Stearoyl CoA Desaturase-1 Silencing in Glioblastoma Cells: Phospholipid Remodeling and Cytotoxicity Enhanced upon Autophagy Inhibition

Modulation of lipid metabolism is a well-established cancer hallmark, and SCD1 has been recognized as a key enzyme in promoting cancer cell growth, including in glioblastoma (GBM), the deadliest brain tumor and a paradigm of cancer resistance. The central goal of this work was to identify, by MS, the phospholipidome alterations resulting from the silencing of SCD1 in human GBM cells, in order to implement an innovative therapy to fight GBM cell resistance. With this purpose, RNAi technology was employed, and low serum-containing medium was used to mimic nutrient deficiency conditions, at which SCD1 is overexpressed. Besides the expected increase in the saturated to unsaturated fatty acid ratio in SCD1 silenced-GBM cells, a striking increase in polyunsaturated chains, particularly in phosphatidylethanolamine and cardiolipin species, was noticed and tentatively correlated with an increase in autophagy (evidenced by the increase in LC3BII/I ratio). The contribution of autophagy to mitigate the impact of SCD1 silencing on GBM cell viability and growth, whose modest inhibition could be correlated with the maintenance of energetically associated mitochondria, was evidenced by using autophagy inhibitors. In conclusion, SCD1 silencing could constitute an important tool to halt GBM resistance to the available treatments, especially when coupled with a mitochondria disrupter chemotherapeutic.     

Patient-Derived Multiple Myeloma 3D Models for Personalized Medicine—Are We There Yet?

Despite the wide variety of existing therapies, multiple myeloma (MM) remains a disease with dismal prognosis. Choosing the right treatment for each patient remains one of the major challenges. A new approach being explored is the use of ex vivo models for personalized medicine. Two-dimensional culture or animal models often fail to predict clinical outcomes. Three-dimensional ex vivo models using patients’ bone marrow (BM) cells may better reproduce the complexity and heterogeneity of the BM microenvironment. Here, we review the strengths and limitations of currently existing patient-derived ex vivo three-dimensional MM models. We analyze their biochemical and biophysical properties, molecular and cellular characteristics, as well as their potential for drug testing and identification of disease biomarkers. Furthermore, we discuss the remaining challenges and give some insight on how to achieve a more biomimetic and accurate MM BM model. Overall, there is still a need for standardized culture methods and refined readout techniques. Including both myeloma and other cells of the BM microenvironment in a simple and reproducible three-dimensional scaffold is the key to faithfully mapping and examining the relationship between these players in MM. This will allow a patient-personalized profile, providing a powerful tool for clinical and research applications.     

Alteration in the Synaptic and Extrasynaptic Organization of AMPA Receptors in the Hippocampus of P301S Tau Transgenic Mice

Tau pathology is a hallmark of Alzheimer’s disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR expression, density, and subcellular distribution in the hippocampus of P301S mice using immunoblot, histoblot, and quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL). Histoblot and immunoblot showed differential regulation of GluA1 and GluA2 in the hippocampus of P301S mice. The GluA2 subunit was downregulated in the hippocampus at 3 months while both GluA1 and GluA2 subunits were downregulated at 10 months. However, the total amount of GluA1-4 was similar in P301S mice and in age-matched wild-type mice. Using quantitative SDS-FRL, we unraveled the molecular organization of GluA1-4 in various synaptic connections at a high spatial resolution on pyramidal cell spines and interneuron dendrites in the CA1 field of the hippocampus in 10-month-old P301S mice. The labeling density for GluA1-4 in the excitatory synapses established on spines was significantly reduced in P301S mice, compared to age-matched wild-type mice, in the strata radiatum and lacunosum-moleculare but unaltered in the stratum oriens. The density of synaptic GluA1-4 established on interneuron dendrites was significantly reduced in P301S mice in the three strata. The labeling density for GluA1-4 at extrasynaptic sites was significantly reduced in several postsynaptic compartments of CA1 pyramidal cells and interneurons in the three dendritic layers in P301S mice. Our data demonstrate that the progressive accumulation of phospho-tau is associated with alteration of AMPARs on the surface of different neuron types, including synaptic and extrasynaptic membranes, leading to a decline in the trafficking and synaptic transmission, thereby likely contributing to the pathological events taking place in AD.     

Mass Spectrometry-Based Proteomic and Metabolomic Profiling of Serum Samples for Discovery and Validation of Tuberculosis Diagnostic Biomarker Signature

Tuberculosis (TB) is a transmissible disease listed as one of the 10 leading causes of death worldwide (10 million infected in 2019). A swift and precise diagnosis is essential to forestall its transmission, for which the discovery of effective diagnostic biomarkers is crucial. In this study, we aimed to discover molecular biomarkers for the early diagnosis of tuberculosis. Two independent cohorts comprising 29 and 34 subjects were assayed by proteomics, and 49 were included for metabolomic analysis. All subjects were arranged into three experimental groups—healthy controls (controls), latent TB infection (LTBI), and TB patients. LC-MS/MS blood serum protein and metabolite levels were submitted to univariate, multivariate, and ROC analysis. From the 149 proteins quantified in the discovery set, 25 were found to be differentially abundant between controls and TB patients. The AUC, specificity, and sensitivity, determined by ROC statistical analysis of the model composed of four of these proteins considering both proteomic sets, were 0.96, 93%, and 91%, respectively. The five metabolites (9-methyluric acid, indole-3-lactic acid, trans-3-indoleacrylic acid, hexanoylglycine, and N-acetyl-L-leucine) that better discriminate the control and TB patient groups (VIP > 1.75) from a total of 92 metabolites quantified in both ionization modes were submitted to ROC analysis. An AUC = 1 was determined, with all samples being correctly assigned to the respective experimental group. An integrated ROC analysis enrolling one protein and four metabolites was also performed for the common control and TB patients in the proteomic and metabolomic groups. This combined signature correctly assigned the 12 controls and 12 patients used only for prediction (AUC = 1, specificity = 100%, and sensitivity = 100%). This multiomics approach revealed a biomarker signature for tuberculosis diagnosis that could be potentially used for developing a point-of-care diagnosis clinical test.     

Revealing the Phenotypic and Genomic Background for PHA Production from Rapeseed-Biodiesel Crude Glycerol Using Photobacterium ganghwense C2.2

Polyhydroxyalkanoates (PHA) are promising biodegradable and biocompatible bioplastics, and extensive knowledge of the employed bacterial strain’s metabolic capabilities is necessary in choosing economically feasible production conditions. This study aimed to create an in-depth view of the utilization of Photobacterium ganghwense C2.2 for PHA production by linking a wide array of characterization methods: metabolic pathway annotation from the strain’s complete genome, high-throughput phenotypic tests, and biomass analyses through plate-based assays and flask and bioreactor cultivations. We confirmed, in PHA production conditions, urea catabolization, fatty acid degradation and synthesis, and high pH variation and osmotic stress tolerance. With urea as a nitrogen source, pure and rapeseed-biodiesel crude glycerol were analyzed comparatively as carbon sources for fermentation at 20 °C. Flask cultivations yielded 2.2 g/L and 2 g/L PHA at 120 h, respectively, with molecular weights of 428,629 g/mol and 81,515 g/mol. Bioreactor batch cultivation doubled biomass accumulation (10 g/L and 13.2 g/L) in 48 h, with a PHA productivity of 0.133 g/(L·h) and 0.05 g/(L·h). Thus, phenotypic and genomic analyses determined the successful use of Photobacterium ganghwense C2.2 for PHA production using urea and crude glycerol and 20 g/L NaCl, without pH adjustment, providing the basis for a viable fermentation process.     

11C-Methionine PET/CT in Assessment of Multiple Myeloma Patients: Comparison to 18F-FDG PET/CT and Prognostic Value

Multiple myeloma (MM) is the second most common haematological malignancy and remains incurable despite therapeutic advances. ¹⁸F-FDG (FDG) PET/CT is a relevant tool MM for staging and it is the reference imaging technique for treatment evaluation. However, it has limitations, and investigation of other PET tracers is required. Preliminary results with L-methyl-[¹¹C]- methionine (MET), suggest higher sensitivity than ¹⁸F-FDG. This study aimed to compare the diagnostic accuracy and prognostic value of ¹FDG and MET in MM patients. We prospectively compared FDG and MET PET/CT for assessment of bone disease and extramedullary disease (EMD) in a series of 52 consecutive patients (8 smoldering MM, 18 newly diagnosed MM and 26 relapsed MM patients). Bone marrow (BM) uptake patterns and the detection of focal lesions (FLs) and EMD were compared. Furthermore, FDG PET parameters with known MM prognostic value were explored for both tracers, as well as total lesion MET uptake (TLMU). Median patient age was 61 years (range, 37–83 years), 54% were male, 13% of them were in stage ISS (International Staging System) III, and 31% had high-risk cytogenetics. FDG PET/CT did not detect active disease in 6 patients, while they were shown to be positive by MET PET/CT. Additionally, MET PET/CT identified a higher number of FLs than FDG in more than half of the patients (63%). For prognostication we focussed on the relapsed cohort, due to the low number of progressions in the two other cohorts. Upon using FDG PET/CT in relapsed patients, the presence of more than 3 FLs (HR 4.61, p = 0.056), more than 10 FLs (HR 5.65, p = 0.013), total metabolic tumor volume (TMTV) p50 (HR 4.91, p = 0.049) or TMTV p75 (HR 5.32, p = 0.016) were associated with adverse prognosis. In MET PET/CT analysis, TMTV p50 (HR 4.71, p = 0.056), TMTV p75 (HR 6.27, p = 0.007), TLMU p50 (HR 8.8, p = 0.04) and TLMU p75 (HR 6.3, p = 0.007) adversely affected PFS. This study confirmed the diagnostic and prognostic value of FDG in MM. In addition, it highlights that MET has higher sensitivity than FDG PET/CT for detection of myeloma lesions, including FLs. Moreover, we show, for the first time, the prognostic value of TMTV and TLMU MET PET/CT in the imaging evaluation of MM patients.     

How Can We Improve Vaccination Response in Old People? Part I: Targeting Immunosenescence of Innate Immunity Cells

Vaccination, being able to prevent millions of cases of infectious diseases around the world every year, is the most effective medical intervention ever introduced. However, immunosenescence makes vaccines less effective in providing protection to older people. Although most studies explain that this is mainly due to the immunosenescence of T and B cells, the immunosenescence of innate immunity can also be a significant contributing factor. Alterations in function, number, subset, and distribution of blood neutrophils, monocytes, and natural killer and dendritic cells are detected in aging, thus potentially reducing the efficacy of vaccines in older individuals. In this paper, we focus on the immunosenescence of the innate blood immune cells. We discuss possible strategies to counteract the immunosenescence of innate immunity in order to improve the response to vaccination. In particular, we focus on advances in understanding the role and the development of new adjuvants, such as TLR agonists, considered a promising strategy to increase vaccination efficiency in older individuals.     

Gain-of-Function Dynamin-2 Mutations Linked to Centronuclear Myopathy Impair Ca2+-Induced Exocytosis in Human Myoblasts

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 μM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.