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This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3′–5′ exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5′–3′ exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SβαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SβαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3′–5′ exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SβαR motif, was first determined to 2.19 Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

Bacterial exonuclease III (ExoIII) is a multifunctional enzyme that uses a single active site to perform two conspicuous activities: (i) apurinic/apyrimidinic (AP)-endonuclease and (ii) 3′→5′ exonuclease activities. The AP endonuclease activity results in AP site incision, while the exonuclease activity results in the continuous excision of 3′ terminal nucleobases to generate a partial duplex for recruiting the downstream DNA polymerase during the base excision repair process (BER). The key determinants of functional selection between the two activities are poorly understood. Here, we use a series of mutational analyses and single-molecule imaging to unravel the pivotal rules governing these endo- and exonuclease activities at the single amino acid level. An aromatic residue, either W212 or F213, recognizes AP sites to allow for the AP endonuclease activity, and the F213 residue also participates in the stabilization of the melted state of the 3′ terminal nucleobases, leading to the catalytically competent state that activates the 3′→5′ exonuclease activity. During exonucleolytic cleavage, the DNA substrate must be maintained as a B-form helix through a series of phosphate-stabilizing residues (R90, Y109, K121 and N153). Our work decouples the AP endonuclease and exonuclease activities of ExoIII and provides insights into how this multifunctional enzyme controls each function at the amino acid level.

In higher eukaryotes, mitochondria play multiple roles in energy production, signaling, and biosynthesis. Mitochondria possess multiple copies of mitochondrial DNA (mtDNA), which encodes 37 genes that are essential for mitochondrial and cellular function. When mtDNA is challenged by endogenous and exogenous factors, mtDNA undergoes repair, degradation, and compensatory synthesis. mtDNA degradation is an emerging pathway in mtDNA damage response and maintenance. A key factor involved is the human mitochondrial genome maintenance exonuclease 1 (MGME1). Despite previous biochemical and functional studies, controversies exist regarding the polarity of MGME1-mediated DNA cleavage. Also, how DNA sequence may affect the activities of MGME1 remains elusive. Such information is not only fundamental to the understanding of MGME1 but critical for deciphering the mechanism of mtDNA degradation. Herein, we use quantitative assays to examine the effects of substrate structure and sequence on the DNA-binding and enzymatic activities of MGME1. We demonstrate that MGME1 binds to and cleaves from the 5'-end of single-stranded DNA substrates, especially in the presence of 5'-phosphate, which plays an important role in DNA binding and optimal cleavage by MGME1. In addition, MGME1 tolerates certain modifications at the terminal end, such as a 5'-deoxyribosephosphate intermediate formed in base excision repair. We show that MGME1 processes different sequences with varying efficiencies, with dT and dC sequences being the most and least efficiently digested, respectively. Our results provide insights into the enzymatic properties of MGME1 and a rationale for the coordination of MGME1 with the 3'-5' exonuclease activity of DNA polymerase γ in mtDNA degradation.

Post-transcriptional modification of nucleosides is observed in almost all elements of RNA. Modified nucleosides finely tune the structure of RNA molecules and affect vital functions, such as the modified wobble position 34 of transfer RNAs expanding the reading preference of anticodons to codons. Recent investigations have revealed that the modification species and their frequencies in an RNA element are not stable but vary with specific cellular factors including metabolites and particular proteins (writers, readers, and erasers). To understand the link between dynamic RNA modifications and biological processes, sensitive and reliable methods for determining modified nucleosides are required. In this study, micro-flow (8 μL/min) hydrophilic interaction liquid chromatography was coupled with triple quadrupole mass spectrometry for the simultaneous determination of adenosine, uridine, cytidine, guanosine, and 20 modified nucleosides. The method was calibrated using 0.1-1000 nM standards (∼0.03-300 ng/mL) and successfully applied to the determination of transfer RNA modifications in the model cyanobacterium Synechococcus elongatus PCC 7942. A protocol for the isolation of a clean transfer RNA pool was optimized, requiring only 25 ng for the identification and quantification of transfer RNA modifications. This micro-flow liquid chromatography-tandem mass spectrometry method constitutes the first step toward monitoring dynamic ribonucleoside modifications in a limited RNA sample.

We present a structural and functional analysis of the DNA polymerase of thermophilic

The widespread CBASS (cyclic oligonucleotide‐based anti‐phage signaling system) immune systems in bacteria protect their hosts from bacteriophage infection by triggering programmed cell death. CBASS systems all encode a cyclic oligonucleotide synthase related to eukaryotic cGAS but use diverse regulators and effector proteins including nucleases, phospholipases, and membrane‐disrupting proteins to effect cell death. Cap18 is a predicted 3′‐5′ exonuclease associated with hundreds of CBASS systems, whose structure, biochemical activities, and biological roles remain unknown. Here we show that Cap18 is a DEDDh‐family exonuclease related to the bacterial exonucleases RNase T and Orn and has nonspecific 3′‐5′ DNA exonuclease activity. Cap18 is commonly found in CBASS systems with associated CapW or CapH+CapP transcription factors, suggesting that it may coordinate with these proteins to regulate CBASS transcription in response to DNA damage. These data expand the repertoire of enzymatic activities associated with bacterial CBASS systems and provide new insights into the regulation of these important bacterial immune systems.

Apurinic/apyrimidinic (AP) endonucleases are the key enzymes in the DNA base excision repair, as they hydrolyze the phosphodiester bond in the AP site formed after removal of the damaged base. Major human AP endonuclease APEX1 also possesses the 3'-phosphodiesterase and 3'→5' exonuclease activities. The biological role of the latter has not been established yet; it is assumed that it corrects DNA synthesis errors during DNA repair. If DNA is damaged at the 3'-side of 5-methylcytosine (mC) residue, the 3'→5' exonuclease activity can change the epigenetic methylation status of the CpG dinucleotide. It remains unclear whether the 3'→5' exonuclease activity of APEX1 contributes to the active epigenetic demethylation or, on the contrary, is limited in the case of methylated CpG dinucleotides in order to preserve the epigenetic status upon repair of accidental DNA damage. Here, we report the results of the first systematic study on the efficiency of removal of 3'-terminal nucleotides from the substrates modeling DNA repair intermediates in the CpG dinucleotides. The best substrates for the 3'→5' exonuclease activity of APEX1 were oligonucleotides with the 3'-terminal bases non-complementary to the template, while the worst substrates contained mC. The presence of mC in the complementary strand significantly reduced the reaction rate even for the non-complementary 3'-ends. Therefore, the efficiency of the 3'→5' exonuclease reaction catalyzed by APEX1 is limited in the case of the methylated CpG dinucleotides, which likely reflects the need to preserve the epigenetic status during DNA repair.

Background Bioinformatic genome surveys indicate that self-cleaving ribonucleic acids (ribozymes) appear to be widespread among all domains of life, although the functions of only a small number have been validated by biochemical methods. Alternatively, cell-based reporter gene assays can be used to validate ribozyme function. However, reporter activity can be confounded by phenomena unrelated to ribozyme-mediated cleavage of RNA. Results We established a ribozyme reporter system in Escherichia coli in which a significant reduction of reporter activity is manifest when an active ribozyme sequence is fused to the reporter gene and the expression of a foreign Bacillus subtilis RNaseJ1 5′ exonuclease is induced from a chromosomally-integrated gene in the same cell. Conclusions The reporter system could be useful for validating ribozyme function in candidate sequences identified from bioinformatics.

The wide use of whole-genome sequencing approach in the modern genomic era has opened a great opportunity to reveal the prospective applications of halophilic bacteria. Robertkochia marina CC-AMO-30D(T) is one of the halophilic bacteria that was previously taxonomically identified without any inspection on its biotechnological potential from a genomic aspect. In this study, we present the whole-genome sequence of R. marina and demonstrated the ability of this bacterium in solubilizing phosphate by producing phosphatase. The genome of R. marina has 3.57 Mbp and contains 3107 predicted genes, from which 3044 are protein coding, 52 are non-coding RNAs, and 11 are pseudogenes. Several phosphatases such as alkaline phosphatases and pyrophosphatases were mined from the genome. Further genomic study (phylogenetics, sequence analysis, and functional mechanism) and experimental data suggested that the alkaline phosphatase produced by R. marina could potentially be utilized in promoting plant growth, particularly for plants on saline-based agricultural land. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-020-00401-2.

Background The rise in Plasmodium falciparum resistance to dihydroartemisinin–piperaquine (DHA–PPQ) treatment has been documented in the Greater Mekong Subregion with associations with mutations in the P. falciparum chloroquine resistance transporter (pfcrt) and plasmepsin 2 (pfpm2) genes. However, it is unclear whether other genes also play a role with PPQ resistance, such as the E415G mutation in the exonuclease (pfexo) gene. The aim of this study was to investigate the role of this mutation in PPQ resistance by generating transgenic parasites expressing the pfexo-E415G mutant allele. Methods Transgenic parasite clones carrying the E415G mutation in PfEXO of the B5 isolate were derived by CRISPR-Cas9 gene editing and verified using PCR and gene sequencing. Polymorphisms of pfkelch-13, pfcrt, and pfexo were examined by PCR while the copy number variations of pfpm2 were examined by both relative quantitative real-time PCR and the duplication breakpoint assay. Drug sensitivity against a panel of antimalarials, the ring-stage survival assay (RSA), the PPQ survival assay (PSA), and bimodal dose-response curves were used to evaluate antimalarial susceptibility. Results The transgenic line, B5-rexo-E415G-B8, was successfully generated. The PPQ-IC90, %PPQ survival, and the bimodal dose-response clearly showed that E415G mutation in PfEXO of B5 isolate remained fully susceptible to PPQ. Furthermore, growth assays demonstrated that the engineered parasites grew slightly faster than the unmodified parental isolates whereas P. falciparum isolates harbouring pfkelch-13, pfcrt, and pfexo mutations with multiple copies of pfpm2 grew much more slowly. Conclusions Insertion of the E415G mutation in PfEXO did not lead to increased PPQ-IC90 and %PPQ survival, suggesting that this mutation alone may not be associated with PPQ resistance, but could still be an important marker if used in conjunction with other markers for monitoring PPQ-resistant parasites. The results also highlight the importance of monitoring and evaluating suspected genetic mutations with regard to parasite fitness and resistance.

Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5′ exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3′-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain—especially the O helix—to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3′ exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO–POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.

A rare and autosomal recessive premature aging disorder, Werner syndrome (WS) is characterized by the early onset of aging-associated diseases, including shortening stature, alopecia, bilateral cataracts, skin ulcers, diabetes, osteoporosis, arteriosclerosis, and chromosomal instability, as well as cancer predisposition. WRN, the gene responsible for WS, encodes DNA helicase with a 3′ to 5′ exonuclease activity, and numerous studies have revealed that WRN helicase is involved in the maintenance of chromosome stability through actions in DNA, e.g., DNA replication, repair, recombination, and epigenetic regulation via interaction with DNA repair factors, telomere-binding proteins, histone modification enzymes, and other DNA metabolic factors. However, although these efforts have elucidated the cellular functions of the helicase in cell lines, they have not been linked to the treatment of the disease. Life expectancy has improved for WS patients over the past three decades, and it is hoped that a fundamental treatment for the disease will be developed. Disease-specific induced pluripotent stem (iPS) cells have been established, and these are expected to be used in drug discovery and regenerative medicine for WS patients. In this article, we review trends in research to date and present some perspectives on WS research with regard to the application of pluripotent stem cells. Furthermore, the elucidation of disease mechanisms and drug discovery utilizing the vast amount of scientific data accumulated to date will be discussed.

Aims Hepatic ischemia reperfusion injury (HIRI) is a complication of liver surgery and liver transplantation. Adipose-derived stem cells (ADSCs) can inhibit oxidative stress and inflammation through a paracrine effect. This study aimed to determine the optimal time window of ADSCs transplantation to restore liver function after HIRI. Main methods A rat model of hepatic ischemia reperfusion combined with partial hepatectomy (HIR/PH) was established. The animals were injected intravenously with 2 × 106 rat ADSCs 2 h before, immediately after, or 6 h after surgery. Liver tissues and blood samples were collected for routine histological and biochemical assays. The molecular changes were analyzed by qRT-PCR and western blotting. Key findings ADSCs significantly improved liver tissue structure and decreased the levels of AST, ALT and ALP, which was indicative of functional recovery. In addition, transplantation of ADSCs immediately after operation decreased the levels of inflammation-related cytokines such as TNF-α, IL-1β and IL-6, and significantly increased the activity of antioxidant enzymes. At the same time, the expression of MDA was decreased. Mechanistically, ADSCs activated the Keap1/Nrf2 pathway in the injured liver. Transplantation of ADSCs pre- and 6 h post-operation did not significantly affect some indices such as mRNA and protein expression of HO-1, and protein expression of NQO1. Significance Transplanting ADSCs immediately after surgery accelerated tissue repair and functional recovery of the liver by activating the Keap1/Nrf2 pathway, which inhibited hepatic inflammation and oxidative stress, and restored the hepatic microenvironment.

Here, we aim at exploring the effect of CST5 on bone resorption and activation of osteoclasts in osteoporosis (OP) rats through the NF‐κB pathway. Microarray analysis was used to screen the OP‐related differentially expressed genes. Osteoporosis was induced in rats by intragastric retinoic acid administration. The serum levels of tartrate‐resistant acid phosphatase (TRAP), bone alkaline phosphatase (BALP) and osteocalcin (OC) and the expression of CD61 on the surface of osteoclasts were examined. The number of osteoclasts and the number and area of resorption pits were detected. Besides, the pathological changes and bone mineral density in bone tissues of rats were assessed. Also, the relationship between CST5 and the NF‐κB pathway was identified through determining the expression of CST5, RANKL, RANK, OPG, p65 and IKB. Poorly expressed CST5 was indicated to affect the OP. CST5 elevation and inhibition of the NF‐κB pathway decreased serum levels of TRAP, BALP and OC and expression of CD61 in vivo and in vitro. In OP rats, CST5 overexpression increased trabecular bones and bone mineral density of bone tissues, but decreased trabecular separation, fat within the bone marrow cavities and the number of osteoclasts through inhibiting the NF‐κB pathway. In vivo experiments showed that CST5 elevation inhibited growth in number and area of osteoclastic resorption pits and restrained osteoclastic bone absorption by inhibiting the NF‐κB pathway. In summary, overexpression of CST5 suppresses the activation and bone resorption of osteoclasts by inhibiting the activation of the NF‐κB pathway.

Damage to noradrenergic neurons in the Locus coeruleus (LC) occurs contributes to neuropathology and behavioral deficits in Alzheimer’s disease (AD); methods to reduce LC damage may therefore be of benefit. We previously showed that vindeburnol, a derivative of the plant alkaloid vincamine, reduced neuroinflammation, amyloid burden, and LC damage in a mouse model of AD; however, effects on behavior were not tested. We now tested the effects of vindeburnol on anxiety-like behavior in 5xFAD mice which develop robust amyloid burden at early ages. During novel object recognition testing, we observed that 5xFAD mice spent more time exploring than wildtype littermates, and that time was reduced by vindeburnol. Vindeburnol also reduced hyperlocomotion in the 5xFAD mice which may have contributed to their increased exploration times. In an open field test, vindeburnol normalized the increase of time spent in the center, and the decrease of time spent near the walls in 5xFAD mice. Vindeburnol reduced amyloid burden in the hippocampus and cortex, areas that contribute to regulation of anxiety-like behavior. In vitro, vindeburnol increased neuronal BDNF expression in a cAMP-dependent manner; and inhibited phosphodiesterase activity with an EC50 near 50 μM. These findings suggest that cAMP-mediated increases in neurotrophic factors contribute to beneficial effects of vindeburnol within the context of LC damage, which may be of value for treatment of some neuropsychiatric symptoms of AD.

Cyclic nucleotide phosphodiesterases (PDEs) are superfamily of enzymes that regulate the spatial and temporal relationship of second messenger signaling in the cellular system. Among the 11 different families of PDEs, phosphodiesterase 1 (PDE1) sub-family of enzymes hydrolyze both 3’,5’-cyclic adenosine monophosphate (cAMP) and 3’,5’-cyclic guanosine monophosphate (cGMP) in a mutually competitive manner. The catalytic activity of PDE1 is stimulated by their binding to Ca(2+)/calmodulin (CaM), resulting in the integration of Ca(2+) and cyclic nucleotide-mediated signaling in various diseases. The PDE1 family includes three subtypes, PDE1A, PDE1B and PDE1C, which differ for their relative affinities for cAMP and cGMP. These isoforms are differentially expressed throughout the body, including the cardiovascular, central nervous system and other organs. Thus, PDE1 enzymes play a critical role in the pathophysiology of diseases through the fundamental regulation of cAMP and cGMP signaling. This comprehensive review provides the current research on PDE1 and its potential utility as a therapeutic target in diseases including the cardiovascular, pulmonary, metabolic, neurocognitive, renal, cancers and possibly others.

The continuous monitoring of any aquatic medium is very important in the evaluation, management and legislative process for the renewal, restoration, safety and protection of the aquatic environment...

Bone histomorphometry remains the gold standard in the assessment and distinction of the different types of renal osteodystrophy. It reflects th sskeletal part of the mineral and bone disorder of chronic kidney disease (CKD-MBD), which also comprises disturbances of mineral metabolism and soft-tissue calcifications. Unfortunately, bone histomorphometry relies on bone biopsies, an invasive procedure. None of the other presently available diagnostic approaches such as non-invasive imaging techniques or circulating biomarkers allows a similarly precise assessment of bone turnover and mineralization [...]

Self-synthesizing materials, in which supramolecular structuring enhances the formation of new molecules that participate to the process, represent an intriguing notion to account for the first appearance of biomolecules in an abiotic Earth. We present here a study of the abiotic formation of interchain phosphodiester bonds in solutions of short RNA oligomers in various states of supramolecular arrangement and their reaction kinetics. We found a spectrum of conditions in which RNA oligomers self-assemble and phase separate into highly concentrated ordered fluid liquid crystal (LC) microdomains. We show that such supramolecular state provides a template guiding their ligation into hundred-bases long chains. The quantitative analysis presented here demonstrates that nucleic acid LC boosts the rate of end-to-end ligation and suppresses the formation of the otherwise dominant cyclic oligomers. These results strengthen the concept of supramolecular ordering as an efficient pathway toward the emergence of the RNA World in the primordial Earth.

The aim of this study was to assess osteogenic potential of three groups of biopolymeric hydrogel-based surfaces made of plain collagen, chitosan or collagen/chitosan, crosslinked with genipin or all three biopolymers modified with silica particles of two sizes (S1 = 240 nm and S2 = 450 nm). Biocompatibility and osteoinductive properties of the resulting composites were analyzed in the human bone marrow-derived mesenchymal stromal cells (hBMSCs) in vitro cultures. It was revealed that all tested materials are biocompatible and significantly enhance ALP activity in hBMSCs which was particularly pronounced for collagen/chitosan based hybrids. Gene expression (RUNX-2, COL-I, OC and VEGF mRNA) analyses performed in hBMSCs cultured at collagen/chitosan materials showed that ColChS1 hybrid the most effectively promotes osteogenic differentiation of hBMSCs. SEM and EDS analyses of materials carried out after 20 days of hBMSCs culturing on ColCh-based hydrogels revealed that the hybrid materials enhanced hBMSCs-mediated mineralization of ECM. Our studies revealed that collagen/chitosan hydrogels modified with silica particles of smaller sizes (ColChS1) exhibit high pro-osteogenic properties without the need of applying any additional osteogenic inducers. That suggests that ColChS1 having the intrinsic osteoinductive activity holds great potential as material of choice for bone regeneration procedures, especially in regeneration of small bone losses.

Mycobacterium smegmatis FenA is a nucleic acid phosphodiesterase with flap endonuclease and 5′ exonuclease activities. The 1.8 Å crystal structure of FenA reported here highlights as its closest homologs bacterial FEN-family enzymes ExoIX, the Pol1 exonuclease domain and phage T5 Fen. Mycobacterial FenA assimilates three active site manganese ions (M1, M2, M3) that are coordinated, directly and via waters, to a constellation of eight carboxylate side chains. We find via mutagenesis that the carboxylate contacts to all three manganese ions are essential for FenA’s activities. Structures of nuclease-dead FenA mutants D125N, D148N and D208N reveal how they fail to bind one of the three active site Mn2+ ions, in a distinctive fashion for each Asn change. The structure of FenA D208N with a phosphate anion engaged by M1 and M2 in a state mimetic of a product complex suggests a mechanism for metal-catalyzed phosphodiester hydrolysis similar to that proposed for human Exo1. A distinctive feature of FenA is that it does not have the helical arch module found in many other FEN/FEN-like enzymes. Instead, this segment of FenA adopts a unique structure comprising a short 310 helix and surface β-loop that coordinates a fourth manganese ion (M4).

The recent discovery of reversible chemical modifications on messenger RNA (mRNA) has opened a new era of post-transcriptional gene regulation in eukaryotes. Among the 15 types of modifications identified in mRNA of eukaryotes, N7-methylguanosine (m7G) is unique ow-ing to its presence in the 5’ cap structure. It remains unknown whether m7G is also present in-ternally in mRNA, and this is largely attributed to the lack of an appropriate analytical method to differentiate internal m7G in mRNA from that in the 5’ cap. To address this analytical chal-lenge, we developed a novel strategy of combining differential enzymatic digestion with liquid chromatography-tandem mass spectrometry analysis to quantify the levels of these two types of m7G modifications in mRNA. In particular, we found that S1 nuclease and phosphodiesterase I exhibit differential activities toward internal and 5’-terminal m7G. By using this method, we found that internal m7G was present in mRNA of cultured human cells as well as plants and ra...

Background: Chronic kidney disease (CKD) is closely related to the cardiovascular events in vascular calcification (VC). However, little has known about the characteristics of kidney injury caused by VC. Fibroblast growth factor 21 (FGF21) is an endocrine factor, which takes part in various metabolic actions with the potential to alleviate metabolic disorder diseases. Even FGF21 has been regarded as a biomarker in CKD, the role of FGF21 in CKD remains unclear. Therefore, in this study, we evaluate the FGF21 on the kidney injury in VC rats. Methods: The male Sprague-Dawley rats were divided into three groups: (1) control group, (2) Vitamin D3 plus nicotine (VDN)-induced VC group, (3) FGF21-treated VDN group. After 4 weeks, the rats were killed and the blood was collected for serum creatinine, urea nitrogen, calcium, and phosphate measurement. Moreover, the renal tissues were homogenized for alkaline phosphatases (ALPs) activity and calcium content. The levels of FGF21 protein were measured by radioimmunoassay. The levels of β-Klotho and FGF receptor 1 (FGFR1) protein were measured by enzyme-linked immunosorbent assay (ELISA). The structural damage and calcifications in aortas were stained by Alizarin-red S. Moreover, the structure of kidney was observed by hematoxylin and eosin staining. Results: The renal function impairment caused by VDN modeling was ameliorated by FGF21 treatment, inhibited the elevated serum creatinine and urea level by 20.5% (34.750 ± 4.334 μmol/L vs. 27.630 ± 2.387 μmol/L) and 4.0% (7.038 ± 0.590 mmol/L vs. 6.763 ± 0.374 mmol/L; P < 0.01), respectively, together with the structural damages of glomerular atrophy and renal interstitial fibrosis. FGF21 treatment downregulated the ALP activity, calcium content in the kidney of VC rats by 42.1% (P < 0.01) and 11.7% (P < 0.05) as well as ameliorated the aortic injury and calcification as compared with VDN treatment alone group, indicating an ameliorative effect on VC. ELISA assays showed that the expression of β-Klotho, a component of FGF21 receptor system, was increased in VDN-treated VC rats by 37.4% (6.588 ± 0.957 pg/mg vs. 9.054 ± 0.963 pg/mg; P < 0.01), indicating an FGF21-resistant state. Moreover, FGF21 treatment downregulated the level of β-Klotho in renal tissue by 16.7% (9.054 ± 0.963 pg/mg vs. 7.544 ± 1.362 pg/mg; P < 0.05). However, the level of FGFR1, the receptor of FGF21, kept unchanged under VDN and VDN plus FGF21 administration (0.191 ± 0.0376 ng/mg vs. 0.189 ± 0.032 ng/mg vs. 0.181 ± 0.034 ng/mg; P > 0.05). Conclusions: In the present study, FGF21 was observed to ameliorate the kidney injury in VDN-induced VC rats. FGF21 might be a potential therapeutic factor in CKD by cutting off the vicious circle between VC and kidney injury.

Lysophosphatidate (LPA) is emerging as a potent mediator of cancer progression in the tumor microenvironment. Strategies for targeting LPA signaling have recently entered clinical trials for fibrosis. These therapies have potential to improve the efficacies of existing chemotherapies and radiotherapy by attenuating chronic inflammation, irrespective of diverse mutations within cancer cells.

There are no published results focusing on the study of hardy kiwifruit as a supplementation to the atherogenic diet. We hypothesized that hardy kiwifruit (Actinidia arguta (A. arguta)) from Poland possess better pro-healthy action than two Asian varieties (Hayward and Bidan). We tested this hypothesis by measuring the metabolic reactions of rats loaded with 1% cholesterol and supplemented with 5% of hardy kiwifruit (A. arguta), Hayward, or Bidan in their diets. The experiment was performed on 71 male Wistar rats. Cholesterol showed a significant impact on the rise of liver somatic index, while lipid profile improved by decreasing the levels of TC, LDL-C, TC/HDL-C, AI, TG, and increasing HDL-C in the serum of rats (P

A novel extracellular alkaline phosphatase/phosphodiesterase from the structural protein family PhoD that encoded by the genome sequence of the marine bacterium Cobetia amphilecti KMM 296 (CamPhoD) has been expressed in Escherichia coli cells. The calculated molecular weight, the number of amino acids, and the isoelectric point (pI) of the mature protein’s subunit are equal to 54832.98 Da, 492, and 5.08, respectively. The salt-tolerant, bimetal-dependent enzyme CamPhoD has a molecular weight of approximately 110 kDa in its native state. CamPhoD is activated by Co2+, Mg2+, Ca2+, or Fe3+ at a concentration of 2 mM and exhibits maximum activity in the presence of both Co2+ and Fe3+ ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn2+, Cu2+, and Mn2+, as well as chelating agents EDTA and EGTA, do not have an appreciable effect on the CamPhoD activity. The temperature optimum for the CamPhoD activity is 45 °C. The enzyme catalyzes the cleavage of phosphate mono- and diester bonds in nucleotides, releasing inorganic phosphorus from p-nitrophenyl phosphate (pNPP) and guanosine 5′-triphosphate (GTP), as determined by the Chen method, with rate approximately 150- and 250-fold higher than those of bis-pNPP and 5′-pNP-TMP, respectively. The Michaelis–Menten constant (Km), Vmax, and efficiency (kcat/Km) of CamPhoD were 4.2 mM, 0.203 mM/min, and 7988.6 S−1/mM; and 6.71 mM, 0.023 mM/min, and 1133.0 S−1/mM for pNPP and bis-pNPP as the chromogenic substrates, respectively. Among the 3D structures currently available, in this study we found only the low identical structure of the Bacillus subtilis enzyme as a homologous template for modeling CamPhoD, with a new architecture of the phosphatase active site containing Fe3+ and two Ca2+ ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.

Nucleic acid detection and quantification have been known to be important at various fields, from genetically modified organisms and gene expression to virus detection. For DNA molecules, digital PCR has been developed as an absolute quantification method which is not dependent on external calibrators. While when it comes to RNA molecules, reverse transcription (RT) step must be taken before PCR amplification to obtain cDNA. With different kinds of reverse transcriptase (RTase) and RT reaction conditions being used in laboratory assays, the efficiency of RT process differs a lot which led variety in quantification results of RNA molecules. In this study, we developed HPLC method combined with enzymatic digestion of RNA to nucleotides for quantification of RNA without RT process. This method was metrologically traceable to four nuceloside monophosphate (NMP) Certification Reference Materials of National Institute of Metrology, China (NIMC) for insurance of accuracy. The established method was used to evaluate the reverse transcription digital polymerase chain reaction (RT-dPCR) of three target genes of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) RNA, including open reading frame 1ab (ORF1ab), nucleocapsid protein (N) and envelope protein (E) gene. Three available RT kits had been evaluated and disparities were observed for the RT efficiency varied from 9% to 182%. It is thus demonstrated that HPLC combined with enzymatic digestion could be a useful method to quantify RNA molecules and evaluate RT efficiency. It is suggested that RT process should be optimized and identified in RNA quantification assays., Graphical abstract Image 1

Structure-based DNA modification analysis provides accurate and important information on genomic DNA changes from epigenetic modifications to various DNA lesions. However, genomic DNA strands are often required to be efficiently digested into single nucleosides. It is an arduous task because of the involvement of multiple enzymes with different catalytic acitivities. Here we constructed a three-enzyme cascade capillary monolithic bioreactor that consists of immobilized deoxyribonuclease I (DNase I), snake venom phosphodiesterase (SVP), and alkaline phosphatase (ALPase). By the use of this cascade capillary bioreactor, genomic DNA can be efficiently digested into single nucleosides with an increasing rate of ∼20 folds. The improvement is mainly attributed to dramatically increase enzymatic capacity and activity. With a designed macro-porous structure, genomic DNA of 5-30 Kb (∼1.6-10 million Daltons) can be directly passed through the bioreactor simply by hand pushing or a low-pressure microinjection pump. By coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further developed a sensitive assay for detection of an oxidative stress biomarker 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in DNA. The proposed three-enzyme cascade bioreactor is also potentially applicable for fast identification and quantitative detection of other lesions and modifications in genomic DNA.

Large-conductance Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) function. We aimed to investigate phosphodiesterase type 1 (PDE1) interactions with BK channels in human DSM to determine the mechanism by which PDE1 regulates human urinary bladder physiology. A combined electrophysiological, functional, and pharmacological approach was applied using human DSM specimens obtained from open bladder surgeries. The perforated whole cell patch-clamp technique was used to record transient BK currents (TBKCs) and the cell membrane potential in freshly isolated human DSM cells in combination with the selective PDE1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX). Isometric DSM tension recordings were used to measure spontaneous phasic and electrical field stimulation-induced contractions in human DSM isolated strips. Selective pharmacological inhibition of PDE1 with 8MM-IBMX (10 μM) increased TBKC activity in human DSM cells, which was abolished by subsequent inhibition of protein kinase A (PKA) with H-89 (10 μM). The stimulatory effect of 8MM-IBMX on TBKCs was reversed upon activation of muscarinic acetylcholine receptors with carbachol (1 μM). 8MM-IBMX (10 μM) hyperpolarized the DSM cell membrane potential, an effect blocked by PKA inhibition. 8MM-IBMX significantly decreased spontaneous phasic and nerve-evoked contractions of human DSM isolated strips. The results reveal a novel mechanism that pharmacological inhibition of PDE1 attenuates human DSM excitability and contractility by activating BK channels via a PKA-dependent mechanism. The data also suggest interactions between PDE1 and muscarinic signaling pathways in human DSM. Inhibition of PDE1 can be a novel therapeutic approach for the treatment of overactive bladder associated with detrusor overactivity.

We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca(2+)]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca(2+) infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 µM) and SKF-96365 (20 µM) signifi cantly inhibited K49-PLA2-induced [Ca(2+)]i increase. These results suggest that K49-PLA2 modulates [Ca(2+)]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.

Adenosine triphosphate (ATP) probes modified with fluorescence dyes that change their fluorescence properties upon cleavage are an interesting tool for monitoring enzymatic ATP turnover. As a readout parameter, fluorescence lifetime is attractive because it is nearly independent of concentration. In our study, we synthesised and investigated fifteen different ATP analogues, in which the fluorophores were attached to the γ-phosphate of ATP. All analogues showed distinctly different fluorescence lifetimes compared to the corresponding values of the free fluorophores. Both increases and decreases in fluorescence lifetime were observed upon attachment to ATP. To shed light on the photophysical processes governing the lifetime changes, we performed photoelectron spectroscopy in air (PESA) to determine HOMO energy levels and time-resolved fluorescence spectroscopy to obtain rate constants. We present evidence that fluorescence quenching in the compounds tested is dynamic and attributed to photoinduced electron transfer (PET), whereas fluorescence lifetime increases are caused by stacking interactions between chromophore and the nucleobase reducing non-radiative relaxation. Finally, we demonstrate that enzymatic cleavage of the ATP analogues presented can be followed by continuous monitoring of fluorescence lifetime changes. published

Background Messenger RNA (mRNA) represents a small percentage of RNAs in a cell, with ribosomal RNA (rRNA) making up the bulk of it. To isolate mRNA from eukaryotes, typically poly-A selection is carried out. Recently, a 5´-phosphate-dependent, 5´→3´ processive exonuclease called Terminator has become available. It will digest only RNA that has a 5´-monophosphate end and therefore it is very useful to eliminate most of rRNAs in cell. Results We have found that in the pathogenic yeast Candida albicans, while 18S and 25S components isolated from yeast in robust growth phase are easily eliminated by Terminator, those isolated from cells in the nutritionally diminished stationary phase, become resistant to digestion by this enzyme. Additional digestions with alkaline phosphatase, tobacco pyrophosphatase combined with Terminator point toward the 5′-prime end of 18S and 25S as the source of this resistance. Inhibition of TOR by rapamycin also induces resistance by these molecules. We also find that these molecules are incorporated into the ribosome and are not just produced incidentally. Finally, we show that three other yeasts show the same behavior. Conclusions Digestion of RNA by Terminator has revealed 18S and 25S rRNA molecules different from the accepted processed ones seen in ribosome generation. The reason for these molecules and the underlying mechanism for their formation is unknown. The preservation of this behavior across these yeasts suggests a useful biological role for it, worthy of further inquiry.