Intrathecal non-viral interleukin-10 gene therapy ameliorates neuropathic pain as measured by both classical static allodynia and a novel supra-spinally mediated pain assay, the Two-Arm Rodent Somatosensory (TARS) task.

Intrathecal delivery of interleukin-10 (IL-10) gene therapy has been reported to be effective in suppressing pain enhancement in a variety of rodent models. However, all publications that have tested this treatment have relied upon measures of static allodynia (von Frey test) and thermal hyperalgesia (Hargreaves test). As this plasmid DNA IL-10 (pDNA-IL10) therapeutic approach is now in human clinical trials for multiple pain indications, including intrathecal delivery for human neuropathic pain, it is important to consider the recent concerns raised in the pain field that such tests reflect spinal rather than supraspinal processing of, and responsivity to, noxious stimuli. Consequently, this raises the question of whether intrathecal pDNA-IL10 can reverse established neuropathic pain when assessed by a test requiring supraspinal, rather than solely spinal, mediation of the behavioral response. The present study utilizes the rat sciatic chronic constriction injury (CCI) model of neuropathic pain to compare the expression of static allodynia with that of cognitively controlled choice behavior in a two-arm maze, adapted from Hayashida et al. (2019). This modification, termed the Two-Arm Rodent Somatosensory (TARS) task, provides rats free choice to reach a desired goal box via a short "arm" of the maze with tactile probes as flooring versus a longer "arm" of the maze with a smooth surface. Here we demonstrate that static allodynia and avoidance of the nociceptive flooring in TARS develop in parallel over time, and that both behaviors also resolve in parallel following intrathecal pDNA-IL10 gene therapy. Details for the construction and use of this new maze design are also provided. Together, this study documents both: (a) the important finding that intrathecal IL-10 gene therapy does indeed resolve neuropathic pain as measured by a supraspinally-mediated behavioral task, and (b) a new, supraspinally-mediated task that allows behavioral assessments across weeks and allows the analysis of both development and resolution of neuropathic pain by therapeutic interventions. As such, the TARS operant behavior task is an improvement over other approaches such as the mechanical conflict-avoidance system which have difficulties demonstrating development and reversal of pain behavior in a within-subject design.

The Transatlantic HbA1c gap: differences in glycaemic control across the lifespan between people included in the US T1D Exchange Registry and those included in the German/Austrian DPV registry.

AIM: To compare HbA1c levels across the lifespan in people with type 1 diabetes in the USA with those in Germany/Austria, and to examine potential differences in HbA1c levels between sexes, insulin delivery methods and minority status. METHODS: Data were extracted from the US T1D Exchange Registry (n=18 381 participants from 73 sites) and from the German/Austrian Prospective Diabetes Follow-up Registry, the DPV (n=32 643 participants from 362 sites). Mean HbA1c was calculated for each year of age for individuals aged ≤25 years, and at 2-year age intervals for individuals aged >25 years. Curves for mean HbA1c by age were estimated using locally weighted scatterplot smoothing. HbA1c differences between registries, sexes, insulin delivery methods, and minority status were assessed by age group using multiple linear regression. RESULTS: In both registries, mean HbA1c increased by ~11 mmol/mol (1.0%) between the ages of 9 and 18 years, although at quite different absolute levels: from 66 mmol/mol (8.2%) to 77 mmol/mol (9.2%) in the T1D Exchange Registry, and from 56 mmol/mol (7.3%) to 66 mmol/mol (8.2%) in the DPV. Sex differences were observed in the DPV only. In the T1D Exchange Registry, injection users had higher mean HbA1c than pump users across the lifespan, whereas in the DPV higher HbA1c levels in injection users were observed in the age groups 6 to <12 years, 12 to <18 years, and 30 to <50 years (P < 0.001). Minority status was significantly associated with higher HbA1c in most age groups in both registries. CONCLUSIONS: Significant differences in HbA1c were noted between the USA and Germany/Austria, with disparities more pronounced in early childhood through to young adulthood. Further studies should identify causes for these disparities.

Efficient transfection of MG-63 osteoblasts using magnetic nanoparticles and oscillating magnetic fields.

AIMS: To examine the potential of magnetic nanoparticles (MNPs) in transfecting human osteosarcoma fibroblasts (MG-63) and investigate the effects of a novel non-viral oscillating nanomagnetic gene transfection system (magnefect-nano™) in enhancing transfection efficiency (TE). METHODS: MG-63 cells were transfected using MNPs coupled with a GFP-carrying plasmid. The magnefect-nano system was evaluated for transfection efficiency and potential associated effects on cell viability. RESULTS: MG-63 cells were efficiently transfected using MNPs and the magnefect-nano system significantly enhanced overall transfection efficiency. MNPs were not found to affect cell viability and/or function of the cells. CONCLUSION: Non-viral transfection using MNPs and the magnefect-nano system can be used to transfect MG-63 cells and assist reporter gene delivery on a single cell basis, highlighting the wide potential of nanomagnetic gene transfection in gene therapy.

Evaluation of the magnetic field requirements for nanomagnetic gene transfection.

UNLABELLED: The objective of this work was to examine the effects of magnet distance (and by proxy, field strength) on nanomagnetic transfection efficiency. METHODS: non-viral magnetic nanoparticle-based transfection was evaluated using both static and oscillating magnet arrays. RESULTS: Fluorescence intensity (firefly luciferase) of transfected H292 cells showed no increase using a 96-well NdFeB magnet array when the magnets were 5 mm from the cell culture plate or nearer. At 6 mm and higher, fluorescence intensity decreased systematically. CONCLUSION: In all cases, fluorescence intensity was higher when using an oscillating array compared to a static array. For distances closer than 5 mm, the oscillating system also outperformed Lipofectamine 2000™.

Description of a fertilization-independent obligate apomictic species: Corunastylis apostasioides Fitzg.

The Australian midge orchid Corunastylis apostasioides of the tribe Diurideae has completely eliminated any male contribution in the process of seed formation, which occurs directly from the maternal tissue by a process termed apomixis. Here, we report C. apostasioides to be an obligate apomictic species devoid of any sexuality and compare its development to a close sexual relative C. fimbriata (R. Br.) D.L. Jones & M.A. Clem. Apomictic characteristics in C. apostasioides include production of seed in absence of fertilization, frequently closed flowers, production of immature pollen in non-dehiscent anthers, expansion of ovaries despite the lack of fertilization and the absence of a citronella scent that is found in C. fimbriata produced to attract pollinating vinegar flies (Jones 2006). The nature of apomixis in C. apostasioides was examined by ovule histology and amplified fragment length polymorphism (AFLP) in each case drawing comparison with sexual C. fimbriata. In C. apostasioides the central megaspore mother cell undergoes diplosporic apomixis, while additional embryos are derived from nucellar or integument initials formed by sporophytic apomixis. Typical of apomicts, C. apostasioides is polyploid compared to the sexual C. fimbriata. The divergences of C. apostasioides from sexuality to apomictic development are discussed.

Quantitation of serum prostate-specific membrane antigen by a novel protein biochip immunoassay discriminates benign from malignant prostate disease.

The lack of a sensitive immunoassay for quantitating serum prostate-specific membrane antigen (PSMA) hinders its clinical utility as a diagnostic/prognostic biomarker. An innovative protein biochip immunoassay was used to quantitate and compare serum PSMA levels in healthy men and patients with either benign or malignant prostate disease. PSMA was captured from serum by anti-PSMA antibody bound to ProteinChip arrays, the captured PSMA detected by surface-enhanced laser desorption/ionization mass spectrometry, and quantitated by comparing the mass signal integrals to a standard curve established using purified recombinant PSMA. The average serum PSMA value for prostate cancer (623.1 ng/ml) was significantly different (P < 0.001) from that for benign prostate hyperplasia (117.1 ng/ml) and the normal groups (age <50, 272.9 ng/ml; age >50, 359.4 ng/ml). These initial results suggest that serum PSMA may be a more effective biomarker than prostate-specific antigen for differentiating benign from malignant prostate disease and warrants additional evaluation of the surface-enhanced laser desorption/ionization PSMA immunoassay to determine its diagnostic utility.

Development and clinical evaluation of a recombinant-antigen-based cytomegalovirus immunoglobulin M automated immunoassay using the Abbott AxSYM analyzer.

A new microparticle enzyme immunoassay (MEIA), the Cytomegalovirus (CMV) Immunoglobulin M (IgM) test, was developed on the Abbott AxSYM analyzer. This test uses recombinant CMV antigens derived from portions of four structural and nonstructural proteins of CMV: pUL32 (pp150), pUL44 (pp52), pUL83 (pp65), and pUL80a (pp38). A total of 1, 608 specimens from random volunteer blood donors (n = 300), pregnant women (n = 1,118), transplant recipients (n = 6), and patients with various clinical conditions and disease states (n = 184) were tested during development and evaluation of this new assay. In a preliminary clinical evaluation we tested specimens collected prospectively from pregnant women (n = 799) and selected CMV IgM-positive archived specimens from pregnant women (n = 39). The results from the new CMV IgM immunoassay were compared to the results of a consensus interpretation of the results obtained with three commercial CMV IgM immunoassays. The results for specimens with discordant results were resolved by a CMV IgM immunoblot assay. The relative sensitivity, specificity, and agreement for the AxSYM CMV IgM assay were 94.29, 96.28, and 96.19%, respectively, and the resolved sensitivity, specificity, and agreement were 95.83, 97.47, and 97.37%, respectively. We also tested serial specimens from women who experienced seroconversion or a recent CMV infection during gestation (n = 17) and potentially cross-reactive specimens negative for CMV IgM antibody by the consensus tests (n = 184). The AxSYM CMV IgM assay was very sensitive for the detection of CMV IgM during primary CMV infection, as shown by the detection of CMV IgM at the same time as or just prior to the detection of CMV IgG. Specimens from individuals with lupus (n = 16) or parvovirus B19 infection (n = 6) or specimens containing hyper IgM (n = 9), hyper IgG (n = 8), or rheumatoid factor (n = 55) did not cross-react with the AxSYM assay. One specimen each from individuals infected with Epstein-Barr virus (n = 26), measles virus (n = 10), herpes simplex virus (n = 12), or varicella-zoster virus (n = 13) infection, one specimen from an influenza vaccinee (n = 14), and one specimen containing antinuclear antibody cross-reacted with the assay. The overall rate of cross-reactivity of the specimens with the assay was 3.3% (6 of 184). The AxSYM CMV IgM assay is a sensitive and specific assay for the detection of CMV-specific IgM.

A comparison of the structures of the alpha:beta and alpha:gamma dimers of mouse salivary androgen-binding protein (ABP) and their differential steroid binding.

Mouse salivary androgen-binding protein (ABP) is a family of dimeric proteins that may play a pheromonal role in Mus musculus. The protein dimer consists of a common alpha subunit disulfide-bonded to a variable (beta or gamma) subunit. Here we report N-terminal sequences of the beta and gamma subunits, showing that they are very similar to each other while being quite different from the alpha subunit. We demonstrate differential androgen binding by the two dimers. Both bind dihydrotestosterone to about the same extent but the alpha:beta dimer binds significantly more testosterone than the alpha:gamma dimer. We discuss the possible significance of this diversity of androgen binding with respect to the possibility that androgen binding is related to a putative pheromonal role for the protein.

Ceratobium mosaic potyvirus: another virus from orchids.

A potyvirus, which we call ceratobium mosaic virus, has been detected in about one third of more than 100 plants representing c. 33 orchid genera in two collections in Australia. It was detected using RT-PCR with redundant primers that are Potyviridae-specific and have additional sequences corresponding to either the SP6 or T7 bacteriophage promoters at their 5'-termini. Thus the nucleotide sequence of the resulting PCR fragments, consisting of about 1.7 kb of the 3' portion of the viral genome, could be determined directly. Viral sequences obtained from five infected orchids indicate that they contained different isolates of a single potyvirus species most closely related to the bean common mosaic group of potyviruses, but clearly distinct from all whose virion protein genes have been reported to the international gene sequence databases.

Serum levels of endothelial and neural cell adhesion molecules in prostate cancer.

BACKGROUND: Tumorigenesis and progression to metastatic disease are accompanied by changes in the expression of cell adhesion molecules (CAMs). Normally expressed CAMs, such as E-cadherin, are lost, while others, i.e., ICAM-1, VCAM-1, NCAM, and E-selectin, are altered and overexpressed in progressive disease and metastases. Abnormal levels of these latter CAMs have been observed in melanoma and carcinomas of the colon and breast, and NCAM is overexpressed in small-cell lung carcinoma (SCLC). The objective of this study was to determine if serum levels of ICAM-1, VCAM-1, NCAM, and E-selectin could differentiate patients with benign prostate hypertrophy (BPH) from those with prostate carcinoma (CaP) and identify prostate cancers with high potential for progression to metastatic disease. METHODS: Serum levels of these CAMs were determined by ELISA in serum from normal males and females and from patients with BPH and CaP before and after treatment. Sera from patients with breast carcinoma, colon carcinoma, melanoma, and small-cell lung carcinoma were also evaluated, as soluble CAMs have been reported to be elevated in these cancer patients. RESULTS: ICAM-1 levels were elevated in sera from patients with breast carcinoma (P = 0.0004) and melanoma (P = 0.0001). VCAM-1 levels were elevated in sera from patients with colon carcinoma (P = 0.0001). NCAM levels were elevated in the sera of patients with SCLC (P = 0.0001). Normal levels of ICAM-1, E-selectin, and NCAM were found in both BPH and pretreatment CaP patients. Median NCAM levels in hormone-refractive CaP patients were significantly greater than in BPH (P = 0.0005) and CaP patients with pathologically determined organ-confined (P = 0.0014) or nonorgan-confined disease (P = 0.0385). VCAM-1 levels were significantly elevated in both BPH patients (P = 0.0002) and CaP patients (P = 0.0002) when compared with levels for normal age-matched donors. None of the CAMs were found to offer an advantage over prostatic-specific antigen (PSA) for monitoring CaP patients following definitive radiotherapy, radical prostatectomy, or hormonal therapy. CONCLUSIONS: The results of this study indicate that serum ICAM-1, VCAM-1, NCAM, and E-selectin are not clinically useful biomarkers for differentiating CaP from BPH, for predicting progression, for identifying metastatic potential, or for monitoring treatment.

Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is expressed in the adrenal cortex and gonads and regulates the expression of several P450 steroid hydroxylases in vitro. We examined the role of SF-1 in the adrenal glands and gonads in vivo by a targeted disruption of the mouse SF-1 gene. All SF-1-deficient mice died shortly after delivery. Their adrenal glands and gonads were absent, and persistent Mullerian structures were found in all genotypic males. While serum levels of corticosterone in SF-1-deficient mice were diminished, levels of adrenocorticotropic hormone (ACTH) were elevated, consistent with intact pituitary corticotrophs. Intrauterine survival of SF-1-deficient mice appeared normal, and they had normal serum level of corticosterone and ACTH, probably reflecting transplacental passage of maternal steroids. We tested whether SF-1 is required for P450 side-chain-cleavage enzyme (P450scc) expression in the placenta, which expresses both SF-1 and P450scc, and found that in contrast to its strong activation of the P450scc gene promoter in vitro, the absence of SF-1 had no effect on P450scc mRNA levels in vivo. Although the region targeted by our disruption is shared by SF-1 and by embryonal long terminal repeat-binding protein (ELP), a hypothesized alternatively spliced product, we believe that the observed phenotype reflects absent SF-1 alone, as PCR analysis failed to detect ELP transcripts in any mouse tissue, and sequences corresponding to ELP are not conserved across species. These results confirm that SF-1 is an important regulator of adrenal and gonadal development, but its regulation of steroid hydroxylase expression in vivo remains to be established.

Analysis of the glottal excitation of emotionally styled and stressed speech.

The problems of automatic recognition of and synthesis of multistyle speech have become important topics of research in recent years. This paper reports an extensive investigation of the variations that occur in the glottal excitation of eleven commonly encountered speech styles. Glottal waveforms were extracted from utterances of non-nasalized vowels for two speakers for each of the eleven speaking styles. The extracted waveforms were parametrized into four duration-related and two slope-related values. Using these six parameters, the glottal waveforms from the eleven styles of speech were analyzed both qualitatively and quantitatively. The glottal waveforms from each style speech were analyzed both qualitatively and quantitatively. The glottal waveforms from each style of speech have been shown to be significantly and identifiably different from all other styles, thereby confirming the importance of the glottal waveform in conveying speech style information and in causing speech waveform variations. The degree of variation in styled glottal waveforms has been shown to be consistent when trained on one speaker and compared with another.

Deoxyribonucleic acid cytometric analysis of prostate core biopsy specimens: relationship to serum prostate specific antigen and prostatic acid phosphatase, clinical stage and histopathology.

We describe a sampling method of obtaining fresh prostate cells that yields adequate numbers of cells for flow cytometric deoxyribonucleic acid (DNA) analysis and produces histograms of good resolution. Exfoliated cells from 204 prostate biopsy wash specimens obtained by agitation of biopsy cores in saline were fixed and stained for DNA analysis. The mean percentage of hyperdiploid cells was statistically different between the pathologically benign and malignant specimens (p < 0.0001). Hyperdiploid cells of 22% or more exhibited a high degree of specificity for the malignant specimens with only a 1.4% (1 of 69 benign specimens) false-positive rate. However, sensitivity was only 41% (25 of 59 malignant specimens were associated with a flow cytometry analysis of 22% or greater hyperdiploid cells) because of the high false-negative rate (59%, 35 of 59). The percentage of hyperdiploid cells correlated statistically with increasing prostate specific antigen (PSA) levels and approached significance with Gleason grade but did not correlate with prostatic acid phosphatase or clinical stage. When the amount of hyperdiploid cells was 22% or more and serum PSA level was greater than 4.0 ng./ml. a 95% chance of a malignant biopsy was predicted. This result was greater than that predicted by a PSA elevation alone. Only a 5% chance of a malignant biopsy was present for patients with less than 22% hyperdiploid cells and 4.0 ng./ml. or less serum PSA, a decrease over either method separately. This method of DNA assessment permits prospective categorization of tumors by ploidy without interfering with histological assessment. The prognostic importance of ploidy analysis awaits further clinical followup.

Tactile communication of speech: comparison of two computer-based displays.

Two methods of encoding speech for tactile displays were compared in discrimination experiments using speech segments. One display represented the short-term speech spectrum in time-swept mode and used vibration amplitude to encode spectral amplitude. The other represented the linear predictive coding (LPC)-derived vocal tract shape as a filled bar graph in which the number of active vibrators was used to encode cross sectional area. The displays were applied to the thigh via a matrix of vibrators. The vibrators were driven at 250 Hz during voiced segments, and by random noise during unvoiced segments. Overall results show a slight superiority for the spectral display in vowel discrimination. Detailed results were analyzed in terms of an articulatory description of the speech stimuli, a multidimensional scaling (MDS) analysis of confusions, and an ideal receiver analysis. The results of these analyses suggest that the detailed characteristics of the tactile patterns were only crudely discriminated.

Tactile communication of speech: II. Comparison of two spectral displays in a vowel discrimination task.

Vowel discrimination experiments were performed comparing two tactile, Optacon-based, spectral displays: a frequency-amplitude (FA) display and a time-swept (TS) display. The set of vowel pairs tested consisted of all pairs from the set of ten American nondipthongized vowels. One set of materials consisted of natural /b/-V-/t/'s (with each vowel represented by 12 utterances generated by four speakers producing each vowel three times). A second set consisted of synthetic vowels (with each vowel represented by a single waveform). On the average, th score obtained with the second set of materials was substantially better than with the first (92% vs 79%); the score obtained with the TS display was slightly better than with the FA display (87% vs 83%); the feature best differentiated was tenseness, followed by the features high and low, then by round and back, and finally by retroflexion; and the correlation between discrimination performance and the physical parameters duration, amplitude, F1, and F2/F1 (taken singly) was relatively weak. In general, the results of this study, together with those of other studies previously reported, suggest that the two displays studied are far from optimum.

A history of the events which culminated in the first faculty of Veterinary Science in Australia.

A few years ago the University did not cater for properly training the men who would have to conduct the industries of the country. During the past 7 years, however, it had established Schools of Agriculture, Education, Mining Engineering, and had made proper provision for the training of chemists and dentists. The last, and in some respects the greatest step, was the establishment of a School of Veterinary Medicine in connection with the University.