Normal mode analysis (NMA) is a fast and inexpensive approach that is largely used to gain insight into functional protein motions, and more recently to create conformations for further computational ...studies. However, when the protein structure is unknown, the use of computational models is necessary. Here, we analyze the capacity of NMA in internal coordinate space to predict protein motion, its intrinsic flexibility, and atomic displacements, using protein models instead of native structures, and the possibility to use it for model refinement. Our results show that NMA is quite insensitive to modeling errors, but that calculations are strictly reliable only for very accurate models. Our study also suggests that internal NMA is a more suitable tool for the improvement of structural models, and for integrating them with experimental data or in other computational techniques, such as protein docking or more refined molecular dynamics simulations.
Brain Expressed X-linked (BEX) protein family consists of five members in humans and is highly expressed during neuronal development. They are known to participate in cell cycle and in signaling ...pathways involved in neurodegeneration and cancer. BEX3 possess a conserved leucine-rich nuclear export signal and experimental data confirmed BEX3 nucleocytoplasmic shuttling. Previous data revealed that mouse BEX3 auto-associates in an oligomer rich in intrinsic disorder. In this work, we show that human BEX3 (hBEX3) has well-defined three-dimensional structure in the presence of small fragments of tRNA (tRFs). Conversely, the nucleic acids-free purified hBEX3 presented disordered structure. Small-angle X-ray scattering data revealed that in the presence of tRFs, hBEX3 adopts compact globular fold, which is very distinct from the elongated high-order oligomer formed by the pure protein. Furthermore, microscopy showed that hBEX3 undergoes condensation in micron-sized protein-rich droplets in vitro. In the presence of tRFs, biomolecular condensates were smaller and in higher number, showing acridine orange green fluorescence emission, which corroborated with the presence of base-paired nucleic acids. Additionally, we found that over time hBEX3 transits from liquid condensates to aggregates that are reversible upon temperature increment and dissolved by 1,6-hexanediol. hBEX3 assemblies display different morphology in the presence of the tRFs that seems to protect from amyloid formation. Collectively, our findings support a role for tRFs in hBEX3 disorder-to-order transition and modulation of phase transitions. Moreover, hBEX3 aggregation-prone features and the specificity in interaction with tRNA fragments advocate paramount importance toward understanding BEX family involvement in neurodevelopment and cell death.
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•We study the short-term manpower planning problem for transhipment container terminals.•A deterministic optimization model is proposed for this problem.•The model solutions are compared to the ...manpower policy adopted by a real TCT.•We show when the terminal policy is effective or there is room for optimization.•The model can be optimally solved even in the case of huge transhipment container terminals.
This paper investigates the short-term manpower planning problem regarding transhipment container terminals. It consists of determining shifts, tasks and activities of the manpower working in these terminals in order to serve vessels in time intervals, which typically do not overlap with personnel shifts. This complex problem is modelled by an integer linear programming formulation. The optimal solutions of the model are compared with the decisions made in accordance with the manpower policy adopted by a real transhipment container terminal. The experimentation sheds light on when its policy is effective or when there is room for optimisation. The computational tests indicate that the model can be optimally solved even in the case of huge transhipment container terminals.
Regulatory T cells (Tregs) are important controllers of the immune system homeostasis by preventing disproportionate immune responses. In the context of cancer, Tregs contribute to tumor development ...by suppressing other immune cells in the tumor microenvironment (TME). Infiltration of Tregs in the TME has been associated with poor prognosis in cancer patients. Thus, understanding the mechanisms underlying Treg recruitment and suppressive functions is essential for developing cancer immunotherapies to boost antitumor immune responses. While antibody‐based strategies targeting Tregs have shown promise, small molecule inhibitors offer distinct advantages, including oral bioavailability and the ability to penetrate the TME and target intracellular proteins. Here, we provide an overview of small molecule inhibitors that have demonstrated efficacy in modulating Tregs activity in cancer and highlight the need for phenotypic assays to characterize therapeutic compounds.
Small molecule inhibitors modulating different pathways involved in regulatory T cells infiltration into the tumor microenvironment, metabolism, epigenome, stability or their suppressive functions can be attractive approaches for the treatment of cancer. The combination of Treg inhibitors with other immunotherapies may become a promising strategy to boost antitumor immunity.
Key message
NADP-ME2 from
Arabidopsis thaliana
exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and effector concentrations. In ...this work, we used molecular modeling approach and site-directed mutagenesis to characterized the NADP-ME2 structural determinants necessary for allosteric regulation providing new insights for enzyme optimization.
Structure–function studies contribute to deciphering how small modifications in the primary structure could introduce desirable characteristics into enzymes without affecting its overall functioning. Malic enzymes (ME) are ubiquitous and responsible for a wide variety of functions. The availability of a high number of ME crystal structures from different species facilitates comparisons between sequence and structure. Specifically, the structural determinants necessary for fumarate allosteric regulation of ME has been of particular interest. NADP-ME2 from
Arabidopsis thaliana
exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and effector concentrations. However, the 3D structure for this enzyme is not yet reported. In this work, we characterized the NADP-ME2 allosteric site by structural modeling, molecular docking, normal mode analysis and mutagenesis. The regulatory site model and its docking analysis suggested that other C4 acids including malate, NADP-ME2 substrate, could also fit into fumarate’s pocket. Besides, a non-conserved cluster of hydrophobic residues in the second sphere of the allosteric site was identified. The substitution of one of those residues, L62, by a less flexible residue as tryptophan, resulted in a complete loss of fumarate activation and a reduction of substrate affinities for the active site. In addition, normal mode analysis indicated that conformational changes leading to the activation could originate in the region surrounding L62, extending through the allosteric site till the active site. Finally, the results in this work contribute to the understanding of structural determinants necessary for allosteric regulation providing new insights for enzyme optimization.
Graphic abstract
Intrahepatic oxidative stress is a key driver of inflammation and fibrogenesis in non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the role of extracellular Nicotinamide ...phosphoribosyltransferase (eNAMPT) and extracellular nicotinic acid phosphoribosyltransferase (eNAPRT) for the detection of advanced fibrosis. eNAMPT and eNAPRT were tested in 180 consecutive biopsy-proven NAFLD patients and compared with liver stiffness (LS) and the FIB-4 score. eNAMPT was similarly distributed across fibrosis stages, whereas eNAPRT was increased in patients with advanced fibrosis (
= 0.036) and was associated with advanced fibrosis (OR 1.08,
= 0.016). A multiple stepwise logistic regression model containing significant variables for advanced fibrosis (eNAPRT, type 2 diabetes, age, male sex, ALT) had an area under the curve (AUC) of 0.82 (Se 89.6%, Sp 67.3%, PPV 46.7%, NPV 93.8%) when compared to that of LS (0.79; Se 63.5%, Sp 86.2%, PPV 66.0%, NPV 84.8%) and to that of the FIB-4 score (0.73; Se 80.0%, Sp 56.8%, PPV 44.9%, NPV 86.6%). The use of eNAPRT in clinical practice might allow for the better characterization of NAFLD patients at higher risk of disease progression.
•BKV Ia genotype exhibited higher viral loads in urine and mutations at VP1 region.•The few patients who developed BKV nephropathy were infected with genotype Ia.•The NCCR architecture was similar ...between BKV Ia and Ib1 genotypes.•The possible association between genotype Ia and BKVAN should be further investigated.
BK polyomavirus (BKV) is an opportunist agent associated with nephropathy (BKVAN) in 1–10% of kidney transplant recipients. BKV is classified into genotypes or subgroups according to minor nucleotidic variations with unknown biological implications. Studies assessing the possible association between genotypes and the risk of BKVAN in kidney transplant patients have presented conflicting results. In these studies, genotype Ia, which is highly prevalent in Brazil, was less frequently found and, thus, comparative data on the biological properties of this genotype are lacking. In this study, BKV Ia and Ib1 genotypes were compared according to their viral load, genetic evolution (VP1 and NCCR) – in a cohort of renal transplant recipients. The patients infected with Ia (13/23; 56.5%) genotype exhibited higher viral loads in urine >1.4 log over Ib1 (10/23; 43.5%); p=0.025. In addition, genotype Ia was associated with diverse mutations at VP1 loops and sites under positive selection outside loops, which were totally absent in Ib1. Although the number of viremic patients was similar, the three patients who had BK nephropathy (BKVAN) were infected with Ia genotype. NCCR architecture (ww or rr) were not distinctive between Ia and Ib1 genotypes. Ia genotype, which is rare in other published BKV cohorts, presented some diverse biological properties in transplanted recipients in comparison to Ib1.
Patients diagnosed with T-cell leukemias and T-cell lymphomas (TCLs) still have a poor prognosis and an inadequate response to current therapies, highlighting the need for targeted treatments. We ...have analyzed the potential therapeutic value of the farnesyltransferase inhibitor, tipifarnib, in 25 TCL cell lines through the identification of genomic and/or immunohistochemical markers of tipifarnib sensitivity. More than half of the cell lines (60%) were considered to be sensitive. Tipifarnib reduced cell viability in these T-cell leukemia and TCL cell lines, induced apoptosis and modified the cell cycle. A mutational study showed TP53, NOTCH1 and DNMT3 to be mutated in 84.6%, 69.2% and 30.0% of sensitive cell lines, and in 62.5%, 0% and 0% of resistant cell lines, respectively. An immunohistochemistry study showed that p-ERK and RelB were associated as potential biomarkers of tipifarnib sensitivity and resistance, respectively. Data from RNA-seq show that tipifarnib at IC
after 72 h downregulated a great variety of pathways, including those controlling cell cycle, metabolism, and ribosomal and mitochondrial activity. This study establishes tipifarnib as a potential therapeutic option in T-cell leukemia and TCL. The mutational state of NOTCH1, p-ERK and RelB could serve as potential biomarkers of tipifarnib sensitivity and resistance.
Most carcinomas present some form of chromosome instability in combination with spindle defects. Numerical instability is likely caused by spindle aberrations, but the origin of breaks and ...translocations remains elusive. To determine whether one mechanism can bring about both types of instability, we studied the relationship between DNA damage and spindle defects. Although lacking apparent repair defects, primary Dido mutant cells formed micronuclei containing damaged DNA. The presence of centromeres showed that micronuclei were caused by spindle defects, and cell cycle markers showed that DNA damage was generated during mitosis. Although the micronuclei themselves persisted, the DNA damage within was repaired during S and G2 phases. DNA breaks in Dido mutant cells regularly colocalized with centromeres, which were occasionally distorted. Comparable defects were found in APC mutant cell lines, an independent system for spindle defects. On the basis of these results, we propose a model for break formation in which spindle defects lead to centromere shearing.
Background and objectives
Nonalcoholic fatty liver disease (NAFLD) management is focused on lifestyle modifications, but long‐term maintenance is a challenge for many individuals. This study aimed to ...evaluate the long‐term effects of two personalized energy‐restricted dietary strategies on weight loss, metabolic and hepatic outcomes in overweight/obese subjects with NAFLD.
Methods
Ninety‐eight subjects from the Fatty Liver in Obesity (FLiO) study (NCT03183193) were randomly assigned to the American Heart Association (AHA) or the FLiO dietary group in a 2‐year controlled trial. Anthropometry, body composition (DXA), biochemical parameters and hepatic status (ultrasonography, Magnetic Resonance Imaging, and elastography) were assessed at baseline, 6, 12 and 24 months.
Results
Both the AHA and FLiO diets significantly reduced body weight at 6 (−9.7% vs −10.1%), 12 (−6.7% vs −9.6%), and 24 months (−4.8% vs −7.6%) with significant improvements in body composition, biochemical and liver determinations throughout the intervention. At the end of the follow‐up, the FLiO group showed a greater decrease in ALT, liver stiffness and Fatty Liver Index, among others, compared to AHA group, although these differences were attenuated when the analyses were adjusted by weight loss percentage. The FLiO group also showed a greater increase in adiponectin compared to AHA group.
Conclusions
The AHA and FLiO diets were able to improve body weight and body composition, as well as metabolic and hepatic status of participants with overweight/obesity and NAFLD within a 2‐year follow‐up. These findings show that both strategies are suitable alternatives for NAFLD management. However, the FLiO strategy may provide more persistent benefits in metabolic and hepatic parameters.
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