Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites

BACKGROUND: Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. RESULTS: To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. CONCLUSION: The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics

Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization

(GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. Conclusion: 14-3-3 proteins can potentially engage around 0.6 % of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been 1Samuel Lunenfeld Research Institute validated by the broad inhibition of 14-3-3 phosphoryla-Mount Sinai Hospital tion-dependent binding in vivo and by the specific analy-600 University Avenue sis of AKAP-Lbc, a RhoGEF that is controlled by it

Broadband unidirectional scattering and improved refractive index sensitivity in all-dielectric core-shell nanoantennae

by Sarang Anant KulkarniM.Tech

Enantioselective synthesis of largazole, development of multicomponent reactions, and design and synthesis of novel HIV -1 protease inhibitors

The dissertation describes the total synthesis of (+)-largazole, the development of multicomponent reactions, and the design and synthesis of novel HIV-1 protease inhibitors incorporating macrocyclic P1\u27-P 2\u27 ligands. Chapter 1 of the dissertation details the total synthesis of (+)-largazole. Largazole is a highly potent and selective antiproliferative agent. The synthesis involves a lipase catalyzed resolution of β-hydroxy ester, a biomimetic formation of thiazoline-thiazole fragment, a cross metathesis reaction. Two equally dense fragments were assembled by a HATU/HOAt coupling. The final macrolactamization was achieved by a HATU/HOAt macrocyclization. The synthesis also features chemical modifications in presence of sensitive thioester functionality. Chapter 2 of the dissertation describes the development of asymmetric multicomponent reactions. A novel asymmetric three component reaction using optically active phenyldihydrofuran, α-imino ester or β-keto ester and a suitable nucleophile is described. The reaction is highly efficient and leads to formation of a densely functionalized tetrahydrofuran with formation of two new C-C bonds and establishes three new chiral centers in a stereoselective fashion in a single step. An unexpected temperature dependent rearrangement of the furans to pyrrolidines has been discussed. An application of this methodology in synthesis of functionalized cyclopentenes is described. Chapter 3 of the dissertation describes the design and synthesis of HIV-1 protease inhibitors containing a macrocycle as a P1\u27-P2\u27 ligand. The macrocyclic inhibitors were synthesized from dienes by ring closing metathesis. A number of ring sizes (9- to 15-membered) were synthesized. The effect of the ring size and unsaturation within the macrocyclic ring on the biological activity was studied. The enzyme inhibitory and cellular potency of the acyclic and cyclic unsaturated and saturated macrocycles were compared

Note on Breeding of Andaman Teal Anas Gibberifrons in South Andaman Islands, India

Volume: 100Start Page: 112End Page: 11

Clip-on wireless wearable microwave sensor for ambulatory cardiac monitoring

We present a new type of non-contact sensor for use in ambulatory cardiac monitoring. The sensor operation is based on a microwave Doppler technique; however, instead of detecting the heart activity from a distance, the sensor is placed on the patient's chest over the clothing. The microwave sensor directly measures heart movement rather than electrical activity, and is thus complementary to ECG. The primary advantages of the microwave sensor includes small size, light weight, low power, low-cost, and the ability to operate through clothing. We present a sample sensor design that incorporates a 2.4 GHz Doppler circuit, integrated microstrip patch antenna, and microntroller with 12-bit ADC data sampling. The prototype sensor also includes a wireless data link for sending data to a remote PC or mobile phone. Sample data is shown for several subjects and compared to data from a commercial portable ECG device. Data collected from the microwave sensor exhibits a significant amount of features, indicating possible use as a tool for monitoring heart mechanics and detection of abnormalities such as fibrillation and akinesia.MIT Media Lab Consortiu

Wearable Doppler radar with integrated antenna for patient vital sign monitoring

A 2.45 GHz wearable Doppler radar unit with radio data link is presented for use in portable patient monitoring and emergency response. Unlike portable Electrocardiograms (ECG) or Photoplethysmography (PPG), the near-field Doppler unit enables monitoring of the person's heart rate without the need for electrical contact or optical access to the patient's skin. The Doppler unit is designed to be embedded in a clothing garment such as a shirt or vest, or used by medical emergency personal in an instrumented blanket or medical stretcher. Since the Doppler unit is placed directly on or behind the patient's torso, the extraneous signals due to relative motion artifacts is greatly reduced. Low-cost design is achieved by employing PWB microstrip elements for the integrated patch antenna, microwave oscillator, and tuning elements. Also, since the distance between the Doppler unit and the patient is fixed, it was possible to tune the detection phase to enable the use of a single mixer diode and eliminate the need for quadrature detection. Measured heart data from this technique shows clear waveform substructure similar to PQRST complex features found in captured ECG data

Refractive-index sensing using hybrid all-dielectric nanoantennae

by Sarang Kulkarni Anant and Ravi Hegd