Assessing venous thrombus in renal cell carcinoma: preliminary results for unenhanced 3D-SSFP MRI.

AIM: To test the potential of unenhanced cardiac- and respiratory-motion-corrected three-dimensional steady-state free precession (3D-SSFP) magnetic resonance imaging (MRI) for the assessment of inferior vena cava (IVC) thrombus in patients with clear-cell renal cell carcinoma (cRCC), compared to standard contrast-enhanced (CE)-MRI and CE-computed tomography (CT). MATERIALS AND METHODS: Eighteen patients with cRCC and IVC thrombus, who received CE-MRI and 3D-SSFP at 1.5 T between June 2015 and December 2017, were included. The diagnostic performance of 3D-SSFP in determining the level of thrombus extension, contrast-to-noise ratio (CNR), and image quality were compared with standard MRI/CT and validated against intraoperative and histopathology results. RESULTS: There was 100% agreement between 3D-SSFP, 83.3% agreement between CE-MRI, and 71.4% agreement between CE-CT and surgical findings regarding the level of IVC thrombus. In addition, 3D-SSFP showed a slightly superior estimate of pathological IVC volume. 3D-SSFP reached a significantly higher CNR in the supra- and infrarenal IVC compared to the morphological sequence T2-weighted half-Fourier axial single-shot fast spin-echo (T2-HASTE) and all phases of CE-MRI. More specifically, 3D-SSFP showed a significantly higher CNR in the infrarenal IVC (mean CNR of 10.09±5.74 versus 4.21±2.33 in the delayed phase, p≤0.001) and in the suprarenal IVC (mean CNR of 9.22±4.11 versus 4.84±5.74 in the late arterial phase, p=0.015). CE-CT also was significantly inferior to 3D-SSFP (p≤0.01) and slightly inferior to CE-MRI (p>0.05). The thrombus delineation score for 3D-SSFP (4.38±0.67) was higher compared to CE-MRI (3.76±0.56, p=0.005). CONCLUSION: This preliminary study indicates that 3D-SSFP can achieve an accurate assessment of IVC thrombus in cRCC patients without the need for contrast medium administration, being superior to standard MRI and CT.

Towards characterization of photo-excited electron transfer and catalysis in natural and artificial systems using XFELs.

The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open capabilities for studying the structure and dynamics of a wide variety of biological and inorganic systems beyond what is possible at synchrotron sources. Although the structure and chemistry at the catalytic sites have been studied intensively in both biological and inorganic systems, a full understanding of the atomic-scale chemistry requires new approaches beyond the steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure at ambient conditions, while overcoming X-ray damage to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by using the intense and ultra-short femtosecond X-ray pulses from an XFEL, where sample is probed before it is damaged. We have developed methodology for simultaneously collecting X-ray diffraction data and X-ray emission spectra, using an energy dispersive spectrometer, at ambient conditions, and used this approach to study the room temperature structure and intermediate states of the photosynthetic water oxidizing metallo-protein, photosystem II. Moreover, we have also used this setup to simultaneously collect the X-ray emission spectra from multiple metals to follow the ultrafast dynamics of light-induced charge transfer between multiple metal sites. A Mn-Ti containing system was studied at an XFEL to demonstrate the efficacy and potential of this method.

Duration of action of hypertonic saline on mucociliary clearance in the normal lung.

Inhalation of hypertonic saline (HS) acutely enhances mucociliary clearance (MC) in both health and disease. In patients with cystic fibrosis (CF), repeated use of HS causes a sustained improvement in MC as well as clinical benefit. The pharmacodynamic duration of activity on MC may be an important determinant of its therapeutic potential in other airways diseases. Before moving toward testing the clinical benefits of HS for non-CF indications, we sought to assess the duration of pharmacodynamic effects of HS in healthy subjects by performing radiotracer clearance studies at baseline, 30-min post-HS administration, and 4-h post-HS administration. Indeed, acceleration of MC was observed when measured 30 min after HS inhalation. This acceleration was most pronounced in the first 30 min after inhaling the radiotracer in the central lung region (mean Ave30Clr = 15.5 vs. 8.6% for 30-min post-HS treatment vs. mean baseline, respectively, P < 0.005), suggesting that acute HS effects were greatest in the larger bronchial airways. In contrast, when MC was measured 4 h after HS administration, all indices of central lung region MC were slower than at baseline: Ave30Clr = 5.9% vs. 8.6% (P = 0.10); Ave90Clr = 12.4% vs. 16.8% (P < 0.05); clearance through 3 h = 29.4 vs. 43.7% (P < 0.002); and clearance through 6 h = 39.4 vs. 50.2% (P < 0.02). This apparent slowing of MC in healthy subjects 4-h post-HS administration may reflect depletion of airway mucus following acute HS administration.

Simulations of the two-dimensional electronic spectroscopy of the photosystem II reaction center.

We report simulations of the two-dimensional electronic spectroscopy of the Q(y) band of the D1-D2-Cyt b559 photosystem II reaction center at 77 K. We base the simulations on an existing Hamiltonian that was derived by simultaneous fitting to a wide range of linear spectroscopic measurements and described within modified Redfield theory. The model obtains reasonable agreement with most aspects of the two-dimensional spectra, including the overall peak shapes and excited state absorption features. It does not reproduce the rapid equilibration from high energy to low energy excitonic states evident by a strong cross-peak below the diagonal. We explore modifications to the model to incorporate new structural data and improve agreement with the two-dimensional spectra. We find that strengthening the system-bath coupling and lowering the degree of disorder significantly improves agreement with the cross-peak feature, while lessening agreement with the relative diagonal/antidiagonal width of the 2D spectra. We conclude that two-dimensional electronic spectroscopy provides a sensitive test of excitonic models of the photosystem II reaction center and discuss avenues for further refinement of such models.

Phylogenetic analysis of spring virema of carp virus reveals distinct subgroups with common origins for recent isolates in North America and the UK.

Genetic relationships between 35 spring viremia of carp virus (SVCV) genogroup Ia isolates were determined based on the nucleotide sequences of the phosphoprotein (P) gene and glycoprotein (G) genes. Phylogenetic analysis based on P gene sequences revealed 2 distinct subgroups within SVCV genogroup Ia, designated SVCV Iai and Iaii, and suggests at least 2 independent introductions of the virus into the USA in 2002. Combined P- and G-sequence data support the emergence of SVCV in Illinois, USA, and in Lake Ontario, Canada, from the initial outbreak in Wisconsin, USA, and demonstrate a close genetic link to viruses isolated during routine import checks on fish brought into the UK from Asia. The data also showed a genetic link between SVCV isolations made in Missouri and Washington, USA, in 2004 and the earlier isolation made in North Carolina, USA, in 2002. However, based on the close relationship to a 2004 UK isolate, the data suggest than the Washington isolate represents a third introduction into the US from a common source, rather than a reemergence from the 2002 isolate. There was strong phylogenetic support for an Asian origin for 9 of 16 UK viruses isolated either from imported fish, or shown to have been in direct contact with fish imported from Asia. In one case, there was 100% nucleotide identity in the G-gene with a virus isolated in China.

Cloning and sequencing hybrid striped bass (Morone saxatilis x M. chrysops) transforming growth factor-beta (TGF-beta), and development of a reverse transcription quantitative competitive polymerase chain reaction (RT-qcPCR) assay to measure TGF-beta mRNA of teleost fish.

A transforming growth factor (TGF)-beta was isolated and cloned from hybrid striped bass (Morone saxatilis x M. chrysops) anterior kidney mononuclear cells. This isolate (Genbank accession number AF140363) contains an open reading frame of 1146 bases coding for a 382 amino acid protein most similar to rainbow trout TGF-beta (57.3 and 78.6% identity with precursor and active protein, respectively) and rat TGF-beta 1 (41.1 and 68.8% identity with precursor and active protein, respectively). Consensus primers were demonstrated to amplify specifically by polymerase chain reaction (PCR), a TGF-beta segment from 14 species of teleost fish comprising 10 taxonomic families in 7 orders. A reverse transcription quantitative competitive polymerase chain reaction (RT-qcPCR) assay was devised to measure TGF-beta mRNA expression in teleost fish. Higher levels of TGF-beta mRNA expression were detected in mononuclear cells of peripheral blood than from spleen or anterior kidney.

Out-of-hospital succinylcholine-assisted endotracheal intubation by paramedics.

STUDY OBJECTIVE: To describe the effectiveness and safety of succinylcholine administration by paramedics to out-of-hospital patients requiring endotracheal intubation. METHODS: A consecutive case series was collected by retrospective review of patient care records from a large, private ambulance agency serving an urban and rural county of 600,000 residents over a 40-month period. All patients for whom endotracheal intubation was facilitated by succinylcholine use were identified. Data were systematically collected on demographics, paramedic diagnosis, intubation attempts, and complications. RESULTS: Succinylcholine was used in 150 patients with a mean age (+/-SD) of 50+/-23 years. Paramedic diagnosis was coma in 43% (64/150; 95% confidence interval [CI] 35% to 51%), trauma in 26% (39/150; 95% CI 19% to 33%), and respiratory failure in 31% (47/150; 95% CI 23% to 39%). No patient was in cardiac arrest at the time of succinylcholine use. The intubation success rate was 92% (138/150; 95% CI 88% to 96%), with success achieved on the first attempt in 82% of patients (123/150; 95% CI 76% to 88%). Four patients who received succinylcholine developed cardiac arrest, and 3 others had symptomatic bradycardia. CONCLUSION: The use of succinylcholine by paramedics to assist intubation appears to aid in intubation of patients who are not in cardiac arrest. However, significant cardiac complications can occur.

Nucleotide sequence and vaccinia expression of the nucleoprotein of a highly virulent, neurotropic strain of Newcastle disease virus.

The nucleoprotein (NP) of Newcastle disease virus (NDV) was selected to study the relative importance of an internal structural protein in the avian immune response. The NP gene of the virulent, neurotropic NDV Texas GB (TGB) strain was cloned and sequenced. Nucleotide sequence data for the NP gene allowed comparison of the deduced amino acid sequences for the NP genes of NDV-TGB and the avirulent duck isolate NDV-D26. These comparisons demonstrated an 89% nucleotide sequence homology and a 97% homology between the deduced amino acid sequences. The NDV-TGB NP expressed in recombinant vaccinia virus (rVAC) was electrophoretically and immunologically identical to the wild-type NDV-TGB. Although inoculation of chickens with the recombinant vaccinia virus expressing the NDV NP gene elicited anti-NDV antibodies in higher titers than in birds inoculated with live LaSota NDV, this strong anti-NDV response did not protect against lethal challenge with NDV-TGB.

Sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3' untranslated region identifies the virus as a close relative of infectious bronchitis virus.

The 3' end of the turkey coronavirus (TCV) genome (1740 bases) including the nucleocapsid (N) gene and 3' untranslated region (UTR) were sequenced and compared with published sequences of other avian and mammalian coronaviruses. The deduced sequence of the TCV N protein was determined to be 409 amino acids with a molecular mass of approximately 45 kDa. The TCV N protein was identical in size and had greater than 90% amino acid identity with published N protein sequences of infectious bronchitis virus (IBV); less than 21% identity was observed with N proteins of bovine coronavirus and transmissible gastroenteritis virus. The 3' UTR showed some variation among the three TCV strains examined, with two TCV strains, Minnesota and Indiana, containing 153 base segments which are not present in the NC95 strain. Nucleotide sequence identity between the 3' UTRs of TCV and IBV was greater than 78%. Similarities in both size and sequence of TCV and IBV N proteins and 3' UTRs provide additional evidence that these avian coronaviruses are closely related.

Paramedic decisions with placement of out-of-hospital intravenous lines.

To determine the incidence of unused out-of-hospital intravenous line (IV) placements, we prospectively studied IV placement in emergency medical services (EMS) patients. Unused IV placement was defined as any patient having an EMS initiated IV that was not used for fluid bolus or medication administration in the field or in the emergency department (ED). Data were analyzed on placement and use of IV lines in the field and in the ED, transport time, years of paramedic practice, and paramedic student presence. Of 290 patients, 165 had an IV initiated (147) or attempted (18). Twenty-nine percent (84 of 290) of the patients received an unused EMS IV. One hundred twenty-five patients had no IV initiated by EMS. Seven subsequently had an IV started and used in the ED, for an undertreatment rate of 2.4% (7 of 290). The presence of a paramedic student increased the odds of an unused IV 1.4 (95% CI, 1.1 to 2.0). IVs are frequently started and not used.

Sequence analysis of the matrix/nucleocapsid gene region of turkey coronavirus.

A reverse transcriptase, polymerase chain reaction (RT-PCR) procedure was used to amplify a segment of the genome of turkey coronavirus (TCV) spanning portions of the matrix and nucleocapsid (MN) protein genes (approximately 1.1 kb). The MN gene region of three epidemiologically distinct TCV strains (Minnesota, NC95, Indiana) was amplified, cloned into pUC19, and sequenced. TCV MN gene sequences were compared with published sequences of other avian and mammalian coronaviruses. A high degree of similarity (>90%) was observed between the nucleotide, matrix protein, and nucleocapsid protein sequences of TCV strains and published sequences of infectious bronchitis virus (IBV). The matrix and nucleocapsid protein sequences of TCV had limited homology (<30%) with MN sequences of mammalian coronaviruses. These results demonstrate a close genetic relationship between the avian coronaviruses, IBV and TCV.

Platelets from thrombocytopenic ponies acutely infected with equine infectious anemia virus are activated in vivo and hypofunctional.

Thrombocytopenia is a consistent finding and one of the earliest hematological abnormalities in horses acutely infected with equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus. Multifactorial mechanisms, including immune-mediated platelet destruction and impaired platelet production, are implicated in the pathogenesis of EIAV-associated thrombocytopenia. This study was undertaken to investigate whether regenerative thrombopoiesis and platelet destruction occurred in ponies acutely infected with EIAV. Circulating large, immature platelets were increased in ponies acutely infected with EIAV late in the infection when platelet count was at a nadir. Morphometric analysis of bone marrow from acutely infected ponies revealed significant increased in megakaryocyte area and megakaryocyte nuclear area. A trend toward increased numbers of megakaryocytes was also observed. Platelets from acutely infected ponies had increased surface-bound fibrinogen and ultrastructural changes consistent with in vivo platelet activation. Platelets also had hypofunctional aggregation responses to three agonists in vitro. We conclude that thrombocytopenia in ponies acutely infected with EIAV is regenerative and suggest that bone marrow platelet production is not severely compromised in these ponies. Our findings reveal that in vivo platelet activation occurs in ponies acutely infected with EIAV, and as a result platelets are hypofunctional in vitro. Activation of platelets in vivo may cause platelet degranulation or formation of platelet aggregates, which would result in removal of these damages platelets from circulation. This may represent a form of nonimmune-mediated platelet destruction in ponies acutely infected with EIAV.

Domains determining ligand specificity for Ca2+ receptors.

The Ca2+ receptor is a G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to changes in the level of extracellular Ca2+. The Ca2+ receptor is a member of a family of G protein-coupled receptors that includes metabotropic glutamate receptors (mGluRs), gamma-aminobutyric acidB receptors, and putative pheromone receptors. As a family, these receptors are characterized by limited sequence homology and an unusually large putative extracellular domain (ECD). The ECD of the mGluRs is believed to determine agonist selectivity, but the functions of the structural domains of the Ca2+ receptor are not known. To identify structural determinants for cation recognition and activation of the Ca2+ receptor (and to further study the mGluRs), two chimeric receptors were constructed in which the large ECD of the Ca2+ receptor and the mGluR1 were interchanged. When expressed in Xenopus laevis oocytes, one of these chimeras, named CaR/mGluR1 [ECD of the Ca2+ receptor and transmembrane domain (TMD) of the mGluR1], responded to cation agonists (Gd3+, Ca2+, neomycin) of the Ca2+ receptor at concentrations similar to those necessary for activation of the native Ca2+ receptor. A reciprocal construct, named mGluR1/CaR (ECD of the mGluR1 and TMD of the Ca2+ receptor), was responsive to mGluR agonists but was much less sensitive to two of three cation agonists known to activate the Ca2+ receptor. A deletion construct of the Ca2+ receptor (DeltantCaR), which lacked virtually the entire ECD, was only activated by one of three agonists tested. These results suggest that the primary determinants for agonist activation of both the Ca2+ receptor and the mGluRs are found in the large ECD and that the Ca2+ receptor is possibly distinguished from the mGluRs in that it may contain sites in the TMD that permit activation by certain cation agonists.

Long terminal repeat sequences of equine infectious anaemia virus are a major determinant of cell tropism.

The Wyoming strain of equine infectious anaemia virus (EIAV) is a highly virulent field strain that replicates to high titre in vitro only in primary equine monocyte-derived macrophages. In contrast, Wyoming-derived fibroblast-adapted EIAV strains (Malmquist virus) replicate in primary foetal equine kidney and equine dermis cells as well as in the cell lines FEA and Cf2Th. Wyoming and Malmquist viruses differ extensively both in long terminal repeat (LTR) and envelope region sequences. We have compared the promoter activities of the Wyoming LTR with those of LTRs derived from fibroblast-adapted viruses by examining their abilities to drive a luciferase reporter gene as well as by construction of infectious molecular clones differing only in LTR sequence. Our results indicate that LTR sequences are a major restriction for growth of the Wyoming strain of EIAV in fibroblasts.

Disease induction by virus derived from molecular clones of equine infectious anemia virus.

Equine infectious anemia virus (EIAV), a macrophage-tropic lentivirus, causes persistent infections of horses. A number of biologic features, including the rapid development of acute disease, the episodic nature of chronic disease, the propensity for viral genetic variation, and the ability for many infected animals to eventually control virus replication, render EIAV a potentially useful model system for the testing of antiretroviral therapies and vaccine strategies. The utility of the EIAV system has been hampered by the lack of proviral clones that encode promptly pathogenic viral stocks. In this report, we describe the generation and characterization of two infectious molecular clones capable of causing acute clinical syndromes similar to those seen in natural infections. Virus derived from clone p19/wenv17 caused severe debilitating disease at 5 to 7 days postinfection; initial febrile episodes were fatal in two of three infected animals. Virus derived from a second clone, p19/wenv16, caused somewhat milder primary febrile episodes by 10 to 12 days postinfection in two of two infected animals. Virus derived from both clones caused persistent infections such that some animals exhibited chronic equine infectious anemia, characterized by multiple disease episodes. The two virulent clones differ in envelope and rev sequences.

The dynamic nature of ST-segment and T-wave changes during acute MI.

Obtaining a prehospital 12-lead electrocardiogram (ECG) diagnostic of acute myocardial injury has been demonstrated to hasten the administration of thrombolytic agents in the emergency department. This case demonstrates that a prehospital electrocardiogram diagnostic of acute anterior wall infarction can become non-diagnostic following routine administration of oxygen, nitroglycerin, and morphine by paramedics. Although this phenomenon has been observed in the in-hospital setting, it has not been reported in patients with a prehospital ECG.

Ca2+ receptor mRNA and protein increase in the rat parathyroid gland with advancing age.

Parathyroid hormone (PTH) release is regulated by extracellular calcium through a Ca2+ receptor (CaR) located on the surface of the parathyroid cell. With advancing age, the serum concentration of PTH increases, and evidence suggests that the calcium set-point for PTH release may also increase. To determine whether these changes are linked to a change in CaR expression, we quantitated mRNA and protein for the receptor in parathyroid glands of 6-week-, 6-month- and 24-month-old rats. Thyroid and kidney tissue were also studied. Between 6 weeks and 24 months of age, CaR mRNA in the parathyroid gland increased 11.4- and 3.3-fold as measured by competitive reverse transcription PCR and solution hybridization assays respectively. Message levels for the receptor also increased in the thyroid but not in the kidney. Coincident with the increase in message levels, receptor protein concentration in the parathyroid increased 7-fold between 6 weeks and 24 months of age. These results suggest that the altered relationship between extracellular calcium and PTH release observed in aging is associated with dramatic changes in CaR metabolism. That PTH secretion is increased despite increased receptor concentration suggests that aging may impair calcium binding or coupling between the CaR and down-stream effector elements in the pathway regulating PTH release.

Characterization and mutational studies of equine infectious anemia virus dUTPase.

The macrophage tropic lentivirus, equine infectious anemia virus (EIAV), encodes a dUTPase in the pol gene that is required for efficient replication in macrophages. Two naturally occurring variants of the enzyme were expressed as recombinant proteins in Escherichia coli; metal chelate affinity chromatography was used to purify histidine-tagged recombinant enzymes to greater than 80% homogeneity in a single chromatographic step. Biochemical and enzymatic analyses of these preparations suggest that this method yields dUTPase that is suitable for detailed mutational analysis. Specific activities of preparations ranged from 4 x 10(3) to 5 x 10(4) units/mg. Recombinant EIAV dUTPase was highly specific for dUTP with a Km in the range of 3 to 8 microM. The enzyme was sensitive to inhibition by dUDP with little inhibition by other nucleotides or the reaction products, dUMP and PPi. The subunit organization of recombinant EIAV dUTPase was probed by gel filtration, glycerol gradient centrifugation, and chemical cross-linking, and is a trimer. We have begun mutational analyses by targeting a conserved domain present at the carboxyl terminus of all dUTPases that shares high homology to the phosphate binding loops (P-loops) of a number of ATP- and GTP-binding phosphatases. The P-loop-like motif of dUTPases is glycine rich but lacks the invariant lysine found in authentic P-loops. Deletion of this motif leads to loss of dUTPase activity; a series of point mutations that have been shown to inactivate authentic P-loops also abolish EIAV dUTPase activity.