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The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is ▵ F508, and this causes cystic fibrosis (CF). New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse ▵ F508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human ▵ F508-CFTR. The processing behavior of ferret WT- and ▵ F508-CFTR proteins remains unknown, and such information is important to predicting the utility of a▵ F508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and ▵ F508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret ▵ F508-CFTR processing was negligible, whereas low levels of processing of ▵ F508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not ▵ F508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTR▵ F508/▵ F508 animal model may be useful. [ABSTRACT FROM AUTHOR]

In cystic fibrosis (CF), a lack of functional CF transmembrane conductance regulator (CFTR) chloride channels causes defective secretion by submucosal glands (SMGs), leading to persistent bacterial infection that damages airways and necessitates tissue repair. SMGs are also important niches for slow-cycling progenitor cells (SCPCs) in the proximal airways, which may be involved in disease-related airway repair. Here, we report that calcitonin gene-related peptide (CGRP) activates CFTR-dependent SMG secretions and that this signaling pathway is hyperactivated in CF human, pig, ferret, and mouse SMGs. Since CGRP-expressing neuroendocrine cells reside in bronchiolar SCPC niches, we hypothesized that the glandular SCPC niche may be dysfunctional in CF. Consistent with this hypothesis, CFTR-deficient mice failed to maintain glandular SCPCs following airway injury. In wild-type mice, CGRP levels increased following airway injury and functioned as an injury-induced mitogen that stimulated SMG progenitor cell proliferation in vivo and altered the proliferative potential of airway progenitors in vitro. Components of the receptor for CGRP (RAMP1 and CLR) were expressed in a very small subset of SCPCs, suggesting that CGRP indirectly stimulates SCPC proliferation in a non-cell-autonomous manner. These findings demonstrate that CGRP-dependent pathways for CFTR activation are abnormally upregulated in CF SMGs and that this sustained mitogenic signal alters properties of the SMG progenitor cell niche in CF airways. This discovery may have important implications for injury/repair mechanisms in the CF airway. [ABSTRACT FROM AUTHOR]

The C2-WW-HECT-type ubiquitin ligases Smurf1 and Smurf2 play a critical role in embryogenesis and adult bone homeostasis via regulation of bone morphogenetic protein, Wnt, and RhoA signaling pathways. The intramolecular interaction between C2 and HECT domains autoinhibits the ligase activity of Smurf2. However, the role of the Smurf1 C2 domain remains elusive. Here, we show that the C2-HECT autoinhibition mechanism is not observed in Smurf1, and instead its C2 domain functions in substrate selection. The Smurf1 C2 domain exerts a key role in localization to the plasma membrane and endows Smurf1 with differential activity toward RhoA versus Smad5 and Runx2. Crystal structure analysis reveals that the Smurf1 C2 domain possesses a typical anti-parallel β-sandwich fold. Examination of the sulfate-binding site analysis reveals two key lysine residues, Lys-28 and Lys-85, within the C2 domain that are important for Smurf1 localization at the plasma membrane, regulation on cell migration, and robust ligase activity toward RhoA, which further supports a Ca2+-independent localization mechanism for Smurf1. These findings demonstrate a previously unidentified role of the Smurf1 C2 domain in substrate selection and cellular localization. [ABSTRACT FROM AUTHOR]

The multi-functional NS1 protein of influenza A virus is a viral virulence determining factor. The last four residues at the Cterminus of NS1 constitute a type I PDZ domain binding motif (PBM). Avian influenza viruses currently in circulation carry an NS1 PBM with consensus sequence ESEV, whereas human influenza viruses bear an NS1 PBM with consensus sequence RSKV or RSEV. The PBM sequence of the influenza A virus NS1 is reported to contribute to high viral pathogenicity in animal studies. Here, we report the identification of PDlim2 as a novel binding target of the highly pathogenic avian influenza virus H5N1 strain with an NS1 PBM of ESEV (A/Chicken/Henan/12/2004/H5N1, HN12-NS1) by yeast two-hybrid screening. The interaction was confirmed by in vitro GST pull-down assays, as well as by in vivo mammalian two-hybrid assays and bimolecular fluorescence complementation assays. The binding was also confirmed to be mediated by the interaction of the PDlim2 PDZ domain with the NS1 PBM motif. Interestingly, our assays showed that PDlim2 bound specifically with HN12- NS1, but exhibited no binding to NS1 from a human influenza H1N1 virus bearing an RSEV PBM (A/Puerto Rico/8/34/H1N1, PR8-NS1). A crystal structure of the PDlim2 PDZ domain fused with the C-terminal hexapeptide from HN12-NS1, together with GST pull-down assays on PDlim2 mutants, reveals that residues Arg16 and Lys31 of PDlim2 are critical for the binding between PDlim2 and HN12-NS1. The identification of a selective binding target of HN12-NS1 (ESEV), but not PR8-NS1 (RSEV), enables us to propose a structural mechanism for the interaction between NS1 PBM and PDlim2 or other PDZ-containing proteins. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 are heme monooxygenases that receive electrons from NADH via Arx, a [2Fe-2S] ferredoxin, and ArR, a ferredoxin reductase. These systems show fast NADH turnovers (kcat = 39-91 s-1) that are efficiently coupled to product formation. The three-dimensional structures of ArR, Arx, and CYP101D1, which form a physiological class I P450 electron transfer chain, have been resolved by x-ray crystallography. The general structural features of these proteins are similar to their counterparts in other class I systems such as putidaredoxin reductase (PdR), putidaredoxin (Pdx), and CYP101A1 of the camphor hydroxylase system from Pseudomonas putida, and adrenodoxin (Adx) of the mitochondrial steroidogenic CYP11 and CYP24A1 systems. However, significant differences in the proposed protein-protein interaction surfaces of the ferredoxin reductase, ferredoxin, and P450 enzyme are found. There are regions of positive charge on the likely interaction face of ArR and CYP101D1 and a corresponding negatively charged area on the surface of Arx. The [2Fe-2S] cluster binding loop in Arx also has a neutral, hydrophobic patch on the surface. These surface characteristics are more in common with those of Adx than Pdx. The observed structural features are consistent with the ionic strength dependence of the activity. [ABSTRACT FROM AUTHOR]

Ccenzyme F420, a hydride carrier, is found in Archaea and some bacteria and has crucial roles in methanogenesis, antibiotic biosynthesis, DNA repair, and activation of antitubercular compounds. CofD, 2-phospho-L-lactate transferase, catalyzes the last step in the biosynthesis of F420-O (F420 without polyglutamate), by transferring the lactyl phosphate moiety of Iactyl(2)diphospho-(5')guanosine to 7,8-didemethyl-8-hydroxy-5-deazariboflavin ribitol (Fo). CofD is highly conserved among F420-producing organisms, and weak sequence homologs are also found in non-F420-producing organisms. This superfamily does not share any recognizable sequence conservation with other proteins. Here we report the first crystal structures of Cofi), the free enzyme and two ternary complexes, with Fo and P1 or with Fo and GDP, from Methanosarcina mazei. The active site is located at the C-terminal end of a Rossmann fold core, and three large insertions make significant contributions to the active site and dimer formation. The observed binding modes of Fo and GDP can explain known biochemical properties of CofD and are also supported by our binding assays. The structures pro- vide significant molecular insights into the biosynthesis of the F420 coenzyme. Large structural differences in the active site region of the non-F420-producing CofD homologs suggest that they catalyze a different biochemical reaction. [ABSTRACT FROM AUTHOR]

The AP-1 transcription factor modulates a wide range of cellular processes, including cellular proliferation, programmed cell death, and survival. JunD is a major component of the AP-1 complex following liver ischemia/reperfusion (I/R) injury; however, its precise function in this setting remains unclear. We investigated the functional significance of JunD in regulating AP-1 transcription following partial lobar I/R injury to the liver, as well as the downstream consequences for hepatocellular remodeling. Our findings demonstrate that JunD plays a protective role, reducing I/R injury to the liver by suppressing acute transcriptional activation of AP-1. In the absence of JunD, c-Jun phosphorylation and AP-1 activation in response to I/R injury were elevated, and this correlated with increased caspase activation, injury, and alterations in hepatocyte proliferation. The expression of dominant negative JNK1 inhibited c-Jun phosphorylation, AP-1 activation, and hepatic injury following I/R in JunD-/- mice but, paradoxically, led to an enhancement of AP-1 activation and liver injury in JunD+/- littermates. Enhanced JunD/JNK1-dependent liver injury correlated with the acute induction of diphenylene iodonium-sensitive NADPH-dependent superoxide production by the liver following I/R. In this context, dominant negative JNK1 expression elevated both Nox2 and Nox4 mRNA levels in the liver in a JunD-dependent manner. These findings suggest that JunD counterbalances JNK1 activation and the downstream redox-dependent hepatic injury that results from I/R, and may do so by regulating NADPH oxidases. [ABSTRACT FROM AUTHOR]

IκB proteins play an important role in regulating NF-κB induction following a diverse range of environmental injuries. Studies evaluating IκBβ knock-in mice (AKBI), in which the IκBα gene is replaced by the IκBβ cDNA, have uncovered divergent properties of IκBα and IκBβ that influence their ability to activate hepatic NF-κB and subsequent downstream proinflammatory processes in a stimulus-specific manner. While AKBI mice demonstrated identical levels of hepatic NF-κB activation in response to endotoxin, a significantly reduced level of hepatic NF-κB activation was observed in AKBI mice after liver ischemia/reperfusion (I/R) injury. This reduced level of NF-κB activation in AKBI mice after liver I/R also correlated with decreased induction of serum TNF-α, reduced hepatic inflammation, and increased survival. In contrast, no differences in any of these indicators were observed between AKBI mice and WT littermates after a lethal injection of LPS. Molecular studies suggest that the specificity of IκBα, but not IκBβ, to properly regulate NF-κB induction during the acute phase of I/R injury is due to injury context-specific activation of c-Src and subsequent tyrosine phosphorylation of IκBα on Tyr42. These results demonstrate that IκBα and IκBβ play unique injury context-specific roles in activating NF-κB-mediated proinflammatory responses and suggest that strategies aimed at inhibiting IκBα gene expression may be of potential therapeutic benefit in hepatic I/R injury. [ABSTRACT FROM AUTHOR]

A new method for the fabrication of an integrated microelectrode for electrochemical detection (ECD) on an electrophoresis microchip is described. The pattern of the microelectrode was directly made on the surface of a microscope slide through an electroless deposition procedure. The surface of the slide was first selectively coated with a thin layer of sodium silicate through a micromoldlng in capillary technique provided by a poly(dimethylsiloxane) (PDMS) microchannel; this left a rough patterned area for the anchoring of catalytic particles. A metal layer was deposited on the pattern guided by these catalytic particles and was used as the working electrode. Factors influencing the fabrication procedure were discussed. The whole chip was built by reversibly sealing the slide to another PDMS layer with electrophoresis microchannels at room temperature. This approach eliminates the need of clean room facilities and expensive apparatus such as for vacuum deposition or sputtering and makes it possible to produce patterned electrodes suitable for ECD on microchip under ordinary chemistry laboratory conditions. Also once the micropattern is ready, it allows the researchers to rebuild the electrode in a short period of time when an electrode failure occurs. Copper and gold microelectrodes were fabricated by this technique. Glucose, dopamine, and catechol as model analytes were tested. [ABSTRACT FROM AUTHOR]

Background: DNA methylation is a form of epigenetic modification that impacts gene expression without modifying the DNA sequence, thereby exerting control over gene function and cellular development. The prediction of DNA methylation is vital for understanding and exploring gene regulatory mechanisms. Currently, machine learning algorithms are primarily used for model construction. However, several challenges remain to be addressed, including limited prediction accuracy, constrained generalization capability, and insufficient learning capacity., Results: In response to the aforementioned challenges, this paper leverages the similarities between DNA sequences and time series to introduce a time series-based hybrid ensemble learning model, called Multi2-Con-CAPSO-LSTM. The model utilizes multivariate and multidimensional encoding approach, combining three types of time series encodings with three kinds of genetic feature encodings, resulting in a total of nine types of feature encoding matrices. Convolutional Neural Networks are utilized to extract features from DNA sequences, including temporal, positional, physicochemical, and genetic information, thereby creating a comprehensive feature matrix. The Long Short-Term Memory model is then optimized using the Chaotic Accelerated Particle Swarm Optimization algorithm for predicting DNA methylation., Conclusions: Through cross-validation experiments conducted on 17 species involving three types of DNA methylation (6 mA, 5hmC, and 4mC), the results demonstrate the robust predictive capabilities of the Multi2-Con-CAPSO-LSTM model in DNA methylation prediction across various types and species. Compared with other benchmark models, the Multi2-Con-CAPSO-LSTM model demonstrates significant advantages in sensitivity, specificity, accuracy, and correlation. The model proposed in this paper provides valuable insights and inspiration across various disciplines, including sequence alignment, genetic evolution, time series analysis, and structure-activity relationships., (© 2023. The Author(s).)

Objective: To investigate the clinical effect of microsurgical scleral drainage and trabeculectomy combined with scleral flap adjustable suture technique in the treatment of primary glaucoma., Methods: One hundred primary glaucoma patients (120 eyes) in Xinyu People's Hospital of Jiangxi province were selected from July 2014 to June 2016. The patients were randomly divided into control group and study group. The control group was treated with compound trabeculectomy, while the study group was treated with microsurgical scleral drainage and trabeculectomy combined with scleral flap adjustable suture technique. In both groups of patients, intraocular pressure, functional filtering bleb formation, and complications before and after surgery were monitored for three days, one week, one month, three months, six months and one year, while anterior chamber depth was determined one week after operation. The extent of success of operation was compared between the two groups., Results: At three days, one week, one month, three months, six months and one year after surgery, intraocular pressure of study group was significantly lower than that of the control group (P<0.05). There was 93.33% formation of functional filtering blebs in the study group, which was significantly higher than that in the control group (60.00%, P<0.001). Moreover, normal anterior chamber formation was significantly higher in the study group (91.67%) than in the control group (71.67%, P<0.01). There was 95.00% operation success in the study group, relative to 68.33% success in the control group (P<0.001)., Conclusion: Microsurgical scleral drainage and trabeculectomy combined with scleral flap adjustable suture technique has better curative effect on primary glaucoma than compound trabeculectomy. Moreover, it does not exacerbate complications. Therefore, the combination treatment technique merits clinical application., (Copyright: © Pakistan Journal of Medical Sciences.)