Development Of Gamma-Modified Atp Analogs To Study Kinase-Catalyzed Phosphorylations

Kinase-catalyzed protein phosphorylation is one of the most important post-translational modifications that controls cascades of biochemical reactions. Irregularities in phosphorylation result in many diseases, such as diabetes mellitus, Parkinsons, and cancer. The development of new methods to monitor kinase-catalyzed phosphorylation is needed to decipher details of normal and diseased cell signaling. The Pflum lab recently developed several -modified ATP analogs to study kinase catalyzed phosphorylation reactions. The -modified ATP analogs have different tags, such as biotin for substrate labeling or aryl-azide for kinase substrates identification. Unfortunately, use of -modified ATP analogs was limited to in vitro studies due to the cell impermeability of ATP analogs. Here, we report the first cell permeable ATP analog compatible with kinase-catalyzed labeling. Cell permeable ATP-biotin showed in vitro protein labeling similar to the previously reported ATP-biotin. Importantly, biotin labeling of kinase substrates in living cells was also observed. Also, we report an alternative method to permeabilize ATP analogs in cases where synthesis of cell permeable ATP analog is not feasible. Permeabilizing ATP analogs will aid in monitoring kinase-catalyzed phosphorylation in living cells, which will enhance studies on cell signaling cascades and disease formation. Furthermore, we developed new crosslinking ATP analogs to identify substrates to kinases. We report affinity-based crosslinking ATP analogs, ATP-acrylamides. ATP-acrylamides crosslinked cysteine containing kinases to their substrates through specific proximal cysteine residues on kinases. ATP-acrylamides will solve problems accompanied with the previously reported ATP-Ar-azide that suffer from nonspecific crosslinking due to the very reactive crosslinking azide group. ATP-acrylamides will provide the details of cell signaling of specific kinases, which is supporting biomedical studies. Lastly, we are studying the effect of different bonds between the -phosphate and the attached group on kinase cosubstrate promiscuity phenomenon. This study revealed the electronic effect of several atoms on the ability of ATP-analogs to act as kinase-cosubstrates. Understanding the factors governing the ability of ATP analogs to act as cosubstrate will lead to development of more suitable ATP analogs for kinase catalyzed phosphorylation studies

Subtraction images: A really helpful tool in non-vascular MRI

AbstractBackgroundSubtraction imaging is a technique whereby an unenhanced T1-weighted sequence is digitally subtracted from the identical sequence performed after gadolinium administration.Aim of the workThis study will highlight the role of subtraction imaging for non-vascular MRI applications.Subjects and methodsThe study included 40 patients presenting with lesions in different parts of the body, that are initially hyperintense on T1W sequences.We used post-processing software (Osirix) to digitally subtract the pre-enhancement from the post enhancement sequences.ResultsBased on subtraction imaging findings 5 patients were diagnosed with intraocular melanomas, two patients were diagnosed with hemorrhagic cysts in the masseter muscle, two patients were diagnosed with hemorrhagic cysts in the parotid gland, two patients were diagnosed with dysplastic hepatic nodules, 5 patients were diagnosed with post-ablation necrosis following hepatocellular carcinoma (HCC) treatment, two patients showed recurrent HCC after chemoembolisation, 3 patients showed chemoembolised HCC without recurrence, 4 patients showed intraluminal gall bladder sludge, 4 patients showed hemorrhagic renal cysts, one patient showed solid papillary neoplasm of the pancreas, 6 patients showed chocolate ovarian cysts, two patient showed ovarian cystadenoma and two patients showed ovarian cystadenocarcinoma.ConclusionSubtraction MRI is very helpful tool in non-vascular MRI applications

Understanding the Molecular Mechanisms Underlying \u3cem\u3eRsv1\u3c/em\u3e Mediated Resistance to SMV in Soybean

Like humans, viral diseases also affect plants. Of these, viruses belonging to the potyvirus genus are the most prolific. The potyvirus soybean mosaic virus (SMV) is an important pathogen of the crop plant soybean. SMV causes mosaic symptoms (yellow areas alternate with dark green areas on the leaves of the plant) and can affect yield by reducing seed quality. Few cultivars from soybean can resist different SMV strains. To understand soybean defense mechanisms to SMV, I identified soybean proteins that interact with the helper component protease (HC-Pro) of SMV, which also functions as the suppressor of host RNA silencing and thereby contributes to viral virulence. A genome wide yeast two hybrid screen identified two HC-Pro interactors; BRI1-associated receptor kinase 1 (BAK1) and ubiquitin conjugating enzyme 2 (UBC2). Interactions with HC-Pro were confirmed using bimolecular florescence complementation (BiFC), and co-immunoprecipitations (Co-IP) assays. HC-Pro showed co-localization with both BAK1 and UBC2 in planta. Six isoforms of BAK1 were identified in soybean (BAK1 a, b, c, d, e, and f). Functional analysis showed that silencing the gene encoding BAK1a resulted in breakdown of resistance derived from the resistance (R) locus Rsv1, against SMV. Consistent with the fact that BAK1 is well known regulator of plant basal immunity, soybean plants silenced for BAK1 exhibited enhanced susceptibility to the bacterial pathogen Pseudomonas syringae. BAK1, a receptor-like kinase, functions as a co-receptor in plant defense signaling as well as brassinosteroid-derived signaling during plant growth. My data indicates that HC-Pro is phosphorylated in the presence of BAK1 and this requires the T341 residue which regulates virus avirulence in Rsv1 plants. This is an important finding because although BAK1 is well known to phosphorylate BRI1 and other defense-related receptors, its involvement in phosphorylating pathogen-derived proteins has not been reported. My work raises the possibility that BAK1-derived phosphorylation of HC-Pro may be important to trigger Rsv1-mediated resistance against SMV

64 MS-CTU: Review of techniques and spectrum of the ureteric diseases

Objective: The study aims to clarify the sensitivity of the CTU, and if is it coast effective and time effective to be used as first and the one-stop shop imaging modality for the diagnosis of the different ureteric diseases.Patients and methods: 400 patients with different urinary tract complaints (hematuria and/or renal colic) did triphasic CTU examinations, for diagnosis of suspected obstructive or traumatic ureteric uropathy from January 2014 to October 2016. These patients were filtered from a larger number of patients – who were presented with urinary tract complaints by plain KUB X ray and US, which showed no explaining kidneys or urinary bladder pathology.Results: Ureteric duplication was detected in 5 (1.25%) patients, ectopic ureter in one patient (0.25%), UPJ stricture in 4 patients (1%), PUJ vascular impression in 2 patients (0.5%), ureteric calculus in 103 patients (25.75%), pyogenic ureteritis in 8 patients (2%), ureteritis cystica in one patient (0.25%) TCC in 3 patients (0.75%), PRPF in one patient (0.025%) and Trauma in one patient (0.025%).Conclusion: CTU is very sensitive tool of imaging and could be confidently considered the one-stop shop imaging tool for accurate diagnosis of the different ureteric lesions

Enhancing Exports through Managing Logistics Performance: Evidence From Middle East and African Countries

Logistics activities and its cost affect daily businesses operations and eventually the country’s economy. If countries are able to manage logistics performance efficiently it will be able to compete better internationally. The key objective of this study is to investigate the relationship between the logistics performance index and its components and exports focusing on Middle East and African countries using a regression model. Results of this study indicated that the logistics performance index positively enhance countries exports. In addition, logistics performance index components namely; customs, infrastructure, international shipments and timeliness have a significant impact individually, except for logistics quality, competence, tracking and tracing. The paper aims at supporting the economic policy maker to improve the economic decision in the field of logistics