in existing classification to improve prognosis estimation and patient selection for adjuvant treatment

The Serum Metabolome of Moderate and Severe COVID-19 Patients Reflects Possible Liver Alterations Involving Carbon and Nitrogen Metabolism

COVID-19 is a global threat that has spread since the end of 2019, causing severe clinical sequelae and deaths, in the context of a world pandemic. The infection of the highly pathogenetic and infectious SARS-CoV-2 coronavirus has been proven to exert systemic effects impacting the metabolism. Yet, the metabolic pathways involved in the pathophysiology and progression of COVID-19 are still unclear. Here, we present the results of a mass spectrometry-based targeted metabolomic analysis on a cohort of 52 hospitalized COVID-19 patients, classified according to disease severity as mild, moderate, and severe. Our analysis defines a clear signature of COVID-19 that includes increased serum levels of lactic acid in all the forms of the disease. Pathway analysis revealed dysregulation of energy production and amino acid metabolism. Globally, the variations found in the serum metabolome of COVID-19 patients may reflect a more complex systemic perturbation induced by SARS-CoV-2, possibly affecting carbon and nitrogen liver metabolism