Targeted therapy for non-small-cell lung cancer: New insights into regulated cell death combined with immunotherapy.

Non-small-cell lung cancer (NSCLC), which has a high rate of metastatic spread and drug resistance, is the most common subtype of lung cancer. Therefore, NSCLC patients have a very poor prognosis and a very low chance of survival. Human cancers are closely linked to regulated cell death (RCD), such as apoptosis, autophagy, ferroptosis, pyroptosis, and necroptosis. Currently, small-molecule compounds targeting various types of RCD have shown potential as anticancer treatments. Moreover, RCD appears to be a specific part of the antitumor immune response; hence, the combination of RCD and immunotherapy might increase the inhibitory effect of therapy on tumor growth. In this review, we summarize small-molecule compounds used for the treatment of NSCLC by focusing on RCD and pharmacological systems. In addition, we describe the current research status of an immunotherapy combined with an RCD-based regimen for NSCLC, providing new ideas for targeting RCD pathways in combination with immunotherapy for patients with NSCLC in the future.

Oenothera biennis with strong copper toxicity resistance enriches trace copper in seeds under copper pollution soil.

Excess copper (Cu) imparts negative effects on plant growth and productivity in soil. To develop the ability of O. biennis to govern pollution soil containing excessive Cu, we investigated seed germination, seedling growth, and seed yield. Furthermore, Cu content and the expression levels of Cu transport related genes in different tissues were measured under exogenous high concentration Cu. O. biennis seeds were sensitive to excess Cu, with an observed reduction in the germination rate, primary root length, fresh weight, and number of seeds germinated daily. Consecutive Cu stress did not cause fatal damage to evening primrose, yet it slowed down plant growth slightly by reducing the leaf water, chlorophyll, plant yield, and seed oil contents while increasing the soluble sugar, proline, malondialdehyde, and H2O2 contents. The Cu content in different organs of O. biennis was disrupted by excess Cu. In particular, the Cu content in O. biennis seeds and seed oil increased and subsequently decreased with the increase of exogenous Cu, reaching a peak under 600 mg·kg-1 consecutive Cu. Furthermore, the 4-month 900 mg·kg-1 Cu treatment did not induce the excessive accumulation of Cu in peels, seeds, and seed oil, maintaining the Cu content within the range required by the Chinese National Food Safety Standards. The treatment also resulted in an upregulation of Cu-uptake (ObCOPT5, ObZIP4, and ObYSL2) and vigorous efflux (ObHMA1) of transport genes, of which expression levels were significant positive correlation (p < 0.05) with the Cu content. Among all organs, the stem replaced the root as the organ exhibited the greatest ability to absorb and store Cu, and even the Cu transport genes could still function continuously in stem under excess Cu. This work identified a species that can tolerate high Cu content in soil while maintaining a high yield. Furthermore, the results revealed the enrichment of Cu to occur primarily in the O. biennis stem rather than the seeds and peel under excess Cu.

A novel approach to achieving more efficient production of the mature form of human IL-37 in plants.

Interleukin-37 is a newly discovered cytokine that plays a pivotal role in suppressing innate inflammation and acquired immunity. We have recently expressed both the mature(mat-) and pro-forms of human IL-37b in plants and demonstrated that while both forms of the plant-made hIL-37b are functional, pmat-hIL37b exhibited significantly greater activity than ppro-IL-37b. Compared to ppro-hIL-37b, on the other hand, the expression level of pmat-hIL-37b was substantially lower (100.5 µg versus 1.05 µg/g fresh leaf mass or 1% versus 0.01% TSP). Since the difference between ppro-hIL-37b and pmat-hIL-37b is that ppro-hIL-37b contains a signal sequence not cleavable by plant cells, we reasoned that this signal sequence would play a key role in stabilizing the ppro-hIL-37b protein. Here, we describe a novel approach to enhancing pmat-hIL-37b production in plants based on incorporation of a gene sequence encoding tobacco etch virus (TEV) protease between the signal peptide and the mature hIL-37b, including a TEV cleavage site at the C-termini of TEV protease. The rationale is that when expressed as a sp-TEV-matIL-37b fusion protein, the stabilizing properties of the signal peptide of pro-hIL-37b will be awarded to its fusion partners, resulting in increased yield of target proteins. The fusion protein is then expected to cleave itself in vivo to yield a mature pmat-hIL-37b. Indeed, when a sp-TEV-matIL-37b fusion gene was expressed in stable-transformed plants, a prominent band corresponding to dimeric pmat-hIL-37b was detected, with expression yields reaching 42.5 µg/g fresh leaf mass in the best expression lines. Bioassays demonstrated that plant-made mature pmat-hIL-37b is functional.

Waterborne Tebuconazole Exposure Induces Male-Biased Sex Differentiation in Zebrafish (Danio rerio) Larvae via Aromatase Inhibition.

Tebuconazole is a widely used fungicide for various crops that targets sterol 14-α-demethylase (CYP51) in fungi. However, attention has shifted to aromatase (CYP19) due to limited research indicating its reproductive impact on aquatic organisms. Herein, zebrafish were exposed to 0.5 mg/L tebuconazole at different developmental stages. The proportion of males increased significantly after long-term exposure during the sex differentiation phase (0-60, 5-60, and 19-60 days postfertilization (dpf)). Testosterone levels increased and 17ß-estradiol and cyp19a1a expression levels decreased during the 5-60 dpf exposure, while the sex ratio was equally distributed on coexposure with 50 ng/L 17ß-estradiol. Chemically activated luciferase gene expression bioassays determined that the male-biased sex differentiation was not caused by tebuconazole directly binding to sex hormone receptors. Protein expression and phosphorylation levels were specifically altered in the vascular endothelial growth factor signaling pathway despite excluding the possibility of tebuconazole directly interacting with kinases. Aromatase was selected for potential target analysis. Molecular docking and aromatase activity assays demonstrated the interactions between tebuconazole and aromatase, highlighting that tebuconazole poses a threat to fish populations by inducing a gender imbalance.

Genome-Wide Identification and Characterization of Aldo-Keto Reductase (AKR) Gene Family in Response to Abiotic Stresses in Solanum lycopersicum.

Tomato is one of the most popular and nutritious vegetables worldwide, but their production and quality are threatened by various stresses in the environment in which they are grown. Thus, the resistance and tolerance of tomatoes to various biotic and abiotic stresses should be improved. Aldo-keto reductases (AKR) are a superfamily of NAD(P)(H)-dependent oxidoreductases that play multiple roles in abiotic and biotic stress defenses by detoxification and reactive oxygen species (ROS) clearance pathways. Here, 28 identified AKR family genes of tomatoes were identified genome-wide, and their characteristics, including chromosomal location, gene structures, protein motifs, and system evolution, were analyzed. Furthermore, the phylogenetic and syntenic relationships in Arabidopsis thaliana, rice, and tomatoes were compared. Expression patterns at different tissues and in response to abiotic stresses, such as drought and salt, were monitored to further explore the function of SlAKRs. Finally, three SlAKRs candidate genes were silenced by Virus induced gene silencing (VIGS) systems in Solanum lycopersicum, showing sensitivity to drought and salt stresses with low contents of proline (Pro) and peroxidase (POD) and high content of malonaldehyde (MDA). This study provides the characteristics and potential functions of SlAKRs in response to abiotic stresses that will be helpful for further studies in S. lycopersicum.

[Clinical and genetic analysis of a patient with Loeys-Dietz syndrome due to variant of TGFBR2 gene].

OBJECTIVE: To explore the genetic basis of a patient with clinically suspected Loeys-Dietz syndrome (LDS). METHODS: A child who had presented at Beijing Anzhen Hospital in September 2018 was selected as the study subject. Clinical data and family history of the patient were collected, along with peripheral blood samples of the proband and his parents. Whole exome sequencing (WES) was carried out through next-generation sequencing. RESULTS: Candidate variants were searched through bioinformatic analysis focusing on genes associated with hereditary aortic aneurysms. Candidate variant was verified by Sanger sequencing. The patient was found to have cardiovascular abnormalities including early-onset aortic dilatation and coarctation, and LDS syndrome was suspected. WES revealed that he has harbored a heterozygous c.1526G>T missense variant of the TGFBR2 gene. The same variant was not found in either parent and was predicted as likely pathogenic (PM1+PM2_Supporting+ PM6+PP3+PP4) based on the guidelines from the American College for Medical Genetics and Genomics (ACMG). CONCLUSION: The TGFBR2 c.1526G>T variant probably underlay the LDS in this patient and was unreported previously in China. Above finding has enriched the mutational spectrum of the TGFBR2 gene associated with the LDS and provided a basis for the genetic counseling for the patient.

Identification of OSCA gene family in Solanum habrochaites and its function analysis under stress.

BACKGROUND: OSCA (hyperosmolality-gated calcium-permeable channel) is a calcium permeable cation channel protein that plays an important role in regulating plant signal transduction. It is involved in sensing changes in extracellular osmotic potential and an increase in Ca2+ concentration. S. habrochaites is a good genetic material for crop improvement against cold, late blight, planthopper and other diseases. Till date, there is no report on OSCA in S. habrochaites. Thus, in this study, we performed a genome-wide screen to identify OSCA genes in S. habrochaites and characterized their responses to biotic and abiotic stresses. RESULTS: A total of 11 ShOSCA genes distributed on 8 chromosomes were identified. Subcellular localization analysis showed that all members of ShOSCA localized on the plasma membrane and contained multiple stress-related cis acting elements. We observed that genome-wide duplication (WGD) occurred in the genetic evolution of ShOSCA5 (Solhab04g250600) and ShOSCA11 (Solhab12g051500). In addition, repeat events play an important role in the expansion of OSCA gene family. OSCA gene family of S. habrochaites used the time lines of expression studies by qRT-PCR, do indicate OSCAs responded to biotic stress (Botrytis cinerea) and abiotic stress (drought, low temperature and abscisic acid (ABA)). Among them, the expression of ShOSCAs changed significantly under four stresses. The resistance of silencing ShOSCA3 plants to the four stresses was reduced. CONCLUSION: This study identified the OSCA gene family of S. habrochaites for the first time and analyzed ShOSCA3 has stronger resistance to low temperature, ABA and Botrytis cinerea stress. This study provides a theoretical basis for clarifying the biological function of OSCA, and lays a foundation for tomato crop improvement.

The Sm gene conferring resistance to gray leaf spot disease encodes an NBS-LRR (nucleotide-binding site-leucine-rich repeat) plant resistance protein in tomato.

KEY MESSAGES: Gray leaf spot (GLS) resistance in tomato is controlled by one major dominant locus, Sm. Sm was fine mapped, and the nucleotide-binding site-leucine-rich repeat (NBS-LRR) gene Solyc11g020100 was identified as a candidate gene for Sm. Further functional analysis indicated that this gene confers high resistance to Stemphylium lycopersici in tomato. Tomato (Solanum Lycopersicum) is widely consumed and cultivated in the world. Gray leaf spot (GLS), caused by Stemphylium lycopersici (S. lycopersici), is one of the most devastating diseases in tomato production. To date, only one resistance gene, Sm, which confers high resistance against GLS disease, has been identified in the wild tomato species Solanum pimpinellifolium. This resistance locus (comprising the Sm gene) has been transferred into the cultivated variety 'Motelle'. Although several studies have reported the mapping of the Sm gene, it has not been cloned, limiting the utilization in tomato breeding. Here, we cloned Sm using a map-based cloning strategy. The Sm gene was mapped in a region of 160 kb at chromosome 11 between two markers, namely, M390 and M410, by using an F2 population from a cross between the resistant cultivar 'Motelle' (Mt) and susceptible line 'Moneymaker' (Mm). Three clustered NBS-LRR (nucleotide-binding site-leucine-rich repeat) resistance genes, namely, Solyc11g020080 (R1), Solyc11g020090 (R2), and Solyc11g020100 (R3) were identified in this interval. Nonsynonymous SNPs were identified in only the open reading frame (ORF) of R3, suggesting it as a strong candidate for the Sm gene. Furthermore, gene silencing of R3 abolished the high resistance to S. lycopersici in Motelle, demonstrating that this gene confers high resistance to S. lycopersici. The cloning of Sm may speed up its utilization for breeding resistant tomato varieties and represents an important step forward in our understanding of the mechanism underlying the resistance to GLS.

Facile synthesis of core\shell Fe3O4@mSiO2(Hb) and its application for organic wastewater treatment.

Treatment of organic wastewater is a challenging task. Biological techniques using biocatalysts have shown their benefits in organic wastewater treatment. In this research, a novel biocatalyst was developed by encapsulation of Fe3O4 microspheres and haemoglobin (Hb) with mesoporous silica, named Fe3O4@mSiO2(Hb). Fe3O4@mSiO2(Hb) exhibited typical mesoporous characteristics (mesoporous silica), magnetic feature (Fe3O4) and peroxidase activity (Hb). The results showed that the immobilization of Hb into Fe3O4@mSiO2 did not affect its activity. In addition, Fe3O4@mSiO2(Hb) exhibited a higher efficiency in the peroxidation of aromatic compounds than free Hb. The peroxidase activity of the synthesized biocatalyst was estimated to be 120 Ug-1, which was almost four times greater than that of previously reported immobilized Hb. Also, the Km of Fe3O4@mSiO2(Hb) was similar to that of the free Hb and it was estimated to be 4.3 × 10-4 µM, indicating that the activity of the Hb in the immobilized enzyme was not affected after immobilization. The immobilized enzyme was also found to be stable, recyclable and reusable. Taken together, these results indicate that the Fe3O4@mSiO2(Hb) has good potential to be used for treating organic wastewater containing aromatic compounds. The magnetically separable novel biocatalyst developed in this study provided not only a more suitable microenvironment for retaining the activity of Hb, but also demonstrated enhanced stability and activity under unfavorable conditions.

Human skin long noncoding RNA WAKMAR1 regulates wound healing by enhancing keratinocyte migration.

An increasing number of studies reveal the importance of long noncoding RNAs (lncRNAs) in gene expression control underlying many physiological and pathological processes. However, their role in skin wound healing remains poorly understood. Our study focused on a skin-specific lncRNA, LOC105372576, whose expression was increased during physiological wound healing. In human nonhealing wounds, however, its level was significantly lower compared with normal wounds under reepithelialization. We characterized LOC105372576 as a nuclear-localized, RNAPII-transcribed, and polyadenylated lncRNA. In keratinocytes, its expression was induced by TGF-ß signaling. Knockdown of LOC105372576 and activation of its endogenous transcription, respectively, reduced and increased the motility of keratinocytes and reepithelialization of human ex vivo skin wounds. Therefore, LOC105372576 was termed "wound and keratinocyte migration-associated lncRNA 1" (WAKMAR1). Further study revealed that WAKMAR1 regulated a network of protein-coding genes important for cell migration, most of which were under the control of transcription factor E2F1. Mechanistically, WAKMAR1 enhanced E2F1 expression by interfering with E2F1 promoter methylation through the sequestration of DNA methyltransferases. Collectively, we have identified a lncRNA important for keratinocyte migration, whose deficiency may be involved in the pathogenesis of chronic wounds.

Identification of CDPK Gene Family in Solanum habrochaites and Its Function Analysis under Stress.

Tomato is an important vegetable crop. In the process of tomato production, it will encounter abiotic stress, such as low temperature, drought, and high salt, and biotic stress, such as pathogen infection, which will seriously affect the yield of tomato. Calcium-dependent protein kinase (CDPK) is a class of major calcium signal receptor which has an important regulatory effect on the perception and decoding of calcium signals. CDPK plays a key role in many aspects of plant growth, such as the elongation of pollen tubes, plant growth, and response to biotic and abiotic stress. While some studies have concentrated on Arabidopsis and pepper, Solanum habrochaites is a wild species relative of cultivated tomato and there is no report on CDPK in Solanum habrochaites to date. Using tomato genomic data, this study identified 33 members of the CDPK gene family. Evolutionary analysis divides family members into four Asian groups, of which the CDPK family members have 11 gene replication pairs. Subcellular location analysis showed that most proteins were predicted to be located in the cytoplasm, and less protein existed on the cell membrane. Not all CDPK family members have a transmembrane domain. Cis regulatory elements relating to light, hormones, and drought stress are overrepresented in the promoter region of the CDPK genes in Solanum habrochaites. The expression levels of each gene under biotic stress and abiotic stress were quantified by qRT-PCR. The results showed that members of the CDPK family in Solanum habrochaites respond to different biotic and abiotic stresses. Among them, the expression of ShCDPK6 and ShCDPK26 genes change significantly. ShCDPK6 and ShCDPK26 genes were silenced using VIGS (virus-induced gene silencing), and the silenced plants illustrated reduced stress resistance to Botrytis cinerea, cold, and drought stress. The results of this study will provide a basis for the in-depth study of the CDPK gene family in Solanum habrochaites, laying the foundation for further analysis of the function of the gene family.

Ectopic expression of a rice plasma membrane intrinsic protein (OsPIP1;3) promotes plant growth and water uptake.

Plasma membrane intrinsic proteins (PIPs) are known to be major facilitators of the movement of a number of substrates across cell membranes. From a drought-resistant cultivar of Oryza sativa (rice), we isolated an OsPIP1;3 gene single-nucleotide polymorphism (SNP) that is mostly expressed in rice roots and is strongly responsive to drought stress. Immunocytochemistry showed that OsPIP1;3 majorly accumulated on the proximal end of the endodermis and the cell surface around the xylem. Expression of GFP-OsPIP1;3 alone in Xenopus oocytes or rice protoplasts showed OsPIP1;3 mislocalization in the endoplasmic reticulum (ER)-like neighborhood, whereas co-expression of OsPIP2;2 recruited OsPIP1;3 to the plasma membrane and led to a significant enhancement of water permeability in oocytes. Moreover, reconstitution of 10×His-OsPIP1;3 in liposomes demonstrated water channel activity, as revealed by stopped-flow light scattering. Intriguingly, by patch-clamp technique, we detected significant NO3- conductance of OsPIP1;3 in mammalian cells. To investigate the physiological functions of OsPIP1;3, we ectopically expressed the OsPIP1;3 gene in Nicotiana benthamiana (tobacco). The transgenic tobacco plants exhibited higher photosynthesis rates, root hydraulic conductivity (Lpr ) and water-use efficiency, resulting in a greater biomass and a higher resistance to water deficit than the wild-type did. Further experiments suggested that heterologous expression of OsPIP1;3 in cyanobacterium altered bacterial growth under different conditions of CO2 gas supply. Overall, besides shedding light on the multiple functions played by OsPIP1;3, this work provides insights into the translational value of plant AQPs.

Morphological and anatomical characteristics of exserted stigma sterility and the location and function of SlLst (Solanum lycopersicum Long styles) gene in tomato.

KEY MESSAGE: Anatomical changes in and hormone roles of the exserted stigma were investigated, and localization and functional analysis of SlLst for the exserted stigma were performed using SLAF-BSA-seq, parental resequencing and overexpression of SlLst in tomato. Tomato accession T431 produces stigmas under relatively high temperatures (> 27 °C, the average temperature in Harbin, China, in June-August), so pollen can rarely reach the stigma properly. This allows the percentage of male sterility exceed 95%, making the use of this accession practical for hybrid seed production. To investigate the mechanism underlying the exserted stigma male sterility, the morphological changes of, anatomical changes of, and comparative endogenous hormone (IAA, ABA, GA3, ZT, SA) changes in flowers during flower development of tomato accessions DL5 and T431 were measured. The location and function of genes controlling exserted stigma sterility were analyzed using super SLAF-BSA-seq, parental resequencing, comparative genomics and the overexpression of SlLst in tomato. The results showed that an increase in cell number mainly caused stigma exsertion. IAA played a major role, while ABA had an opposite effect on stigma exertion. Moreover, 26 candidate genes related to the exserted stigma were found, located on chromosome 12. The Solyc12g027610.1 (SlLst) gene was identified as the key candidate gene by functional analysis. A subcellular localization assay revealed that SlLst is targeted to the nucleus and cell membrane. Phenotypic analysis of SlLst-overexpressing tomato showed that SlLst plays a crucial role during stigma exsertion.

The α-Subunit of the Chloroplast ATP Synthase of Tomato Reinforces Resistance to Gray Mold and Broad-Spectrum Resistance in Transgenic Tobacco.

Chloroplast ATP synthase (cpATPase) is responsible for ATP production during photosynthesis. Our previous studies showed that the cpATPase CF1 α subunit (AtpA) is a key protein involved in Clonostachys rosea-induced resistance to the fungus Botrytis cinerea in tomato. Here, we show that expression of the tomato atpA gene was upregulated by B. cinerea and Clonostachys rosea. The tomato atpA gene was then isolated, and transgenic tobacco lines were obtained. Compared with untransformed plants, atpA-overexpressing tobacco showed increased resistance to B. cinerea, characterized by reduced disease incidence, defense-associated hypersensitive response-like reactions, balanced reactive oxygen species, alleviated damage to the chloroplast ultrastructure of leaf cells, elevated levels of ATP content and cpATPase activity, and enhanced expression of genes related to carbon metabolism, photosynthesis, and defense. Incremental Ca2+ efflux and steady H+ efflux were observed in transgenic tobacco after inoculation with B. cinerea. In addition, overexpression of atpA conferred enhanced tolerance to salinity and resistance to the fungus Cladosporium fulvum. Thus, AtpA is a key regulator that links signaling to cellular redox homeostasis, ATP biosynthesis, and gene expression of resistance traits to modulate immunity to pathogen infection and provides broad-spectrum resistance in plants in the process.

Identification and Functional Analysis of Tomato TPR Gene Family.

Tomato (Solanum lycopersicum) as an important vegetable grown around the world is threatened by many diseases, which seriously affects its yield. Therefore, studying the interaction between tomato and pathogenic bacteria is biologically and economically important. The TPR (Tetratricopeptide repeat) gene family is a class of genes containing TPR conserved motifs, which are widely involved in cell cycle regulation, gene expression, protein degradation and other biological processes. The functions of TPR gene in Arabidopsis and wheat plants have been well studied, but the research on TPR genes in tomato is not well studied. In this study, 26 TPR gene families were identified using bioinformatics based on tomato genome data, and they were analyzed for subcellular localization, phylogenetic evolution, conserved motifs, tissue expression, and GO (Gene Ontology) analysis. The qRT-PCR was used to detect the expression levels of each member of the tomato TPR gene family (SlTPRs) under biological stress (Botrytis cinerea) and abiotic stress such as drought and abscisic acid (ABA). The results showed that members of the tomato TPR family responded to various abiotic stresses and Botrytis cinerea stress, and the SlTPR2 and SlTPR4 genes changed significantly under different stresses. Using VIGS (Virus-induced gene silencing) technology to silence these two genes, the silenced plants showed reduced disease resistance. It was also shown that TPR4 can interact with atpA which encodes a chloroplast ATP synthase CF1 α subunit. The above results provide a theoretical basis for further exploring the molecular mechanism of TPR-mediated resistance in disease defense, and also provide a foundation for tomato disease resistance breeding.

Genome-Wide Identification and Analysis of CC-NBS-LRR Family in Response to Downy Mildew and Black Rot in Chinese Cabbage.

The nucleotide-binding site-leucine-rich repeat (NBS-LRR) gene family is the largest group of plant disease resistance (R) genes widespread in response to viruses, bacteria, and fungi usually involved in effector triggered immunity (ETI). Forty members of the Chinese cabbage CC type NBS-LRR family were investigated in this study. Gene and protein characteristics, such as distributed locations on chromosomes and gene structures, were explored through comprehensive analysis. CC-NBS-LRR proteins were classified according to their conserved domains, and the phylogenetic relationships of CC-NBS-LRR proteins in Brassica rapa, Arabidopsis thaliana, and Oryza sativa were compared. Moreover, the roles of BrCC-NBS-LRR genes involved in pathogenesis-related defense were studied and analyzed. First, the expression profiles of BrCC-NBS-LRR genes were detected by inoculating with downy mildew and black rot pathogens. Second, sensitive and resistant Chinese cabbage inbred lines were screened by downy mildew and black rot. Finally, the differential expression levels of BrCC-NBS-LRR genes were monitored at 0, 1, 3, 6, 12 and 24 h for short and 0, 3, 5, 7, 10 and 14 days for long inoculation time. Our study provides information on BrCC-NBS-LRR genes for the investigation of the functions and mechanisms of CC-NBS-LRR genes in Chinese cabbage.

A Study of the Interaction, Morphology, and Structure in Trypsin-Epigallocatechin-3-Gallate Complexes.

Understanding the interaction between proteins and polyphenols is of significance to food industries. The aim of this research was to investigate the mode of aggregation for trypsin-EGCG (Epigallocatechin-3-gallate) complexes. For this, the complex was characterized by fluorescence spectroscopy, circular dichroism (CD) spectra, small-angel X-ray scattering (SAXS), and atomic force microscope (AFM) techniques. The results showed that the fluorescence intensity of trypsin-EGCG complexes decreased with increasing the concentration of EGCG, indicating that the interaction between trypsin and EGCG resulted in changes in the microenvironment around fluorescent amino acid residues. The results of CD analysis showed conformational changes in trypsin after binding with EGCG. The results from SAXS analysis showed that the addition of EGCG results in the formation of aggregates of trypsin-EGCG complexes, and increasing the concentration of EGCG resulted in larger aggregates. AFM images showed that the trypsin-EGCG complex formed aggregates of irregular ellipsoidal shapes with the size of about 200 × 400 × 200 nm, with EGCG interconnecting the trypsin particles. Overall, according to these results, it was concluded that the large aggregates of trypsin-EGCG complexes are formed from several small aggregates that are interconnected. The results of this study shed some light on the interaction between digestive enzymes and EGCG.

LDkit: a parallel computing toolkit for linkage disequilibrium analysis.

BACKGROUND: Linkage disequilibrium (LD) analysis is broadly utilized in genetics to understand the evolutionary and demographic history and helps geneticists identify genes associated with interested inherited traits, such as diseases. There are some tools for linkage disequilibrium analysis either in a local or online way; however, there has been no such tool supporting both graphical user interface (GUI) and parallel computing. RESULTS: We developed a GUI software called LDkit for LD analysis, which supports parallel computing. The LDkit supports both variant call format (VCF) and PLINK 'ped + map' format. At the same time, users could also just analyze a subset of individuals from the whole population. The LDkit reads the data by block and then paralleled the computation process by monitoring the usage of processes. Assessment on the Human 1000 genome data showed that when paralleled with 32 threads, the running time was reduced to less than 6 minutes from ~77 minutes using the chromosome 22 dataset with 1,103,547 SNPs and 2504 individuals. CONCLUSIONS: The software LDkit can be effectively used to calculate and plot LD decay, LD block, and linkage disequilibrium analysis between a site and a given region. Most importantly, both graphical user interface (GUI) and stand-alone packages are available for users' convenience. LDkit was written in JAVA language under cross-platform support.

Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. iNap sensors enabled quantification of cytosolic and mitochondrial NADPH pools that are controlled by cytosolic NAD+ kinase levels and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase and AMP kinase. Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response in vivo. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live cells and gaining new insights into cell metabolism.

GsSnRK1 interplays with transcription factor GsERF7 from wild soybean to regulate soybean stress resistance.

Although the function and regulation of SnRK1 have been studied in various plants, its molecular mechanisms in response to abiotic stresses are still elusive. In this work, we identified an AP2/ERF domain-containing protein (designated GsERF7) interacting with GsSnRK1 from a wild soybean cDNA library. GsERF7 gene expressed dominantly in wild soybean roots and was responsive to ethylene, salt, and alkaline. GsERF7 bound GCC cis-acting element and could be phosphorylated on S36 by GsSnRK1. GsERF7 phosphorylation facilitated its translocation from cytoplasm to nucleus and enhanced its transactivation activity. When coexpressed in the hairy roots of soybean seedlings, GsSnRK1(wt) and GsERF7(wt) promoted plants to generate higher tolerance to salt and alkaline stresses than their mutated species, suggesting that GsSnRK1 may function as a biochemical and genetic upstream kinase of GsERF7 to regulate plant adaptation to environmental stresses. Furthermore, the altered expression patterns of representative abiotic stress-responsive and hormone-synthetic genes were determined in transgenic soybean hairy roots after stress treatments. These results will aid our understanding of molecular mechanism of how SnRK1 kinase plays a cardinal role in regulating plant stress resistances through activating the biological functions of downstream factors.