Biochemical studies of inositol N-acetylglucosaminyltransferase involved in mycothiol biosynthesis in Corynebacterium diphtheria.

Mycothiol (MSH) is the predominant low molecular weight thiol produced by actinomycetes, and it plays a pivotal role in the bacterial detoxication process. 1L-myo-Inositol-1-phosphate (1L-Ins-1-P) α-N-acetylglucosaminyltransferase (GlcNAc-T), known as MshA, is the only glycosyltransferase involved in MSH biosynthesis. In this work, the MshA from Corynebacterium diphtheria, named as CdMshA, was expressed, purified, and studied in detail. Its enzymatic activity to transfer GlcNAc to 1L-Ins-1-P was confirmed by the isolation and rigorous characterization of its reaction product 3-phospho-1-d-myo-inositol-2-acetamido-2-deoxy-α-d-glucopyranoside. CdMshA was shown to accept only UDP-GlcNAc and 1L-Ins-1-P as its substrates among various tested glycosyl donors, such as UDP-GlcNAc, UDP-Gal, UDP-Glc, UDP-GalNAc and UDP-GlcA, and glycosyl acceptors, such as myo-inositol, 1L-Ins-1-P and 1D-Ins-1-P. The results have demonstrated the strict substrate selectivity of CdMshA. Furthermore, its reaction kinetics with UDP-GlcNAc and 1L-Ins-1-P as substrates were characterized, while site-directed mutagenesis of CdMshA disclosed that its amino acid residues N28, K81 and R157 were essential for its enzymatic activity.

The Production Splitting Method of Offshore Multilayer Combined Water Flooding Gas Wells with Gas Dissolving.

Offshore gas reservoirs are characterized by thin interlayers, high production, few wells, etc., and are often exploited by multilayer combined mining, whereas the production dynamics of multilayer gas reservoirs are very different from those of single-layer gas reservoirs. Therefore, clarifying the gas production contribution of each layer in multilayer combined gas reservoirs is an important prerequisite for analyzing the potential of gas reservoirs and realizing efficient development. In this paper, unlike the past method of evaluating the gas production contribution of each layer by using the KH attribute of the reservoir, we combined the modified B-L equation considering CO2 dissolution and the multilayer multizone seepage equation to establish a dynamic split model of the production dynamics of multilayer water-driven gas reservoirs, verified the reliability of the model through the numerical model and the results of the production well logging, quantitatively analyzed the degree of influence of each parameter on the contribution of the layered gas production, and designed the orthogonal experiments. The main controlling factors of the gas production contribution of each layer were determined. The results of the study show that (1) the main controlling factors for the gas production contribution of each layer in the early stage of WDG are, in order, permeability, thickness, outer boundary distance, porosity, CO2 content, and total gas production rate; however, the main controlling factors for the gas production contribution of each layer in the late stage of WDG are, in order, thickness, permeability, outer boundary distance, porosity, CO2 content, and total gas production rate; and the combined view shows that the permeability and thickness have the greatest influence. (2) In multilayer production, the conditions of high permeability, close gas-water boundary, poor gas content, and low CO2 content will reduce the gas production contribution of the layer with the increase of production time. (3) Compared with the results of production logging and numerical simulation, the split model can better predict the gas production of each layer, and the prediction error is no more than 10%. (4) By comparing with the numerical simulation results, the model can realize the prediction of the time of seeing water in the layer with stronger water body capability. (5) The model takes into account the effect of the CO2 content, better reflects the actual gas composition of each layer, and can improve the production prediction accuracy by up to 4%. Considering the high cost of production logging in offshore oil and gas fields, the inability of the KH method to reflect the dynamic changes of gas production in each layer, the poor application of stratified sampling to dry gas reservoirs, and other limitations, the model in this paper can be utilized to simulate the multilayer water-driven gas drive process when the energy of the water body is strong by using the geological parameters of the reservoir and the fluid parameters, and the simulation results of this model provide directions for offshore multilayer water-driven gas reservoirs to improve the recovery rate, and for plugging and regulating the water and exploiting the potential of gas wells that have seen water.

Segmentation of Breast Tubules in H&E Images Based on a DKS-DoubleU-Net Model.

The formation of breast tubules plays an important role in the pathological grading of breast cancer. Breast tubules surrounded by a large number of epithelial cells are located in the subcutaneous tissue of the chest. The shapes of breast tubules are various, including tubular, round, and oval, which makes the process of breast tubule segmentation a difficult task. Deep learning technology, capable of learning complex data structures via efficient representation, could help pathologists accurately detect breast tubules in hematoxylin and eosin (H&E) stained images. In this paper, we propose a deep learning model named DKS-DoubleU-Net to accurately segment breast tubules with complex appearances in H&E images. The proposed DKS-DoubleU-Net model suggests using a DenseNet module as the encoder of the second subnetwork of DoubleU-Net, which utilizes dense features between layers and strengthens the propagation of features extracted in all previous layers, in order to better discover the intrinsic characteristics of breast tubules with complex structures and diverse shapes. Moreover, a feature fusing module called Kernel Selecting Module (KSM) is inserted before each output layer of the two U-Net branches of the DoubleU-Net, to implement a multiscale feature fusion via a self-adaptive kernel selecting for the sake of accurate segmentation of breast tubules in different sizes. The experiments on the public BRACS dataset and a private clinical dataset have shown that our model achieves better segmentation performance, compared to the state-of-art models of U-Net, DoubleU-Net, ResUnet++, HRNet, and DeepLabV3+. Specifically, on the public BRACS dataset, our method produced an F1-Score of 92.98%, which outperforms the F1-Score of U-Net, DoubleU-Net, and HRNet by 4.24%, 0.37%, and 1.68%, respectively, and is much better than performances of DeepLabV3+ and ResUnet++ by 7.83% and 23.84%, respectively. On the private clinic dataset, the proposed model achieved an F1-Score of 73.13%, which has shown an improvement of 10.31%, 1.89%, 4.88%, 15.47%, and 31.1% to the performances of the U-Net, DoubleU-Net, HRNet, DeepLabV3+, and ResUnet++, respectively. Superior performance could also be observed when comparing the proposed DKS-DoubleU-Net with the others using the metrics of Dice and mIou.

Influence of natural and anthropogenic controls on runoff in the Keriya River, central Tarim Basin, China.

The potential impact of natural factors on the runoff of intermittent rivers and ephemeral streams (IRES) has been largely ignored in the Tarim Basin, China. A representative example is the Keriya River. To quantify the long-term dynamic variations in lower reach surface runoff of IRES, river length, defined as the distance between a selected fix point along the perennial river segment to its dynamic, ephemeral end, was used as an indicator. Using a total of 272 remote sensing images, we digitized and measured the distance (river length) between the center of Yutian County and the river's end point on each image, and then calculated monthly inter-annual and intra-annual variations in length of the lower Keriya River from 2000 to 2019. Hydrometeorological data were combined with descriptors of anthropogenic disturbances to assess the relative influence of natural factors and anthropogenic disturbances on lower reach river runoff. The results showed that intra-annual variations in river length fluctuated seasonally, with the minimum value occurring in June; two main peaks occurred in March and August. The minimum June value in river length was closely linked to an increase in agricultural water demand and a decrease in upper reach runoff. The August peak in river length was related to the peak values in upper reach runoff and agricultural water demand; upper reach runoff made a significant contribution because the former was about 20% more than the latter in summer. The March peak corresponded to elevated lower reach groundwater levels and to the melting of ice along river channels. Inter-annual variations in river length were due to inter-annual variations in upper reach runoff and middle reach agricultural water use which increased slightly during the study period. Inter-annual variations in frequency and amplitude of the fluctuations in river length were mainly controlled by changes in upper reach runoff. The minimum in river length in 2009 was consistent with the low in upper reach runoff of the Keriya River and other rivers in the Tarim Basin. The most significant factors controlling variations in river length are natural in origin.

Responses of two dominant desert plant species to the changes in groundwater depth in hinterland natural oasis, Tarim Basin.

Groundwater is increasingly becoming a permanent and steady water source for the growth and reproduction of desert plant species due to the frequent channel cutoff events in arid inland river basins. Although it is widely acknowledged that the accessibility of groundwater has a significant impact on plant species maintaining their ecological function, little is known about the water use strategies of desert plant species to the groundwater availability in Daryaboyi Oasis, Central Tarim Basin. This study initially determined the desirable and stressing groundwater depths based on ecological and morphological parameters including UAV-based fractional vegetation cover (FVC) images and plant growth status. Then, leaf δ13C values of small- and big-sized plants were analyzed to reveal the water use strategies of two dominant woody species (Populus euphratica and Tamarix ramosissima) in response to the groundwater depth gradient. The changes in FVC and growth status of plants suggested that the actual groundwater depth should be kept at an appropriate range of about 2.1-4.3 m, and the minimum groundwater depth should be less than 7 m. This will ensure the protection of riparian woody plants at a normal growth state and guarantee the coexistence of both plant types. Under a desirable groundwater condition, water alternation (i.e., flooding and rising groundwater depth) was the main factor influencing the variation of plant water use efficiency. The obtained results indicated that big-sized plants are more salt-tolerant than small ones, and T. ramosissima has strong salt palatability than P. euphratica. With increasing groundwater depth, P. euphratica continuously decreases its growth status to maintain hydraulic efficiency in drought condition, while T. ramosissima mainly increases its water use efficiency first and decreases its growth status after then. Besides, in a drought condition, T. ramosissima has strong adaptability than P. euphratica. This study will be informative for ecological restoration and sustainable management of Daryaboyi Oasis and provides reference materials for future research programs.

Galactosylation of caffeic acid by an engineered ß-galactosidase.

Glycosylation is useful for improving the chemical properties and physiological functions of biologically and pharmacologically important compounds. The glycosylation of phenolic compounds can increase their solubility and stability in water. The addition of galactose residue has special meaning as it facilitates targeted delivery of drugs to the liver cancer cells with abundant galactose acceptors on the cell surface. In this work, the engineered ß-galactosidase W980F from Lactobacillus bulgaricus L3 was utilized for the glycosylation of caffeic acid, a well-known phenolic phytochemical with broad bioactivities. The reaction was performed by incubation of the enzyme with 200 mM of lactose and 100 mM of caffeic acid at 45°C for 1 h. The product was purified and analyzed by MS and NMR spectra. The MS revealed a signal of [M-H]‒ at m/z 341.09, suggesting monogalactosylated products of caffeic acid (Mr 342). The NMR spectra further identified the products to be caffeic acid 3'-O-ß- galactopyranoside and caffeic acid 4'-O-ß-galactopyranoside in a ratio of 1:3. This was the first discovery that caffeic acid could be galactosylated by the engineered glycosidase.

Glycosylation of phenolic compounds by the site-mutated ß-galactosidase from Lactobacillus bulgaricus L3.

ß-Galactosidases can transfer the galactosyl from lactose or galactoside donors to various acceptors and thus are especially useful for the synthesis of important glycosides. However, these enzymes have limitations in the glycosylation of phenolic compounds that have many physiological functions. In this work, the ß-galactosidase from Lactobacillus bulgaricus L3 was subjected to site-saturation mutagenesis at the W980 residue. The recombinant pET-21b plasmid carrying the enzyme gene was used as the template for mutation. The mutant plasmids were transformed into Escherichia coli cells for screening. One recombinant mutant, W980F, exhibited increased yield of glycoside when using hydroquinone as the screening acceptor. The enzyme was purified and the effects of the mutation on enzyme properties were determined in detail. It showed improved transglycosylation activity on novel phenolic acceptors besides hydroquinone. The yields of the glycosides produced from phenol, hydroquinone, and catechol were increased by 7.6% to 53.1%. Moreover, it generated 32.3% glycosides from the pyrogallol that could not be glycosylated by the wild-type enzyme. Chemical structures of these glycoside products were further determined by MS and NMR analysis. Thus, a series of novel phenolic galactosides were achieved by ß-galactosidase for the first time. This was a breakthrough in the enzymatic galactosylation of the challenging phenolic compounds of great values.

Purification of fructooligosaccharides by immobilized yeast cells and identification of ethyl ß-D-fructofuranoside as a novel glycoside formed during the process.

A yeast strain (XS1) capable of selective utilization of fructooligosaccharides (FOSs) syrup was identified as Wickerhamomyces anomala. Cells of W. anomala XS1 were immobilized in calcium alginate and incubated with an FOS mixture at 30 °C. The purity of the FOS increased from 54.4% to 80.1% (w/w) as 93.6% of monosaccharides were metabolized while the oligosaccharides were not affected. The immobilized yeast cells could be recycled 10 times and the corresponding batch treatments achieved FOS purities around 80%. Thus, the method could be promising for large-scale purification of FOS syrup at low cost. A byproduct formed by the yeast was identified as ethyl ß-D-fructofuranoside by MS and NMR spectroscopy.