Efficient splicing of the CPE intein derived from directed evolution of the Cryptococcus neoformans PRP8 intein.

Intein-mediated protein splicing has been widely used in protein engineering; however, the splicing efficiency and extein specificity usually limit its further application. Thus, there is a demand for more general inteins that can overcome these limitations. Here, we study the trans-splicing of CPE intein obtained from the directed evolution of Cne PRP8, which shows that its splicing rate is ~29- fold higher than that of the wild-type. When the +1 residue of C-extein is changed to cysteine, CPE also shows high splicing activity. Faster association and higher affinity may contribute to the high splicing rate compared with wild-type intein. These findings have important implications for the future engineering of inteins and provide clues for fundamental studies of protein structure and folding.

Mitophagy in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the most common cause of end-stage kidney disease worldwide and is the main microvascular complication of diabetes. The increasing prevalence of diabetes has increased the need for effective treatment of DKD and identification of new therapeutic targets for better clinical management. Mitophagy is a highly conserved process that selectively removes damaged or unnecessary mitochondria via the autophagic machinery. Given the important role of mitophagy in the increased risk of DKD, especially with the recent surge in COVID-19-associated diabetic complications, in this review, we provide compelling evidence for maintaining homeostasis in the glomeruli and tubules and its underlying mechanisms, and offer new insights into potential therapeutic approaches for treatment of DKD.

Improved protein purification system based on C-terminal cleavage of Npu DnaE split intein.

A purification system was constructed with the N-segment of the Npu DnaE split intein as an affinity ligand immobilized onto an epoxy-activated medium and the C-segment used as the cleavable tag fusing target protein. The affinity properties of C-tagged proteins adsorbed on IN affinity chromatography medium were studied with GFP as a model target protein. The saturated adsorption capacity and dynamic adsorption capacity reached 51.9-21.0 mg mL-1, respectively. With this system, two model proteins, GFP and alcohol dehydrogenase (ADH), has been successfully taglessly purified with regulation of Zn2+ and DTT. The yield, purification factor and purity of purified tagless GFP reached 39, 11.7 and 97%, respectively; while these values for purified tagless ADH were 38.2, 6.8 and 91%, respectively. These results showed that the system for Npu DnaE split intein-mediated affinity adsorption and in situ cleavage is a potential platform for recombinant protein production.

Characteristics of airborne bacterial communities in indoor and outdoor environments during continuous haze events in Beijing: Implications for health care.

There is solid evidence that haze pollution threatens human health owing to the abiotic pollutants it contains. However, the characteristics of airborne bacterial communities in indoor and outdoor environments exhibiting haze occurrence are still unknown. Thus, we examined variations in both indoor and outdoor airborne bacterial communities in Beijing from December 9-27, 2016, a period which included three haze events. The outdoor airborne bacterial communities were clustered into two main groups (Groups I and II), and they shifted between two typical bacterial communities regardless of the haze event. The Chao1, Shannon, and phylogenetic diversity indexes and abundance of dominant classes changed significantly, as did airborne bacterial community type. The indoor airborne bacterial community closely tracked the outdoor bacterial community type, forming two obvious groups supported by Adonis analysis, changes in dominant classes, and bacterial diversity compared to the outdoor group. Furthermore, we found that the airborne bacterial community type could affect the morbidity of respiratory diseases. Daily pneumonia cases were significantly higher in Group I (p = 0.035), whereas daily amygdalitis cases were significantly higher in Group II (p = 0.025). Interestingly, the enriched classes in the indoor environment were quite different from those in the typical airborne bacterial community environment, except for Clostridia, which had significantly higher abundance in both indoor environments. In conclusion, we found that the two indoor and outdoor airborne bacterial community types changed independently of haze events, and the special airborne bacterial community type was closely related to the incidence of pneumonia in the heavy haze season.

Crystal structures of a natural DNA polymerase that functions as an XNA reverse transcriptase.

Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2'-deoxy-2'-fluoro-ß-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.

Comprehensive analysis of hippocampal miRNAomes in humans and mice.

AIM: This study aims to explore the similarity and difference of hippocampal miRNAomes between humans and mice. MATERIALS & METHODS: A systematic comparison of the miRNAomes between healthy human and mouse hippocampi was performed using high-throughput sequencing followed by bioinformatic analyses. RESULTS: A novel miRNA termed novel-21-5p and a human-specific miR-656-3p were identified in human hippocampi, which were expressed ubiquitously and predicted to be associated with neural activities. Compared with mouse, abundantly expressed miRNAs in human hippocampus were notably enriched in pathways pertaining to neural activities, such as neurotrophin TRK receptor signaling pathway, axon guidance and synaptic transmission. Expression pattern of orthologous miRNAs between human and mouse hippocampi was conserved. Meanwhile, the expression conservation was positively correlated with the sequence conservation. CONCLUSION: Hippocampal miRNAomes between humans and mice were overall comparable; the differences in expression or function across species should be considered when constructing mouse models.

A Convenient Split-Intein Tag Method for the Purification of Tagless Target Proteins.

In this work, we describe a novel self-cleaving tag technology based on a highly modified split-intein cleaving element. In this system, the N-terminal segment of an engineered split intein is expressed in E. coli and covalently immobilized onto a capture resin, while the smaller C-terminal intein segment is fused to the N-terminus of the desired target protein. The tagged target can then be expressed in an appropriate expression system, without concern for premature intein cleaving. During the purification, strong association between the intein segments effectively captures the tagged target onto the capture resin while simultaneously generating a cleaving-competent intein complex. Once the complex is purified by washing the column, intein-mediated cleavage and release of the tagless target is induced with a simple shift in buffer pH from 8.5 to 6.2. The result is a convenient and effective method for the purification of traceless and tagless target proteins, which can be used in characterization and functional studies. © 2018 by John Wiley & Sons, Inc.

Structural basis for TNA synthesis by an engineered TNA polymerase.

Darwinian evolution experiments carried out on xeno-nucleic acid (XNA) polymers require engineered polymerases that can faithfully and efficiently copy genetic information back and forth between DNA and XNA. However, current XNA polymerases function with inferior activity relative to their natural counterparts. Here, we report five X-ray crystal structures that illustrate the pathway by which α-(L)-threofuranosyl nucleic acid (TNA) triphosphates are selected and extended in a template-dependent manner using a laboratory-evolved polymerase known as Kod-RI. Structural comparison of the apo, binary, open and closed ternary, and translocated product detail an ensemble of interactions and conformational changes required to promote TNA synthesis. Close inspection of the active site in the closed ternary structure reveals a sub-optimal binding geometry that explains the slow rate of catalysis. This key piece of information, which is missing for all naturally occurring archaeal DNA polymerases, provides a framework for engineering new TNA polymerase variants.

Altered Gut Microbiota in a Mouse Model of Alzheimer's Disease.

The topic of gut microbiota is currently attracting considerable interest as a potential factor in Alzheimer's disease (AD). However, the extent and time course of alterations in the gut microbiota, and their effects on AD pathology remain uncertain. Herein, we compared the fecal microbiomes and fecal short chain fatty acid composition (SCFAs) between wild-type and AD model mice at different ages under strictly controlled specific pathogen free conditions, and also conducted microscopic investigations of intestinal structures. Our results showed that the microbiota composition and diversity were perturbed and the level of SCFAs was reduced in AD mice, predicting alterations in more than 30 metabolic pathways, which may be associated with amyloid deposition and ultrastructural abnormalities in AD mouse intestine. These findings indicate that AD pathology might not only affect brain function directly, but also exacerbate cognitive deficits through reducing the level of SCFAs via alterations of gut microbiota induced by intestinal amyloid deposition. Our data may support a role of gut microbiota, and suggest a novel route for therapeutic intervention in AD.

Synthesis and polymerase activity of a fluorescent cytidine TNA triphosphate analogue.

Threose nucleic acid (TNA) is an artificial genetic polymer capable of undergoing Darwinian evolution to produce aptamers with affinity to specific targets. This property, coupled with a backbone structure that is refractory to nuclease digestion, makes TNA an attractive biopolymer system for diagnostic and therapeutic applications. Expanding the chemical diversity of TNA beyond the natural bases would enable the development of functional TNA molecules with enhanced physiochemical properties. Here, we describe the synthesis and polymerase activity of a fluorescent cytidine TNA triphosphate analogue (1,3-diaza-2-oxo-phenothiazine, tCfTP) that maintains Watson-Crick base pairing with guanine. Polymerase-mediated primer-extension assays reveal that tCfTP is efficiently added to the growing end of a TNA primer. Detailed kinetic assays indicate that tCfTP and tCTP have comparable rates for the first nucleotide incorporation step (kobs1). However, addition of the second nucleotide (kobs2) is 700-fold faster for tCfTP than tCTP due the increased effects of base stacking. Last, we found that TNA replication using tCfTP in place of tCTP exhibits 98.4% overall fidelity for the combined process of TNA transcription and reverse transcription. Together, these results expand the chemical diversity of enzymatically generated TNA molecules to include a hydrophobic base analogue with strong fluorescent properties that is compatible with in vitro selection.

Long non-coding RNAs in brain development, synaptic biology, and Alzheimer's disease.

Long non-coding RNAs (lncRNAs), which are long transcripts without apparent protein-coding roles, interfere with gene expression and signaling events at various stages. Increasing evidence has suggested that lncRNAs function in the regulation of tissue homeostasis and under pathophysiologic conditions. In the nervous system, the expression of lncRNAs has been detected and characterized under normal physiologic conditions and in disease states. Some lncRNAs regulate brain development and synaptic plasticity. In Alzheimer's disease (AD), several lncRNAs have been demonstrated to regulate ß-amyloid production/generation, synaptic impairment, neurotrophin depletion, inflammation, mitochondrial dysfunction, and stress responses. This review summarizes data on lncRNA expression and focuses on neural lncRNAs that may function in AD. Although our understanding of lncRNAs remains in its infancy, this review provides insight into the contribution of lncRNAs to AD.

The correlation between the length of repetitive domain and mechanical properties of the recombinant flagelliform spidroin.

Spider silk is an attractive biopolymer with numerous potential applications due to its remarkable characteristics. Among the six categories of spider silks, flagelliform (Flag) spider silk possesses longer and more repetitive core domains than others, therefore performing the highest extensibility. To investigate the correlation between the recombinant spidroin size and the synthetic fiber properties, four recombinant proteins with different sizes [N-Scn-C (n=1-4)] were constructed and expressed using IMPACT system. Subsequently, different recombinant spidroins were spun into fibers through wet-spinning via a custom-made continuous post-drawing device. Mechanical tests of the synthetic fibers with four parameters (maximum stress, maximum extension, Young's modulus and toughness) demonstrated that the extensibility of the fibers showed a positive correlation with spidroin size, consequently resulting in the extensibility of N-Sc4-C fiber ranked the highest (58.76%) among four fibers. Raman data revealed the relationship between secondary structure content and mechanical properties. The data here provide a deeper insight into the relationship between the function and structure of Flag silk for future design of artificial fibers.

Purification of Microbially Expressed Recombinant Proteins via a Dual ELP Split Intein System.

Fusions of elastin-like peptide (ELP) purification tags and self-cleaving inteins provide a powerful platform for purifying tagless recombinant proteins without the need for conventional packed-bed columns. A drawback to this method has been premature cleaving of the ELP tag during expression, before the purification procedure can take place. Here we demonstrate a split-intein method, where the self-cleaving intein is divided into two inactive segments during expression and purification. Spontaneous assembly of the purified intein segments then restores self-cleaving activity to deliver the tagless target protein.

Phloroglucinol protects the urinary bladder via inhibition of oxidative stress and inflammation in a rat model of cyclophosphamide-induced interstitial cystitis.

BACKGROUND: Phloroglucinol plays an important role in oxidative stress and inflammatory responses. The effects of phloroglucinol have been proven in various disease models. The aim of the present study was to investigate the efficacy and possible mechanisms of phloroglucinol in the treatment of interstitial cystitis (IC). METHODS: Thirty-two female Sprague-Dawley (SD) rats were used in this study. IC was induced by intraperitoneal injection of cyclophosphamide (CYP). Rats were randomly allocated to one of four groups (n = 8 per group): A control group, which was injected with saline (75 mg/kg; i.p.) instead of CYP on days 1, 4, and 7; a chronic IC group, which was injected with CYP (75 mg/kg; i.p.) on days 1, 4, and 7; a high-dose (30 mg/kg) phloroglucinol-treated group; and a low-dose (15 mg/kg) phloroglucinol-treated group. On day 8, the rats in each group underwent cystometrography (CMG), and the bladders were examined for evidence of oxidative stress and inflammation. Statistical analysis was performed by analysis of variance (ANOVA) followed by least square difference multiple comparison post-hoc test. RESULTS: Histological evaluation showed that bladder inflammation in CYP-treated rats was suppressed by phloroglucinol. CMG revealed that the CYP treatment induced overactive bladder in rats that was reversed by phloroglucinol. Up-regulated tumor necrosis factor-α and interleukin-6 expression in the CYP-treated rats were also suppressed in the phloroglucinol treated rats. CYP treatment significantly increased myeloperoxidase activity as well as the decreased activities of catalase of the bladder, which was reversed by treatment with phloroglucinol. CONCLUSIONS: The application of phloroglucinol suppressed oxidative stress, inflammation, and overactivity in the bladder. This may provide a new treatment strategy for IC.

A general purification platform for toxic proteins based on intein trans-splicing.

Many important functional proteins often exhibit toxicity when overexpressed in heterologous hosts. Unfortunately, this toxicity can complicate the production of these proteins in recombinant systems, which can slow their characterization. Although a number of engineered expression strains and plasmids have been developed to optimize toxic protein expression, many targets remain recalcitrant in these systems due to extreme toxicity to the expression host. In this work, we have developed a novel protein purification platform based on intein trans-splicing, with special relevance for proteins that are extremely toxic to recombinant host cells. The toxic protein is split into two inactive fragments, which are separately expressed in fusion to the segments of a split intein. The N-terminal intein segment is first immobilized onto an affinity column and washed, followed by addition of the C-terminal segment and purification of the complex. The assembled intein controllably splices to deliver the mature target protein, simultaneously releasing the purified target from the affinity column. To optimize this method, we generated a hybrid split intein consisting of the N-terminus of the Npu DnaE intein and the C-terminus of the Ssp DnaE intein. This hybrid intein tolerates a wider range of amino acids at the +2 site of the C-terminal splicing junction than the Npu intein alone. In the production of the highly toxic homing endonuclease I-TevI, the yield from the hybrid intein is 50 % higher than the native Npu DnaE intein, while the I-TevI protein purified from both inteins showed native activity.

Analysis of the roles of mutations in thyroid hormone receptor-ß by a bacterial biosensor system.

Mutations in thyroid hormone receptors (TRs) often lead to metabolic and developmental disorders, but patients with these mutations are difficult to treat with existing thyromimetic drugs. In this study, we analyzed six clinically observed mutations in the ligand-binding domain of the human TRß using an engineered bacterial hormone biosensor. Six agonist compounds, including triiodothyronine (T3), thyroxine (T4), 3,5,3'-triiodothyroacetic acid (Triac), GC-1, KB-141, and CO-23, and the antagonist NH-3 were examined for their ability to bind to each of the TRß mutants. The results indicate that some mutations lead to the loss of ability to bind to native ligands, ranging from several fold to several hundred fold, while other mutations completely abolish the ability to bind to any ligand. Notably, the effect of each ligand on each TRß mutant in this bacterial system is highly dependent on both the mutation and the ligand; some ligands were bound well by a wide variety of mutants, while other ligands lost their affinity for all but the WT receptor. This study demonstrates the ability of our bacterial system to differentiate agonist compounds from antagonist compounds and shows that one of the TRß mutations leads to an unexpected increase in antagonist ability relative to other mutations. These results indicate that this bacterial sensor can be used to rapidly determine ligand-binding ability and character for clinically relevant TRß mutants.

A dual ELP-tagged split intein system for non-chromatographic recombinant protein purification.

Self-cleaving elastin-like protein (ELP) tags provide a very promising tool for recombinant protein purification. With this method, the target protein is purified by simple ELP-mediated precipitation steps, followed by self-cleavage and removal of the ELP tag. Unfortunately, however, inteins usually experience some level of pre-cleavage during protein expression, which can significantly decrease final yields. In this study, we solve this problem by splitting the intein into two ELP-tagged segments. Each segment is incapable of pre-cleavage alone, but the assembled segments release the target protein rapidly when assembled in vitro. The result is the very tight control of the tag cleaving reaction, combined with the simplicity of the ELP purification method. Using this system, we successfully purified four different sizes of target proteins with final yields comparable to or higher than our original contiguous intein-ELP system. Further, we demonstrate a streamlined split intein method, where cells expressing the tagged intein segments are combined prior to cell lysis, allowing the segments to be co-purified in a single reaction mixture.

The impact of TGF-ß1 on the mRNA expression of TßR I, TßR II, Smad4 and the invasiveness of the JEG-3 placental choriocarcinoma cell line.

Human choriocarcinoma is one of the most aggressive malignant tumors characterized by early hematogenous spread to lung and brain tissues, and may be a cause of death in patients. Choriocarcinoma may occur following pregnancy and during implantation; however, trophoblastic invasion in human pregnancy is tightly regulated. The transforming growth factor-beta 1 (TGF-ß1) has been suggested to play a role in controlling this process. In this study, we investigated the impact of TGF-ß1 on invasion, as well as its sites of action in the TGF-ß1/Smad pathway using a JEG-3 choriocarcinoma cell line. Following the treatment of cells with different doses of TGF-ß1, cell invasion was observed. We also detected the expression of TGF-ß receptor type I (TßR I) and TGF-ß receptor type II (TßR II), Smad4, matrix metalloprotease (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 in JEG-3 cells. Our data demonstrated that TGF-ß1 promoted the invasive capability of JEG-3 cells depending on the downregulation of TßR I, TßR II, Smad4 and the upregulation of MMP-9 and TIMP-1. These observations suggest that TGF-ß1 may play a critical role in the initiation of the trophoblastic invasion process.

Interleukin-12 inhibits cell invasion in choriocarcinoma.

Gestational trophoblastic disease (GTD) is a unique disease that arises from allografting of the conceptus, and has a characteristic morphology and biological behavior. It encompasses a spectrum of interrelated diseases, including hydatidiform mole, invasive mole, choriocarcinoma and placental-site trophoblastic tumor, but its pathogenesis remains unrevealed. Particularly, choriocarcinoma is a highly malignant tumor with poor prognosis. In this study, we cultured the human choriocarcinoma cell line JEG-3 in vitro. After treating the cells with different doses of interleukin (IL)-12, the cell invasion was observed. We also detected the expression of matrix metalloproteinases (MMP)-9 and tissue inhibitors of metalloproteinases (TIMP)-1 and the cell cycle of JEG-3 cells. Our data indicated that IL-12 inhibits cell invasion in a dose- and time-dependent manner through regulating the expression of MMP-9 and TIMP-1. In addition, treatment with IL-12 redistributes the phases of the cell cycle in JEG-3 cells. These findings suggest an antitumor role of IL-12 in choriocarcinoma, with far reaching possibilities for understanding the mechanisms of IL-12.

Effects of electromagnetic radiation on spatial memory and synapses in rat hippocampal CA1.

In this study, we investigated the effects of mobile phone radiation on spatial learning, reference memory, and morphology in related brain regions. After the near-field radiation (0.52-1.08 W/kg) was delivered to 8-week-old Wistar rats 2 hours per day for 1 month, behavioral changes were examined using the Morris water maze. Compared with the sham-irradiated rats, the irradiated rats exhibited impaired performance. Morphological changes were investigated by examining synaptic ultrastructural changes in the hippocampus. Using the physical dissector technique, the number of pyramidal neurons, the synaptic profiles, and the length of postsynaptic densities in the CA1 region were quantified stereologically. The morphological changes included mitochondrial degenerations, fewer synapses, and shorter postsynaptic densities in the radiated rats. These findings indicate that mobile phone radiation can significantly impair spatial learning and reference memory and induce morphological changes in the hippocampal CA1 region.