Partial Retention Research Articles

P-089: Identification of novel targets in multiple myeloma for “undruggable” RAS/CDK signaling cascade

<h3>Background</h3> RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis; both pathways are undruggable. We evaluated molecular changes associated with pathway-level responses after RAS/CDK inhibition to identify novel molecular targets. <h3>Method</h3> in our previous studies MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to target RAS/CDK pathways. Our studies indicate strong synergistic (IC<0.5) MM cytotoxicity triggered by Ei+Ci treatment, which in a dose-dependent manner arrested MM cells in G0/G1 phase and activated mitochondrial apoptotic signaling. Ei+Ci treatment decreased Erk1/2, CDK4/6, and p-Erk1/2 levels in MM cells, and also induced inhibition of key targets (c-myc, p-RSK,-RB, E2F1) of RAS/CDK cascade. Our studies in patient samples indicate that MM cells co-cultured with or without autologous BM stromal cells remain equally sensitive to Ei+Ci, suggesting that Ei+Ci combination can overcome the protective effects of the MM BM milieu. An in vivo study demonstrated a significant (P=0.0004) MM burden decrease in Ei+Ci-treated mice. Our studies therefore suggesting on-target activity of these inhibitors in vitro/vivo. <h3>Results</h3> We evaluated mRNA splicing changes in MM cells, with and without Erk1/2 knockdown or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. Upregulation in response to Erk1/2 knockdown and downregulation due to treatment with Ei+Ci were considered spliced gene signatures linked to RAS/CDK modulation. Gene/pathway enrichment analyses of these genes showed their involvement in cell proliferation and regulation of epigenetic networks in MM. Importantly, these analyses suggest that overexpression of RAVER1/SNRPB core splicing regulator genes are associated with RAS/CDK pathway regulation. These genes encode subunits of U1/2/4/5 spliceosome complexes and are involved in intron retention processes, a marker of malignant transformation. We evaluated expressions of RAVER1 and SNRPB in 558 MM patent samples and 10 normal donor BM PCs and observed significant (p<2e-11) upregulation of both genes in clonal PCs with progression from MGUS to sMM, and to overt MM. SNRPB overexpression is associated with shorter overall patient survival (p<0.01), while RAVER1 has a trend toward poor outcomes. SNRPB proteins are also overexpressed in MM cells. Evaluating SNRPB effects on RNA splicing showed upregulation of transcripts with full intron retention or transcripts with cryptic stop codons utilizing intronic sequences causing their partial retention. Thus, SNRPB overexpression contributes to aberrant transcriptome splicing associated with RAS/CDK cascade in MM. <h3>Conclusions</h3> Our studies show an association between RNA processing and RAS-CDK pathways in MM, identify a core splicing protein, SNRPB, as a novel target for modulating this undruggable cascade, and suggest that targeting spliceosome complexes is a promising therapy.

Germline polymorphisms and alternative splicing of human immunoglobulin light chain genes

SummaryInference of germline polymorphisms in immunoglobulin genes from B cell receptor repertoires is complicated by somatic hypermutations, sequencing/PCR errors, and by varying length of reference alleles. The light chain inference is particularly challenging owing to large gene duplications and absence of D genes. We analyzed the light chain cDNA sequences from naïve B cell receptor repertoires from 100 individuals. We optimized light chain allele inference by tweaking parameters of the TIgGER functions, extending the germline reference sequences, and establishing mismatch frequency patterns at polymorphic positions to filter out false-positive candidates. We identified 48 previously unreported variants of light chain variable genes. We selected 14 variants for validation and successfully validated 11 by Sanger sequencing. Clustering of light chain 5′UTR, L-PART1, and L-PART2 revealed partial intron retention in 11 kappa and 9 lambda V alleles. Our results provide insight into germline variation in human light chain immunoglobulin loci.

The decellularized ovary as a potential scaffold for maturation of preantral ovarian follicles of prepubertal mice

ABSTRACT In the current study, a decellularized ovary scaffold was used for the encapsulation and development of preantral follicles. Maturation and the expression of the related genes were evaluated and compared against in vivo conditions. Mature rat ovaries were decellularized and decellularization was confirmed using hematoxylin/eosin and Hoechst stains. Collagen and glycosaminoglycan (GAG) preservation and the extracellular matrix (ECM) architecture were evaluated by Masson’s trichrome staining, Alcian blue staining, and scanning electron microscopy, respectively. Raman confocal microscopy and DNA quantification were performed to show DNA and cell debris depletion and ECM retention. Preantral follicles of mice were cultured for 12 days in the decellularized (3D) and two-dimensional (2D) conditions. Finally, the survival rate, antrum formation rate and diameters of follicles, levels of estradiol (E2) and progesterone (P4), and expression of the ZP2, Gdf9, Bmp6, and Bmp15 genes were evaluated in the 2D and 3D culture systems. On day 13 of culture, oocyte maturation was induced by hCG. Although histochemical tests showed partial retention of ECM, Raman confocal microscopy showed some extent of ECM washing out along with the depletion of DNA and cell debris. However, SEM showed the preservation of the ovarian ECM’s ultrastructure. The survival rate of follicles was similar, but the antrum formation rate, concentrations of E2 and P4, and oocyte maturation rate were significantly higher in 3D compared with 2D. The level of ZP2, Gdf9, Bmp6, and Bmp15 gene expression in follicles matured in 2D, 3D, and in vivo was statistically similar. In all matured follicles, their gene expression levels significantly declined relative to preantral follicles. Ovarian follicles that matured in the decellularized ovaries demonstrated the same follicular growth and maturation gene expression levels as those that grew in the natural ovary. This is consistent with using the decellularized ovary as a scaffold suitable for ovarian reconstruction. Abbreviations GAG: glycosaminoglycan; ECM: extracellular matrix; 2D: two-dimensional; E2: estradiol; P4: progesterone; BMP15: bone morphogenetic protein 15; GDF9: growth differentiation factor 9; ZP2: zona pellucida 2; Gdf9: growth/differentiation factor-9; Bmp6: bone morphogenetic protein 6; Bmp15: bone morphogenetic protein 15.

Impact of long term conservation agriculture on soil quality under cereal based systems of North West India

Soil quality is of utmost essential for yield sustainability of intensive cereal based cropping system in North West Indo Gangetic plains of India. Hence, we evaluated long-term (10 years) effect of conservation agriculture (CA) practices on soil quality improvement under six different cropping scenarios (Sc), i.e. Sc1-represented by transplanted puddled rice (TPR) followed by conventional tilled broadcasted wheat (CT-wheat) with residue removal, Sc2-TPR rice followed by zero tillage (ZT) wheat and ZT-mung bean with partial residue retention, Sc3-direct seeded rice (DSR) followed by ZT-wheat and ZT-mung bean with full residue retention, Sc4-DSR is replaced by ZT-maize followed by ZT-wheat and ZT-mung bean, Sc5 and Sc6 were – Sc3 integrated with sub surface drip irrigation (SDI) and Sc4 + SDI, respectively. Soil samples were collected from 0 to 5, 5–15 and 15–30 cm soil depth from each scenario after harvesting of wheat in 2019. Results showed that, reduction in bulk density (BD), soil penetration resistance (SPR) and enhancement of water holding capacity and infiltration were associated with CA based scenarios (Sc3–Sc6). Scenario 3 recorded lowest BD of 1.39 and 1.58 g cm−3 at 0–5 and 5–15 cm soil depth, respectively. CA based Sc6 recorded highest infiltration rate (1.48 cm hr−1) and lowest was associated with Sc1 (0.5 cm hr−1). The enrichment of soil organic carbon (SOC) content, stock, available nitrogen and potassium was mainly confined to upper surface soil layer (0–5 cm). The SOC content and stock in CA based scenarios (average of Sc3 to Sc6) was 41–57 and 69–94% higher than Sc1 at 0–5 cm soil layer. Available nitrogen was increased by 23–50 and 64–98% and available potassium increased by 13–28 and 42–71% in 0–5 and 5–15 cm soil depth, respectively in CA based scenarios over Sc1. Similarly, microbial biomass carbon (MBC) and dehydrogenase (DHA) activity in top soil layer under CA based scenarios was increased by 177–195 and 67–107% over Sc1, respectively. The maximum SQI was registered with Sc6 (0.91) followed by Sc4 (0.89) and least was recorded in Sc1 (0.65) at 0–5 cm soil depth. Maize-wheat based cropping system recorded higher SQI over rice–wheat based cropping system. Sustainable yield index was strongly related with key soil quality indicators and also positively correlated with SQI. Thus our study suggests that CA based maize-wheat-mung bean cropping system should be recommended for better soil quality and yield sustainability in North West India.

Thermal record of the building of an orogen in the retro‐foreland basin: Insight from basement and detrital thermochronology in the eastern Pyrenees and the north Pyrenean basin (France)

AbstractAn understanding of the evolution of foreland basins improves our knowledge of how mountain belts have grown and helps us to decipher events which may not be preserved in the orogen. The infilling of the north Pyrenean retro‐foreland basin (Aquitaine Basin, France) during the main exhumation of the Pyrenees and its corresponding thermal history have not been fully investigated. We applied apatite fission track (AFT) and (U‐Th‐Sm)/He (AHe) methods coupled with inverse thermal modelling on both the detrital Eocene (47 to 33 Ma) syn‐orogenic Palassou conglomerates of the eastern part of the Aquitaine Basin and basement samples from the North Pyrenean Zone and the Axial Zone of the Pyrenees. Apatite crystals were separated from granitic cobbles found in the conglomerates. AFT ages for detrital samples range from 27 ± 2 to 43 ± 4 Ma, and AHe ages from 13 ± 1 to 76 ± 5 Ma. For in situ massifs AFT ages range from 35 ± 2 to 90 ± 17 Ma and AHe ages from 39 ± 2 and 80 ± 5 Ma. AFT ages for detrital samples are close to deposition ages, whereas AHe ages are older and younger than deposition ages and show a partial thermal resetting due to burial. A detailed analysis of the ages obtained and thermal histories derived from modelling shows that ages reflect (a) exhumation from 70 to 55 Ma revealed by a long stay in the partial retention zone (PRZ), (b) a Palaeocene–Eocene cooling in the Pyrenees, (c) a post‐depositional episode of moderate heating of the sediments in the basin represented by partially reset young AHe and AFT ages compared to deposition ages and (d) an early to mid‐Miocene final exhumation of the basin deposits as evidenced by young AHe ages and geological constrains. These results reflect a common event with the south Pyrenean foreland basin that is characterized by high piedmont aggradation from the late Eocene to the Miocene. The aggradation of sediments is possibly connected with well‐known high elevation low relief surfaces in the core of the Pyrenees and followed by a Miocene exhumation event that is already observed on the southern flank. However, the timing of the aggradation and exhumation events could be different between the north and the south. Erosion occurred most probably during the early to mid‐Miocene in the north and during the late Miocene–early Pliocene in the south.

Controlled preparation and properties of acrylic acid epoxy‐acrylate composite emulsion for self‐crosslinking coatings

AbstractA stable epoxy‐acrylate composite latex was successfully prepared through emulsion polymerization of modified epoxy acrylic (EPAC) oligomer with acrylate monomer. The EPAC oligomer was obtained using active acrylic acid (AA) to react with epoxy resin. And by regulating the reaction degree of the active hydrogen of AA and epoxide group, there is the acquisition of terminal double bond that gives EPAC reactivity, together with the partial retention of the epoxide group that enables self‐crosslinking during film formation. The structural conformation of the oligomer was ascertained by Fourier transform infrared (FTIR) spectroscopy. The factors influencing the stability of the epoxy‐acrylate composite latex were investigated. The epoxy‐acrylate composite latex was the most stable when methyl acrylic acid was 1.5 wt% and modified EPAC oligomer was 15 wt% of the total monomer weight. The morphology and property of the composite latex films were characterized by scanning electron microscopy, transmission electron microscopy (TEM), and temperature‐modulated differential scanning calorimetry (TOPEM‐DSC). The results confirm that there is successful emulsion copolymerization between modified EPAC oligomer and acrylate monomer. TEM show that the particles of epoxy‐acrylate composite have a core‐shell structure, and there is no free epoxy resin. The FTIR and TOPEM‐DSC results reveal that the copolymer emulsion possesses self‐crosslinking ability. During film formation, self‐crosslinking reaction occurs between epoxide groups with carboxyl groups, giving exothermal phenomena. The thermal stability as well as the corrosion resistance of the films was analyzed. The results show outstanding thermal stability as well as corrosion resistance attributable to the crosslinking reticulation structure. It is envisaged that the epoxy‐acrylate composite latex has great potential in the development of high‐performance aqueous coatings.

Synthesis of Enantioenriched Amines by Iron‐Catalysed Amination of Alcohols Employing at Least One Achiral Substrate

AbstractThe synthesis of a broad range of enantioenriched amines by the direct Fe‐catalysed coupling of amines with alcohols through the borrowing hydrogen strategy, while at least one of these substrates is achiral is reported. When starting from α‐chiral amines and achiral alcohols, a wide range of enantioenriched amine products, including N‐heterocyclic moieties can be obtained with complete retention of stereochemistry and the power of this method is demonstrated in the one‐step synthesis of known pharmaceuticals from commercially available, simple enantiopure primary amines and achiral alcohols. It was also found that the use of β‐branched enantioenriched primary alcohols and achiral amines as reaction partners leads to a partial loss of stereochemical integrity in the final product, however, a systematic optimization enabled partial retention of enantiopurity and possible parameters effecting for racemization were identified.magnified image

Biogeochemical Mobility of Contaminants from a Replica Radioactive Waste Trench in Response to Rainfall-Induced Redox Oscillations.

Results of investigations into factors influencing contaminant mobility in a replica trench located adjacent to a legacy radioactive waste site are presented in this study. The trench was filled with nonhazardous iron- and organic matter (OM)-rich components, as well as three contaminant analogues strontium, cesium, and neodymium to examine contaminant behavior. Imposed redox/water-level oscillations, where oxygen-laden rainwater was added to the anoxic trench, resulted in marked biogeochemical changes including the removal of aqueous Fe(II) and circulation of dissolved carbon, along with shifts to microbial communities involved in cycling iron (Gallionella, Sideroxydans) and methane generation (Methylomonas, Methylococcaceae). Contaminant mobility depended upon element speciation and rainfall event intensity. Strontium remained mobile, being readily translocated under hydrological perturbations. Strong ion-exchange reactions and structural incorporation into double-layer clay minerals were likely responsible for greater retention of Cs, which, along with Sr, was unaffected by redox oscillations. Neodymium was initially immobilized within the anoxic trenches, due to either secondary mineral (phosphate) precipitation or via the chemisorption of organic- and carbonate-Nd complexes onto variably charged solid phases. Oxic rainwater intrusions altered Nd mobility via competing effects. Oxidation of Fe(II) led to partial retention of Nd within highly sorbing Fe(III)/OM phases, whereas pH decreases associated with rainwater influxes resulted in a release of adsorbed Nd to solution with both pH and OM presumed to be the key factors controlling Nd attenuation. Collectively, the behavior of simulated contaminants within this replica trench provided unique insights into trench water biogeochemistry and contaminant cycling in a redox oscillatory environment.

Unraveling synonymous and deep intronic variants causing aberrant splicing in two genetically undiagnosed epilepsy families

BackgroundVariants identified through parent–child trio-WES yield up to 28–55% positive diagnostic rate across a variety of Mendelian disorders, there remain numerous patients who do not receive a genetic diagnosis. Studies showed that some aberrant splicing variants, which are either not readily detectable by WES or could be miss-interpreted by regular detecting pipelines, are highly relevant to human diseases.MethodsWe retrospectively investigated the negative molecular diagnostics through trio-WES for 15 genetically undiagnosed patients whose clinical manifestations were highly suspected to be genetic disorders with well-established genotype–phenotype relationships. We scrutinized the synonymous variants from WES data and Sanger sequenced the suspected intronic region for deep intronic variants. The functional consequences of variants were analyzed by in vitro minigene experiments.ResultsHere, we report two abnormal splicing events, one of which caused exon truncating due to the activation of cryptic splicing site by a synonymous variant; the other caused partial intron retention due to the generation of splicing sites by a deep intronic variant.ConclusionsWe suggest that, despite initial negative genetic test results in clinically highly suspected genetic diseases, the combination of predictive bioinformatics and functional analysis should be considered to unveil the genetic etiology of undiagnosed rare diseases.

Hydraulic performance of horizontal constructed wetlands for stormwater treatment: A pilot-scale study in the Mediterranean

Horizontal subsurface flow (HSSF) constructed wetlands (CWs) are widely adopted as a highly effective technique for stormwater treatment. However, the cumulative clogging phenomenon in HSSF is a complex and challenging operative issue affecting removal efficiencies and the lifespan of CWs. This paper aims to evaluate the reliability of a combined approach for assessing the occurrence of clogging and its potential spatial and temporal evolution in two pilot-scale HSSF-CW. The experimental CW system was fed with stormwater runoff from a parking area during the rainy season and sequential batch reactor (SBR) wastewater (WW) during the dry season. The falling head (FH) method and a drainage equation were used to determine the hydraulic conductivity at saturation (Ks) of the porous medium and investigate the hydraulic performance of the system. A residence time distribution (RTD) analysis was also performed to evaluate the hydraulic flow dynamics and define the crucial parameters characterising the CW. The applied hydraulic measurement techniques highlighted that clogging was in the initial stages of development after 2.5 years of operation, likely associated with the presence of a pond and the partial retention of total suspended solids (TSS) by the HSSF system. Overall, the good consistency in the results of the applied methods supports their future application for predicting the occurrence of clogging phenomena in HSSF CWs.

Morpho-Physiological Testing of NaCl Sensitivity of Tobacco Plants Overexpressing Choline Oxidase Gene.

In this study the transgenic lines (TLs) of tobacco (Nicotiana tabacum L.), which overexpress the heterologous gene encoding the bacterial enzyme choline oxidase were evaluated. The goal of our work is to study the effect of choline oxidase gene expression on the sensitivity of plant tissues to the action of NaCl. The regenerative capacity, rhizogenesis, the amount of photosynthetic pigments and osmotically active compounds (proline and glycine betaine) were assessed by in vitro cell culture methods using biochemical and morphological parameters. Transgenic lines with confirmed expression were characterized by high regeneration capacity from callus in the presence of 200 mmol NaCl, partial retention of viability at 400 mmol NaCl. These data correlated with the implicit response of regenerants and whole plants to the harmful effects of salinity. They turned out to be less sensitive to the presence of 200 mmol NaCl in the cultivation medium, in contrast to the WT plants.

Confirming the Diagnosis and Classifying Placenta Accreta Spectrum (PAS) Disorders: Minutes of 2020 Online International Workshop on PAS in Beijing

Confirming the Diagnosis and Classifying Placenta Accreta Spectrum (PAS) Disorders: Minutes of 2020 Online International Workshop on PAS in Beijing

In situ monitoring of microstructure evolution during thermal processing of uranium-zirconium alloys using laser-generated ultrasound

Laser-generated ultrasound was used to monitor microstructure evolution during thermal processing of as-cast, polycrystalline binary uranium-zirconium metallic fuel alloys with compositions of U-20wt.%Zr (U-20Zr), U-50wt.%Zr (U-50Zr) and U-80wt.%Zr (U-80Zr). Ultrasonic waveforms were recorded during heating and cooling the samples from room temperature to >973 K and back. A phase transition temperature for all three compositions was estimated from the temperature at which an abrupt and rapid reduction in ultrasonic velocities was observed. Microstructural features on the length scale of tens of micrometers were inferred from the observation of scattering of ultrasonic waves by elastic heterogeneities above ~823 K in U-20Zr, while a hysteresis in the ultrasonic velocities of U-80Zr upon cooling was attributed to a partial retention of the high temperature phase following thermal annealing. The U-50Zr alloy exhibited a reversible viscoelastic response above 933 K, as evidenced by the observation of high frequency attenuation of the shear component of the waveforms at high temperature. Ultrasonic measurements were supplemented by in situ transmission electron microscopy (TEM). The TEM images revealed that the δ-U-Zr matrix in the three compositions underwent a spinodal decomposition above ~823 K into nanoscale regions. The ultrasonic measurements revealed larger, micron-scale structure evolution in the U-20Zr alloy at the same temperature. This large-scale structure is associated with heterogeneous regions having different Zr content. Our findings show the potential heating rate dependence of microstructural evolution in U-Zr alloys and highlight differences in the thermomechanical response and associated length scales during thermal annealing between single- and dual-phase compositions. These results demonstrate the utility of laser ultrasonics to rapidly and efficiently scan phase boundaries and monitor micrometer-scale structure evolution in metallic fuel alloys.

Foreland migration of orogenic exhumation during nappe stacking: Inferences from a high-resolution thermochronological profile over the Southeast Carpathians

The exhumation mechanism of orogens showing one structural vergence, or single-sided orogens, is still not fully understood due to their common transition from periods of high convergence to slab retreat. The overprinting of inherited nappe stacks by subsequent back-arc extension makes it difficult to assess the low amounts of contractional exhumation observed, which are furthermore difficult to interpret due to long residence times in partial retention zones of typical low-temperature thermochronology markers. This is the case in many orogens worldwide and is often observed in the Mediterranean area as well. One notable exception is the Romanian segment of the Carpathian Mountains, where the back-arc extension took place far from the location where the nappe stack formed. To understand the mechanics of contractional exhumation in this single-sided orogen, we generated a new high-resolution low-temperature (apatite fission-track and U-Th/He) thermochronological transect across the Southeast Carpathian nappe stack and its foreland. This new dataset and its correlation with previous exhumation and kinematic studies demonstrate that the gradual accretion of sediments and crustal material was associated with an exhumation that migrated progressively towards the foreland throughout the entire contractional history. The exhumation style changed when the thin-skinned deformation was interrupted by periods of crustal accretion, associated either with the late Eocene onset of continental collision or with the late Miocene locking of the subduction system. The generally accepted moments of onset and termination of slab retreat are not directly visible in available exhumation data of the Southeast Carpathians. Our results are in agreement with modelling studies, which inferred that the formation of single-sided collisional wedges is primarily controlled by the pre-existing rheology of continental plates and is associated with large amounts of continental subduction. • Formation of the Southeast Carpathian orogen is associated with a pattern of migration of exhumation towards the foreland; • The initial exhumation pattern is modified by the onset of continental collision and locking of the subduction system; • The formation of such orogens is primarily controlled by plates rheology and prolonged periods of continental subduction.

In Vitro and In Vivo Characterization of MCT1 Inhibitor AZD3965 Confirms Preclinical Safety Compatible with Breast Cancer Treatment.

Simple SummaryThe vast majority of tumors originate in tissues that use different substrates and oxygen to produce energy. However, tumors are disorganized structurally and functionally, which creates areas where oxygen and nutrients are poorly available. To survive and proliferate, cancer cells adapt by switching their metabolism to lactic fermentation. Their fate is further optimized by intercellular cooperation, but this creates a weakness that can be exploited therapeutically. Indeed, AZD3965 is a new drug currently tested in clinical trials that inhibits a cooperation based on lactate swapping for glucose between fermenting and respiring cells. It inhibits lactate transporter monocarboxylate transporter 1. Here, using malignant and nonmalignant cells representative of the breast tissue and several behavioral tests in mice, we establish that AZD3965 is safe for therapeutic use against cancer. The only side effect that we detected was a short-term memory retention defect that transiently perturbed the orientation of mice in space.To survive and proliferate in solid tumors, cancer cells adapt and evolve rapidly in microenvironments where oxygen and substrate bioavailability fluctuates over time and space. This creates metabolic heterogeneity. Cancer cells can further cooperate metabolically, for example by swapping glycolytic end-product lactate for blood-borne glucose. This type of cooperation can be targeted therapeutically, since transmembrane lactate exchanges are facilitated by lactate-proton symporters of the monocarboxylate (MCT) family. Among new drugs, AZD3965 is a first-in-class selective MCT1 inhibitor currently tested in Phase I/II clinical trials for patients with different types of cancers. Because MCT1 can function bidirectionally, we tested here whether and how malignant and nonmalignant cells adapt their metabolism and MCT repertoire when AZD3965 inhibits either lactate import or export. Using breast-associated malignant and nonmalignant cell lines as models, we report that AZD3965 is not directly cytotoxic. In the presence of glucose and glutamine, oxidative cells can survive when lactate uptake is blocked, and proliferating cells compensate MCT1 inhibition by overexpressing MCT4, a specialized facilitator of lactate export. Phenotypic characterization of mice focusing on metabolism, muscle and brain physiology found partial and transient memory retention defect as sole consequence of MCT1 inhibition by AZD3965. We therefore conclude that AZD3965 is compatible with anticancer therapy.

Understanding the Mechanistic Requirements for Efficient and Stereoselective Alkene Epoxidation by a Cytochrome P450 Enzyme

The cytochrome P450 (CYP) family of heme monooxygenase enzymes commonly catalyzes enantioselective hydroxylation and epoxidation reactions. Epoxidation reactions have been hypothesized to proceed via multiple mechanisms involving different reactive intermediates. Here, we use activity, spectroscopic, structural, and molecular dynamics data to investigate the activity and stereoselectivity of 4-vinylbenzoic acid epoxidation by the bacterial enzyme CYP199A4 from Rhodopseudomonas palustris HaA2. The epoxidation of 4-vinylbenzoic acid by CYP199A4 proceeded with high enantioselectivity, giving the (S)-epoxide in 99% ee at an activity of 220 nmol nmol-CYP–1 min–1. Optical and EPR spectroscopy, redox potential measurements, and the crystal structure of 4-vinylbenzoic acid-bound CYP199A4 indicated the partial retention of an aqua ligand at the heme center in the presence of the substrate, providing a justification of the lower activity (∼20%) compared to the oxidative demethylation of 4-methoxybenzoic acid. Mutagenesis at the conserved acid–alcohol pair (D251/T252), which perturbs the generation of the reactive oxygen intermediates, was employed to investigate their role in epoxidation reactions. The T252A mutant increased the rate of turnover of the catalytic cycle, but an elevation in hydrogen peroxide generation via uncoupling resulted in a similar rate of epoxide formation. The activity of epoxidation significantly reduced with the D251N mutant. The chemoselectivity and stereoselectivity of the epoxidation reaction were maintained in the turnovers by these mutants. Overall, there was little evidence that other intermediates, aside from the archetypal reactive ferryl porphyrin cation radical, Compound I, contributed significantly to the epoxidation reaction. The observation of the high selectivity for the (S)-enantiomer was rationalized by molecular dynamics simulations. When the arrangement of the alkene and the active intermediate approached an ideal transition state structure for epoxidation, one face of the alkene was more often exposed to the iron oxo unit.

Comparative analysis of oral cavity sanation in children under general anesthesia and inhalation anesthesia with nitrogen oxide

The aim of this study was to carry out a comparative analysis of the volume of dental intervention under conditions of combined endotracheal anesthesia and inhalation anesthesia of nitrous oxide in pediatric dentistry.Materials and methods. Within the framework of this study, the results of oral cavity sanitation were analyzed in 300 patients aged 2 to 5 years. The median age was 3.7 years. The participants were divided into 2 equal samples: 150 patients underwent sanitation under general anesthesia (anesthesia group), 150 under conditions of inhalation anesthesia of nitrous oxide (sedation group). In the sedation group, there were 83 (55.3%) boys and 67 (44.7%) girls, in the anesthesia group – 89 participants (59.3%) were male, and 61 (40.7%) were female. The patients included in this study had comparable dental status. Also, when performing dental interventions in both groups, according to indications, local anesthesia was performed: children under 4 years old – lidocaine, children over 4 years old – articaine.Results. Based on the results of the analysis of the data obtained, it was found that the number of visits required for full-fledged dental treatment was statistically significantly lower in the anesthesia group compared to the sedation group: thus, all patients who underwent combined endotracheal anesthesia required only one visit to the clinic for necessary treatment. But it was also found that the total time of dental treatment differed depending on the anesthetic aid used: in the anesthesia group, it usually took from 1 to 2 hours (in 98% of patients), during this time a complete sanitation of the oral cavity was carried out, and in the sedation group varied more widely, with half of the patients in the range from 30 minutes to 1 hour (14% and 36.7% of children, respectively), but the number of visits for complete oral cavity sanitation increased to 8.Conclusions. In the present work, it was revealed that dental treatment under general anesthesia in children was more effective than the same treatment under conditions of inhalation anesthesia of nitrous oxide with preserved consciousness. The use of combined endotracheal anesthesia allows to reduce the number of visits to the clinic and avoid refusals from further dental treatment. General anesthesia was also more effective than sedation in terms of the number of cured teeth with caries, chronic pulpitis and extracted teeth. Nitrous oxide inhalation anesthesia provides partial retention of consciousness, is more manageable and better tolerated by children.

The Great Falls Tectonic Zone after the assembly of Laurentia: evidence for long-term tectonic stability from xenolith apatite

Combined U-Pb geochronology, and trace element analysis (Th, U, Y, Sr, rare earth elements: REE) by LA-Q-ICPMS and halogen chemistry (F, Cl, OH) by SEM-EDX are applied to apatite from a suite of amphibolite- and granulite-facies xenoliths from the Bearpaw Mountains (Montana, USA), to constrain cooling of Paleoproterozoic mid- to lower crust. Xenoliths hosting fluorapatite and hydroxyfluorapatite were emplaced at 54 to 50 Ma within K-rich, host lamprophyres (“minettes”). Granulite xenoliths yield apatite with highly dispersed U-Pb isotopic compositions. But discordia fit through their youngest populations yield Neoproterozoic-Cambrian (c. 650–500 Ma) lower intercepts. A single amphibolite xenolith from a shallower depth contains apatite with older single-grain apatite ages in the range of 1200–890 Ma. These lower intercept ages are much younger than previously reported zircon U--Pb ages from the same samples, which register a thermal maximum in the Palaeoproterozoic (c. 1700–1800 Ma). Trace element analysis of apatite confirms that most grains did not undergo retrogressive recrystallization. Additionally, there is a statistically significant link between apatite size and single grain ages in at least one sample. Our interpretation is that these apatite U-Pb ages represent slow cooling through the apatite Pb partial retention zone (c. 375–450 °C). Whether these data represent a distinct cooling event, or simple protracted cooling from a Palaeoproterozoic thermal peak, cannot be strictly constrained by these data. However, previous evidence from rutile U-Pb in xenoliths from nearby locations implies slow cooling from a Palaeoproterozoic peak. If a slow cooling scenario is assumed, by comparing apatite cooling ages to previous results from Ti-in-zircon thermometry on metamorphic zircon from the same xenoliths, a cooling rate of 0.14--0.33 °C/Myr can be estimated for these granulite xenoliths. Our apatite data thus contribute to a growing consensus on the geodynamic history of the northern Wyoming Craton and Medicine Hat Block, wherein lithospheric stability was maintained on a giga-year timescale from a granulite-facies metamorphic event associated with the assembly of Laurentia up until Laramide orogenesis.

Partial Retention of Legacy Trees Protect Mycorrhizal Inoculum Potential, Biodiversity, and Soil Resources While Promoting Natural Regeneration of Interior Douglas-Fir

Clearcutting reduces proximity to seed sources and mycorrhizal inoculum potential for regenerating seedlings. Partial retention of legacy trees and protection of refuge plants, as well as preservation of the forest floor, can maintain mycorrhizal networks that colonize germinants and improve nutrient supply. However, little is known of overstory retention levels that best protect mycorrhizal inoculum while also providing sufficient light and soil resources for seedling establishment. To quantify the effect of tree retention on seedling regeneration, refuge plants, and resource availability, we compared five harvesting methods with increasing retention of overstory trees (clearcutting (0% retention), seed tree (10% retention), 30% patch retention, 60% patch retention, and 100% retention in uncut controls) in an interior Douglas-fir-dominated forest in British Columbia. Regeneration increased with proximity to legacy trees in partially cut forests, with increasing densities of interior Douglas-fir, western redcedar, grand fir, and western hemlock seedlings with overstory tree retention. Clearcutting reduced cover of ectomycorrhizal refuge plants (from 80 to 5%) while promoting arbuscular mycorrhizal plants the year after harvest. Richness of shrubs, herbs, and mosses declined with increasing harvesting intensity, but tree richness remained at control levels. The presence of legacy trees in all partially cut treatments mitigated these losses. Light availability declined with increasing overstory cover and proximity to leave trees, but it still exceeded 1,000 W m−2 in the clearcut, seed tree and 30% retention treatments. Increasing harvesting intensity reduced aboveground and belowground C stocks, particularly in live trees and the forest floor, although forest floor losses were also substantial where thinning took place in the 60% retention treatment. The loss of forest floor carbon, along with understory plant richness with intense harvesting was likely associated with a loss of ectomycorrhizal inoculum potential. This study suggests that dispersed retention of overstory trees where seed trees are spaced ~10–20 m apart, and aggregated retention where openings are &amp;lt;60 m (2 tree-lengths) in width, will result in an optimal balance of seed source proximity, inoculum potential, and resource availability where seedling regeneration, plant biodiversity, and carbon stocks are protected.

Cement selection criteria for full coverage restorations: A comprehensive review of literature.

BackgroundProper cement selection in fixed prosthesis plays a determinative role in providing long-term serviceability, retention, caries prevention, and patient satisfaction. This study, reviews different luting agent characteristics and their application based on different clinical situations and different types of full coverage restorations. Material and MethodsAn electronic search was conducted through PubMed, Medline, and Google scholar using following keywords or combinations: restoration, full coverage, PFM, porcelain fused to metal, all ceramic, zirconia, ceramic, casting, fixed partial denture, cement*, dental cement, cement selection, and retention. The most related articles were selected for review. ResultsChoosing a proper luting agent is highly dependent on scientific knowledge regarding the characteristics of restorative materials and luting agents. Conventional cements could be indicated in various situations; however, some restorative materials or clinical situations call for resin-cements to provide predictable retention, support, and durability. ConclusionsConscious selection of retentive cement for each type of restoration/material is necessary to provide predictable successful treatment and reduce the potential complications. Key words:Adhesive cement, dental bridgework, dental cements, dental crowns, dental porcelain, prostheses and implants.