Structural and functional insights into yeast Tbf1 as an atypical telomeric repeat-binding factor.

Telomeric repeat-binding factor 1 (Tbf1) has a similar architecture as the TRF family of telomeric proteins and plays important roles in both telomere homeostasis and ribosome regulation. However, the molecular basis of why Tbf1 has such different functions compared to other TRFs remains unclear. Here, we present the crystal structures of the TRF homology (TRFH) and Myb-L domains from Schizosaccharomyces pombe Tbf1 (spTbf1). TRFH-mediated homodimerization is essential for spTbf1 stability. Importantly, spTbf1TRFH lacks the conserved docking motif for interactions with telomeric proteins, explaining why spTbf1 does not participate in the assembly of the shelterin complex. Finally, structural and biochemical analyses demonstrate that TRFH and Myb-L domains as well as the loop region of spTbf1 coordinate to recognize S. pombe telomeric double-stranded DNA. Overall, our findings provide structural and functional insights into how fungi Tbf1 acts as an atypical telomeric repeat-binding factor, which helps to understand the evolution of TRFH-containing telomeric proteins.

Automatic Recognition and Quantification Feeding Behaviors of Nursery Pigs Using Improved YOLOV5 and Feeding Functional Area Proposals.

A novel method is proposed based on the improved YOLOV5 and feeding functional area proposals to identify the feeding behaviors of nursery piglets in a complex light and different posture environment. The method consists of three steps: first, the corner coordinates of the feeding functional area were set up by using the shape characteristics of the trough proposals and the ratio of the corner point to the image width and height to separate the irregular feeding area; second, a transformer module model was introduced based on YOLOV5 for highly accurate head detection; and third, the feeding behavior was recognized and counted by calculating the proportion of the head in the located feeding area. The pig head dataset was constructed, including 5040 training sets with 54,670 piglet head boxes, and 1200 test sets, and 25,330 piglet head boxes. The improved model achieves a 5.8% increase in the mAP and a 4.7% increase in the F1 score compared with the YOLOV5s model. The model is also applied to analyze the feeding pattern of group-housed nursery pigs in 24 h continuous monitoring and finds that nursing pigs have different feeding rhythms for the day and night, with peak feeding periods at 7:00-9:00 and 15:00-17:00 and decreased feeding periods at 12:00-14:00 and 0:00-6:00. The model provides a solution for identifying and quantifying pig feeding behaviors and offers a data basis for adjusting the farm feeding scheme.

[Enzymatic properties and degradation characterization of a bis(2-hydroxyethyl) terephthalate hydrolase from Saccharothrix sp.]

The discovery of new enzymes for poly(ethylene terephthalate) (PET) degradation has been a hot topic of research globally. Bis-(2-hydroxyethyl) terephthalate (BHET) is an intermediate compound in the degradation of PET and competes with PET for the substrate binding site of the PET-degrading enzyme, thereby inhibiting further degradation of PET. Discovery of new BHET degradation enzymes may contribute to improving the degradation efficiency of PET. In this paper, we discovered a hydrolase gene sle (ID: CP064192.1, 5085270-5086049) from Saccharothrix luteola, which can hydrolyze BHET into mono-(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA). BHET hydrolase (Sle) was heterologously expressed in Escherichia coli using a recombinant plasmid, and the highest protein expression was achieved at a final concentration of 0.4 mmol/L of isopropyl-ß-d-thiogalactoside (IPTG), an induction duration of 12 h and an induction temperature of 20 ℃. The recombinant Sle was purified by nickel affinity chromatography, anion exchange chromatography, and gel filtration chromatography, and its enzymatic properties were also characterized. The optimum temperature and pH of Sle were 35 ℃ and 8.0, and more than 80% of the enzyme activity could be maintained in the range of 25-35 ℃ and pH 7.0-9.0 and Co2+ could improve the enzyme activity. Sle belongs to the dienelactone hydrolase (DLH) superfamily and possesses the typical catalytic triad of the family, and the predicted catalytic sites are S129, D175, and H207. Finally, the enzyme was identified as a BHET degrading enzyme by high performance liquid chromatography (HPLC). This study provides a new enzyme resource for the efficient enzymatic degradation of PET plastics.

The predictive effect of different machine learning algorithms for pressure injuries in hospitalized patients: A network meta-analyses.

Background: Pressure injury has always been a focus and difficulty of nursing. With the development of nursing informatization, a large amount of structured and unstructured data has been generated, and it is difficult for traditional methods to utilize these data. With the intersection of artificial intelligence and nursing, it has become a new trend to apply machine learning algorithms to build pressure injury prediction models to manage pressure injuries. However, there is no evidence on the effectiveness of the method and which of a large number of algorithms for machine learning is more applicable to pressure injuries. Objective: This review aims to systematically synthesize existing evidence to determine the effectiveness of applying machine learning algorithms for pressure injury management, to further evaluate and compare pressure injury prediction models constructed by numerous machine learning algorithms, and to derive evidence for the best algorithms for predicting and managing pressure injuries. Design: Systematic review and network meta-analysis. Methods: A systematic electronic search was conducted in the EBSCO, Embase, PubMed, and Web of Science databases. We included all retrospective diagnostic accuracy trials and prospective diagnostic accuracy trials constructing a predictive model by machine learning for pressure injuries up to December 2021. Two review authors independently selected relevant studies and extracted data using the Cochrane handbook for systematic reviews of diagnostic test accuracy. The network meta-analysis was conducted using statistical software R and STATA. The certainty of the evidence was rated using the QUADAS-2 tool. Result: Twenty-five clinical diagnostic trials with a total of 237397 participants were identified in this review. The results of our study revealed that pressure injury machine learning models can effectively predict these injuries. Combining the algorithms separately yields the main results: decision trees (sensitivity: 0.66, 95% CI: 0.42 to 0.84, specificity: 0.90, 95% CI: 0.78 to 0.96, diagnostic odds ratio [DOR]: 18, 95% CI: 7 to 49, AUC: 0.88, 95% CI: 0.85 to 0.91), logistic regression (sensitivity: 0.71, 95% CI: 0.60 to 0.80, specificity: 0.83, 95% CI: 0.75 to 0.89, DOR: 12, 95% CI: 9 to 17, AUC: 0.84, 95% CI: 0.81 to 0.87), neural networks (sensitivity: 0.73, 95% CI: 0.55 to 0.86, specificity: 0.78, 95% CI: 0.65 to 0.87, DOR: 9, 95% CI: 5 to 19, AUC: 0.82, 95% CI: 0.79 to 0.85), random forests (sensitivity: 0.72, 95% CI: 0.26 to 0.95, specificity: 0.96, 95% CI: 0.80 to 0.99, DOR: 56, 95% CI: 3 to 1258, AUC: 0.95, 95% CI: 0.93 to 0.97), support vector machines (sensitivity: 0.81, 95% CI: 0.69 to 0.90, specificity: 0.81, 95% CI: 0.59 to 0.93, DOR: 19, 95% CI: 6 to 54, AUC: 0.88, 95% CI: 0.85 to 0.90). According to the analysis of ROC and AUC values, random forest is the best algorithm for the prediction model of pressure injury. Conclusions: This review revealed that machine learning algorithms are generally effective in predicting pressure injuries, and after data merging, the random forest algorithm is the best algorithm for pressure injury prediction. Further well-designed diagnostic controlled trials are recommended to strengthen the current evidence. Registration number PROSPERO: CRD42021276993.

Basis and current state of computed tomography perfusion imaging: a review.

Computed tomography perfusion (CTP) is a functional imaging that allows for providing capillary-level hemodynamics information of the desired tissue in clinics. In this paper, we aim to offer insight into CTP imaging which covers the basics and current state of CTP imaging, then summarize the technical applications in the CTP imaging as well as the future technological potential. At first, we focus on the fundamentals of CTP imaging including systematically summarized CTP image acquisition and hemodynamic parameter map estimation techniques. A short assessment is presented to outline the clinical applications with CTP imaging, and then a review of radiation dose effect of the CTP imaging on the different applications is presented. We present a categorized methodology review on known and potential solvable challenges of radiation dose reduction in CTP imaging. To evaluate the quality of CTP images, we list various standardized performance metrics. Moreover, we present a review on the determination of infarct and penumbra. Finally, we reveal the popularity and future trend of CTP imaging.

Numerical Analysis on Heat Characteristics of the Ventilation Basket for Fresh Tea Leaves.

Plastic baskets are commonly used as containers for fresh tea leaves during storage and transport after harvest. Nevertheless, there are significant challenges in controlling the core temperature of the basket since fresh tea leaves still maintain a certain degree of respiration after being harvested, with extremely high temperatures being the major factor for the color change of fresh tea leaves. A numerical model was developed to improve the temperature control of the plastic basket, by which the influence of different structural parameters on the core temperature in the plastic baskets with fresh tea leaves was analyzed. The accuracy of the model in predicting airflow and temperature distributions was validated against experimental data. The maximum RMSE was 1.158 °C and the maximum MRE was 5.410% between the simulated and test temperature value. The maximum deviation between the simulated velocity and test velocity was 0.11 m/s, the maximum RE was 29.05% and the maximum SD was 0.024. The results show that a plastic basket with a ventilation duct efficiently decreased the temperature of the fresh tea leaves and significantly affected the heat transfer between the fresh tea leaves and the ambient air compared to the plastic basket without a ventilation duct. Furthermore, the effect on the heat transfer was further expanded by the use of a plastic basket with a ventilation duct when the plastic baskets were stacked. The maximum temperature differences were 0.52 and 0.40 according to the stacked and single-layer products, respectively. The ambient temperature and the bulk density of the fresh tea leaves have a significant influence on the core temperature.

Structural insights into Pot1-ssDNA, Pot1-Tpz1 and Tpz1-Ccq1 Interactions within fission yeast shelterin complex.

The conserved shelterin complex caps chromosome ends to protect telomeres and regulate telomere replication. In fission yeast Schizosaccharomyces pombe, shelterin consists of telomeric single- and double-stranded DNA-binding modules Pot1-Tpz1 and Taz1-Rap1 connected by Poz1, and a specific component Ccq1. While individual structures of the two DNA-binding OB folds of Pot1 (Pot1OB1-GGTTAC and Pot1OB2-GGTTACGGT) are available, structural insight into recognition of telomeric repeats with spacers by the complete DNA-binding domain (Pot1DBD) remains an open question. Moreover, structural information about the Tpz1-Ccq1 interaction requires to be revealed for understanding how the specific component Ccq1 of S. pombe shelterin is recruited to telomeres to function as an interacting hub. Here, we report the crystal structures of Pot1DBD-single-stranded-DNA, Pot1372-555-Tpz1185-212 and Tpz1425-470-Ccq1123-439 complexes and propose an integrated model depicting the assembly mechanism of the shelterin complex at telomeres. The structure of Pot1DBD-DNA unveils how Pot1 recognizes S. pombe degenerate telomeric sequences. Our analyses of Tpz1-Ccq1 reveal structural basis for the essential role of the Tpz1-Ccq1 interaction in telomere recruitment of Ccq1 that is required for telomere maintenance and telomeric heterochromatin formation. Overall, our findings provide valuable structural information regarding interactions within fission yeast shelterin complex at 3' ss telomeric overhang.

Reshaping the binding channel of a novel GH113 family ß-mannanase from Paenibacillus cineris (PcMan113) for enhanced activity.

Endo-ß-mannanases are important enzymes for degrading lignocellulosic biomass to generate mannan, which has significant health effects as a prebiotic that promotes the development of gut microbiota. Here, a novel endo-ß-mannanase belonging to glycoside hydrolase (GH) family 113 from Paenibacillus cineris (PcMan113) was cloned, expressed and characterized, as one of only a few reported GH113 family ß-mannanases. Compared to other functionally and structurally characterized GH113 mannanases, recombinant PcMan113 showed a broader substrate spectrum and a better performance. Based on a structural homology model, the highly active mutant PcMT3 (F110E/N246Y) was obtained, with 4.60- and 5.53-fold increases of enzyme activity (towards KG) and catalytic efficiency (kcat/Km, against M5) compared with the WT enzyme, respectively. Furthermore, molecular dynamics (MD) simulations were conducted to precisely explore the differences of catalytic activity between WT and PcMT3, which revealed that PcMT3 has a less flexible conformation, as well as an enlarged substrate-binding channel with decreased steric hindrance and increased binding energy in substrate recognition. In conclusion, we obtained a highly active variant of PcMan113 with potential for commercial application in the manufacture of manno-oligosaccharides.

Posture Detection of Individual Pigs Based on Lightweight Convolution Neural Networks and Efficient Channel-Wise Attention.

In this paper, a lightweight channel-wise attention model is proposed for the real-time detection of five representative pig postures: standing, lying on the belly, lying on the side, sitting, and mounting. An optimized compressed block with symmetrical structure is proposed based on model structure and parameter statistics, and the efficient channel attention modules are considered as a channel-wise mechanism to improve the model architecture.The results show that the algorithm's average precision in detecting standing, lying on the belly, lying on the side, sitting, and mounting is 97.7%, 95.2%, 95.7%, 87.5%, and 84.1%, respectively, and the speed of inference is around 63 ms (CPU = i7, RAM = 8G) per postures image. Compared with state-of-the-art models (ResNet50, Darknet53, CSPDarknet53, MobileNetV3-Large, and MobileNetV3-Small), the proposed model has fewer model parameters and lower computation complexity. The statistical results of the postures (with continuous 24 h monitoring) show that some pigs will eat in the early morning, and the peak of the pig's feeding appears after the input of new feed, which reflects the health of the pig herd for farmers.

Structural and Molecular Dynamics Analysis of Plant Serotonin N-Acetyltransferase Reveal an Acid/Base-Assisted Catalysis in Melatonin Biosynthesis.

Serotonin N-acetyltransferase (SNAT) is the key rate-limiting enzyme in melatonin biosynthesis. It mediates melatonin biosynthesis in plants by using serotonin and 5-methoxytryptamine (5-MT), but little is known of its underlying mechanisms. Herein, we present a detailed reaction mechanism of a SNAT from Oryza sativa through combined structural and molecular dynamics (MD) analysis. We report the crystal structures of plant SNAT in the apo and binary/ternary complex forms with acetyl-CoA (AcCoA), serotonin, and 5-MT. OsSNAT exhibits a unique enzymatically active dimeric fold not found in the known structures of arylalkylamine N-acetyltransferase (AANAT) family. The key residues W188, D189, D226, N220, and Y233 located around the active pocket are important in catalysis, confirmed by site-directed mutagenesis. Combined with MD simulations, we hypothesize a novel plausible catalytic mechanism in which D226 and Y233 function as catalytic base and acid during the acetyl-transfer reaction.

Crystal structure of a S-adenosyl-L-methionine-dependent O-methyltransferase-like enzyme from Aspergillus flavus.

S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) are widely distributed among almost all organisms and often characterized with conserved Rossmann fold, TIM barrel, and D×G×G×G motif. However, some MTases show no methyltransferase activity. In the present study, the crystal structure of LepI, one MTase-like enzyme isolated from A. flavus that catalyzes pericyclic reactions, was investigated to determine its structure-function relationship. The overall structure of LepI in complex with the SAM mimic S-adenosyl-L-homocysteine (SAH) (PDB ID: 6IV7) indicated that LepI is a tetramer in solution. The residues His133, Arg197, Arg295, and Asp296 located near the active site can form hydrogen bonds with the substrate, thus participating in catalytic reactions. The binding of SAH in LepI is almost identical to that in other resolved MTases; however, the location of catalytic residues differs significantly. Phylogenetic trials suggest that LepI proteins share a common ancestor in plants and algae, which may explain the conserved SAM-binding site. However, the accelerated evolution of A. flavus has introduced both functional and structural changes in LepI. More importantly, the residue Arg295, which is unique to LepI, might be a key determinant for the altered enzymatic behavior. Collectively, the differences in the composition of catalytic residues, as well as the unique tetrameric form of LepI, define its unique enzymatic behavior. The present work provides an additional understanding of the structure-function relationship of MTases and MTase-like enzymes.

Merging C-H Vinylation with Switchable 6π-Electrocyclizations for Divergent Heterocycle Synthesis.

Pyridinium-containing polyheterocycles exhibit distinctive biological properties and interesting electrochemical and optical properties and thus are widely used as drugs, functional materials, and photocatalysts. Here, we describe a unified two-step strategy by merging Rh-catalyzed C-H vinylation with two switchable electrocyclizations, including aza-6π-electrocyclization and all-carbon-6π-electrocyclization, for rapid and divergent access to dihydropyridoisoquinoliniums and dihydrobenzoquinolines. Through computation, the high selectivity of aza-electrocyclization in the presence of an appropriate "HCl" source under either thermal conditions or photochemical conditions is shown to result from the favorable kinetics and symmetries of frontier orbitals. We further demonstrated the value of this protocol by the synthesis of several complex pyridinium-containing polyheterocycles, including the two alkaloids berberine and chelerythrine.

Crystal structure of Oryza sativa TDC reveals the substrate specificity for TDC-mediated melatonin biosynthesis.

Plant tryptophan decarboxylase (TDC) is a type II Pyridoxal-5'-phosphate-dependent decarboxylase (PLP_DC) that could be used as a target to genetically improve crops. However, lack of accurate structural information on plant TDC hampers the understanding of its decarboxylation mechanisms. In the present study, the crystal structures of Oryza sativa TDC (OsTDC) in its complexes with pyridoxal-5'-phosphate, tryptamine and serotonin were determined. The structures provide detailed interaction information between TDC and its substrates. The Y359 residue from the loop gate is a proton donor and forms a Lewis acid-base pair with serotonin/tryptamine, which is associated with product release. The H214 residue is responsible for PLP binding and proton transfer, and its proper interaction with Y359 is essential for OsTDC enzyme activity. The extra hydrogen bonds formed between the 5-hydroxyl group of serotonin and the backbone carboxyl groups of F104 and P105 explain the discrepancy between the catalytic activity of TDC in tryptophan and in 5-hydroxytryptophan. In addition, an evolutionary analysis revealed that type II PLP_DC originated from glutamic acid decarboxylase, potentially as an adaptive evolution of mechanism in organisms in extreme environments. This study is, to our knowledge, the first to present a detailed analysis of the crystal structure of OsTDC in these complexes. The information regarding the catalytic mechanism described here could facilitate the development of protocols to regulate melatonin levels and thereby contribute to crop improvement efforts to improve food security worldwide.

GmMAX2-D14 and -KAI interaction-mediated SL and KAR signaling play essential roles in soybean root nodulation.

Despite of important functions of strigolactones (SLs) and karrikins (KARs) in plant development, plant-parasite and plant-fungi interactions, their roles in soybean-rhizobia interaction remain elusive. SL/KAR signaling genes GmMAX2a, GmD14s, and GmKAIs are activated by rhizobia infection. GmMAX2a restored atmax2 root hair defects and soybean root hairs were changed in GmMAX2a overexpression (GmMAX2a-OE) or knockdown (GmMAX2a-KD) mutants. GmMAX2a-KD gave fewer, whereas GmMAX2a-OE produced more nodules than GUS hairy roots. Mutation of GmMAX2a in its KD or OE transgenic hairy roots affected the rhizobia infection-induced increases in early nodulation gene expression. Both mutant hairy roots also displayed the altered auxin, jasmonate and abscisic acid levels, as further verified by transcriptomic analyses of their synthetic genes. Overexpression of an auxin synthetic gene GmYUC2a also affected SL and KAR signaling genes. GmMAX2a physically interacted with SL/KAR receptors GmD14s, GmKAIs, and GmD14Ls with different binding affinities, depending on variations in the critical amino acids, forming active D14/KAI-SCFMAX2 complexes. The knockdown mutant roots of the nodule-specifically expressing GmKAIs and GmD14Ls gave fewer nodules, with altered expression of several early nodulation genes. The expression levels of GmKAIs, and GmD14Ls were markedly changed in GmMAX2a mutant roots, so did their target repressor genes GmD53s and GmSMAX1s. Thus, SL and KAR signaling were involved in soybean-rhizobia interaction and nodulation partly through interactions with hormones, and this may explain the different effects of MXA2 orthologs on legume determinate and indeterminate nodulation. The study provides fresh insights into the roles of GmMAX2-mediated SL/KAR signaling in soybean root hair and nodule formation.

Structure-based tailoring of the first coumarins-specific bergaptol O-methyltransferase to synthesize bergapten for depigmentation disorder treatment.

Bergapten has long been used in combination with ultraviolet A irradiation to treat depigmentation disorder. However, extremely low bergapten contents in plants and difficulties in synthesizing bergapten have limited its application. Here, we developed an alternative bergapten-production method. We first determined the crystal structures of bergaptol O-methyltransferase from Peucedanum praeruptorum (PpBMT) and the ternary PpBMT-S-adenosyl-L-homocysteine (SAH)-bergaptol complex to identify key residues involved in bergaptol binding. Then, structure-based protein engineering was performed to obtain PpBMT mutants with improved catalytic activity towards bergaptol. Subsequently, a high-activity mutant was used to produce bergapten for pharmacological-activity analysis. Key PpBMT amino acids involved in bergaptol binding and substrate specificity were identified, such as Asp226, Asp246, Ser265, and Val320. Site-directed mutagenesis and biochemical analysis revealed that the V320I mutant efficiently transformed bergaptol to produce bergapten. Pharmacological-activity analysis indicated that bergapten positively affected hair pigmentation in mice and improved pigmentation levels in zebrafish embryos. This report provides the first description of the catalytic mechanism of coumarins-specific O-methyltransferase. The high-activity V320I mutant protein could be used in metabolic engineering to produce bergapten in order to treat depigmentation disorder. This structure-function study provides an alternative synthesis method and important advances for treating depigmentation disorders.

The Molecular and Structural Basis of O-methylation Reaction in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn.

Methoxylated coumarins represent a large proportion of officinal value coumarins while only one enzyme specific to bergaptol O-methylation (BMT) has been identified to date. The multiple types of methoxylated coumarins indicate that at least one unknown enzyme participates in the O-methylation of other hydroxylated coumarins and remains to be identified. Combined transcriptome and metabonomics analysis revealed that an enzyme similar to caffeic acid O-methyltransferase (COMT-S, S is short for similar) was involved in catalyzing all the hydroxylated coumarins in Peucedanum praeruptorum. However, the precise molecular mechanism of its substrate heterozygosis remains unsolved. Pursuing this question, we determined the crystal structure of COMT-S to clarify its substrate preference. The result revealed that Asn132, Asp271, and Asn325 govern the substrate heterozygosis of COMT-S. A single mutation, such as N132A, determines the catalytic selectivity of hydroxyl groups in esculetin and also causes production differences in bergapten. Evolution-based analysis indicated that BMT was only recently derived as a paralogue of caffeic acid O-methyltransferase (COMT) via gene duplication, occurring before the Apiaceae family divergence between 37 and 100 mya. The present study identified the previously unknown O-methylation steps in coumarin biosynthesis. The crystallographic and mutational studies provided a deeper understanding of the substrate preference, which can be used for producing specific O-methylation coumarins. Moreover, the evolutionary relationship between BMT and COMT-S was clarified to facilitate understanding of evolutionary events in the Apiaceae family.

Divergent total syntheses of five illudalane sesquiterpenes and assignment of the absolute configuration.

Concise, divergent total syntheses of five bioactive illudalane sesquiterpenes have been achieved. Our synthesis features an intermolecular [2+2+2] cycloaddition, and a lactone-directed aromatic C-H oxygenation to generate a temporary phenolic hydroxyl group which enables regioselective methylation. Furthermore, the absolute configuration of radulactone was assigned by chemical synthesis.

Identification and analysis of adenine N6-methylation sites in the rice genome.

DNA N6-methyladenine (6mA) is a non-canonical DNA modification that is present at low levels in different eukaryotes1-8, but its prevalence and genomic function in higher plants are unclear. Using mass spectrometry, immunoprecipitation and validation with analysis of single-molecule real-time sequencing, we observed that about 0.2% of all adenines are 6mA methylated in the rice genome. 6mA occurs most frequently at GAGG motifs and is mapped to about 20% of genes and 14% of transposable elements. In promoters, 6mA marks silent genes, but in bodies correlates with gene activity. 6mA overlaps with 5-methylcytosine (5mC) at CG sites in gene bodies and is complementary to 5mC at CHH sites in transposable elements. We show that OsALKBH1 may be potentially involved in 6mA demethylation in rice. The results suggest that 6mA is complementary to 5mC as an epigenomic mark in rice and reinforce a distinct role for 6mA as a gene expression-associated epigenomic mark in eukaryotes.

FKBP12.6 protects heart from AngII-induced hypertrophy through inhibiting Ca2+ /calmodulin-mediated signalling pathways in vivo and in vitro.

We previously observed that disruption of FK506-binding protein 12.6 (FKBP12.6) gene resulted in cardiac hypertrophy in male mice. Studies showed that overexpression of FKBP12.6 attenuated thoracic aortic constriction (TAC)-induced cardiac hypertrophy in mice, whereas the adenovirus-mediated overexpression of FKBP12.6 induced hypertrophy and apoptosis in cultured neonatal cardiomyocytes, indicating that the role of FKBP12.6 in cardiac hypertrophy is still controversial. In this study, we aimed to investigate the roles and mechanisms of FKBP12.6 in angiotensin II (AngII)-induced cardiac hypertrophy using various transgenic mouse models in vivo and in vitro. FKBP12.6 knockout (FKBP12.6-/- ) mice and cardiac-specific FKBP12.6 overexpressing (FKBP12.6 TG) mice were infused with AngII (1500 ng/kg/min) for 14 days subcutaneously by implantation of an osmotic mini-pump. The results showed that FKBP12.6 deficiency aggravated AngII-induced cardiac hypertrophy, while cardiac-specific overexpression of FKBP12.6 prevented hearts from the hypertrophic response to AngII stimulation in mice. Consistent with the results in vivo, overexpression of FKBP12.6 in H9c2 cells significantly repressed the AngII-induced cardiomyocyte hypertrophy, seen as reductions in the cell sizes and the expressions of hypertrophic genes. Furthermore, we demonstrated that the protection of FKBP12.6 on AngII-induced cardiac hypertrophy was involved in reducing the concentration of intracellular Ca2+ ([Ca2+ ]i), in which the protein significantly inhibited the key Ca2+ /calmodulin-dependent signalling pathways such as calcineurin/cardiac form of nuclear factor of activated T cells 4 (NFATc4), calmodulin kinaseII (CaMKII)/MEF-2, AKT/Glycogen synthase kinase 3ß (GSK3ß)/NFATc4 and AKT/mTOR signalling pathways. Our study demonstrated that FKBP12.6 protects heart from AngII-induced cardiac hypertrophy through inhibiting Ca2+ /calmodulin-mediated signalling pathways.

Conformational Bias by a Removable Silyl Group: Construction of Bicyclo[n.3.1]alkenes by Ring Closing Metathesis.

Herein, we report a novel strategy based on a conformationally controlled RCM by a removable silyl group, which allows the facile synthesis of various bicyclo[n.3.1]alkenes, especially a set of highly strained bicyclo[5.3.1]alkenes. Further derivatizations of the silyl group and the resultant double bond of bicyclo[5.3.1]undecene 2 f enabled a concise synthesis of A-B-C ring skeleton of taxol. Density functional theory (DFT) calculations suggest that the introduction of a bulky silyl group at C-5 position of the 1,3-dialkenylcyclohexanol substrates dramatically lowers the energy bias gap between diaxial conformers (to RCM) and diequatorial conformers (to cross metathesis), thereby favoring the expected RCM reaction to give the challenging bridged molecules.