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As higher density formats become more and more common in HTS labs, the expectations for maintaining faster, lower cost screens puts great pressure on traditional 96-well screens. In some cases higher density formats are not compatible with the assay. This seems especially true in cell-based assays. In our case, the nature of the cells' response forced us to remain in 96-well plates. In this paper, we describe the development of a luminescence reporter assay and its performance in two detection modes, flash and glow. The advantages in cost and throughput for each technique are explored, along with automation considerations. An additional new technology, the use of pins for low-volume transfers, is also briefly described because of its dramatic effect on our screen's throughput. However, it will be more thoroughly presented in a future publication. Comparing the technologies available for HTS aids in designing automated systems that meet the unique needs of each assay.

Cytoskeletal protein expression in sensory neurons and sciatic nerve axonal growth were examined in type 1 diabetic BB/Wor rats after sciatic nerve crush injury. Diabetic male rats were subjected to sciatic nerve crush at 6 wk of diabetes. L4 and L5 dorsal root ganglia (DRG) mRNA expression of low and medium molecular weight neurofilaments (NF-L, NF-M), betaII- and betaIII-tubulin as well as protein expression of NF-L, NF-M, and beta-tubulin were examined at various time points following crush injury and compared with age- and sex-matched non-diabetic BB/Wor rats. Steady state mRNA expression of NF-L, NF-M, betaII- and betaIII-tubulin were decreased in diabetic DRG. NF-L and NF-M proteins were also decreased in DRG of uncrushed diabetic animals. After crush injury, betaII- and betaIII-tubulin mRNA were upregulated in control animals at day 2 and day 6, respectively, and beta-tubulin protein showed similarly increased expression after crush injury, while such upregulations did not occur in diabetic animals. Conversely, mRNA and protein expressions of NF-L, NF-M were downregulated to a lesser extent in diabetic animals compared to control rats. These changes were associated with impaired axonal elongation and caliber growth of regenerating fibers in diabetic rats. We propose that upregulation of tubulin has a negative feedback on NF expression in response to nerve injury, as seen in control rats. The absence of this upregulation in diabetic animals may impair its regulatory effect on NF expression and contribute to perturbed nerve regeneration seen in diabetic nerve.

Mixed epithelial and stromal tumor of the kidney is a recently recognized neoplasm that occurs almost exclusively in perimenopausal women. Because it frequently contains areas of smooth muscle in which epithelial structures are embedded, some have concluded that it is the adult form of congenital mesoblastic nephroma. Others have concluded that the morphology and epidemiology of mixed epithelial and stromal tumor indicate that it is unrelated to congenital mesoblastic nephroma. Although the genetic alterations of mixed epithelial and stromal tumor have not been previously elucidated, much is known about the genetic alterations of cellular congenital mesoblastic nephroma. The present study was undertaken to determine if mixed epithelial and stromal tumors have any of the genetic alterations recognized as typical of cellular congenital mesoblastic nephroma. RNA extraction was performed on formalin-fixed, paraffin-embedded tissue from 7 mixed epithelial and stromal tumors followed by reverse-transcription polymerase chain reaction to detect the ETV6-NTRK3 gene fusion. Fluorescent in situ hybridization with centromere-specific probes for chromosomes 8, 11, and 17 was performed to evaluate polyploidy of these chromosomes in 11 cases of mixed epithelial and stromal tumor. None of the mixed epithelial and stromal tumors showed any of these genetic alterations. We conclude that mixed epithelial and stromal tumor of the kidney lacks the genetic alterations typical of cellular congenital mesoblastic nephroma, is unrelated to it, and the appellation "adult mesoblastic nephroma" should not be used for these tumors.

In order to explore the neuroprotective and cross-species activities of C-peptide on type 1 diabetic neuropathy, spontaneously diabetic BB/W-rats were given increasing doses of human recombinant C-peptide (hrC-peptide). Diabetic rats received 10, 100, 500, or 1000 microg of hrC-peptide/kg body weight/day from onset of diabetes. After 2 months of hrC-peptide administration, 100 microg and greater doses completely prevented the nerve conduction defect, which was associated with a significant but incomplete prevention of neural Na+/K+-ATPase activity in diabetic rats with 500 microg or greater C-peptide replacement. Increasing doses of hrC-peptide showed increasing prevention of early structural abnormalities such as paranodal swelling and axonal degeneration and an increasing frequency of regenerating sural nerve fibers. We conclude that hrC-peptide exerts a dose dependent protection on type 1 diabetic neuropathy in rats and that this effect is probably mediated by the partially conserved sequence of the active C-terminal pentapeptide.