Substrate Specificity of SAMHD1 Triphosphohydrolase Activity Is Controlled by Deoxyribonucleoside Triphosphates and Phosphorylation at Thr592

The sterile alpha motif (SAM) and histidine-aspartate (HD) domain containing protein 1 (SAMHD1) constitute a triphosphohydrolase that converts deoxyribonucleoside triphosphates (dNTPs) into deoxyribonucleosides and triphosphates. SAMHD1 exists in multiple states. The monomer and apo- or GTP-bound dimer are catalytically inactive. Binding of dNTP at allosteric site 2 (AS2), adjacent to GTP-binding allosteric site 1 (AS1), induces formation of the tetramer, the catalytically active form. We have developed an enzyme kinetic assay, tailored to control specific dNTP binding at each site, allowing us to determine the kinetic binding parameters of individual dNTPs at both the AS2 and catalytic sites for all possible combinations of dNTP binding at both sites. Here, we show that the apparent <i>K</i>_m values of dNTPs at AS2 vary in the order of dCTP < dGTP < dATP < dTTP. Interestingly, dCTP binding at AS2 significantly reduces the dCTP hydrolysis rate, which is restored to a rate comparable to that of other dNTPs upon dGTP, dATP, or dTTP binding at AS2. Strikingly, a phosphomimetic mutant, Thr592Asp SAMHD1 as well as phospho-Thr592, show a significantly altered substrate specificity, with the rate of dCTP hydrolysis being selectively reduced regardless of which dNTP binds at AS2. Furthermore, cyclin A2 binding at the C-terminus of SAMHD1 induces the disassembly of the SAMHD1 tetramer, suggesting an additional layer of SAMHD1 activity modulation by cyclin A2/CDK2 kinase. Together, our results reveal multiple allosteric mechanisms for controlling the rate of dNTP destruction by SAMHD1

An Optical Biosensor-Based Quantification of the Microcystin Synthetase A Gene: Early Warning of Toxic Cyanobacterial Blooming

The monitoring and control of toxic cyanobacterial strains, which can produce microcystins, is critical to protect human and ecological health. We herein reported an optical-biosensor-based quantification of the microcystin synthetase A (mcyA) gene so as to discriminate microcystin-producing strains from nonproducing strains. In this assay, the mcyA-specific ssDNA probes were designed in silico with an on-line tool and then synthesized to be covalently immobilized on an optical-fiber surface. Production of fluorescently modified target DNA fragment amplicons was accomplished through the use of Cy5-tagged deoxycytidine triphosphates (dCTPs) in the polymerase chain reaction (PCR) method, which resulted in copies with internally labeled multiple sites per DNA molecule and delivered great sensitivity. With a facile surface-based hybridization process, the PCR amplicons were captured on the optical-fiber surface and were induced by an evanescent-wave field into fluorescence emission. Under the optimum conditions, the detection limit was found to be 10 pM (S/N ratio = 3) and equaled 10³ gene copies/mL. The assay was triumphantly demonstrated for PCR amplicons of mcyA detection and showed satisfactory stability and reproducibility. Moreover, the sensing system exhibited excellent selectivity with quantitative spike recoveries from 87 to 102% for <i>M. aeruginosa</i> species in the mixed samples. There results confirmed that the method would serve as an accurate, cost-effective, and rapid technique for in-field testing of toxic <i>Microcystis</i> sp. in water, giving early information for water quality monitoring against microcystin-producing cyanobacteria

Table1_Recruitment of the cardiac conduction system for optimal resynchronization therapy in failing heart.pdf

Heart failure (HF) is a leading health burden around the world. Although pharmacological development has dramatically advanced medication therapy in the field, hemodynamic disorders or mechanical desynchrony deteriorated by intra or interventricular conduction abnormalities remains a critical target beyond the scope of pharmacotherapy. In the past 2 decades, nonpharmacologic treatment for heart failure, such as cardiac resynchronization therapy (CRT) via biventricular pacing (BVP), has been playing an important role in improving the prognosis of heart failure. However, the response rate of BVP-CRT is variable, leaving one-third of patients not benefiting from the therapy as expected. Considering the non-physiological activation pattern of BVP-CRT, more efforts have been made to optimize resynchronization. The most extensively investigated approach is by stimulating the native conduction system, e.g., His-Purkinje conduction system pacing (CSP), including His bundle pacing (HBP) and left bundle branch area pacing (LBBAP). These emerging CRT approaches provide an alternative to traditional BVP-CRT, with multiple proof-of-concept studies indicating the safety and efficacy of its utilization in dyssynchronous heart failure. In this review, we summarize the mechanisms of dyssynchronous HF mediated by conduction disturbance, the rationale and acute effect of CSP for CRT, the recent advancement in clinical research, and possible future directions of CSP.</p

Free-Energy-Driven Lock/Open Assembly-Based Optical DNA Sensor for Cancer-Related microRNA Detection with a Shortened Time-to-Result

Quantification of cancer biomarker microRNAs (miRs) by exquisitely designed biosensors with a short time-to-result is of great clinical significance. With immobilized capture probes (CPs) and fluorescent-labeled signal probes (SPs), surface-involved sandwich-type (SST) biosensors serve as powerful tools for rapid, highly sensitive, and selective detection of miR in complex matrices as opposed to the conventional techniques. One key challenge for such SST biosensors is the existence of false-negative signals when the amount of miRs exceeds SPs in solution phase for a surface with a limited number of CP. To meet this challenge, a dynamic lock/open DNA assembly was designed to rationally program the pathway for miR/SP hybrids. Based on secondary structure analysis and free-energy assessment, a “locker” strand that partially hybridizes with target miR by two separated short arms was designed to stabilize target miR, preventing possible false-negative signals. The strategy was demonstrated on a fiber-based fluorescent DNA-sensing platform. CP/miR/SP sandwiches formed on the fiber surface would generate fluorescent signals for quantitative analysis. The developed SST biosensor was able to detect miR Hsa <i>let-7a</i> with a detection limit of 24 pM. The applicability of this free-energy-driven lock/open assembly-based optical DNA sensor was further confirmed with spiked human urine and serum samples

Empty adenoviral capsid is sufficient to induce keratitis.

<p>(A) Silver stained polyacrylamide gel of proteins from intact HAdV-37 (V) or empty capsid (EC). First lane (M) shows protein standards. Arrows on the right point to capsid proteins missing from the empty capsid; capsid proteins V and VII are marked by the second and fourth arrows from the top, respectively. (B) Mouse cornea injected with Cy3 dye-labeled empty capsid (EC). Intracellular virus position was visualized with confocal microscopy at 90 min pi (n = 3 corneas). Red: Cy3-labeled empty capsid. Green: intracellular actin (phalloidin stain). Blue: nuclei (TO-PRO3 stain). Scale bar 20 µM. (C) Clinical appearance and (D) histopathology of mouse corneas at 4 dpi. Corneas were injected with virus free buffer (M), intact virus (V), or empty capsid (EC) (n = 5 mice/group).</p

Adenoviral genomic DNA is not sufficient to induce keratitis in mice.

<p>(A) Confocal microscopy of mouse corneal stroma at 1 day after mock treatment with transfection reagent alone (M), HAdV-5 vector expressing eGFP (V), or plasmid vector EGFP-C1 (90 or 500 ng DNA). Photographs are representative of three corneas in each group. Scale bar, 200 µM. (B) Flow cytometric analysis of corneas at 1 day after injection with transfection reagent alone (M), plasmid vector (90 or 500 ng DNA) and transfection reagent, or HAdV-5 (V) vector expressing eGFP. Numbers in histograms denote percentage of total cells expressing eGFP. (C and D) C57BL/6J mouse corneas were injected with virus free buffer (M), HAdV-37 (V) or 90 ng and 500 ng of HAdV-37 genomic DNA with transfection reagent and observed up to 4 dpi. Representative photographs (C) and histopathology sections (D) of corneas at 4 dpi are shown (n = 5 mice/group).</p

Empty viral capsid induces chemokine expression and infiltration of leukocytes into the cornea.

<p>(A) Infiltrating leukocytes were quantified using flow cytometry in corneas 4 days after injection with virus free buffer (M), intact HAdV-37 (V), or empty viral capsid (EC) (n = 6 corneas/group). Data represents the mean of three separate experiments, and error bars denote SD. (B) Myeloperoxidase (MPO) levels were quantified in mouse corneas 2 days after injection with virus free buffer (M), intact virus (V), or empty capsid (EC) (n = 6 corneas/group). Data represents the mean of two separate experiments, and error bars denote SD. (C–E) Cytokine protein levels as measured by ELISA in corneas 16 hours after injection with virus free buffer (M), intact virus (V), or empty capsid (EC). CXCL1 (C), CCL2 (D), and IL-6 (E) protein levels are shown (n = 9 corneas/group). Data shown represents the mean of three independent experiments, and error bars represent SD. * p<.05, ANOVA.</p

UV-inactivated adenovirus induces leukocyte infiltration and cytokine expression.

<p>(A) Representative dot plots of single cell suspensions prepared from corneas at 4 dpi stained with Gr1 and F4/80 and gated on CD45^high labeled cells. Corneas were infected with virus free buffer (M), or intact (V), UV-inactivated (UV), and heat-inactivated (H) HAdV-37. (B) Quantification of average numbers of Gr1 and F4/80 stained corneal cells in intact (V), UV-inactivated (UV), or heat-inactivated (H) virus injected corneas at 4 dpi (n = 6 mice/group). Data is derived from three separate experiments, and error bars represent SD. (C) Myeloperoxidase (MPO) levels assessed 24 hours post injection with virus free buffer (M), intact virus (V), UV-inactivated virus (UV), or heat-inactivated virus (H) are shown (n = 9 mice/group). Data represents mean of three independent experiments ± SD. (D–F) Cytokine expression in corneas after injection with virus free buffer (M), intact virus (V), UV-inactivated virus (UV), or heat-inactivated virus (H) as measured at 16 hpi by ELISA for CXCL1 (D), CCL2 (E), and IL-6 (F) protein (n = 9 mice/group). Data represents mean of three independent experiments ± SD. * p<.05, ANOVA.</p

Adenoviral genomic DNA induces differential expression of cytokines but does not cause infiltration of leukocytes into the cornea.

<p>(A) Flow cytometric analysis of Gr1 and F4/80 positive cells in mouse corneas at 4 days after injection with virus free buffer (M), virus free buffer and transfection reagent (M+T), intact HAdV-37 (V), intact HAdV-37 and transfection reagent (V+T), and 90 ng or 500 ng of HAdV-37 genomic DNA with transfection reagent (n = 6 corneas/group). Data shown represents the mean of three independent experiments, and error bars represent SD. (B–E) Protein levels of cytokines IL-6 (B), CXCL1 (C), CXCL2 (D) and CCL2 (E) in mouse corneas at 16 hpi. Corneas were injected with virus free buffer (M), virus free buffer and transfection reagent (M+T), intact HAdV-37 (V), intact HAdV-37 and transfection reagent (V+T), and 90 ng or 500 ng of HAdV-37 genomic DNA with transfection reagent (n = 9 corneas/group). Data shown represents the mean of three separate experiments, and error bars denote SD. * p<.05, ANOVA.</p

Viral gene expression is not essential for adenovirus keratitis.

<p>(A) Real-time PCR for the relative expression of viral transcript E1A10S at 4 hpi in mock (M), intact (V), UV-inactivated (UV), or heat-inactivated (H) HAdV-37 infected A549 cells. Data represents mean of three separate experiments ± SD. (B) Mouse corneas injected with Cy3-labeled intact (V), UV- inactivated (UV), or heat-inactivated (H) virus were analyzed by confocal microscopy at 90 min pi (n = 5 corneas/group). Red: Cy3-labeled virus. Green: intracellular actin (phalloidin stain). Blue: nuclei (TO-PRO3 stain). Scale bar 20 µM. (C) Representative photographs and (D) hematoxylin and eosin stained histopathological sections of mice corneas at 4 dpi, infected with virus free buffer (M), intact virus (V), UV-inactivated virus (UV), or heat-inactivated virus (H) (n = 5 mice/group).</p