Heparin versus aspirin thromboprophylaxis adds independent value to IMPEDE-VTE score for venous thrombosis prediction in multiple myeloma.

Multiple myeloma (MM) is associated to an increased incidence of venous thromboembolism (VTE). IMPEDE-VTE score constitutes a valuable risk assessment tool for VTE prediction in first-line MM patients. Nevertheless, refinement of the primary thromboprophylaxis category of this score (which pools aspirin and heparin) seems desirable. To investigate the role of the type of thromboprophylaxis, within IMPEDE-VTE score, for VTE prediction in MM patients. Retrospective analysis of a single-center cohort of 438 MM patients receiving first-line antimyeloma treatment (1991-2020). IMPEDE-VTE score was calculated. Primary thromboprophylaxis was additionally stratified into aspirin- and heparin-based regimen subgroups. VTE risk was analyzed by Cox regression. Median follow-up during first-line antimyeloma treatment was 6.0 months (IQR 4.1-9.0 months). Twenty-three patients developed VTE (5.3%, 95%CI 3.4-7.8%). IMPEDE-VTE score showed a notable predictive value (area under the ROC curve: 0.70, 95%CI 0.60-0.80). Cox analysis confirmed that 1-point increase in the score resulted in a 1.3-fold increase in VTE risk (HR 1.30, 95%CI 1.13-1.53, p < 0.001). In the multivariable analysis, the type of primary thromboprophylaxis (heparin versus aspirin) was an independent predictive factor (HR 0.15, 95% CI 0.05-0.47, p = 0.001). The combined model showed a higher goodness-of-fit (Akaike Information Criterion [AIC]: 99) than IMPEDE-VTE separately (AIC:235). Our analysis contributes to the external validation of IMPEDE-VTE score for the prediction of VTE in MM. But more interestingly, our results demonstrate that among those patients receiving thromboprophylaxis, the type of regimen (heparin versus aspirin) adds independent predictive value and should be explored for a more accurate risk assessment.

Systems toxicology of chemically induced liver and kidney injuries: histopathology-associated gene co-expression modules.

Organ injuries caused by environmental chemical exposures or use of pharmaceutical drugs pose a serious health risk that may be difficult to assess because of a lack of non-invasive diagnostic tests. Mapping chemical injuries to organ-specific histopathology outcomes via biomarkers will provide a foundation for designing precise and robust diagnostic tests. We identified co-expressed genes (modules) specific to injury endpoints using the Open Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System (TG-GATEs) - a toxicogenomics database containing organ-specific gene expression data matched to dose- and time-dependent chemical exposures and adverse histopathology assessments in Sprague-Dawley rats. We proposed a protocol for selecting gene modules associated with chemical-induced injuries that classify 11 liver and eight kidney histopathology endpoints based on dose-dependent activation of the identified modules. We showed that the activation of the modules for a particular chemical exposure condition, i.e., chemical-time-dose combination, correlated with the severity of histopathological damage in a dose-dependent manner. Furthermore, the modules could distinguish different types of injuries caused by chemical exposures as well as determine whether the injury module activation was specific to the tissue of origin (liver and kidney). The generated modules provide a link between toxic chemical exposures, different molecular initiating events among underlying molecular pathways and resultant organ damage. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Journal of Applied Toxicology published by John Wiley & Sons, Ltd.

Adenine nucleotide translocase is acetylated in vivo in human muscle: Modeling predicts a decreased ADP affinity and altered control of oxidative phosphorylation.

Proteomics techniques have revealed that lysine acetylation is abundant in mitochondrial proteins. This study was undertaken (1) to determine the relationship between mitochondrial protein acetylation and insulin sensitivity in human skeletal muscle, identifying key acetylated proteins, and (2) to use molecular modeling techniques to understand the functional consequences of acetylation of adenine nucleotide translocase 1 (ANT1), which we found to be abundantly acetylated. Eight lean and eight obese nondiabetic subjects had euglycemic clamps and muscle biopsies for isolation of mitochondrial proteins and proteomics analysis. A number of acetylated mitochondrial proteins were identified in muscle biopsies. Overall, acetylation of mitochondrial proteins was correlated with insulin action (r = 0.60; P < 0.05). Of the acetylated proteins, ANT1, which catalyzes ADP-ATP exchange across the inner mitochondrial membrane, was acetylated at lysines 10, 23, and 92. The extent of acetylation of lysine 23 decreased following exercise, depending on insulin sensitivity. Molecular dynamics modeling and ensemble docking simulations predicted the ADP binding site of ANT1 to be a pocket of positively charged residues, including lysine 23. Calculated ADP-ANT1 binding affinities were physiologically relevant and predicted substantial reductions in affinity upon acetylation of lysine 23. Insertion of these derived binding affinities as parameters into a complete mathematical description of ANT1 kinetics predicted marked reductions in adenine nucleotide flux resulting from acetylation of lysine 23. Therefore, acetylation of ANT1 could have dramatic physiological effects on ADP-ATP exchange. Dysregulation of acetylation of mitochondrial proteins such as ANT1 therefore could be related to changes in mitochondrial function that are associated with insulin resistance.

Incidence and prognostic impact of U2AF1 mutations and other gene alterations in myelodysplastic neoplasms with isolated 20q deletion.

BACKGROUND: In myelodysplastic neoplasms (MDS), the 20q deletion [del(20q)] is a recurrent chromosomal abnormality that it has a high co-occurrence with U2AF1 mutations. Nevertheless, the prognostic impact of U2AF1 in these MDS patients is uncertain and the possible clinical and/or prognostic differences between the mutation type and the mutational burden are also unknown. METHODS: Our study analyzes different molecular variables in 100 MDS patients with isolated del(20q). RESULTS & CONCLUSIONS: We describe the high incidence and negative prognostic impact of U2AF1 mutations and other alterations such as in ASXL1 gene to identify prognostic markers that would benefit patients to receive earlier treatment.

Predicting the effects of amino acid replacements in peptide hormones on their binding affinities for class B GPCRs and application to the design of secretin receptor antagonists.

Computational prediction of the effects of residue changes on peptide-protein binding affinities, followed by experimental testing of the top predicted binders, is an efficient strategy for the rational structure-based design of peptide inhibitors. In this study we apply this approach to the discovery of competitive antagonists for the secretin receptor, the prototypical member of class B G protein-coupled receptors (GPCRs). Proteins in this family are involved in peptide hormone-stimulated signaling and are implicated in several human diseases, making them potential therapeutic targets. We first validated our computational method by predicting changes in the binding affinities of several peptides to their cognate class B GPCRs due to alanine replacement and compared the results with previously published experimental values. Overall, the results showed a significant correlation between the predicted and experimental ΔΔG values. Next, we identified candidate inhibitors by applying this method to a homology model of the secretin receptor bound to an N-terminal truncated secretin peptide. Predictions were made for single residue replacements to each of the other nineteen naturally occurring amino acids at peptide residues within the segment binding the receptor N-terminal domain. Amino acid replacements predicted to most enhance receptor binding were then experimentally tested by competition-binding assays. We found two residue changes that improved binding affinities by almost one log unit. Furthermore, a peptide combining both of these favorable modifications resulted in an almost two log unit improvement in binding affinity, demonstrating the approximately additive effect of these changes on binding. In order to further investigate possible physical effects of these residue changes on receptor binding affinity, molecular dynamics simulations were performed on representatives of the successful peptide analogues (namely A17I, G25R, and A17I/G25R) in bound and unbound forms. These simulations suggested that a combination of the α-helical propensity of the unbound peptide and specific interactions between the peptide and the receptor extracellular domain contribute to their higher binding affinities.

Lactam constraints provide insights into the receptor-bound conformation of secretin and stabilize a receptor antagonist.

The natural ligands for family B G protein-coupled receptors are moderate-length linear peptides having diffuse pharmacophores. The amino-terminal regions of these ligands are critical for biological activity, with their amino-terminal truncation leading to production of orthosteric antagonists. The carboxyl-terminal regions of these peptides are thought to occupy a ligand-binding cleft within the disulfide-bonded amino-terminal domains of these receptors, with the peptides in amphipathic helical conformations. In this work, we have characterized the binding and activity of a series of 11 truncated and lactam-constrained secretin(5-27) analogues at the prototypic member of this family, the secretin receptor. One peptide in this series with lactam connecting residues 16 and 20 [c[E(16),K(20)][Y(10)]sec(5-27)] improved the binding affinity of its unconstrained parental peptide 22-fold while retaining the absence of endogenous biological activity and competitive antagonist characteristics. Homology modeling with molecular mechanics and molecular dynamics simulations established that this constrained peptide occupies the ligand-binding cleft in an orientation similar to that of natural full-length secretin and provided insights into why this peptide was more effective than other truncated conformationally constrained peptides in the series. This lactam bridge is believed to stabilize an extended α-helical conformation of this peptide while in solution and not to interfere with critical residue-residue approximations while docked to the receptor.

Importance of each residue within secretin for receptor binding and biological activity.

Secretin is a linear 27-residue peptide hormone that stimulates pancreatic and biliary ductular bicarbonate and water secretion by acting at its family B G protein-coupled receptor. While, like other family members, the carboxyl-terminal region of secretin is most important for high affinity binding and its amino-terminal region is most important for receptor selectivity and receptor activation, determinants for these activities are distributed throughout the entire length of this peptide. In this work, we have systematically investigated changing each residue within secretin to alanine and evaluating the impact on receptor binding and biological activity. The residues most critical for receptor binding were His1, Asp3, Gly4, Phe6, Thr7, Ser8, Leu10, Asp15, Leu19, and Leu23. The residues most critical for biological activity included His1, Gly4, Thr7, Ser8, Glu9, Leu10, Leu19, Leu22, and Leu23, with Asp3, Phe6, Ser11, Leu13, Asp15, Leu26, and Val27 also contributing. While the importance of residues in positions analogous to His1, Asp3, Phe6, Thr7, and Leu23 is conserved for several closely related members of this family, Leu19 is uniquely important for secretin. We, therefore, have further studied this residue by molecular modeling and molecular dynamics simulations. Indeed, the molecular dynamics simulations showed that mutation of Leu19 to alanine was destabilizing, with this effect greater than that observed for the analogous position in the other close family members. This could reflect reduced contact with the receptor or an increase in the solvent-accessible surface area of the hydrophobic residues in the carboxyl terminus of secretin as bound to its receptor.

UNDERSTANDING STRUCTURAL EFFECTS OF MULTIPOLE MOMENTS ON AQUEOUS SOLVATION OF IONS USING THE SOFT-STICKY DIPOLE-QUADRUPOLE-OCTUPOLE WATER MODEL.

The effects of water multipole moments on the aqueous solvation of ions were determined in Monte Carlo simulations using soft-sticky dipole-quadrupole-octupole (SSDQO) water. Water molecules formed linear hydrogen bonds to Cl(-) using the new SSDQO1 parameters, similar to multi-site models. However, the dipole vector was tilted rather than parallel to the oxygen-Na(+) internuclear vector as in most multi-site model, while experiment and ab initio molecular dynamics simulations generally indicate a range of values between tilted and parallel. By varying the multipoles in SSDQO, the octupole was found to determine the orientation around Na(+). Moreover, analysis of the multipoles of more conventional models is predictive of their performance as solvents.

Solvation of Glucose, Trehalose, and Sucrose by the Soft Sticky Dipole-Quadrupole-Octupole Water Model.

Water structure around sugars modeled by partial charges is compared for soft-sticky dipole-quadrupole-octupole (SSDQO), a fast single-site multipole model, and commonly used multi-site models in Monte Carlo simulations. Radial distribution functions and coordination numbers of all the models indicate similar hydration by hydrogen-bond donor and acceptor waters. However, the new optimized SSDQO1 parameters as well as TIP4P-Ew and TIP5P predict a "lone-pair" orientation for the water accepting the sugar hydroxyl hydrogen bond that is more consistent with the limited experimental data than the "dipole" orientation in SPC/E, which has important implications for studies of the cryoprotectant properties of sugars.

Solvation of Biomolecules by the Soft Sticky Dipole-Quadrupole-Octupole Water Model.

The soft sticky dipole-quadrupole-octupole (SSDQO) potential energy function represents a water molecule by a single site with a van der Waals sphere and point multipoles. Previously, SSDQO was shown to give good properties for liquid water and solvation of simple ions and is faster than three point models. Here, SSDQO is assessed for solvating biologically relevant molecules having a multi-site, partial charge description. Monte Carlo simulations of ethanol, benzene, and N-methylacetamide in SSDQO with SPC/E moments showed the water structure was as good as in SPC/E. Thus, SSDQO is potentially useful for simulations of biological macromolecules in aqueous solution.

Temperature and pressure dependence of the optimized soft-sticky dipole-quadrupole-octupole water model.

The soft-sticky dipole-quadrupole-octupole (SSDQO) potential energy function for a coarse-grained single-site water model has Lennard-Jones interactions and an approximate multipole expansion for the electrostatics. Here, the Lennard-Jones parameters and multipole moments of SSDQO were optimized so that the structural, thermodynamic, dynamic, and dielectric properties agreed with experimental values of liquid water at ambient conditions. Using these parameters, the temperature and pressure dependence of various properties were shown to be in good agreement with experiment, including a temperature of maximum density at approximately 260 K. This new parametrization, referred to as SSDQO1, is both computationally faster and generally more accurate over a wide range of conditions than traditional three-site water models, which demonstrates that a model with a single dipole, quadrupole, and octupole on each water molecule can reproduce the tetrahedral hydrogen bonded network of water.

IgM monoclonal component associated with type I Gaucher disease resolved after enzyme replacement therapy: a case report.

The frequency of monoclonal gammopathy of undetermined significance (MGUS) is higher in patients with type I Gaucher disease (GD I) than in the general population. Although enzyme replacement therapy is effective in the control of the disease, its effect on MGUS is still controversial. We present the case of a 65-year-old woman with extensive GD I associated with IgM MGUS, in whom enzyme replacement therapy succeeded in eradicating the monoclonal component. This observation further supports the idea that enzyme replacement therapy decreases the chronic antigenic stimulation responsible for gammopathies in Gaucher disease.

Assessment of the bioavailability of an antibiotic prophylactic protocol in patients undergoing third molar surgery.

The aim of this prospective study was to determine the antibiotic bioavailability of a prophylactic protocol in patients undergoing third molar surgery. Samples from 25 patients were analysed (average age 21 ± 3.89 years, range 18-33 years; 14 female). The patients received single-dose prophylaxis of 2 g amoxicillin orally 1 hour prior to third molar surgery. Venous blood (1.5 ml) and blood from the third molar socket (1.50 ml) were obtained. The amoxicillin plasma concentration was determined in both samples by high performance liquid chromatography with a diode-array detector (HPLC/DAD). Their associations with demographic variables (age, height, weight, body mass index (BMI), sex) and antibiotic exposure time were analyzed using linear regression models. The mean amoxicillin plasma level detected in the venous blood was 1.21 ± 1.17 µg/ml (range 0.49-6.34 µg/ml) and in the third molar socket was 4.14 ± 2.24 µg/ml (range 0.86-7.46 µg/ml) (P < 0.001). No relationship was observed between the bioavailability of the drug and the patient biometric indices evaluated. The prophylactic administration of 2 g amoxicillin in third molar surgery showed greater bioavailability in the molar socket than the concentrations established as necessary to inhibit the growth of microorganisms that cause oral infections. The results show the need to review the current infection control protocols in oral surgery in light of the overestimated doses observed.

An increased percentage of myeloid CD34+ bone marrow cells stratifies intermediate IPSS-R myelodysplastic syndrome patients into prognostically significant groups.

INTRODUCTION: The Revised International Prognostic Scoring System (IPSS-R) for myelodysplastic syndromes (MDS) has established an intermediate category where a disease-modifying intervention is a matter of debate. Flow cytometry allows us to determine a fraction of immature myeloid cells in a semiautomated procedure. The aim of this study, mirroring IPSS-R study inclusion criteria, was to test whether bone marrow (BM) CD34+My percentage has independent prognostic value in the MDS setting. METHODS: BM CD34+My cells were quantified, at diagnosis, selecting CD34+/CD45+/CD11b±/CD13+. Patients were excluded when receiving treatment for altering the natural course of the disease and when IPSS-R could not be calculated due to the lack of metaphases. Finally, Cox analyses were performed, on a series of 260 patients, for overall survival (OS) and time to acute myeloid leukemia (AML) transformation. RESULTS: By analyzing ROC curves, the most accurate prognostic variable, regarding blasts by cytology and CD34+ by cytometry, was the percentage of blasts by microscopy. The percentage of CD34+My in BM showed an AUC of 0.767 and 0.576 for time to AML transformation and OS, respectively. When performing a multivariate regression including the IPSS-R and the percentage of BM CD34+My cells >1%, both factors predicted for a shorter time to AML transformation. In addition, CD34+My percentage successfully stratified the intermediate IPSS-R category into two prognostic groups with a relative risk of 5.73 (95% CI [1.2-27.8]; P = .03). CONCLUSION: We found that BM CD34+My percentage has an independent value concerning the IPSS-R, especially relevant for the prediction of transformation to AML and within the intermediate group.

Genome-wide gene expression changes associated with exposure of rat liver, heart, and kidney cells to endosulfan.

Endosulfan was once the most commonly used pesticide in agriculture and horticulture. It is an environmentally persistent organochlorine compound with the potential to bioaccumulate as it progresses through the food chain. Its acute and chronic toxicity to mammals, including humans, is well known, but the molecular mechanisms of its toxicity are not fully understood. To gain insight to these mechanisms, we examined genome-wide gene expression changes of rat liver, heart, and kidney cells induced by endosulfan exposure. We found that among the cell types examined, kidney and liver cells were the most sensitive and most resilient, respectively, to endosulfan insult. We acquired RNA sequencing information from cells exposed to endosulfan to identify differentially expressed genes, which we further examined to determine the cellular pathways that were affected. In kidney cells, exposure to endosulfan was uniquely associated with altered expression levels of genes constituting the hypoxia-inducible factor-1 (HIF-1) signaling pathway. In heart and liver cells, exposure to endosulfan altered the expression levels of genes for many members of the extracellular matrix (ECM)-receptor interaction pathway. Because both HIF-1 signaling and ECM-receptor interaction pathways directly or indirectly control cell growth, differentiation, proliferation, and apoptosis, our findings suggest that dysregulation of these pathways is responsible for endosulfan-induced cell death.

Neuroprotective mechanisms activated in non-seizing rats exposed to sarin.

Exposure to organophosphate (OP) nerve agents, such as sarin, may lead to uncontrolled seizures and irreversible brain injury and neuropathology. In rat studies, a median lethal dose of sarin leads to approximately half of the animals developing seizures. Whereas previous studies analyzed transcriptomic effects associated with seizing sarin-exposed rats, our study focused on the cohort of sarin-exposed rats that did not develop seizures. We analyzed the genomic changes occurring in sarin-exposed, non-seizing rats and compared differentially expressed genes and pathway activation to those of seizing rats. At the earliest time point (0.25 h) and in multiple sarin-sensitive brain regions, defense response genes were commonly expressed in both groups of animals as compared to the control groups. All sarin-exposed animals activated the MAPK signaling pathway, but only the seizing rats activated the apoptotic-associated JNK and p38 MAPK signaling sub-pathway. A unique phenotype of the non-seizing rats was the altered expression levels of genes that generally suppress inflammation or apoptosis. Importantly, the early transcriptional response for inflammation- and apoptosis-related genes in the thalamus showed opposite trends, with significantly down-regulated genes being up-regulated, and vice versa, between the seizing and non-seizing rats. These observations lend support to the hypothesis that regulation of anti-inflammatory genes might be part of an active and sufficient response in the non-seizing group to protect against the onset of seizures. As such, stimulating or activating these responses via pretreatment strategies could boost resilience against nerve agent exposures.

Prognostic signature and clonality pattern of recurrently mutated genes in inactive chronic lymphocytic leukemia.

An increasing numbers of patients are being diagnosed with asymptomatic early-stage chronic lymphocytic leukemia (CLL), with no treatment indication at baseline. We applied a high-throughput deep-targeted analysis, especially designed for covering widely TP53 and ATM genes, in 180 patients with inactive disease at diagnosis, to test the independent prognostic value of CLL somatic recurrent mutations. We found that 40/180 patients harbored at least one acquired variant with ATM (n=17, 9.4%), NOTCH1 (n=14, 7.7%), TP53 (n=14, 7.7%) and SF3B1 (n=10, 5.5%) as most prevalent mutated genes. Harboring one 'sub-Sanger' TP53 mutation granted an independent 3.5-fold increase of probability of needing treatment. Those patients with a double-hit ATM lesion (mutation+11q deletion) had the shorter median time to first treatment (17 months). We found that a genomic variable: TP53 mutations, most of them under the sensitivity of conventional techniques; a cell phenotypic factor: CD38-positive expression; and a classical marker as ß2-microglobulin, remained as the unique independent predictors of outcome. The high-throughput determination of TP53 status, particularly in this set of patients frequently lacking high-risk chromosomal aberrations, emerges as a key step, not only for prediction modeling, but also for exploring mutation-specific therapeutic approaches and minimal residual disease monitoring.

The relationship of TP53 R72P polymorphism to disease outcome and TP53 mutation in myelodysplastic syndromes.

Nonsynonymous TP53 exon 4 single-nucleotide polymorphism (SNP), R72P, is linked to cancer and mutagen susceptibility. R72P associations with specific cancer risk, particularly hematological malignancies, have been conflicting. Myelodysplastic syndrome (MDS) with chromosome 5q deletion is characterized by erythroid hypoplasia arising from lineage-specific p53 accumulation resulting from ribosomal insufficiency. We hypothesized that apoptotically diminished R72P C-allele may influence predisposition to del(5q) MDS. Bone marrow and blood DNA was sequenced from 705 MDS cases (333 del(5q), 372 non-del(5q)) and 157 controls. Genotype distribution did not significantly differ between del(5q) cases (12.6% CC, 38.1% CG, 49.2% GG), non-del(5q) cases (9.7% CC, 44.6% CG, 45.7% GG) and controls (7.6% CC, 37.6% CG, 54.8% GG) (P=0.13). Allele frequency did not differ between non-del(5q) and del(5q) cases (P=0.91) but trended towards increased C-allele frequency comparing non-del(5q) (P=0.08) and del(5q) (P=0.10) cases with controls. Median lenalidomide response duration increased proportionate to C-allele dosage in del(5q) patients (2.2 (CC), 1.3 (CG) and 0.89 years (GG)). Furthermore, C-allele homozygosity in del(5q) was associated with prolonged overall and progression-free survival and non-terminal interstitial deletions that excluded 5q34, whereas G-allele homozygozity was associated with inferior outcome and terminal deletions involving 5q34 (P=0.05). These findings comprise the largest MDS R72P SNP analysis.

An unusual atlanto-axial dislocation. Case report.

A case is presented in which the patient sustained a complete dislocation of the odontoid process in front of the anterior arch of the first cervical vertebra. There was no fracture of the arch of C-1 vertebra. The patient was treated successfully by trans-oral excision of the odontoid process and the anterior arch of C-1. Twenty days later the patient's spine was stabilized posteriorly at C-1 through C-3 with wire and methyl-methacrylate.

The allantoplacenta of Mabuya mabouya (Sauria, Scincidae).

Analysis of placentation in the final stages of development in Mabuya mabouya shows that the placenta is formed by the apposition of the chorioallantois to the uterine mucosa implicating the entire embryonic chamber, because the allantoic vesicle invades all the exocoelom. The chorioallantoic placenta presents the features proper of a type IV allantoplacenta. However, in the mesometrial area peripheral to the placentome, we found that the paraplacentome is an additional zone specialized for histotrophic transfer, and is separated from the rest of the embryonic chamber by a chorionic invagination formed of polymorphic cells. The chorionic areolae are components of the embryonic hemisphere; they are in apposition to an endometrium with columnar epithelial cells and several glands that secrete toward the cavity of the areolae. They are observed only in the preparturition stage, probably operating in maternal-fetal transfer of nutrients during the last embryonic growth stage. The mesometrial hemisphere possesses specializations related to histotrophic nutrition (placentome, paraplacentome, and chorionic areolae), while in the abembryonic hemisphere there is an allantoplacenta of mixed function, with capacity for histotrophic nutrition and for gas exchange. The absorptive plaques are small rounded areas constituted by chorionic cells similar to the paraplacentomal chorionic cells, in intimate apposition with a secretory uterine epithelium. Separating the absorptive plaques are respiratory segments histologically similar to the type I allantoplacenta. The additional histotrophic areas found for this species demonstrate the great specialization of this allantoplacenta, and support the highest degree of matrotrophy among reptiles reached in the Neotropical Mabuya.