A recombineering based approach for high-throughput conditional knockout targeting vector construction

Functional analysis of mammalian genes in vivo is primarily achieved through analysing knockout mice. Now that the sequencing of several mammalian genomes has been completed, understanding functions of all the genes represents the next major challenge in the post-genome era. Generation of knockout mutant mice has currently been achieved by many research groups but only by making individual knockouts, one by one. New technological advances and the refinements of existing technologies are critical for genome-wide targeted mutagenesis in the mouse. We describe here new recombineering reagents and protocols that enable recombineering to be carried out in a 96-well format. Consequently, we are able to construct 96 conditional knockout targeting vectors simultaneously. Our new recombineering system makes it a reality to generate large numbers of precisely engineered DNA constructs for functional genomics studies

500 MS/s 4-Bit Flash ADC with Complementary Architecture

This paper proposes a 500 MS/s 4-bit flash analog-to-digital converter (ADC) featuring a differential input voltage range of 1.2 Vpp operating at a supply voltage of 1.2 V. Although the proposed circuit utilizes a conventional flash ADC structure, its track and hold circuit, driving buffer, and preamp circuits corresponding to the analog stages are designed using complementary architecture to attain a sufficient swing range even at a low supply voltage. Notably, the proposed structure satisfies the error requirements. The error source of the flash ADC, such as the comparator’s input referred offset, did not degrade its performance, while the use of a calibration circuit, characterized by power consumption and area burdens and increased complexity, could also be avoided. Therefore, the proposed flash ADC met the error requirements, such as the comparator’s input referred offset, without the need for calibration circuits. The chip, fabricated using the TSMC 65 nm process, covers an area of 1,160 × 950 μm2 and consumes 78 mW of power. Furthermore, its signal-to-noise and distortion ratio and spurious-free dynamic range were measured to be 23.36 dB and 30.26 dB, respectively, at a sampling frequency of 500 MHz

Stress-induced nuclear translocation of CDK5 suppresses neuronal death by downregulating ERK activation via VRK3 phosphorylation

Although extracellular signal-related kinase 1/2 (ERK 1/2) activity is generally associated with cell survival, prolonged ERK activation induced by oxidative stress also mediates neuronal cell death. Here we report that oxidative stress-induced cyclin-dependent kinase 5 (CDK5) activation stimulates neuroprotective signaling via phosphorylation of vaccinia-related kinase 3 (VRK3) at Ser 108. The binding of vaccinia H1-related (VHR) phosphatase to phosphorylated VRK3 increased its affinity for phospho-ERK and subsequently downregulated ERK activation. Overexpression of VRK3 protected human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced apoptosis. However the CDK5 was unable to phosphorylate mutant VRK3, and thus the mutant forms of VRK3 could not attenuate apoptotic process. Suppression of CDK5 activity results in increase of ERK activation and elevation of proapoptotic protein Bak expression in mouse cortical neurons. Results from VRK3-deficient neurons were further confirmed the role of VRK3 phosphorylation in H2O2-evoked ERK regulation. Importantly, we showed an association between phospho-VRK3 levels and the progression of human Alzheimer’s disease (AD) and Parkinson’s disease (PD). Together our work reveals endogenous protective mechanism against oxidative stress-induced neuronal cell death and suggest VRK3 as a potential therapeutic target in neurodegenerative diseases.1186Ysciescopu

The T2-Shortening Effect of Gadolinium and the Optimal Conditions for Maximizing the CNR for Evaluating the Biliary System: a Phantom Study

OBJECTIVE: Clear depiction of the common bile duct is important when evaluating neonatal cholestasis in order to differentiate biliary atresia from other diseases. During MR cholangiopancreatography, the T2-shortening effect of gadolinium can increase the contrast-to-noise ratio (CNR) of the bile duct and enhance its depiction. The purpose of this study was to confirm, by performing a phantom study, the T2-shortening effect of gadolinium, to evaluate the effect of different gadolinium chelates with different gadolinium concentrations and different magnetic field strengths for investigating the optimal combination of these conditions, and for identifying the maximum CNR for the evaluation of the biliary system. MATERIALS AND METHODS: MR imaging using a T2-weighted single-shot fast spin echo sequence and T2 relaxometry was performed with a sponge phantom in a syringe tube. Two kinds of contrast agents (Gd-DTPA and Gd-EOB-DTPA) with different gadolinium concentrations were evaluated with 1.5T and 3T scanners. The signal intensities, the CNRs and the T2 relaxation time were analyzed. RESULTS: The signal intensities significantly decreased as the gadolinium concentrations increased (p < 0.001) with both contrast agents. These signal intensities were higher on a 3T (p < 0.001) scanner. The CNRs were higher on a 1.5T (p < 0.001) scanner and they showed no significant change with different gadolinium concentrations. The T2 relaxation time also showed a negative correlation with the gadolinium concentrations (p < 0.001) and the CNRs showed decrease more with Gd-EOB-DTPA (versus Gd-DTPA; p < 0.001) on a 3T scanner (versus 1.5T; p < 0.001). CONCLUSION: A T2-shortening effect of gadolinium exhibits a negative correlation with the gadolinium concentration for both the signal intensities and the T2 relaxation time. A higher CNR can be obtained with Gd-DTPA on a 1.5T MRI scannerope

Effect of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand on the Reduction of Joint Inflammation in Experimental Rheumatoid Arthritis

ABSTRACT This study focused on the potential therapeutic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on collagen-induced arthritis (CIA) and on the elucidation of the mechanisms involved. DBA/1J mice with established CIA were treated with various amount of recombinant soluble human TRAIL. The effects of TRAIL on the development and severity of CIA in this DBA/1J mouse model were assessed clinically and histologically, and a detailed investigation was conducted on proinflammatory cytokine and anticollagen-specific antibody levels. Cellular immunity was evaluated by investigating the proliferative responses and cytokine release profiles of splenocytes after TRAIL treatment. TRAIL treatment significantly reduced the severity and incidence of CIA, joint swelling, erythema, and edema. Histologic evaluations revealed that inflammatory cell infiltration, cartilage destruction, and bone erosion were significantly reduced in joints of TRAIL-treated mice with dose-dependent manner. TRAIL treatment also strongly decreased and/or normalized the productions of proinflammatory cytokines and of anti-collagen-specific antibodies in the sera of CIA mice. Furthermore, in vitro studies with primary splenocytes showed the cytotoxic effect of TRAIL on activated lymphocytes, with reduction of inflammatory cytokine release. These findings show that TRAIL administration is an effective anti-inflammatory treatment that prevents the development and progression of CIA in DBA/1J mice, and they suggest that TRAIL might be considered a potential treatment for human RA

Recurrent Acute Pancreatitis in a Patient with Type IIb hyperlipoproteinemia: A Case Report and Review of the Literature in Korea

Hyperlipidemia is a rare cause of pancreatitis. It has been believed that free fatty acids released from hydrolyzed serum chylomicrons or triglycerides and chylomicrons induce hyperlipidemic pancreatitis by damaging acinar cells and capillaries. Type I, IV or V hyperlipidemic (Fredrickson's classification) pancreatitides have distinctive features of increased and heightened serum chylomicron and triglyceride levels. In contrast, type IIb hyperlipidemia usually doesn't have increased chylomicrons. It is a dominant inherited genetic disorder and doesn't manifest the subjective symptom before combining vascular complications such as coronary artery disease. Only a few cases of type IIb hyperlipidemic pancreatitis have been reported. We experienced a male patient with recurrent hyperlipidemic pancreatitis combined with type IIb hyperlipidemia. We present the case report and a review of the literature of hyperlipidemic pancreatitis, especially cases in Korea

Rate of Isolation and Trends of Antimicrobial Resistance of Multidrug Resistant Pseudomonas Aeruginosa from Otorrhea in Chronic Suppurative Otitis Media

ObjectivesTo assess the rate of isolation of Pseudomonas aeruginosa (PA) and multidrug-resistant PA (MDR-PA) from patients with chronic suppurative otitis media (CSOM) otorrhea and the annual trend of antibiotic-resistance.MethodsOtorrhea samples were collected aseptically from 1,598 CSOM patients. The rate of bacterial isolation and the results of antibiotic susceptibility testing were evaluated retrospectively.ResultsThe PA isolation rate from CSOM otorrhea was 24.4%. Of the 398 isolated strains tested for their susceptibilities to 10 antibiotics, 395 strains showed definitive results. Of these, 183 (46.3%) were susceptible to whole antibiotics and 212 (53.7%) was resistant to more than 1 antibiotics, with the frequency of antibiotics-resistance increasing significantly over time. Although strains susceptible to all antibiotics decreased over time, the rate of isolation of MDR-PA did not change significantly. Resistance to aminoglycosides and quinolones was higher than to other antibiotics and significantly increased over time, whereas resistance to other antibiotics showed no trend.ConclusionMDR-PA, assessed using five individual antibiotics and six antibiotic-classes, showed no tendency to increase or decrease over time. This may have been due to increased concern about antibiotic-resistant bacterial strains, leading to improved infection control within hospitals and healthcare centers

Amifostine ameliorates recognition memory defect in acute radiation syndrome caused by relatively low-dose of gamma radiation

This study examined whether amifostine (WR-2721) could attenuate memory impairment and suppress hippocampal neurogenesis in adult mice with the relatively low-dose exposure of acute radiation syndrome (ARS). These were assessed using object recognition memory test, the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, and immunohistochemical markers of neurogenesis [Ki-67 and doublecortin (DCX)]. Amifostine treatment (214 mg/kg, i.p.) prior to irradiation significantly attenuated the recognition memory defect in ARS, and markedly blocked the apoptotic death and decrease of Ki-67- and DCX-positive cells in ARS. Therefore, amifostine may attenuate recognition memory defect in a relatively low-dose exposure of ARS in adult mice, possibly by inhibiting a detrimental effect of irradiation on hippocampal neurogenesis