Construction of plasmids for expression of genes in the isoprenoid biosynthetic pathway

honors thesisCollege of ScienceChemistryC. Dale PoulterThomas G. RichmondThe isoprenoid biosynthetic pathway is present in some form in all organisms. Two essential enzymes in the pathway are isopentenyl diphosphate (IPP) isomerase and farnesyl diphosphate (FPP) synthase. IPP isomerase catalyzes an essential activation step in the pathway. FPP synthase catalyzes the synthesis of FPP, which is a key intermediate that serves as a substrate for many important branch-point enzymes in the pathway. The purpose of this project was to construct a synthetic operon containing the IPP isomerase gene and the FPP synthase gene. Translational coupling of the two genes was accomplished by inserting bases in the operon which link genes in the tryptophan operon in E. coli. With such an operon, IPP can quickly be converted to FPP. The operon was constructed by cutting the two genes out of plasmids and inserting them both in the same plasmid with the bases of the tryptophan operon between the genes. The plasmid was inserted into E. coli, and the enzymes encoded by the genes were produced. The activity of the enzymes was measured by adding IPP and determining how much FPP was produced. The operon was successfully constructed, and thus FPP can quickly be synthesized from IPP. Additionally, a fusion protein containing IPP isomerase and FPP synthase will be made from one of the plasmids containing the operon

Squamous Cell Carcinoma of the Thyroid as a Result of Anaplastic Transformation from BRAF-Positive Papillary Thyroid Cancer

Papillary thyroid carcinoma (PTC) is the most common malignant neoplasm of the thyroid. Majority of the PTC carries an excellent prognosis. However, patients with tall cell variant (TCV) of papillary thyroid carcinoma have a worse prognosis than those with the classic variant. On the other hand, squamous cell carcinoma of the thyroid (SCT) is an unusual neoplasm thought to arise as a primary tumor or as a component of an anaplastic or undifferentiated carcinoma. We report a patient with TCV of PTC presenting years later with squamous transformation. In addition, the patient was found to have BRAF mutation. Such dedifferentiation is considered to be a rare phenomenon and has been reported only in the form of case reports in the literature. The relationship between BRAFV600E mutation and squamous cell transformation of papillary thyroid cancer is unknown at this time. Meticulous pathology is needed to identify such variants. Our patient responded to treatment with concurrent chemotherapy with carboplatin and paclitaxel along with radiation

Extraosseous Intradural Chondrosarcoma of the Cervical Spine: A Case Report with Brief Review of Literature

Mesenchymal chondrosarcoma (MCS) is a malignant cancer of the cartilage that accounts for less than 1% of all chondrosarcomas and typically occurs within the bone. One-third of all mesenchymal chondrosarcomas are extraosseous soft tissue sarcomas, rendering this as an uncommon entity. We report a rare case of an extraosseous chondrosarcoma with the cervical spinal canal in a 21-year-old male. The purpose of this case report is to discuss the imaging characteristics of this pathology proven diagnosis

Posterior fossa medulloblastoma in an atypical extra-axial location: A case report

Medulloblastoma is the most common posterior fossa tumor of childhood typically within the fourth ventricle. However, extra-axial medulloblastoma in posterior fossa is an uncommon diagnosis. We report a case in a 33-month-old male who presented with repeated complaints of abdominal pain, intermittent emesis, and diarrhea, and diagnosed with right cerebellar extra-axial medulloblastoma, which was surgically resected. Majority of the reported extra-axial medulloblastoma in posterior fossa in the United States are located in the cerebellopontine angle. However, to the best of our knowledge, our case is the first to document medulloblastoma occurring exclusively in the cerebellar hemispheric extra-axial space rather than the cerebellopontine angle. Although the diagnosis can present as a radiological dilemma, a systematic multimodality imaging approach can aid in narrowing the differential diagnosis and timely management. In this case report, we will discuss the imaging characteristics, differential diagnosis, and management strategies, alongside a brief review of the world literature of extra-axial medulloblastoma. Keywords: Medulloblastoma, Pediatric neoplasm, Posterior fossa tumors, Extra-axia

Final LDRD report : enhanced spontaneous emission rate in visible III-nitride LEDs using 3D photonic crystal cavities.

The fundamental spontaneous emission rate for a photon source can be modified by placing the emitter inside a periodic dielectric structure allowing the emission to be dramatically enhanced or suppressed depending on the intended application. We have investigated the relatively unexplored realm of interaction between semiconductor emitters and three dimensional photonic crystals in the visible spectrum. Although this interaction has been investigated at longer wavelengths, very little work has been done in the visible spectrum. During the course of this LDRD, we have fabricated TiO{sub 2} logpile photonic crystal structures with the shortest wavelength band gap ever demonstrated. A variety of different emitters with emission between 365 nm and 700 nm were incorporated into photonic crystal structures. Time-integrated and time-resolved photoluminescence measurements were performed to measure changes to the spontaneous emission rate. Both enhanced and suppressed emission were demonstrated and attributed to changes to the photonic density of states

Neuronally produced versican V2 renders C-fiber nociceptors IB 4

A subpopulation of nociceptors, the glial cell-line derived neurotrophic factor (GDNF)-dependent, non-peptidergic C-fibers, express a cell-surface glycoconjugate that can be selectively labeled with isolectin B4 (IB(4)), a homotetrameric plant lectin from Griffonia simplicifolia. We show that versican is an IB(4)-binding molecule in rat dorsal root ganglion (DRG) neurons. Using reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and immunofluorescence experiments on rat lumbar DRG, we provide the first demonstration that versican is produced by neurons. In addition, by probing Western blots with splice variant specific antibodies we show that the IB(4)-binding versican contains only the glycosaminoglycan alpha (α GAG) domain. Our data support V2 as the versican isoform that renders this subpopulation of nociceptors IB(4)-positive (+)

RNA-Seq Analysis of IL-1B and IL-36 Responses in Epidermal Keratinocytes Identifies a Shared MyD88-Dependent Gene Signature

IL-36 cytokines have recently emerged as mediators of inflammation in autoimmune conditions including psoriasis vulgaris (PsV) and generalized pustular psoriasis (GPP). This study used RNA-seq to profile the transcriptome of primary epidermal keratinocytes (KCs) treated with IL-1B, IL-36A, IL-36B, or IL-36G. We identified some early IL-1B-specific responses (8 h posttreatment), but nearly all late IL-1B responses were replicated by IL-36 cytokines (24 h posttreatment). Type I and II interferon genes exhibited time-dependent response patterns, with early induction (8 h) followed by no response or repression (24 h). Altogether, we identified 225 differentially expressed genes (DEGs) with shared responses to all 4 cytokines at both time points (8 and 24 h). These involved upregulation of ligands (IL1A, IL1B, and IL36G) and activating proteases (CTSS) but also upregulation of inhibitors such as IL1RN and IL36RN. Shared IL-1B/IL-36 DEGs overlapped significantly with genes altered in PsV and GPP skin lesions, as well as genes near GWAS loci linked to autoimmune and autoinflammatory diseases (e.g., PsV, psoriatic arthritis, inflammatory bowel disease, and primary biliary cholangitis). Inactivation of MyD88 adapter protein using CRISPR/Cas9 completely abolished expression responses of such DEGs to IL-1B and IL-36G stimulation. These results provide a global view of IL-1B and IL-36 expression responses in epidermal KCs with fine-scale characterization of time-dependent and cytokine-specific response patterns. Our findings support an important role for IL-1B and IL-36 in autoimmune or autoinflammatory conditions and show that MyD88 adaptor protein mediates shared IL-1B/IL-36 responses

Aberrant detergent-insoluble excitatory amino acid transporter 2 accumulates in alzheimer disease

Alzheimer disease (AD) is characterized by deposition of amyloid-β, tau, and other specific proteins that accumulate in the brain in detergent-insoluble complexes. Alzheimer disease also involves glutamatergic neurotransmitter system disturbances. Excitatory amino acid transporter 2 (EAAT2) is the dominant glutamate transporter in cerebral cortex and hippocampus. We investigated whether accumulation of detergent-insoluble EAAT2 is related to cognitive impairment and neuropathologic changes in AD by quantifying detergent-insoluble EAAT2 levels in hippocampus and frontal cortex of cognitively normal patients, patients with clinical dementia rating of 0.5 (mildly impaired), and AD patients. Parkinson disease patients served as neurodegenerative disease controls. We found that Triton X-100-insoluble EAAT2 levels were significantly increased in patients withAD compared with controls, whereas Triton X-100-insoluble EAAT2 levels inpatients with clinical dementia rating of 0.5 were intermediately elevatedbetween control and AD subjects. Detergentinsolubility of presenilin-1, a structurally similar protein, did not differ among the groups, thus arguing that EAAT2 detergent insolubility was not causedby nonspecific cellular injury. These findings demonstrate that detergent-insoluble EAAT2 accumulation is a progressive biochemical lesion that correlates with cognitive impairment and neuropathologic changes in AD. These findings lend further support to the idea that dysregulationof the glutamatergic system may play a significant role in AD pathogenesis. © 2010 by the American Association of Neuropathologists, Inc

Cytokine responses in nonlesional psoriatic skin as clinical predictor to anti-TNF agents

BackgroundA major issue with the current management of psoriasis is our inability to predict treatment response.ObjectiveOur aim was to evaluate the ability to use baseline molecular expression profiling to assess treatment outcome for patients with psoriasis.MethodsWe conducted a longitudinal study of 46 patients with chronic plaque psoriasis treated with anti-TNF agent etanercept, and molecular profiles were assessed in more than 200 RNA-seq samples.ResultsWe demonstrated correlation between clinical response and molecular changes during the course of the treatment, particularly for genes responding to IL-17A/TNF in keratinocytes. Intriguingly, baseline gene expressions in nonlesional, but not lesional, skin were the best marker of treatment response at week 12. We identified USP18, a known regulator of IFN responses, as positively correlated with Psoriasis Area and Severity Index (PASI) improvement (P = 9.8 × 10-4) and demonstrate its role in regulating IFN/TNF responses in keratinocytes. Consistently, cytokine gene signatures enriched in baseline nonlesional skin expression profiles had strong correlations with PASI improvement. Using this information, we developed a statistical model for predicting PASI75 (ie, 75% of PASI improvement) at week 12, achieving area under the receiver-operating characteristic curve value of 0.75 and up to 80% accurate PASI75 prediction among the top predicted responders.ConclusionsOur results illustrate feasibility of assessing drug response in psoriasis using nonlesional skin and implicate involvement of IFN regulators in anti-TNF responses