The preparation of native S‐palmitoylated (S‐palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S‐palm membrane ...proteins by removable‐backbone‐modification‐assisted Ser/Thr ligation (RBMGABA‐assisted STL). This method involves two critical steps: 1) synthesis of S‐palm peptides by a new γ‐aminobutyric acid based RBM (RBMGABA) strategy, and 2) ligation of the S‐palm RBM‐modified peptides to give the desired S‐palm product by the STL method. The utility of the RBMGABA‐assisted STL method was demonstrated by the synthesis of rabbit S‐palm sarcolipin (SLN) and S‐palm matrix‐2 (M2) ion channel. The synthesis of S‐palm membrane proteins highlights the importance of developing non‐NCL methods for chemical protein synthesis.
Native proteins by non‐native methods: An RBMGABA‐assisted STL strategy (removable‐backbone‐modification‐assisted serine/threonine ligation) was developed for the chemical synthesis of native S‐palmitoylated membrane proteins, which have not previously been accessible through chemical synthesis. The successful synthesis of S‐palmitoylated sarcolipin and M2 demonstrates the importance of non‐NCL methods for chemical protein synthesis.
Brassinosteroid (BR) has been shown to modulate plant tolerance to various stresses. S‐nitrosoglutathione reductase (GSNOR) is involved in the plant response to environment stress by fine‐turning the ...level of nitric oxide (NO). However, whether GSNOR is involved in BR‐regulated Na+/K+ homeostasis to improve the salt tolerance in halophyte is unknown. Here, we firstly reported that high salinity increases the expression of BR‐biosynthesis genes and the endogenous levels of BR in mangrove Kandelia obovata. Then, salt‐induced BR triggers the activities and gene expressions of GSNOR and antioxidant enzymes, thereafter decrease the levels of malondialdehyde, hydrogen peroxide. Subsequently, BR‐mediated GSNOR negatively regulates NO contributions to the reduction of reactive oxygen species generation and induction of the gene expression related to Na+ and K+ transport, leading to the decrease of Na+/K+ ratio in the roots of K. obovata. Finally, the applications of exogenous BR, NO scavenger, BR biosynthetic inhibitor and GSNOR inhibitor further confirm the function of BR. Taken together, our result provides insight into the mechanism of BR in the response of mangrove K. obovata to high salinity via GSNOR and NO signaling pathway by reducing oxidative damage and modulating Na+/K+ homeostasis.
Summary Statement
Salinity increases brassinosteroid (BR)‐biosynthesis genes expression and endogenous BR in mangrove Kandelia obovata.
BR enhances the activity and gene expression of S‐nitrosoglutathione reductase that negatively regulates nitric oxide signaling and reactive oxygen species level, while, BR regulates antioxidant enzymes and Na+/K+ homeostasis.
SUMMARY
Salicylic acid (SA) is known to enhance salt tolerance in plants. However, the mechanism of SA‐mediated response to high salinity in halophyte remains unclear. Using electrophysiological and ...molecular biological methods, we investigated the role of SA in response to high salinity in mangrove species, Kandelia obovata, a typical halophyte. Exposure of K. obovata roots to high salinity resulted in a rapid increase in endogenous SA produced by phenylalanine ammonia lyase pathway. The application of exogenous SA improved the salt tolerance of K. obovata, which depended on the NADPH oxidase‐mediated H2O2. Exogenous SA and H2O2 increased Na+ efflux and reduced K+ loss by regulating the transcription levels of Na+ and K+ transport‐related genes, thus reducing the Na+/K+ ratio in the salt‐treated K. obovata roots. In addition, exogenous SA‐enhanced antioxidant enzyme activity and its transcripts, and the expressions of four genes related to AsA‐GSH cycle as well, then alleviated oxidative damages in the salt‐treated K. obovata roots. However, the above effects of SA could be reversed by diphenyleneiodonium chloride (the NADPH oxidase inhibitor) and paclobutrazol (a SA biosynthesis inhibitor). Collectively, our results demonstrated that SA‐induced salt tolerance of K. obovata depends on NADPH oxidase‐generated H2O2 that affects Na+/K+ and redox homeostasis in response to high salinity.
Significance Statement
Our study further clarified the role of salicylic acid (SA) in the networks of salt tolerance in mangrove plants. The results are of great scientific significance in complementing the mechanism of SA in salt tolerance of halophyte.
Histamine (HA) is a biogenic amine that can accumulate to high concentration levels in food as a result of microbial activity and can cause toxic effects in consumers. In this work, a portable ...electrochemical immunosensor capable of detecting HA with high sensitivity and selectivity was developed. Prussian blue-chitosan-gold nanoparticle (PB-CS-AuNP) nanocomposite films with excellent biocompatibility were synthesized and characterized by scanning electron microscopy and energy dispersive X-ray analysis. The PB-CS-AuNP were coated onto a screen-printed electrode by one-step electrodeposition and used to conjugate the HA ovalbumin conjugate (HA-Ag). HA was determined by a competition between the coating HA-Ag and the HRP labeled HA antibody (HRP-HA-Ab). After careful optimization of assay conditions and Box-Behnken analysis, the developed immunosensor showed a linear range from 0.01 to 100μg/mL for HA in fish samples. The average recoveries from spiked samples ranged from 97.25% to 105%. The biosensor also showed good specificity, reproducibility, and stability, indicating its potential application in monitoring HA in a simple and low cost manner.
•A portable immunosensor for sensitive detection of histamine was developed.•Prussian blue-chitosan-gold nanoparticles were used as signal amplification materials.•The immunosensor showed a detection limit of 1.25ng/mL for histamine in fish samples.
Anxiety disorders are the most prevalent psychiatric disorders, but their pathogenic mechanism remains poorly understood. Here, we report that transmembrane protein 74 (TMEM74), which contains two ...putative transmembrane domains and exhibits high levels of mRNA in the brain, is closely associated with the pathogenesis of anxiety disorders. TMEM74 was decreased in the serum of patients with anxiety and the basolateral amygdaloid nucleus (BLA) in chronic stress mice. Furthermore, genetic deletion of Tmem74 or selective knockdown of Tmem74 in BLA pyramidal neurons resulted in anxiety-like behaviors in mice. Whole-cell recordings in BLA pyramidal neurons revealed lower hyperpolarization-activated cation current (I
) and greater input resistance and excitability in Tmem74
neurons than in wild-type neurons. Accordingly, surface expression of hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels was also lower in the BLA of Tmem74
mice. The I
current blocker ZD7288 mimicked these effects in BLA pyramidal neurons in wild-type mice but not in Tmem74
mice. Consistent with the improvement in anxiety-like behaviors, Tmem74 overexpression restored HCN1 channel trafficking and pyramidal neuron excitability in the BLA of Tmem74
and chronic stress mice. Mechanistically, we demonstrate that interactions between Tmem74 and HCN1 are physiologically relevant and that transmembrane domain 1 (TM1) is essential for the cellular membrane localization of Tmem74 to enhance I
. Together, our findings suggest that Tmem74 coupling with HCN1 acts as a critical component in the pathophysiology of anxiety and is a potential target for new treatments of anxiety disorders.
Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, ...Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ.
In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined "3n"-, "3n + 1"-, or "3n + 2"-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to "3n + 1"-bp, "3n + 2"-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions.
NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.
Sugarcane is the leading economic crop in China, requires huge quantities of nitrogen in the preliminary plant growth stages. However, the use of an enormous amount of nitrogen fertilizer increases ...the production price, and have detrimental results on the environment, causes severe soil and water pollution. In this study, a total of 175 endophytic strains were obtained from the sugarcane roots, belonging to five different species, i.e.,
Saccharum officinarum
,
Saccharum barberi
,
Saccharum robustum
,
Saccharum spontaneum
, and
Saccharum sinense
. Among these, only 23
Enterobacter
strains were chosen based on nitrogen fixation, PGP traits, hydrolytic enzymes production, and antifungal activities. Also, all selected strains were showed diverse growth range under different stress conditions, i.e., pH (5–10), temperature (20–45°C), and NaCl (7–12%) and 14 strains confirmed positive
nifH
, and 12 strains for
acdS
gene amplification, suggested that these strains could fix nitrogen along with stress tolerance properties. Out of 23 selected strains,
Enterobacter roggenkampii
ED5 was the most potent strain. Hence, this strain was further selected for comprehensive genome analysis, which includes a genome size of 4,702,851 bp and 56.05% of the average G + C content. Genome annotations estimated 4349 protein-coding with 83 tRNA and 25 rRNA genes. The CDSs number allocated to the KEGG, COG, and GO database were 2839, 4028, and 2949. We recognized a total set of genes that are possibly concerned with ACC deaminase activity, siderophores and plant hormones production, nitrogen and phosphate metabolism, symbiosis, root colonization, biofilm formation, sulfur assimilation and metabolism, along with resistance response toward a range of biotic and abiotic stresses.
E. roggenkampii
ED5 strain was also a proficient colonizer in sugarcane (variety GT11) and enhanced growth of sugarcane under the greenhouse. To the best of our knowledge, this is the first information on the whole-genome sequence study of endophytic
E. roggenkampii
ED5 bacterium associated with sugarcane root. And, our findings proposed that identification of predicted genes and metabolic pathways might describe this strain an eco-friendly bioresource to promote sugarcane growth by several mechanisms of actions under multi-stresses.
Blood-brain barrier (BBB) dysfunction has been suggested to play an important role in epilepsy. However, the mechanism mediating the transition from cerebrovascular damage to epilepsy remains ...unknown. Here, we report that endothelial cyclin-dependent kinase 5 (CDK5) is a central regulator of neuronal excitability. Endothelial-specific Cdk5 knockout led to spontaneous seizures in mice. Knockout mice showed increased endothelial chemokine (C-X-C motif) ligand 1 (Cxcl1) expression, decreased astrocytic glutamate reuptake through the glutamate transporter 1 (GLT1), and increased glutamate synaptic function. Ceftriaxone restored astrocytic GLT1 function and inhibited seizures in endothelial Cdk5-deficient mice, and these effects were also reversed after silencing Cxcl1 in endothelial cells and its receptor chemokine (C-X-C motif) receptor 2 (Cxcr2) in astrocytes, respectively, in the CA1 by AAV transfection. These results reveal a previously unknown link between cerebrovascular factors and epileptogenesis and provide a rationale for targeting endothelial signaling as a potential treatment for epilepsy.
With the wide application of preimplantation genetic testing (PGT) with trophectoderm (TE) biopsy, the safety of PGT has always been a concern. Since TE subsequently forms the placenta, it is ...speculated that the removal of these cells was associated with adverse obstetrical or neonatal outcomes after single frozen-thawed blastocyst transfer (FBT). Previous studies report contradictory findings with respect to TE biopsy and obstetric and neonatal outcomes.
We conducted a retrospective cohort study including 720 patients with singleton pregnancies from single FBT cycles who delivered at the same university-affiliated hospital between January 2019 and March 2022. The cohorts were divided into two groups: the PGT group (blastocysts with TE biopsy, n = 223) and the control group (blastocysts without biopsy, n = 497). The PGT group was matched with the control group by propensity score matching (PSM) analysis at a ratio of 1:2. The enrolled sample sizes in the two groups were 215 and 385, respectively.
Patient demographic characteristics were comparable between the groups after PSM except for the proportion of recurrent pregnancy loss, which was significantly higher in the PGT cohort (31.2 vs. 4.2%, P < 0.001). Patients in the PGT group had significantly higher rates of gestational hypertension (6.0 vs. 2.6%, adjusted odds ratio (aOR) 2.91, 95% confidence interval (CI) 1.18-7.18, P = 0.020) and abnormal umbilical cord (13.0 vs. 7.8%, aOR 1.94, 95% CI 1.08-3.48, P = 0.026). However, the occurrence of premature rupture of membranes (PROM) (12.1 vs. 19.7%, aOR 0.59, 95% CI 0.35-0.99, P = 0.047) was significantly lower in biopsied blastocysts than in unbiopsied embryos. There were no significant differences in regard to other obstetric and neonatal outcomes between the two groups.
Trophectoderm biopsy is a safe approach, as the neonatal outcomes from biopsied and unbiopsied embryos were comparable. Furthermore, PGT is associated with higher risks of gestational hypertension and abnormal umbilical cord but may have a protective effect on PROM.
Three rare heteromultinuclear complexes, ∞NiL(4,4′‐bipy)Pr (NO3)3·(CH3)2CHOH (1), {CuLSm (NO3)3}2(4,4′‐bipy)·CH3OH (2) and {CuL (CH3CH2OH)Eu (NO3)3 (3) with a symmetrical salamo‐like hexadentate ...ligand H2L have been synthesized, and characterized by FT‐IR, UV–vis and X‐ray crystallography. Complex 1 is a 1D coordination polymer constructed from heterobimetallic Ni(L)Pr (NO3)3 units which are connected by the exo‐dentate ligand 4,4′‐bipy bearing nitrogen‐donor atoms. Complex 2 is a heterotetranuclear dimer based on Cu(L)Sm (NO3)3 moieties which are linked through the exo‐dentate 4,4′‐bipy hasing nitrogen‐donor atoms. Complex 3 is a heterodinuclear structure, Cu (II) atom is five‐coordinate possessing a distorted square pyramidal geometry, and Eu (III) atom is a deca‐coordinate adopting a distorted bicapped square antiprism. In addition, fluorescence and antimicobial properties of the ligand H2L and its complexes 1–3 have also been discussed.
Three rare heteromultinuclear complexes, ∞NiL(4,4′‐bipy)Pr (NO3)3·(CH3)2CHOH (1), {CuLSm (NO3)3}2(4,4′‐bipy)·CH3OH (2) and {CuL (CH3CH2OH)Eu (NO3)3 (3) with a symmetrical salamo‐like hexadentate ligand H2L have been synthesized, and characterized by FT‐IR, UV–vis and X‐ray crystallography. In addition, fluorescence and antimicobial properties of the ligand H2L and its complexes 1–3 have also been discussed.
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