Structural versatility that serves the function of the HRD motif in the catalytic loop of protein tyrosine kinase, Src

Site‐directed mutagenesis is a traditional approach for structure–function analysis of protein tyrosine kinases, and it requires the generation, expression, purification, and analysis of each mutant enzyme. In this study, we report a versatile high throughput bacterial screening system that can identify functional kinase mutants by immunological detection of tyrosine phosphorylation. Two key features of this screening system are noteworthy. First, instead of blotting bacterial colonies directly from Agar plates to nitrocellulose membrane, the colonies were cultured in 96‐well plates, and then spotted in duplicate onto the membrane with appropriate controls. This made the screening much more reliable compared with direct colony blotting transfer. A second feature is the parallel use of a protein tyrosine phosphatase (PTP)‐expressing host and a non‐PTP‐expressing host. Because high activity Src mutants are toxic to the host, the PTP system allowed the identification of Src mutants with high activity, while the non‐PTP system identified Src mutants with low activity. This approach was applied to Src mutant libraries randomized in the highly conserved HRD motif in the catalytic loop, and revealed that structurally diverse residues can replace the His and Arg residues, while the Asp residue is irreplaceable for catalytic activity

Children’s understanding of reality and possibility and its cultural transmission mechanisms

When learning about concepts that are difficult to experience first-hand, children must rely on information from others. One challenge for young children is that adults may provide differing information, yet few studies have examined how children reconcile conflicting beliefs from different sources. Across three studies, I explored children’s understanding of reality and possibility in natural and supernatural domains from secular and Christian communities in a largely secular society, Mainland China. Two age groups were included, one group before formal schooling (5- to 6-year-olds), where children are mainly exposed to testimony from parents and their immediate circle, and one group after several years of schooling (9- to 11-year-olds), where the testimony from parents may support or conflict with school testimony. Specifically, in Study 1, children and their parents were asked to judge the existence of unobservable scientific and religious entities. Results showed that the ontological judgments of children from both age groups were in strong correspondence with their parents’ beliefs, even when parental testimony may conflict with the testimony children receive in school. Study 2 expanded beyond Study 1 to explore children’s understanding of fact and fiction in counter-intuitive processes. Study 2 also asked whether religious exposure from the immediate circle in a largely secular society may extend Christian children’s understanding of possibility in formal religious contexts to folk religious contexts, fantastical contexts or improbable contexts in general. It was found that with age, Christian Chinese children became less likely to extend their belief in the impossible via God’s intervention to other magical or divine powers. Lastly, Study 3 examined and revealed the specific elements of parental testimony that might alert children to the existence or non-existence of unobservable concepts by analyzing parent-child conversations about unobservable scientific and religious concepts in both high consensus and low consensus domains. Taken together, Study 1 and Study 2 demonstrated the weight of testimony from parents and the immediate community on children’s understanding of possibility and facts when there is conflicting testimony in the larger society. Study 3 provided evidence on parental testimony as one possible cultural transmission mechanism. The final chapter addresses the significance and implications of these findings in the field of developmental science and education

Evidence for activated Lck protein tyrosine kinase as the driver of proliferation in acute myeloid leukemia cell, CTV-1

Acute myeloid leukemia (AML) is a heterogeneous group of fast growing cancers of myeloid progenitor cells, for which effective treatments are still lacking. Identification of signaling inhibitors that block their proliferation could reveal the proliferative mechanism of a given leukemia cell, and provide small molecule drugs for targeted therapy for AML. In this study, kinase inhibitors that block the majority of cancer signaling pathways are evaluated for their inhibition of two AML cell lines of the M5 subtypes, CTV-1 and THP-1. While THP-1 cells do not respond to any of these inhibitors, CTV-1 cells are potently inhibited by dasatinib, bosutinib, crizotinib, A-770041, and WH-4-23, all potent inhibitors for Lck, a Src family kinase. CTV-1 cells contain a kinase activity that phosphorylates an Lck-specific peptide substrate in an Lck inhibitor-sensitive manner. Furthermore, the Lck gene is over-expressed in CTV-1, and it contains four mutations, two of which are located in regions critical for Lck negative regulation, and are confirmed to activate Lck. Collectively, these results provide strong evidence that mutated and overexpressed Lck is driving CTV-1 proliferation. While Lck activation and overexpression is rare in AML, this study provides a potential therapeutic strategy for treating patients with a similar oncogenic mechanism

Optimal treatment of ceftazidime-avibactam and aztreonam-avibactam against bloodstream infections or lower respiratory tract infections caused by extensively drug-resistant or pan drug-resistant (XDR/PDR) Pseudomonas aeruginosa

ObjectiveTo evaluate the efficacy of ceftazidime-avibactam (CZA) and aztreonam-avibactam (AZA) against bloodstream infections (BSIs) or lower respiratory tract infections (LRTIs) – caused by extensive drug-resistant or pan drug-resistant (XDR/PDR) Pseudomonas aeruginosa.MethodThe two-fold dilution method was used to determine the minimum inhibitory concentrations (MICs) of CZA/AZA against XDR/PDR P. aeruginosa. Whole-genome sequencing was used to analyze the resistance determinants of each isolate. Monte Carlo simulations (MCSs) were used to evaluate the probability of target attainment (PTA) and the cumulative fraction of response (CFR) of each CZA/AZA dosing regimen via traditional infusion (TI)/optimized two-step-administration therapy (OTAT).ResultsWe found that XDR/PDR P. aeruginosa may carry some rare MBLs (e.g.: IND-6, SLB-1, THIN-B). P. aeruginosa isolates producing IMP-45, VIM-1, or VIM-2 were inhibited by AZA at a concentration of 2 to 8 mg/L. All isolates producing IND-6 plus other serine β-lactamases were high-level resistant to CZA/AZA (MICs >64 mg/L). All simulated dosing regimens of CZA/AZA against BSIs-causing XDR/PDR P. aeruginosa achieved 100% PTA when the MIC was ≤32 mg/L.ConclusionAZA has been considered as an option for the treatment of infections caused by XDR/PDR P. aeruginosa producing IMP-45, VIM-1, or VIM-2. OTAT with sufficient pharmacodynamic exposure may be an optimal treatment option for XDR/PDR P. aeruginosa with a high-level MIC of CZA/AZA

An Enhanced Drought-Tolerant Method Using SA-Loaded PAMPS Polymer Materials Applied on Tobacco Pelleted Seeds

Drought is one of the most important stress factors limiting the seed industry and crop production. Present study was undertaken to create novel drought-resistant pelleted seeds using the combined materials with superabsorbent polymer, poly(2-acrylamide-2-methyl propane sulfonic acid) (PAMPS) hydrogel, and drought resistance agent, salicylic acid (SA). The optimized PAMPS hydrogel was obtained as the molar ratio of 2-acrylamido-2-methyl-propanesulfonic acid (AMPS) to potassium peroxydisulfate (KPS) and N, N′-methylene-bis-acrylamide (MBA) was 1 : 0.00046 : 0.00134. The hydrogel weight after swelling in deionized water for 24 h reached 4306 times its own dry weight. The water retention ratio (RR) of PAMPS was significantly higher as compared with the control. It could keep as high as 85.3% of original weight after 30 min at 110°C; even at 25°C for 40 d, the PAMPS still kept RR at 33.67%. PAMPS disintegration ratio increased gradually and reached around 30% after embedding in soil or activated sludge for 60 d. In addition, there were better seed germination performance and seedling growth in the pelleted treatments with SA-loaded PAMPS hydrogel under drought stress than control. It suggested that SA-loaded PAMPS hydrogel, a nontoxic superabsorbent polymer, could be used as an effective drought resistance material applied to tobacco pelleted seeds

Arranging Small Molecules with Subnanometer Precision on DNA Origami Substrates for the Single‐Molecule Investigation of Protein–Ligand Interactions

DNA origami nanostructures are versatile substrates for the single‐molecule investigation of biomolecular interactions as they enable the display of molecular species in complex arrangements. Herein, the fundamental limitations of this approach are explored by displaying pairs of small‐molecule ligands of the protein trypsin on DNA origami substrates and adjusting their ligand–ligand spacing with subnanometer precision. Bidentate binding of trypsin to the ligand pairs is investigated by atomic force microscopy (AFM), microscale thermophoresis (MST), and molecular dynamics simulations. Bidentate trypsin binding is strongly affected by the distance of the ligand pairs and the accessibility of the protein's binding pockets. MST cannot resolve the differences in bidentate trypsin binding because of the nonspecific binding of trypsin to the DNA origami substrates, rendering the AFM‐based single‐molecule detection of binding events superior to ensemble measurements. Finally, even monodentate binding to a single ligand may be affected by subnanometer variations in its position, highlighting the importance of local microenvironments that vary even over molecular distances. While this single‐molecule approach can provide viable information on the effects of ligand arrangements on bidentate protein binding, in‐depth investigations into the nature of local microenvironments will be required to exploit its full potential

Inflammatory response triggered by avian hepatitis E virus in vivo and in vitro

Hepatitis E virus (HEV) is relevant to public health worldwide, and it affects a variety of animals. Big liver and spleen disease (BLS) and hepatitis-splenomegaly syndrome (HSS) associated with avian HEV (aHEV) were first reported in 1988 and in 1991, respectively. Here, cell culture–adapted aHEV genotype 3 strain, YT-aHEV (YT strain), a typical genotype isolated in China, was used for basic and applied research. We evaluated liver injury during the early stages of infection caused by the YT strain in vivo. Both in vivo and in vitro experimental data demonstrated that viral infection induces innate immunity, with mRNA expression levels of two key inflammatory factors, interleukin-1β (IL-1β) and IL-18, significantly upregulated. The YT strain infection was associated with the activation of Toll-like receptors (TLRs), nuclear factor kappa B (NF-κB), caspase-1, and NOD-like receptors (NLRs) in the liver and primary hepatocellular carcinoma epithelial cells (LMH). Moreover, inhibiting c-Jun N-terminal kinase, extracellular signal–regulated kinase (ERK1 or 2), P38, NF-κB, or caspase-1 activity has different effects on NLRs, and there is a mutual regulatory relationship between these signaling pathways. The results show that SB 203580, U0126, and VX-765 inhibited IL-1β and IL-18 induced by the YT strain, whereas Pyrrolidinedithiocarbamate (PDTC) had no significant effect on the activity of IL-1β and IL-18. Pretreatment of cells with SP600125 had an inhibitory effect on IL-18 but not on IL-1β. The analysis of inhibition results suggests that there is a connection between Mitogen-activated protein kinase (MAPK), NF-κB, and the NLRs signaling pathways. This study explains the relationship between signaling pathway activation (TLRs, NF-κB, MAPK, and NLR–caspase-1) and viral-associated inflammation caused by YT strain infection, which will help to dynamic interaction between aHEV and host innate immunity