Combinatorial protein dimerization enables precise multi-input synthetic computations.

Bacterial transcription factors (TFs) with helix-turn-helix (HTH) DNA-binding domains have been widely explored to build orthogonal transcriptional regulation systems in mammalian cells. Here we capitalize on the modular structure of these proteins to build a framework for multi-input logic gates relying on serial combinations of inducible protein-protein interactions. We found that for some TFs, their HTH domain alone is sufficient for DNA binding. By fusing the HTH domain to TFs, we established dimerization dependent rather than DNA-binding-dependent activation. This enabled us to convert gene switches from OFF-type into more widely applicable ON-type systems and to create mammalian gene switches responsive to new inducers. By combining both OFF and ON modes of action, we built a compact, high-performance bandpass filter. Furthermore, we were able to show cytosolic and extracellular dimerization. Cascading up to five pairwise fusion proteins yielded robust multi-input AND logic gates. Combinations of different pairwise fusion proteins afforded a variety of 4-input 1-output AND and OR logic gate configurations.

The lncRNA GUSB Pseudogene 11 (GUSBP11) Promotes the Tumor Growth and Metastasis in Lung Adenocarcinoma.

Lung cancer, with lung adenocarcinoma comprising over 40% of cases, presents a global health challenge. Evidence indicates that long non-coding RNAs (lncRNAs), such as GUSBP11, could have therapeutic potential. Thus we explored the role and mechanism of GUSBP11 in lung adenocarcinoma. Bioinformatics analyses demonstrated GUSBP11 was upregulated in lung adenocarcinoma and was correlated with worsening prognosis. Quantitative PCR (qPCR) analysis revealed that of GUSBP11 was highly expressed in 61 paired lung adenocarcinoma patient tumor compared to paracancerous tissue samples. GUSBP11 knockdown suppressed lung adenocarcinoma cells proliferation and metastasis in vitro while promoted cell apoptosis, and the silencing of GUSBP11 impaired in vivo tumor growth in lung adenocarcinoma. Mechanistic insights revealed GUSBP11's role in inhibiting the regulatory functions of KHSRP, a protein essential for lung adenocarcinoma cell proliferation and metastasis. Taken together, our findings underscore the therapeutic and diagnostic potential of targeting the GUSBP11-KHSRP axis in lung adenocarcinoma, paving the way for further exploration in clinical settings.

Hawthorn leaves flavonoids attenuate cardiac remodeling induced by simulated microgravity.

CONTEXT: Hawthorn leaves are a kind of widely used medicinal plant in China. The major ingredient, hawthorn leaves flavonoids (HLF), have cardiotonic, cardioprotective, and vascular protective effects. OBJECTIVE: The study evaluated the protective role of HLF in cardiac remodelling and the underlying mechanisms under simulated microgravity by hindlimb unloading rats. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were divided into control, HLF, HU (hindlimb unloading) and HU + HLF groups (n = 8). After HU and daily intragastric administration at the dose of 100 mg/kg/d for 8 weeks, cardiac function and structure were evaluated by biochemical indices and histopathology. We identified the main active compounds and mechanisms involved in the cardioprotective effects of HLF via bioinformatics and molecular docking analysis, and relative signalling pathway activity was verified by Western blot. RESULTS: HLF treatment could reverse the HU-induced decline in LV-EF (HU, 55.13% ± 0.98% vs. HU + HLF, 71.16% ± 5.08%), LV-FS (HU, 29.44% ± 0.67% vs. HU + HLF, 41.62% ± 4.34%) and LV mass (HU, 667.99 ± 65.69 mg vs. HU + HLF, 840.02 ± 73.00 mg). Furthermore, HLF treatment significantly increased NPRA expression by 135.39%, PKG by 51.27%, decreased PDE5A by 20.03%, NFATc1 by 41.68% and Rcan1.4 by 54.22%. CONCLUSIONS: HLF plays a protective effect on HU-induced cardiac remodelling by enhancing NPRA-cGMP-PKG pathway and suppressing the calcineurin-NFAT pathway, which provides a theoretical basis for use in clinical therapies.

Thioredoxin-interacting protein is a favored target of miR-125b, promoting metastasis and progression of pancreatic cancer via the HIF1α pathway.

MicroRNAs (miRs) are vital in the development of pancreatic cancer (PC) targeting several cellular processes. This study was aimed at evaluating the function of miR-125b and the mechanism involved in PC. Cell migration, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and Bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) study was done to establish the migration capability, cell viability, and cell proliferation, respectively. Binding sites for miR-125b were recognized by luciferase assay, and the expression of protein by Western blot and immunofluorescence assay. In vivo study was done by BALB/c nude xenograft mice for evaluating the function of miR-125b. The study showed that expression of miR-125b was elevated in PC cells and tissues and was correlated to proliferation and migration of cells. Also, overexpression of miR-125b encouraged migration, metastasis, and proliferation of BxPC-3 cells, and suppression reversed it. We also noticed that thioredoxin-interacting protein (TXNIP) was the potential target of miR-125b. The outcomes also suggested that miR-125b governed the expression of TXNIP inversely via directly attaching to the three prime untranslated region (3'-UTR) activating hypoxia-inducible factor 1α (HIF1α). Looking into the relation between HIF1α and TXNIP, we discovered that TXNIP caused the degradation and export of HIF1α by making a complex with it. The miR-125b-TXNIP-HIF1α pathway may serve as a useful strategy for diagnosing and treating PC.

Superwetting pH-Responsive Polyaniline Coatings: Toward Versatile Separation of Complex Oil-Water Mixtures.

Intelligent materials with controlled wettability have caused widespread concern in various sewage applications. In this study, a smart pH-responsive polyaniline (PANI) coating has been synthesized in one step in aqueous media and coated on materials in common use, such as polyester mesh, cotton fabric, and sponge. The PANI coatings can switch their superwettability response to ambient pH and be used in continuous separation of oil-water-oil systems which are frequently found in actual oil leakage accidents. Moreover, bidirectional emulsion separation (water-in-oil and oil-in-water) can be realized on such a coating material. The coated sponge can be used as an oil adsorbent for invertible capture and release by changing pH. Based on excellent antifouling and recyclability, as well as the prominent chemical/mechanical stability, PANI coatings can be applied in actual oily wastewater treatment systems. It is anticipated that the coating materials will show promise in many applications because of the cost-effective and environmentally friendly aqueous media preparation procedure.

Association of IGF-1 gene rs2195239 polymorphism with the risk and clinical features of gastric cancer in a Chinese Han population.

BACKGROUND: Recently, a Japanese study investigated the relationship between insulin-like growth factor-1 (IGF-1) gene rs2195239 polymorphism and gastric cancer (GC) risk and found rs2195239 polymorphism did not relate with the risk of GC. However, no Chinese studies have addressed this relationship until now. Thus, the aims of this study were to demonstrate whether IGF-1 gene rs2195239 polymorphism was linked with the risk and clinical features of GC in a Chinese Han population. METHODS: In order to verify the link between IGF-1 gene rs2195239 polymorphism and GC risk, we recruited 361 GC cases and 418 controls in this case-control study. The genotyping was done by use of a custom-by-design 48-Plex SNP scan TM Kit. RESULTS: This study found that IGF-1 gene rs2195239 polymorphism decreased the risk of GC. Stratified analyses suggested that the significant association was shown in the females, non-smokers, non-drinkers, and age <60 years groups for GC. In addition, IGF-1 gene rs2195239 polymorphism correlated with the tumor size, tumor clinical stage, and pathological types for GC patients. CONCLUSION: To sum up, this study shows that IGF-1 gene rs2195239 polymorphism is associated with the risk and clinical features of GC patients in this Chinese population.

AT1-receptor autoantibody exposure in utero contributes to cardiac dysfunction and increased glycolysis in fetal mice.

Exposure to adverse factors in utero may lead to adaptive changes in cardiac structure and metabolism, which increases the risk of chronic cardiovascular disease later in life. Studies showed that the angiotensin II type 1 receptor autoantibodies (AT1-AAs) are able to cross the placenta into the circulation of pregnant rodents' embryo, which adversely affects embryogenesis. However, the effects of AT1-AA exposure on the fetal heart in utero are still unknown. In this study, we investigated whether intrauterine AT1-AA exposure has adverse effects on fetal heart structure, function and metabolism. AT1-AA-positive pregnant mouse models were successfully established by passive immunity, evidenced by increased AT1-AA content. Morphological and ultrasonic results showed that the fetal mice on embryonic day 18 (E18) of AT1-AA group have loose and disordered myocardial structure, and decreased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), compared with control groups. The myocardium of AT1-AA group fetal mice on E18 exhibited increased expression of the key molecules in the glycolytic pathway, pyruvate and lactic acid content and ATP production, suggesting that the glycolysis rate was enhanced. Furthermore, the enhanced effect of glycolysis caused by AT1-AA is mainly through the PPARß/δ pathway. These data confirmed that fetus exposure to AT1-AA in utero developed left ventricular dysfunction, myocardial structural arrangement disorders, and enhanced glycolysis on E18. Our results support AT1-AA being a potentially harmful factor for cardiovascular disease in fetal mice.

Active immunization using hand-push emulsification method increases the operator's risk of transcutaneous immunization.

Setting up an animal model by using active immunization methods is a common means of studying immune-related diseases or producing antibodies with high titer and high activities. However, the security during the process of pathogen emulsification remains unclear. In a physical examination, we unexpectedly noticed high levels of angiotensin II type 1 receptor autoantibody (AT1-AA) specific to the immunizing antigen in the sera of some researchers who had participated in setting up active immunization animal models, and we were puzzled about the cause of AT1-AA production. In this study, we intended to investigate whether the emulsified antigen was the source of infection in these researchers, and if so, how to prevent it from occurring. AT1-AA was detected by advanced ELISA method. The participants presented higher levels of AT1-AA compared with non-participants of the same laboratory. This phenomenon remained that some factors during the process of rat model establishment may contribute to AT1-AA production. Animal and glove penetration studies indicated the emulsified antigen infection was attributed to neither aerosol or fur touch nor penetrating through gloves. However, AT1-AA level was largely decreased in the participants after they used an automatic emulsification device. Because of the strong permeability of the adjuvant, we speculated that emulsified antigen might get access to the unprotected skin of the participants accidentally during the immunization process. These results demonstrated that accidental contacts of emulsified antigens may infect researchers during the process of traditional hand-push emulsification, resulting in high specific autoantibody levels, which can be prevented by using appropriate tools.

The inhibitory effect of BKCa channels induced by autoantibodies against angiotensin II type 1 receptor is independent of AT1R.

Autoantibodies against angiotensin II Type 1 receptor (AT1-AA) are routinely detected in the serum of preeclampsia patients, which results in an increase in vascular tone and an elevation in intracellular calcium concentration of rat vascular smooth muscle (VSM). The big conductance calcium-activated potassium channels (BKCa channels) account for the dominant outward currents in VSMCs, contributing to membrane hyperpolarization and vasodilation. In the present study, we investigated the effect of AT1-AA on BKCa channels. A preeclampsia model was established by passively immunizing healthy pregnant BALB/c mice with AT1-AA extracted from hybridoma culture supernatant. Blood pressure, serum AT1-AA levels, and urinary protein were measured in the immunized mice. BKCa channel expression was detected using qRT-PCR and immunohistochemical technique. The patch-clamp technique was used to record the single currents of BKCa channels in the HEK293T cells that had been transfected. AT1-AA immunized mice exhibited elevated AT1-AA and urinary protein levels compared with mice of the vehicle group. Systolic blood pressure was also increased in the immunized group. BKCa channel ß1-subunit expression was reduced in the mesenteric arteries of immunized mice. AT1-AA could inhibit the BKCa currents and the inhibitory effects were not completely reversed following the application of valsartan, an inhibitor of AT1 receptor. In conclusion, AT1-AA could decrease BKCa expression and inhibit BKCa activity independent of AT1R. These inhibitory effects are likely to be contributory factors in the promotion of increased vascular tone caused by AT1-AA in preeclampsia.

Validated ¹H and 13C Nuclear Magnetic Resonance Methods for the Quantitative Determination of Glycerol in Drug Injections.

In the current study, we employed high-resolution proton and carbon nuclear magnetic resonance spectroscopy (¹H and 13C NMR) for quantitative analysis of glycerol in drug injections without any complex pre-treatment or derivatization on samples. The established methods were validated with good specificity, linearity, accuracy, precision, stability, and repeatability. Our results revealed that the contents of glycerol were convenient to calculate directly via the integration ratios of peak areas with an internal standard in ¹H NMR spectra, while the integration of peak heights were proper for 13C NMR in combination with an external calibration of glycerol. The developed methods were both successfully applied in drug injections. Quantitative NMR methods showed an extensive prospect for glycerol determination in various liquid samples.

[Angiotensin II type 1 receptor autoantibody induces myocardial fibrosis by activating cardiac fibroblasts].

Myocardial fibrosis (MF) is an important pathological process of cardiac remodeling in patients with heart failure; however its etiology has not been clear. It has been known that the angiotensin II type 1 receptor autoantibody (AT1-AA) is present in patients with heart failure, but it is unclear whether this antibody directly causes MF. In this study, we investigated the role of AT1-AA in MF and its effects on cardiac fibroblasts (CFs). The AT1-AA positive rat model was established by active immunization method, and the measurement of indexes were made in the 8th week after active immunity. The results of heart echocardiography showed that the cardiac systolic and diastolic functions of AT1-AA positive rats were impaired with reduced left ventricular wall thickness and enlarged heart chambers. HE staining results showed that the myocardial fibers were disorganized and ruptured, and Masson staining revealed that the area of collagen fibers around the myocardium and coronary arteries was significantly increased in AT1-AA positive group compared with that of the control group (P < 0.05). Moreover, primary CFs isolated from neonatal rats were cultured and treated with AT1-AA for 48 h. CCK-8 and immunofluorescence staining results showed that AT1-AA enhanced proliferation rate of CFs (P < 0.001), and Western blot results showed that AT1-AA significantly increased expressions of collagen I (Col I), Col III, matrix metalloproteinase-2 (MMP-2) and MMP-9 in CFs (all P < 0.05). Taken together, these results suggest that AT1-AA may induce MF and cardiac dysfunction via activating CFs.

[Compatible principles of Xiebai decoction family based on complex network analysis].

In order to explore the compatible principles of Xiebai decoction family, formulae from ancient and modern Xiebai decoction family were collected and sorted in this study. The compatible characteristics, core herbs, as well as the relativity of herbs nature in Xiebai decoction family were analyzed based on scale free network and other data-mining methods such as association rules, clustering analysis and correspondence analysis. The scale free network results showed that in Xiebai decoction family, Mori Cortex-Lycii Cortex-Glycyrrhizae Radix et Rhizoma was used as the core compatible group and formed the complicated compatible network with other additional herbs; association rules results showed that the core herbs in such formulae included Mori Cortex, Lycii Cortex, Glycyrrhizae Radix et Rhizoma, scutellaria root, Platycodon root, Anemarrhena, and almond, which formed corresponding herbal pairs and compatibility; clustering analysis showed that Mori Cortex was the core herb in Xiebai decoction family, and Mori Cortex-Lycii Cortex-Glycyrrhizae Radix et Rhizoma was its main combination unit, which was always compatible with herbs of clearing heat, reducing phlegm, supplementing Qi and nourishing Yin to form the series prescriptions. The results indicated that the core compatibility features of Xiebai decoction family were clearing heat in lung and relieving cough and asthma, providing a basis for the clinical application of Xiebai decoction family.

A simple and biosafe method for isolation of human umbilical vein endothelial cells.

Human umbilical vein endothelial cells (HUVECS) are used as an irreplaceable tool for the study of vascular diseases. However, the technicians who isolate HUVECs are largely exposed to potential infectious threats. Here we report the development of a specialized instrument to protect researchers from known or unknown infectious agents when they operate on human umbilical cords. This instrument can be assembled by common laboratory supplies and adapted to accommodate umbilical cords of different lengths. When the cord is enclosed within the instrument, the risk of sample contamination and operator infection is greatly reduced. Using our instrument, endothelial cells were successfully isolated from human umbilical veins without contamination. The cells were verified by their cobblestone-like morphology and by immunofluorescence staining (Factor VIII and CD31 positivity and α-SMA negativity). Our instrument simplifies and optimizes the cell extraction process, and most importantly elevates the biosafety to a higher level during the isolation of human umbilical vein endothelial cells.

[Research on application status of Chinese herbal decoction pieces based on clinical survey].

Chinese herbal decoction pieces are the basic approaches for clinical traditional Chinese medicine (TCM), reflecting the features and advantages of TCM. In order to investigate the clinical application status and features of Chinese herbal decoction pieces, the questionnaire on application of commonly used Chinese herbal decoction pieces was designed in this study for analysis of the application situations of Chinese herbal decoction pieces from 56 medical institutions in 10 provinces. The results showed 549 varieties of Chinese herbs and 801 varieties of decoction pieces were used on clinic. They can be classified into 19 categories according to their effects. The varieties of Gancao (Glycyrrhizae Radix et Rhizoma), Huangqi (Astragali Radix), Dihuang (Rehmanniae Radix), Chuanxiong (Chuanxiong Rhizoma), Baizhu (Atractylodis Macrocephale Rhizima), Huangqin (Scutellariae Radix), Danggui (Angelicae Sinenses Radix), Baishao (Paeoniae Radix Alba) and Maidong (Ophiopogonis Radix) were most common ones; the application of Chinese herbal decoction pieces from different medical institutions was differentiated from areas to areas. The survey results reflected the general situation about application of decoction pieces, providing the basic data for recording and completing Chinese herbal decoction pieces in essential drug system, with certain reference significance for the production of Chinese medicinal materials and the allocation of the varieties of Chinese herbal decoction pieces.

Efficient uranium capture by polysulfide/layered double hydroxide composites.

There is a need to develop highly selective and efficient materials for capturing uranium (normally as UO2(2+)) from nuclear waste and from seawater. We demonstrate the promising adsorption performance of S(x)-LDH composites (LDH is Mg/Al layered double hydroxide, [S(x)](2-) is polysulfide with x = 2, 4) for uranyl ions from a variety of aqueous solutions including seawater. We report high removal capacities (q(m) = 330 mg/g), large K(d)(U) values (10(4)-10(6) mL/g at 1-300 ppm U concentration), and high % removals (>95% at 1-100 ppm, or ∼80% for ppb level seawater) for UO2(2+) species. The S(x)-LDHs are exceptionally efficient for selectively and rapidly capturing UO2(2+) both at high (ppm) and trace (ppb) quantities from the U-containing water including seawater. The maximum adsorption coeffcient value K(d)(U) of 3.4 × 10(6) mL/g (using a V/m ratio of 1000 mL/g) observed is among the highest reported for U adsorbents. In the presence of very high concentrations of competitive ions such as Ca(2+)/Na(+), S(x)-LDH exhibits superior selectivity for UO2(2+), over previously reported sorbents. Under low U concentrations, (S4)(2-) coordinates to UO2(2+) forming anionic complexes retaining in the LDH gallery. At high U concentrations, (S4)(2-) binds to UO2(2+) to generate neutral UO2S4 salts outside the gallery, with NO3(-) entering the interlayer to form NO3-LDH. In the presence of high Cl(-) concentration, Cl(-) preferentially replaces [S4](2-) and intercalates into LDH. Detailed comparison of U removal efficiency of S(x)-LDH with various known sorbents is reported. The excellent uranium adsorption ability along with the environmentally safe, low-cost constituents points to the high potential of S(x)-LDH materials for selective uranium capture.

Tm3+ mediated multicolor luminescence of NaYbF4:Er,Tm@NaYF4 for advanced anti-counterfeiting.

Lanthanide doped multicolor luminescent materials have attracted extensive attention due to their advanced anti-counterfeiting properties. However, designing a simple, hard-to-copy and multicolor anti-counterfeiting strategy based on upconversion nanoparticles (UCNPs) remains a huge challenge. Herein, a strategy to modulate luminescence color by altering the mediating action of Tm3+ was proposed. As a proof of concept, the mediating action of Tm3+ was explored in NaYbF4:30%Er,1%Tm@NaYF4 by changing the doping ratio of Yb3+/Er3+/Tm3+, and red, yellow and blue luminescence was successfully obtained. Then, NaYbF4:x%Er,1%Tm@NaYF4 (x = 2, 10, 30, 50, 99), NaYbF4:x%Er@NaYF4 (x = 2, 10, 30, 50, 100) and NaYbF4:1%Tm@NaYF4:x%Er@NaYF4 (x = 2, 10, 30, 50, 100) were synthesized to further identify that the mediating action of Tm3+ was related to the doping ratio and distance between dopant ions. In addition, the luminescence color of NaYbF4:30%Er,1%Tm@NaYF4 changed from red to yellow with the increase of excitation power density. Based on the above, NaYbF4:Er,Tm@NaYF4 UCNPs show excellent performance in anti-counterfeiting of paintings, thus revealing their great potential in advanced anti-counterfeiting applications.

Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells.

Here, we present a gene regulation strategy enabling programmable control over eukaryotic translational initiation. By excising the natural poly-adenylation (poly-A) signal of target genes and replacing it with a synthetic control region harboring RNA-binding protein (RBP)-specific aptamers, cap-dependent translation is rendered exclusively dependent on synthetic translation initiation factors (STIFs) containing different RBPs engineered to conditionally associate with different eIF4F-binding proteins (eIFBPs). This modular design framework facilitates the engineering of various gene switches and intracellular sensors responding to many user-defined trigger signals of interest, demonstrating tightly controlled, rapid and reversible regulation of transgene expression in mammalian cells as well as compatibility with various clinically applicable delivery routes of in vivo gene therapy. Therapeutic efficacy was demonstrated in two animal models. To exemplify disease treatments that require on-demand drug secretion, we show that a custom-designed gene switch triggered by the FDA-approved drug grazoprevir can effectively control insulin expression and restore glucose homeostasis in diabetic mice. For diseases that require instantaneous sense-and-response treatment programs, we create highly specific sensors for various subcellularly (mis)localized protein markers (such as cancer-related fusion proteins) and show that translation-based protein sensors can be used either alone or in combination with other cell-state classification strategies to create therapeutic biocomputers driving self-sufficient elimination of tumor cells in mice. This design strategy demonstrates unprecedented flexibility for translational regulation and could form the basis for a novel class of programmable gene therapies in vivo.

Minimally invasive pyeloplasty versus open pyeloplasty for ureteropelvic junction obstruction in infants: a systematic review and meta-analysis.

Background: To compare the perioperative outcomes and success rates of minimally invasive pyeloplasty (MIP), including laparoscopic and robotic-assisted laparoscopic pyeloplasty, with open pyeloplasty (OP) in infants. Materials and Methods: In September 2022, a systematic search of PubMed, EMBASE, and the Cochrane Library databases was undertaken. The systematic review and meta-analysis were conducted in accordance with PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines, with the study registered prospectively in the PROSPERO database (CRD42022359475). Results: Eleven studies were included. Dichotomous and continuous variables were presented as odds ratios (OR) and standard mean differences (SMD), respectively, with their 95% confidence intervals (CI). Compared to OP, a longer operation time and shorter length of stay were associated with MIP (SMD: 0.96,95% CI: 0.30 to 1.62, p = 0.004, and SMD: -1.12, 95% CI: -1.82 to -0.43, p = 0.002, respectively). No significant differences were found between the MIP and OP in terms of overall postoperative complications (OR:0.84, 95% CI: 0.52 to 1.35, p = 0.47), minor complications (OR: 0.76, 95% CI: 0.40 to 1.42, p = 0.39), or major complications (OR: 1.10, 95% CI: 0.49 to 2.50, p = 0.81). In addition, a lower stent placement rate was related to MIP (OR: 0.09, 95% CI: 0.02 to 0.47, p = 0.004). There was no statistical difference for success rate between the MIP and OP (OR: 1.35, 95% CI: 0.59 to 3.07, p = 0.47). Finally, the results of subgroup analysis were consistent with the above. Conclusions: Our meta-analysis demonstrates that MIP is a feasible and safe alternative to OP for infants, presenting comparable perioperative outcomes and similar success rates, albeit requiring longer operation times. However, it is essential to consider the limitations of our study, including the inclusion of studies with small sample sizes and the combination of both prospective and retrospective research designs.

The landscape of N6-methyladenosine modification patterns and altered transcript profiles in the cardiac-specific deletion of natriuretic peptide receptor A.

The atrial natriuretic peptide (ANP) and the brain natriuretic peptide (BNP) are critical biological makers and regulators of cardiac functions. Our previous results show that NPRA (natriuretic peptide receptor A)-deficient mice have distinct metabolic patterns and expression profiles compared with the control. Still, the molecular mechanism that could account for this observation remains to be elucidated. Here, methylation alterations were detected by mazF-digestion, and differentially expressed genes of transcriptomes were detected by a Genome Oligo Microarray using the myocardium from NPRA-deficient (NPRA-/-) mice and wild-type (NPRA+/+) mice as the control. Comprehensive analysis of m6A methylation data gave an altered landscape of m6A modification patterns and altered transcript profiles in cardiac-specific NPRA-deficient mice. The m6A "reader" igf2bp3 showed a clear trend of increase, suggesting a function in altered methylation and expression in cardiac-specific NPRA-deficient mice. Intriguingly, differentially m6A-methylated genes were enriched in the metabolic process and insulin resistance pathway, suggesting a regulatory role in cardiac metabolism of m6A modification regulated by NPRA. Notably, it was confirmed that the pyruvate dehydrogenase kinase 4 (Pdk4) gene upregulated the gene expression and the hypermethylation level simultaneously, which may be the key factor for the cardiac metabolic imbalance and insulin resistance caused by natriuretic peptide signal resistance. Taken together, cardiac metabolism might be regulated by natriuretic peptide signaling, with decreased m6A methylation and a decrease of Pdk4.

Engineering receptors in the secretory pathway for orthogonal signalling control.

Synthetic receptors targeted to the secretory pathway often fail to exhibit the expected activity due to post-translational modifications (PTMs) and/or improper folding. Here, we engineered synthetic receptors that reside in the cytoplasm, inside the endoplasmic reticulum (ER), or on the plasma membrane through orientation adjustment of the receptor parts and by elimination of dysfunctional PTMs sites. The cytoplasmic receptors consist of split-TEVp domains that reconstitute an active protease through chemically-induced dimerization (CID) that is triggered by rapamycin, abscisic acid, or gibberellin. Inside the ER, however, some of these receptors were non-functional, but their activity was restored by mutagenesis of cysteine and asparagine, residues that are typically associated with PTMs. Finally, we engineered orthogonal chemically activated cell-surface receptors (OCARs) consisting of the Notch1 transmembrane domain fused to cytoplasmic tTA and extracellular CID domains. Mutagenesis of cysteine residues in CID domains afforded functional OCARs which enabled fine-tuning of orthogonal signalling in mammalian cells.