Baculovirus Mediated Expression of Bovine Herpesvirus-1 Glycoprotein-C in Insect Cells

Bovine herpesvirus-1 (BHV-1) glycoprotein C (gC) is an immunodominant protein that is believed to play a role in initial viral attachment. In the present study a recombinant baculovirus was constructed by incorporating BHV-1 gC coding gene to characterize the expression of the glycoprotein in infected Spodoptera frugiperda cells. The BHV-1 complete gC gene was cloned into pENTR/SD/D Directional TOPO vector (entry clone). The purified entry clone plasmid was subjected to LR recombination with the linear baculovirus DNA which was then transfected into S. frugiperda cells. The recombinant baculovirus carrying the gC coding gene was selected against ganciclovirus in the S. frugiperda cells (first passage), serially passaged for two more generations and the third passage viral stock was used to infect fresh S. frugiperda cells for gene expression study. The reactivity of polyclonal antibody with the S. frugiperda cell expressed gC was detected by immunoperoxidase test. The recombinant gC was purified by Ni-NTA column chromatography and immunoprecipitation. Protein bands of molecular weight 54 and 28 kDa were detected consistently with both monoclonal and polyclonal antibodies, when the recombinant protein was subjected to SDS-PAGE and Western blot analysis. Expression of gC gene in the infected S. frugiperda cells was further confirmed by dot-ELISA indicating its potential use as a coating antigen in an indirect ELISA for diagnosis of BHV-1, the causative agent of infectious bovine rhinotracheitis/infectious pustular vulvovaginitis.     

Renal blood flow distribution in septic hyperdynamic pigs

Acute hyperdynamic sepsis in man is associated with increased renal blood flow (RBF) and polyuria, presumably due to high RBF to the medulla causing washout of interstitial osmoles. This presumption was studied in 14 hyperdynamic piglets with hindlimb sepsis and compared to 11 control piglets. RBF distribution was measured by ⁵¹Cr-labeled microspheres previously injected into the left ventricle and compared to cardiac output measured by rate of isotope disappearance.Both septic and control piglets had comparable vital signs at the time of the study. Cardiac output and RBF were significantly increased in septic piglets; cortical flow was also significantly increased whereas medullary flow was only slightly increased. These data demonstrate that polyuria in hyperdynamic septic piglets is not due to interstitial medullary washout secondary to increased medullary flow. Polyuria probably reflects tubular dysfunction unrelated to renal hemodynamic changes.     

Reflectance confocal microscopy of optical phantoms

A reflectance confocal scanning laser microscope (rCSLM) operating at 488-nm wavelength imaged three types of optical phantoms: (1) 100-nm-dia. polystyrene microspheres in gel at 2% volume fraction, (2) solid polyurethane phantoms (INO Biomimic(TM)), and (3) common reflectance standards (Spectralon(TM)). The noninvasive method measured the exponential decay of reflected signal as the focus (z(f)) moved deeper into the material. The two experimental values, the attenuation coefficient μ and the pre-exponential factor ρ, were mapped into the material optical scattering properties, the scattering coefficient μ(s) and the anisotropy of scattering g. Results show that μ(s) varies as 58, 8-24, and 130-200 cm(-1) for phantom types (1), (2) and (3), respectively. The g varies as 0.112, 0.53-0.67, and 0.003-0.26, respectively.     

Structure of the human hexabrachion (tenascin) gene.

The structure of the gene encoding human hexabrachion (tenascin) has been determined from overlapping clones isolated from a human genomic bacteriophage library. The genomic inserts were characterized by restriction mapping, Southern blot analysis, PCR, and DNA sequencing. The coding region of the hexabrachion gene spans approximately 80 kilobases of DNA and consists of 27 exons separated by 26 introns. The exon-intron structure supports a hypothesis based on the cDNA sequence that the hexabrachion gene is an assembly of DNA modules that are also found elsewhere in the genome. Single exons may encode a module, a portion of a module, or a group of modules. The 15 type III units similar to those found in fibronectin are each encoded either by a single exon or by two exons interrupted by an intron. All type III units known to be spliced out of the smaller forms of the protein are encoded by one exon. The fibrinogen-like domain of 210 amino acids is encoded by five exons. The 14.5 epidermal growth factor-like repeats are all encoded by a single exon.     

Gastritis and gastric blood flow in hyperdynamic septic pigs.

The present study was designed to determine the gastric hemodynamics in a septic model that causes both a hyperdynamic state and acute erosive gastritis. Sepsis was established in twelve pigs by the intramuscular shank injection of Pastuerella multocida (42 X 10(8) colonies) in triptose phosphate broth; four pigs received sterile broth injections (shams) and eight received sterile saline injections (controls). After 18 hours, cardiac output was measured by both the cardiogreen dilution technic and the radioactive microsphere dilution technic; cardiac output was then compared to total and regional gastric blood flow measured by the raioactive microsphere entrapment technic. Acute erosive gastritis developed in septic pigs in the fundus and body of the stomach; the antrum was spared. Cardiac output was significantly increased in septic pigs compared with sham and control pigs. Total gastric blood flow and regional blood flow to the fundus, body, and antrum were also increased in direct proportion to the increase in cardiac output. These data suggest that acute erosive gastritis is primarily due to an end-organ cellular insult from sepsis and is not primarily due to a decrease or redistribution in gastric blood flow.     

A protocol for dynamic magnetic resonance imaging of the pelvic floor

Methods of dynamic magnetic resonance imaging (dMRI) of the pelvic floor vary among centers making interpretation investigator-dependent and comparison of results difficult. We describe a protocol utilizing standard MRI equipment, which achieves high image quality while remaining practical and cost-effective. Fifteen patients, with difficulty in evacuation of stool, underwent dMRI. Each patient was trained prior to the procedure. The pelvis was mapped in the sagittal plane using T2-weighted dMRI in rest, strain and evacuation phases with rectal hydro-gel as contrast. Images obtained were used to identify and quantify the dynamics of each pelvic compartment. Acquisition time for each phase was 14 seconds. Extensive patient instruction and T2-weighted dMRI with rectal contrast optimized image quality and efficiency. The evacuation phase yielded data on the extent of dysfunction and compartment prolapse, not seen in the other phases. These findings led to change in management in 67 % (n=10) of patients.     

Carbapenem resistant Gram-negative bacteremia in an Indian intensive care unit: A review of the clinical profile and treatment outcome of 50 patients

Background:

Growing antimicrobial resistance and limited therapeutic options to treat carbapenem-resistant bacteremia prompted us to evaluate the clinical outcomes associated with healthcare-associated bacteremia.

Methods:

This was a retrospective observational study of carbapenem-resistant Gram-negative bacteremia performed at a tertiary care facility in Chennai, India between May 2011 and May 2012.

Results:

In our study, patients had mean 11.76 days of intensive care unit (ICU) care and mean time to onset of bacteremia was 6.4 days after admission. The commonest organism was Klebsiella pneumoniae (44%). Patients with combination treatment had lower mortality (44.8%) compared with colistin monotherapy (66.6%); (P = 0.35).

Conclusion:

Carbapenem resistant bacteremia is a late onset infection in patients with antibiotic exposure in the ICU and carries a 30 days mortality of 60%; K. pneumoniae is the most common organism at our center. Two drug combinations appear to carry a lower mortality compared with monotherapy.     

Unusual Uptake of Prostate Specific Tracer <Superscript>68</Superscript>Ga-PSMA–HBED-CC in a Benign Thyroid Nodule

⁶⁸Ga-Prostate specific membrane antigen- N,N′-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid- positron emission tomography/computed tomography or 68 Ga- HBED-CC-PSMA PET/CT, popularly known as PSMA PET/CT, is able to detect a small volume of recurrent prostate carcinoma (PC) when there is a prostate specific antigen (PSA) rise on follow-up after prostatectomy or other definitive treatment for PC. The use of PSMA PET/CT in the initial staging in PC is uncertain at this time. Clinical studies are underway to define its exact role in the management of the disease. At the same time it is important to be aware of unexpected sites of uptake of this ligand. We present here the case of a 62-year-old male patient who underwent prostatectomy for adenocarcinoma prostate. He also had a long-standing left solitary thyroid nodule (STN). Four months after surgery, he had a rising trend in serum PSA levels on three occasions, but the absolute value was less than 4 at all times. He underwent a ⁶⁸Ga-PSMA-HBED-CC PET/CT, but it did not reveal any recurrent/metastatic site of disease. However, there was increased tracer uptake in the left STN. Fine needle aspiration cytology revealed features of atypia of undetermined significance, Bethesda category III. The patient underwent a left hemithyroidectomy and the histopathology showed features of a follicular adenoma.