Portable optical fiber probe-based spectroscopic scanner for rapid cancer diagnosis: a new tool for intraoperative margin assessment

There continues to be a significant clinical need for rapid and reliable intraoperative margin assessment during cancer surgery. Here we describe a portable, quantitative, optical fiber probe-based, spectroscopic tissue scanner designed for intraoperative diagnostic imaging of surgical margins, which we tested in a proof of concept study in human tissue for breast cancer diagnosis. The tissue scanner combines both diffuse reflectance spectroscopy (DRS) and intrinsic fluorescence spectroscopy (IFS), and has hyperspectral imaging capability, acquiring full DRS and IFS spectra for each scanned image pixel. Modeling of the DRS and IFS spectra yields quantitative parameters that reflect the metabolic, biochemical and morphological state of tissue, which are translated into disease diagnosis. The tissue scanner has high spatial resolution (0.25 mm) over a wide field of view (10 cm × 10 cm), and both high spectral resolution (2 nm) and high spectral contrast, readily distinguishing tissues with widely varying optical properties (bone, skeletal muscle, fat and connective tissue). Tissue-simulating phantom experiments confirm that the tissue scanner can quantitatively measure spectral parameters, such as hemoglobin concentration, in a physiologically relevant range with a high degree of accuracy (<5% error). Finally, studies using human breast tissues showed that the tissue scanner can detect small foci of breast cancer in a background of normal breast tissue. This tissue scanner is simpler in design, images a larger field of view at higher resolution and provides a more physically meaningful tissue diagnosis than other spectroscopic imaging systems currently reported in literatures. We believe this spectroscopic tissue scanner can provide real-time, comprehensive diagnostic imaging of surgical margins in excised tissues, overcoming the sampling limitation in current histopathology margin assessment. As such it is a significant step in the development of a platform technology for intraoperative management of cancer, a clinical problem that has been inadequately addressed to date.     

Bactericidal antibiotics do not appear to cause oxidative stress in Listeria monocytogenes

Oxidative stress can be an important contributor to the lethal effect of bactericidal antibiotics in some bacteria, such as Escherichia coli and Staphylococcus aureus. Thus, despite the different target-specific actions of bactericidal antibiotics, they have a common mechanism leading to bacterial self-destruction by internal production of hydroxyl radicals. The purpose of the present study was to determine if a similar mechanism is involved in antibiotic killing of the infectious human pathogen, Listeria monocytogenes. We treated wild-type L. monocytogenes and oxidative stress mutants (Δsod and Δfri) with three different bactericidal antibiotics and found no difference in killing kinetics. In contrast, wild-type E. coli and an oxidative stress mutant (ΔsodA ΔsodB) differed significantly in their sensitivity to bactericidal antibiotics. We conclude that bactericidal antibiotics did not appear to cause oxidative stress in L. monocytogenes and propose that this is caused by its noncyclic tricarboxylic acid (TCA) pathway. Hence, in this noncyclic metabolism, there is a decoupling between the antibiotic-mediated cellular requirement for NADH and the induction of TCA enzyme activity, which is believed to mediate the oxidative stress reaction.     

1,25-Dihydroxyvitamin D3-mediated alterations in microtubule proteins isolated from chick intestinal epithelium: analyses by isoelectric focusing.

Recent work has indicated that vectorial Ca2+ transport across the intestinal epithelium occurs in vesicles and may involve the participation of microtubules [Nemere et al., 1986]. Since 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) stimulates this Ca2+ transport process, microtubule (MT) isotypes were studied as a potential regulatory point. The effect of 1,25(OH)2D3 status on tubulin isotypes was analyzed by isoelectric focusing (IEF) gels of taxol stabilized MTs prepared from intestinal epithelium of vitamin D-deficient chicks dosed with vehicle (-D) or 1.3 nmoles of 1,25(OH)2D3 (+D) 2.5, 5, 10, 15, or 43 h prior to sacrifice. Four bands, one of which was identified as alpha-tubulin on the basis of Western analysis, increased in Coomassie Blue staining intensity 5-15 h after 1,25(OH)2D3, corresponding to the time course of augmented vesicular Ca2+ transport. Dose-response studies revealed similar changes in tubulin isotype profiles in IEF gels, again corresponding to doses known to elicit enhanced Ca2+ absorption (52-6,500 pmoles of hormone). The role of Ca2+ transport was also examined. Isoelectrically focused intestinal epithelial tubulin from -D chicks allowed to transport Ca2+ for 30 min revealed increased staining of bands relative to nonabsorbing -D controls. By comparison, Ca2+ transport in +D chicks resulted in fainter bands relative to nonabsorbing, +D controls. MTs prepared from fasted or fed chicks revealed similar changes upon IEF, but of much smaller magnitude. Enhanced phosphorylation did not account for the appearance of the more acidic bands, although 1,25(OH)2D3 treatment resulted in decreased 32P content of a presumptive non-tubulin component, relative to preparations from -D controls. Glucocorticoids, which are known to suppress 1,25(OH)2D3-stimulated Ca2+ transport, led to severely diminished levels of total tubulin, as judged by SDS-PAGE, rather than altered tubulin isotypes. Thus, MTs of intestine are subject to regulation by hormonal status, as well as by the amount of Ca2+ available for transepithelial transport.     

Specific inhibition of human cytomegalovirus glycoprotein B-mediated fusion by a novel thiourea small molecule

A novel small molecule inhibitor of human cytomegalovirus (HCMV) was identified as the result of screening a chemical library by using a whole-virus infected-cell assay. Synthetic chemistry efforts yielded the analog designated CFI02, a compound whose potency had been increased about 100-fold over an initial inhibitor. The inhibitory concentration of CFI02 in various assays is in the low nanomolar range. CFI02 is a selective and potent inhibitor of HCMV; it has no activity against other CMVs, alphaherpesviruses, or unrelated viruses. Mechanism-of-action studies indicate that CFI02 acts very early in the replication cycle, inhibiting virion envelope fusion with the cell plasma membrane. Mutants resistant to CFI02 have mutations in the abundant virion envelope glycoprotein B that are sufficient to confer resistance. Taken together, the data suggest that CFI02 inhibits glycoprotein B-mediated HCMV virion fusion. Furthermore, CFI02 inhibits the cell-cell spread of HCMV. This is the first study of a potent and selective small molecule inhibitor of CMV fusion and cell-cell spread.     

Characterization of BK Polyomaviruses from Kidney Transplant Recipients Suggests a Role for APOBEC3 in Driving In-Host Virus Evolution

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Evidence that U5 snRNP recognizes the 3' splice site for catalytic step II in mammals.

The first AG dinucleotide downstream from the branchpoint sequence (BPS) is chosen as the 3'' splice site during catalytic step II of the splicing reaction. The mechanism and factors involved in selection of this AG are not known. Early in mammalian spliceosome assembly, U2AF65 binds to the pyrimidine tract between the BPS and AG. Here we show that U2AF65 crosslinking is replaced by crosslinking of three proteins of 110, 116 and 220 kDa prior to catalytic step II, and we provide evidence that all three proteins are components of U5 snRNP. These proteins interact with pre-mRNA in the region spanning from immediately downstream of U2 snRNP''s binding site at the BPS to just beyond the 3'' splice site. We also demonstrate that there are strict constraints on both the sequence and the distance between the BPS and AG for catalytic step II. Together, these observations suggest that U5 snRNP is positioned on the 3'' splice site by an interaction (direct or indirect) with U2 snRNP bound at the BPS and by a direct interaction with the pyrimidine tract. The functional AG for catalytic step II may be specified, in turn, by its location with respect to the U5 snRNP binding site.     

Association of U2 snRNP with the spliceosomal complex E.

In metazoans, the E complex is operationally defined as an ATP-independent spliceosomal complex that elutes as a single peak on a gel filtration column and can be chased into spliced products in the presence of an excess of competitor pre-mRNA. The A complex is the first ATP-dependent functional spliceosomal complex. U1 snRNP first binds tightly to the 5'splice site in the E complex and U2 snRNP first binds tightly to the branch site in the A complex. In this study, we have generated and characterized a monoclonal antibody (mAb 4G8) directed against SAP 62, a component of U2 snRNP and a subunit of the essential mammalian splicing factor SF3a. We show that this antibody is highly specific for SAP 62, detecting only SAP 62 on Western blots and immunoprecipitating only SAP 62 from nuclear extracts. The anti-SAP 62 antibody also immunoprecipitates U2 snRNP and the A complex. Significantly, however, we find that the E complex is also efficiently immunoprecipitated by the anti-SAP 62 antibody. This antibody does not cross-react with any E complex-specific components, indicating that SAP 62 itself is associated with the E complex. To determine whether other U2 snRNP components are associated with the E complex, we used antibodies to the U2 snRNP proteins B"and SAP 155. These antibodies also specifically immunoprecipitate the E complex. These observations indicate that U2 snRNP is associated with the E complex. However, we find that U2 snRNP is not as tightly bound in the E complex as it is in the A complex. The possible significance of the weak association of U2 snRNP with the E complex is discussed.     

Glomerular function in spontaneously diabetic rats.

This study was undertaken to determine whether hyperfiltration exists at the single nephron level and whether albumin excretion is increased early in the course of diabetes in Biobreeding rats. Diabetic rats were studied at 8-12 weeks after the onset of diabetes. Control animals were age-matched, diabetes-resistant rats. Urinary and tubular fluid albumin concentrations were measured by polyacrylamide gel electrophoresis. Clearance and micropuncture techniques were used to determine whole kidney and single nephron glomerular filtration rate, renal blood flow, and glomerular capillary pressure. The urinary albumin excretion rate (1.3 +/- 0.1 mg/24 hr) and the tubular fluid albumin concentration (4.7 +/- 0.7 mg/dl) in the diabetic group were significantly elevated when compared with urinary albumin excretion (0.9 +/- 0.1 mg/24 hr) and tubular fluid albumin concentration (2.5 +/- 0.5 mg/dl) in the control group. There were no significant differences in glomerular hemodynamics (whole kidney or single nephron glomerular filtration rate or glomerular capillary pressure) between diabetic and control rats. The kidney weight and kidney weight to body weight ratio were significantly higher in diabetic rats when compared with control rats. Early diabetes in Biobreeding rats is characterized by mild albuminuria and increased kidney size, but not glomerular hyperfiltration.     

Changes in courtship behaviour following rejection: The influence of female phenotype in Drosophila melanogaster

Courtship can be costly and so selection should favour individual males that reduce courtship towards female types that have a low probability of resulting in copulation. One way males can do this is by associating previous courtship failure with the traits of particular rejecting females. We characterised changes in male Drosophila melanogaster courtship behaviour following a failed mating attempt with one of the four female phenotypes that varied in size, age or mating status. To do this, we assessed individual courtship behaviour for each male presented again with a female of the same phenotype that previously rejected him. Males reduced subsequent courtship most strongly for recently mated (sexually non‐receptive) females. More interestingly, males also significantly reduced courtship activity following a failed mating experience from old females but did not do so for control (large, young, virgin) or small females. As such, males significantly reduced courtship towards both female types possessing chemical cues associated with their phenotype (age and mating status), but not towards a female phenotype based on physical characteristics (body size). Our results suggest that males are able to modify their courtship behaviour following experience, but that they are better prepared to associate chemical traits that may be more reliable indicators of the likelihood of courtship failure.     

Impact of exotic invasion on the temporal stability of natural annual plant communities

Much work in ecology has focused on understanding how changes in community diversity and composition will affect the temporal stability of communities (the degree of fluctuations in community abundance or biomass over time). While theory suggests diversity and dominant species can enhance temporal stability, empirical work has tended to focus on testing the effect of diversity, often using synthetic communities created with high species evenness. We use a complementary approach by studying the temporal stability of natural plant communities invaded by a dominant exotic, Erodium cicutarium. Invasion was associated with a significant decline in community diversity and change in the identity of the dominant species allowing us to evaluate predictions about how these changes might affect temporal stability. Community temporal stability was not correlated with community richness or diversity prior to invasion. Following invasion, community stability was again not correlated with community richness but was negatively correlated with community diversity. Before and after invasion, community stability was positively correlated with the stability of the most dominant species in the community, even though the identity of the dominant species changed from a native (prior to invasion) to an exotic species. Our results demonstrate that invasion by a dominant exotic species may reduce diversity without negatively affecting the temporal stability of natural communities. These findings add support to the idea that dominant species can strongly affect temporal stability, independent of community diversity.