R2 Region Research Articles

Phleum pratense-pollen adaptive variations and pollen microbiome investigation under different climatic regions and prospects of allergenicity.

Phleum pratense is an allergenic grass that pollinates in spring in Pakistan. Databases Allergenonline.org and Allergen.org record ten P. pratense allergens and their isoforms. Phl P 1, Phlp 5, and Phl p 11 are major P. pratense-pollen allergens with demonstrated basophil activity and skin test reactivity. Little is known about P. pratense pollen adaptive variations in different climatic regions and pollen-associated microbial diversity. In this study, we collected P. pratense-pollen and soils in the spring season 2022. Samples were collected from three climatic regions in Pakistan (R1, R2 and R3) with differences in mean monthly air temperature, mean monthly precipitation and elevation. The morphology of pollen was observed by light microscopy, scanning electron microscopy (SEM), biochemical fingerprint analysis, and composition of pollen were investigated by fourier-transform infrared spectroscopy (FTIR). The pollen-associated bacterial populations were identified through a Biolog GEN III microplate system. The pollen water-soluble proteins were isolated and stabilized in phosphate buffer saline (PBS) and tested for allergenicity responses through dot blots and western blots analysis. The morphology study found difference in pollen biochemical composition. Biolog identified Brevibacterium epidermidis and Pantoea agglomerans from P. pratense pollen. Protein extract quantification and sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) gel found decreased protein expression in R1 region pollen compared to R2 and R3 region pollen. Allergenicity studies found differential expression of beta-expansin and profilin allergens in pollen obtained from the three regions. Beta-expansin and profilin were suppressed in R1 pollen and expressed in compared to R2 and R3 pollen. This is the first study to identify B. epidermidis and P. agglomerans growth on P. pratense pollen. Variable allergen expression in P. pratense pollen has also been observed in different regions. Soil pH, an increase in mean monthly temperature and a decrease in mean monthly precipitation correlated with pollen biochemical composition, and reduced beta-expansin and profilin expression involved in pollen growth and development. The findings of this research are unique, which enhances basic knowledge and understanding of P. pratense-pollen associated microbiota and climate change impacts on the pollen allergen expression.

Kinetic Ensemble of Tau Protein through the Markov State Model and Deep Learning Analysis.

The ordered assembly of Tau protein into filaments characterizes Alzheimer's and other neurodegenerative diseases, and thus, stabilization of Tau protein is a promising avenue for tauopathies therapy. To dissect the underlying aggregation mechanisms on Tau, we employ a set of molecular simulations and the Markov state model to determine the kinetics of ensemble of K18. K18 is the microtubule-binding domain of Tau protein and plays a vital role in the microtubule assembly, recycling processes, and amyloid fibril formation. Here, we efficiently explore the conformation of K18 with about 150 μs lifetimes in silico. Our results observe that all four repeat regions (R1-R4) are very dynamic, featuring frequent conformational conversion and lacking stable conformations, and the R2 region is more flexible than the R1, R3, and R4 regions. Additionally, it is worth noting that residues 300-310 in R2-R3 and residues 319-336 in R3 tend to form sheet structures, indicating that K18 has a broader functional role than individual repeat monomers. Finally, the simulations combined with Markov state models and deep learning reveal 5 key conformational states along the transition pathway and provide the information on the microsecond time scale interstate transition rates. Overall, this study offers significant insights into the molecular mechanism of Tau pathological aggregation and develops novel strategies for both securing tauopathies and advancing drug discovery.

Analysis of Receptor-Type Protein Tyrosine Phosphatase Extracellular Regions with Insights from AlphaFold.

The receptor-type protein tyrosine phosphatases (RPTPs) are involved in a wide variety of physiological functions which are mediated via their diverse extracellular regions. They play key roles in cell-cell contacts, bind various ligands and are regulated by dimerization and other processes. Depending on the subgroup, they have been described as everything from 'rigid rods' to 'floppy tentacles'. Here, we review current experimental structural knowledge on the extracellular region of RPTPs and draw on AlphaFold structural predictions to provide further insights into structure and function of these cellular signalling molecules, which are often mutated in disease and are recognised as drug targets. In agreement with experimental data, AlphaFold predicted structures for extracellular regions of R1, and R2B subgroup RPTPs have an extended conformation, whereas R2B RPTPs are twisted, reflecting their high flexibility. For the R3 PTPs, AlphaFold predicts that members of this subgroup adopt an extended conformation while others are twisted, and that certain members, such as CD148, have one or more large, disordered loop regions in place of fibronectin type 3 domains suggested by sequence analysis.

Genome-wide association study of kernel colour traits and mining of elite alleles from the major loci in maize

BackgroundMaize kernel colour is an important index for evaluating maize quality and value and mainly entails two natural pigments, carotenoids and anthocyanins. To analyse the genetic mechanism of maize kernel colour and mine single nucleotide polymorphisms (SNPs) related to kernel colour traits, an association panel including 244 superior maize inbred lines was used to measure and analyse the six traits related to kernel colour in two environments and was then combined with the about 3 million SNPs covering the whole maize genome in this study. Two models (Q + K, PCA + K) were used for genome-wide association analysis (GWAS) of kernel colour traits.ResultsWe identified 1029QTLs, and two SNPs contained in those QTLs were located in coding regions of Y1 and R1 respectively, two known genes that regulate kernel colour. Fourteen QTLs which contain 19 SNPs were within 200 kb interval of the genes involved in the regulation of kernel colour. 13 high-confidence SNPs repeatedly detected for specific traits, and AA genotypes of rs1_40605594 and rs5_2392770 were the most popular alleles appeared in inbred lines with higher levels. By searching the confident interval of the 13 high-confidence SNPs, a total of 95 candidate genes were identified.ConclusionsThe genetic loci and candidate genes of maize kernel colour provided in this study will be useful for uncovering the genetic mechanism of maize kernel colour, gene cloning in the future. Furthermore, the identified elite alleles can be used to molecular marker-assisted selection of kernel colour traits.

Unveiling the Cosmic Cradle: clustering and massive star formation in the enigmatic Galactic bubble N59

ABSTRACT In this paper, we have conducted an investigation focused on a segment of the Spitzer mid-infrared bubble N59, specifically referred to as R1 within our study. Situated in the inner Galactic plane, this region stands out for its hosting of five 6.7 GHz methanol masers, as well as numerous compact $\mathrm{H}\, \rm {{\small II}}$ regions, massive clumps, filaments, and prominent bright rims. As 6.7 GHz masers are closely linked to the initial phases of high-mass star formation, exploring regions that exhibit a high abundance of these maser detections provides an opportunity to investigate relatively young massive star-forming sites. To characterize the R1 region comprehensively, we utilize multiwavelength (archival) data from optical to radio wavelengths, together with 13CO and C18O data. Utilizing the Gaia DR3 data, we estimate the distance towards the bubble to be 4.66 ± 0.70 kpc. By combining near-infrared (NIR) and mid-infrared (MIR) data, we identify 12 Class I and 8 Class II sources within R1. Furthermore, spectral energy distribution (SED) analysis of selected sources reveals the presence of four embedded high-mass sources with masses ranging from 8.70 to 14.20 M⊙. We also identified several O and B-type stars from radio continuum analysis. Our molecular study uncovers two distinct molecular clouds in the region, which, although spatially close, occupy different regions in velocity space. We also find indications of a potential hub-filament system fostering star formation within the confines of R1. Finally, we propose that the feedback from the $\mathrm{H}\, \rm {{\small II}}$ regions has led to the formation of prominent Bright Rimmed Clouds (BRCs) within our region of interest.

APOE 5'UTR Methylation Pattern Analysis in Blood and Brain Tissue from Alzheimer's Disease Affected Patients.

APOE ε4 allele is the major genetic risk factor for Alzheimer's Disease (AD). Furthermore, APOE methylation pattern has been described to be associated with the disease and to follow a bimodal pattern, with a hypermethylated CpG island and a hypomethylated promoter region. However, little is known about the methylation levels in the APOE 5'UTR region. Here, the methylation of two regions (R1 and R2) within APOE 5'UTR was investigated in both peripheral blood mononuclear cells (PBMCs) and hippocampus (HIC) samples to identify differentially methylated CpG sites and to associate clinical, genetic features and cerebrospinal fluid (CSF) biomarkers levels. DNA was extracted from PBMCs of 20 AD and 20 healthy controls (HC) and from 6 AD and 3 HC HIC samples. The methylation analysis was carried out by means of pyrosequencing. In AD PBMCs we found that R1 region displayed a higher methylation level, while the opposite trend was observed in R2. The presence of ε4 allele highlighted a marked increase in R1 methylation level and a decrease in R2. In AD PBMCs and HIC, age progression resulted to be associated with an increase in the methylation level of R1. Lastly, the methylation of a CpG site in R2 was found to be related to CSF biomarkers. Despite the lack of a statistical significance, the outcome from this exploratory analysis highlighted the presence of a difference in methylation in APOE 5'UTR in PBMCs of AD patients which seemed to be associated also with APOE genotype, age and CSF biomarkers level.

Watershed groundwater level multistep ahead forecasts by fusing convolutional-based autoencoder and LSTM models

The development of deep learning-based groundwater level forecast models can tackle the challenge of high dimensional groundwater dynamics, predict groundwater variation trends accurately, and manage groundwater resources effectively, thereby contributing to sustainable water resources management. This study proposed a novel ConvAE-LSTM model, which fused a Convolutional-based Autoencoder model (ConvAE) and a Long Short-Term Memory Neural Network model (LSTM), to provide accurate spatiotemporal groundwater level forecasts over the next three months. The HBV-light and LSTM models are chosen as benchmarks. An ensemble of point data and the corresponding derived images concerning the past (observations) and the future (forecasts from a conceptual model) of groundwater levels at 33 groundwater wells in Jhuoshuei River basin of Taiwan between 2000 and 2019 constituted the case study. The findings showcase the effectiveness of the ConvAE-LSTM model in extracting crucial features from both point and imagery datasets. This model successfully establishes spatiotemporal dependencies between regional images and groundwater level data over diverse time frames, leading to accurate multi-step-ahead forecasts of groundwater levels. Notably, the ConvAE-LSTM model exhibits a substantial improvement, with the R-squared values showing an increase of more than 18%, 22%, and 49% for the R1, R2, and R3 regions, respectively, compared to the HBV-light model. Additionally, it outperforms the LSTM model in this regard. This study represents a noteworthy milestone in environmental modeling, offering key insights for designing sustainable groundwater management strategies to ensure the long-term availability of this vital resource.

Biochar accelerates the start-up of the anammox process: Phenomenon and potential mechanisms

Biochar was applied to accelerate the establishment of the anaerobic ammonium oxidation (anammox) reactor without temperature control (10 to 37 °C). 5 g/L coconut and apricot charcoal were added in R1 and R2. As a result, it took 121, 85 and 90 days for the start-up of R0, R1 and R2. The anammox pathway related functional gene (hzsB) in the bottom region of R0, R1 and R2 were 4.86 × 1011, 1.31 × 1012 and 5.85 × 1011 copies/g after 215-day operation which was significantly greater than that in the seeding sludge (2.56 × 107 copies/g). Candidatus Kuenenia and C. Brocadia were enriched in all reactors and the maximum nitrogen removal rate of R0, R1 and R1 were 1.29, 1.41 and 1.48 kgN/(m3·d), respectively. Biochar promoted the electron transfer capacity, resulting in the increase of functional gene responsible for the anammox pathway. The anammox microbes were enriched and specific anammox activity and nitrogen removal efficiency were improved eventually. The findings in our study could provide an alternative to establish the anammox process rapidly without temperature control.

Analysis of the correlation between gene copy deletion in the AZFc region and male infertility in Japanese men.

Deletion of the azoospermia factor c (AZFc), located on the long arm of the Y chromosome, is a cause of male infertility. The structure of the Y chromosome is diversified by the copy number of various genes, such as deleted in azoospermia (DAZ), basic protein Y2, chromodomain Y1, testis-specific transcript Y-linked 4, and Golgi autoantigen golgin subfamily a2 like Y, located in the AZF region. In this study, we investigated the deletion of each gene copy and analyzed its relationship with Japanese male infertility. Deletions of single nucleotide variants of each gene copy in 721 proven fertile men as controls, 139 patients with non-obstructive azoospermia (NOA), and 56 patients with oligozoospermia (OS) were analyzed via polymerase chain reaction-restriction fragment length polymorphism analysis. Their association with infertility was analyzed using logistic regression analysis adjusted for the Y-chromosome haplogroup, D1a2a. Deletions of DAZ/II in the r1 region and DAZ/V in the r1 and r2 regions showed significant associations with NOA (odds ratio [OR]=4.15, 95% confidence interval [CI]=1.18-14.6, P=0.026; OR=4.19, 95% CI=1.19-14.7, P=0.025, respectively). They did not show any association with OS. Partial deletion of the AZFc region affects spermatogenesis in Japanese male.

How Botulinum Neurotoxin Light Chain A1 Maintains Stable Association with the Intracellular Neuronal Plasma Membrane.

Botulinum neurotoxin serotype A (BoNT/A) is the most potent protein toxin for humans and is utilized as a therapy for numerous neurologic diseases. BoNT/A comprises a catalytic Light Chain (LC/A) and a Heavy Chain (HC/A) and includes eight subtypes (BoNT/A1-/A8). Previously we showed BoNT/A potency positively correlated with stable localization on the intracellular plasma membrane and identified a low homology domain (amino acids 268-357) responsible for LC/A1 stable co-localization with SNAP-25 on the plasma membrane, while LC/A3 was present in the cytosol of Neuro2A cells. In the present study, steady-state- and live-imaging of a cytosolic LC/A3 derivative (LC/A3V) engineered to contain individual structural elements of the A1 LDH showed that a 59 amino acid region (275-334) termed the MLD was sufficient to direct LC/A3V from the cytosol to the plasma membrane co-localized with SNAP-25. Informatics and experimental validation of the MLD-predicted R1 region (an α-helix, residues 275-300) and R2 region (a loop, α-helix, loop, residues 302-334) both contribute independent steps to the stable co-localization of LC/A1 with SNAP-25 on the plasma membrane of Neuro-2A cells. Understanding how these structural elements contribute to the overall association of LC/A1 on the plasma membrane may identify the molecular basis for the LC contribution of BoNT/A1 to high potency.

Rearrangement with the nkd2 promoter contributed to allelic diversity of the r1 gene in maize (Zea mays).

SUMMARYThe maize red1 (r1) locus regulates anthocyanin accumulation and is a classic model for allelic diversity; changes in regulatory regions are responsible for most of the variation in gene expression patterns. Here, an intrachromosomal rearrangement between the distal upstream region of r1 and the region of naked endosperm 2 (nkd2) upstream to the third exon generated a nkd2 null allele lacking the first three exons, and the R1‐st (stippled) allele with a novel r1 5′ promoter region homologous to 5′ regions from nkd2‐B73. R1‐sc:124 (an R1‐st derivative) shows increased and earlier expression than a standard R1‐g allele, as well as ectopic expression in the starchy endosperm compartment. Laser capture microdissection and RNA sequencing indicated that ectopic R1‐sc:124 expression impacted expression of genes associated with RNA modification. The expression of R1‐sc:124 resembled nkd2‐W22 expression, suggesting that nkd2 regulatory sequences may influence the expression of R1‐sc:124. The r1‐sc:m3 allele is derived from R1‐sc:124 by an insertion of a Ds6 transposon in intron 4. This insertion blocks anthocyanin regulation by causing mis‐splicing that eliminates exon 5 from the mRNA. This allele serves as an important launch site for Ac/Ds mutagenesis studies, and two Ds6 insertions believed to be associated with nkd2 mutant alleles were actually located in the r1 5′ region. Among annotated genomes of teosinte and maize varieties, the nkd2 and r1 loci showed conserved overall gene structures, similar to the B73 reference genome, suggesting that the nkd2‐r1 rearrangement may be a recent event.

Crystal structure of a family VIII β-lactamase fold hydrolase reveals the molecular mechanism for its broad substrate scope.

Family VIII esterases present similarities to class C β-lactamases, which show nucleophilic serines located at the S-X-X-K motif instead of the G-X-S-X-G or G-D-S-(L) motif shown by other carboxylesterase families. Here, we report the crystal structure of a novel family VIII (subfamily VIII. I) esterase (EH7 ; denaturing temperature, 52.6 ± 0.3 °C; pH optimum 7.0-9.0) to deepen its broad substrate range. Indeed, the analysis of the substrate specificity revealed its capacity to hydrolyse nitrocefin as a model chromogenic cephalosporin substrate (40.4 ± 11.4 units·g-1 ), and a large battery of 66 structurally different esters (up to 1730 min-1 ), including bis(2-hydroxyethyl)-terephthalate (241.7 ± 8.5 units·g-1 ) and the mycotoxin T-2 (1220 ± 52 units·g-1 ). It also showed acyltransferase activity through the synthesis of benzyl 3-oxobutanoate (40.4 ± 11.4 units·g-1 ) from benzyl alcohol and vinyl acetoacetate. Such a broad substrate scope is rare among family VIII esterases and lipolytic enzymes. Structural analyses of free and substrate-bound forms of this homooctamer esterase suggest that EH7 presents a more opened and exposed S1 site having no steric hindrance for the entrance of substrates to the active site, more flexible R1, R2 and R3 regions allowing for the binding of a wide spectrum of substrates into the active site, and small residues in the conserved motif Y-X-X containing the catalytic Tyr enabling the entrance of large substrates. These unique structural elements in combination with docking experiments allowed us to gain valuable insights into the substrate specificity of this esterase and possible others belonging to family VIII.

Variations and influencing factors of nitrate accumulation in the deep soil profiles of apple orchards on the Loess Plateau

Nitrate accumulation in the soil profile of uplands is an important fate of surplus nitrogen (N) fertilizer in intensive agricultural regions. A comprehensive understanding of the spatial variation in nitrate accumulation characteristics in the deep (>2 m) soil profile and identification of the key driving factors are important for mitigating N loss and associated environmental problems. We sampled the deep soil profiles (to 8–13 m) in four regions (i.e., R1, R2, R3 and R4) from north to south of the apple-planting region on the Loess Plateau (LP), spanning a highly spatial variation of climates, soils, orchard standing ages, and N fertilizer rates. Regression analysis and random forest analysis were used to identify the characteristics of nitrate accumulation in the vadose zones at the regional scale. The results showed that nitrate accumulation characteristics (represented by four indices: peaks of nitrate content, peak depths, nitrate accumulation depths and nitrate accumulation amounts) were significantly positively correlated with the stand ages of orchards. Nitrate stock in the soil profiles (0–8 m) increased from north to south and averaged 4149 and 13,633 kg N ha−1 in the 25-yr-old orchards for R1 and R4, respectively. Nitrate stock in the soil profiles (0–13 m) of 30-yr-old orchards in R4 averaged 19,283 kg N ha−1, ranging from 12,859 to 25,900 kg N ha−1. The nitrate accumulation depths in the orchards in the R1 and R4 regions were deeper due to the high soil sand content and high water input, respectively. It highlights that the water input rate and soil sand content were key controlling factors for peak depth and nitrate accumulation depth, while stand age and N fertilization rate mainly controlled the peak of nitrate content and nitrate accumulation amount. The southern apple-planting region with a shallow vadose zone and high irrigation is suggested to be designated as the nitrate vulnerable zone.

Functional and molecular characterization of a cold-active lipase from Psychrobacter celer PU3 with potential antibiofilm property

The lipase gene from Psychrobacter celer PU3 was cloned into pET-28a(+) expression vector and overexpressed in E. coli BL21 (DE3) pLysS cells. The purified Psychrobacter celer lipase (PCL) was characterized as an alkaline active enzyme and has a molecular mass of around 30 kDa. The PCL was active even at a low temperature and the optimum range was observed between 10 and 40 °C temperatures. MALDI-TOF and phylogenetic analysis ensured that Psychrobacter celer PU3 lipase (PCL) was closely related to P. aureginosa lipase (PAL). MD simulation results suggest that temperature change did not affect the overall structure of PCL, but it might altered the temperature-dependent PCL functional changes. R1 (129–135 AA) and R2 (187–191 AA) regions could be important for temperature-dependent PCL function and they fluctuated much at 35 °C temperature. PMSF completely inhibited PCL lipase activity and it demonstrates the presence of serine residues in the active site of PCL. PCL is moderately halophilic and most of the tested organic solvents found to be inhibiting the lipase activity except the solvents ethanol and methanol. PCL activity was increased with surfactants (SDS and CTAB) and bleaching agents (hydrogen peroxide). The effect of different metal ions on PCL resulted that only mercuric chloride was found as the enhancer of the lipase activity. Antibiofilm property of PCL was evaluated against pathogenic Vibrio parahaemolyticus isolated from the diseased shrimp and MIC value was 500 U. PCL significantly altered the morphology and biofilm density of V. parahaemolyticus and the same was observed through scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) imaging. RT-PCR analysis revealed that the mRNA expression level of biofilm, colony morphology and major toxin-related (aphA, luxS, opaR, tolC, toxR) genes of V. parahaemolyticus were significantly downregulated with PCL treatment.

Comparison of Protein Concentration in Red and White Muscles in Two Species of Bony Fish

The current study deals with estimating the protein concentration and the effect of fish weight on protein concentration values in red and white muscles in two different regions ( R1 : Anterior region lies 2 cm behind the head and R2: posterior region lies 2cm from caudal fin (in two types of bony fish, namely common carp (Cyprinus carpio) and Nile tilapia (Oreochromis niloticus). Samples were collected from Karmat Ali river- north of Basrah between October 2019 and February 2020. The protein was extracted using protein extraction buffer, the current study show that the average of protein concentration in red muscles of Nile tilapia ranged between 7.74-7.4 mg / ml and ( 6.8-8.85 mg / ml) in R1 and R2 region respectively, while it ranged between 173-334 mg / ml and 127-253 mg / ml in R1and R2 region in white muscles, respectively. In Carp, protein concentration for red muscles was 7.19-9.10 mg / ml and 6.87-8.41 mg / ml in R1 and R2 regions, respectively. On the other hand, protein concentration in white muscles ranged between 98.7-250.2 mg / ml and 61.5-214.1 mg / ml in both R1 and R2 regions respectively. The statistical analysis results of the protein concentration in the red and white muscles in the body regions indicated that there was a significant difference(P<0.05) and non- significant difference (P>0.05) between the protein concentration in red and white muscles in the studied species ,The current study concluded that white muscles contain a higher protein concentration than red muscles and that weight gain has a significant effect on protein concentration in white muscles.

Two-Stage Shrinkage Bayesian Estimators For The Shape Parameter of Pareto Distribution Dependent on Katti’s Regions

This study proposes a two-stage shrinkage Bayesian estimation of the shape parameter of Pareto distribution. Additional information from the past and considered presently in new estimation processes has been receiving considerable attention in the last few decades, especially when a sample unit is costly or difficult to obtain. The proposed two-stage pooling estimation procedure assumes that the prior knowledge of θ can take the form of an initial estimate θ0 of θ. The expressions for bias, bias ratio, mean square error, expected sample size, and relative efficiency are derived based on the two regions of R1 and R2. Certain values of the constants are considered, and the R language is used for statistical programming. The numerical results and conclusion suggest that the proposed estimators have higher relative efficiency compared with the classical Bayesian estimator with respect to a guess value. The effective region of the estimator dependent on R2 is better than that of the estimator dependent dependent on R1.

Establishment of an in vitro flow cytometric detection method for lipid-accumulated cells

Eukaryotic cells can store and converse excess lipid to the cytosolic lipid droplets. Adipogenesis of preadipocytes has been often used to study the molecular basis and the effect of obesity drugs on fat cell conversion. Many methods were developed for the detection of the cytosolic lipid droplets as Nile red, BODIPY 493/503 (4, 4-difluoro-1, 3, 5, 7, 8-pentamethyl-4-bora-3a, 4adiaza-s-indacene), BODIPY 665/676, 1,6-diphenylhexatriene (DPH), DAPI, Hoechst, Sudan III, and Oil-red O. The differences in the spectral properties of these lipophilic dyes and their advantages of each are discussed. In this study, an in vitro flow cytometric detection method was established for the detection of lipid-accumulated cells. Commonly, the longer the period of adipogenic induction, the greater the quantity of lipid in fat cell can accumulate. Thus, to determine whether increasing the fat stored within a cell would result in the greater granularity. 3T3-L1 cells in culture were hormonally induced for adipogenesis. Then, these cells were dissociated and analyzed in a flow cytometer at 0, 5, and 10 days post-induction. After adipogenic induction, the cells had become increasingly heterogeneous in their cellular granularity. The cells containing greater granular structure were markedly increased, and this increase in granularity positively correlated with the time of the post-adipogenic induction. On the other analysis, the 0 and 10 days post adipogenic induction of 3T3-L1 cells were gated for 4 regions. The R1 region contains cells with a level of granularity similar to that seen in the control cells (non-adipogenic induction), whereas R2 to R4 regions contain cells with increasing granularity. According to all data, we have successfully established an in vitro flow cytometric detection method for the detection of lipid-accumulated cells. We wish this method will be applied on the research of obesity drugs and the design of therapeutic strategies for obesity in the future.

Mechanism and Structural Insights Into a Novel Esterase, E53, Isolated From Erythrobacter longus.

Esterases are a class of enzymes that split esters into an acid and an alcohol in a chemical reaction with water, having high potential in pharmaceutical, food and biofuel industrial applications. To advance the understanding of esterases, we have identified and characterized E53, an alkalophilic esterase from a marine bacterium Erythrobacter longus. The crystal structures of wild type E53 and three variants were solved successfully using the X-ray diffraction method. Phylogenetic analysis classified E53 as a member of the family IV esterase. The enzyme showed highest activity against p-nitrophenyl butyrate substrate at pH 8.5–9.5 and 40°C. Based on the structural feature, the catalytic pocket was defined as R1 (catalytic center), R2 (pocket entrance), and R3 (end area of pocket) regions. Nine variants were generated spanning R1–R3 and thorough functional studies were performed. Detailed structural analysis and the results obtained from the mutagenesis study revealed that mutations in the R1 region could regulate the catalytic reaction in both positive and negative directions; expanding the bottleneck in R2 region has improved the enzymatic activity; and R3 region was associated with the determination of the pH pattern of E53. N166A in R3 region showed reduced activity only under alkaline conditions, and structural analysis indicated the role of N166 in stabilizing the loop by forming a hydrogen bond with L193 and G233. In summary, the systematic studies on E53 performed in this work provide structural and functional insights into alkaliphilic esterases and further our knowledge of these enzymes.

A genome‐wide analysis of NPF and NRT2 transporter gene families in bread wheat provides new insights into the distribution, function, regulation and evolution of nitrate transporters

Nitrate transporters (NRT) in plants are mainly encoded by the NPF and NRT2 gene families. We aimed to reveal the chromosome distribution, collinearity, coexpression and evolution of the NPF and NRT2 genes in the genome of wheat (Triticum aestivum). Nitrate transport activity of representative proteins was also verified. Genomic information, collinearity analysis, coexpression network analysis, nitrate transport activity and polymorphism analysis were integrated to identify and characterize the NPF and NRT2 genes. We identified 331 NPF and 46 NRT2 genes in the wheat genome. Tandem duplication was the main driver of the expansion of the NRT2 genes. The NPF genes were mainly distributed on the 2-, 3- and 7- chromosome groups and in the R2b region. The NRT2 genes were mainly distributed on the 6-chromosome group and in the R1 region. Multiple transcription factor families were coexpressed with NPF and NRT2 genes in wheat. Two NPFs and one NRT2 could transport NO3− under either 0.5 mM or 10 mM nitrate concentrations. One hundred and eighty-five NPF and 27 NRT2 genes fit the neutral selection model. Natural variations in NPF genes resulted in differences in the nitrogen uptake of wheat. Duplication events are ubiquitous in NPF and NRT2 gene families in the wheat genome. GRAS may be a previously unrecognized transcription factor that regulates NPF genes expression. It is unreliable to predict the activity of NPF and NRT2 proteins based only on their phylogenetic relationships. Polymorphisms in NPF and NRT2 genes mainly accumulate by random drift.

Comparison of [11C]UCB-J and [18F]FDG PET in Alzheimer’s disease: A tracer kinetic modeling study

[11C]UCB-J PET for synaptic vesicle glycoprotein 2 A (SV2A) has been proposed as a suitable marker for synaptic density in Alzheimer’s disease (AD). We compared [11C]UCB-J binding for synaptic density and [18F]FDG uptake for metabolism (correlated with neuronal activity) in 14 AD and 11 cognitively normal (CN) participants. We assessed both absolute and relative outcome measures in brain regions of interest, i.e., K1 or R1 for [11C]UCB-J perfusion, VT (volume of distribution) or DVR to cerebellum for [11C]UCB-J binding to SV2A; and Ki or KiR to cerebellum for [18F]FDG metabolism. [11C]UCB-J binding and [18F]FDG metabolism showed a similar magnitude of reduction in the medial temporal lobe of AD –compared to CN participants. However, the magnitude of reduction of [11C]UCB-J binding in neocortical regions was less than that observed with [18F]FDG metabolism. Inter-tracer correlations were also higher in the medial temporal regions between synaptic density and metabolism, with lower correlations in neocortical regions. [11C]UCB-J perfusion showed a similar pattern to [18F]FDG metabolism, with high inter-tracer regional correlations. In summary, we conducted the first in vivo PET imaging of synaptic density and metabolism in the same AD participants and reported a concordant reduction in medial temporal regions but a discordant reduction in neocortical regions.