An international review of the characteristics of viral nucleic acid-amplification testing (NAT) reveals a trend towards the use of smaller pool sizes and individual donation NAT.

BACKGROUND AND OBJECTIVES: Nucleic acid-amplification testing (NAT) is used for screening blood donations/donors for blood-borne viruses. We reviewed global viral NAT characteristics and NAT-yield confirmatory testing used by blood operators. MATERIALS AND METHODS: NAT characteristics and NAT-yield confirmatory testing used during 2019 was surveyed internationally by the International Society of Blood Transfusion Working Party Transfusion-Transmitted Infectious Diseases. Reported characteristics are presented herein. RESULTS: NAT was mainly performed under government mandate. Human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B virus (HBV) NAT was performed on all donors and donation types, while selective testing was reported for West Nile virus, hepatitis E virus (HEV), and Zika virus. Individual donation NAT was used for HIV, HCV and HBV by ~50% of responders, while HEV was screened in mini-pools by 83% of responders performing HEV NAT. Confirmatory testing for NAT-yield samples was generally performed by NAT on a sample from the same donation or by NAT and serology on samples from the same donation and a follow-up sample. CONCLUSION: In the last decade, there has been a trend towards use of smaller pool sizes or individual donation NAT. We captured characteristics of NAT internationally in 2019 and provide insights into confirmatory testing approaches used for NAT-yields, potentially benefitting blood operators seeking to implement NAT.

International review of blood donation nucleic acid amplification testing.

BACKGROUND AND OBJECTIVES: Nucleic acid amplification testing (NAT), in blood services context, is used for the detection of viral and parasite nucleic acids to reduce transfusion-transmitted infections. This project reviewed NAT for screening blood donations globally. MATERIALS AND METHODS: A survey on NAT usage, developed by the International Society of Blood Transfusion Working Party on Transfusion-transmitted Infectious Diseases (ISBT WP-TTID), was distributed through ISBT WP-TTID members. Data were analysed using descriptive statistics. RESULTS: Forty-three responses were received from 32 countries. Increased adoption of blood donation viral screening by NAT was observed over the past decade. NAT-positive donations were detected for all viruses tested in 2019 (proportion of donations positive by NAT were 0.0099% for human immunodeficiency virus [HIV], 0.0063% for hepatitis C virus [HCV], 0.0247% for hepatitis B virus [HBV], 0.0323% for hepatitis E virus [HEV], 0.0014% for West Nile virus [WNV] and 0.00005% for Zika virus [ZIKV]). Globally, over 3100 NAT-positive donations were identified as NAT yield or solely by NAT in 2019 and over 22,000 since the introduction of NAT, with HBV accounting for over half. NAT-positivity rate was higher in first-time donors for all viruses tested except WNV. During 2019, a small number of participants performed NAT for parasites (Trypanosoma cruzi, Babesia spp., Plasmodium spp.). CONCLUSION: This survey captures current use of blood donation NAT globally. There has been increased NAT usage over the last decade. It is clear that NAT contributes to improving blood transfusion safety globally; however, there is a need to overcome economic barriers for regions/countries not performing NAT.

Fibre orientation atlas guided rapid segmentation of white matter tracts.

Fibre tract delineation from diffusion magnetic resonance imaging (MRI) is a valuable clinical tool for neurosurgical planning and navigation, as well as in research neuroimaging pipelines. Several popular methods are used for this task, each with different strengths and weaknesses making them more or less suited to different contexts. For neurosurgical imaging, priorities include ease of use, computational efficiency, robustness to pathology and ability to generalise to new tracts of interest. Many existing methods use streamline tractography, which may require expert neuroimaging operators for setting parameters and delineating anatomical regions of interest, or suffer from as a lack of generalisability to clinical scans involving deforming tumours and other pathologies. More recently, data-driven approaches including deep-learning segmentation models and streamline clustering methods have improved reproducibility and automation, although they can require large amounts of training data and/or computationally intensive image processing at the point of application. We describe an atlas-based direct tract mapping technique called 'tractfinder', utilising tract-specific location and orientation priors. Our aim was to develop a clinically practical method avoiding streamline tractography at the point of application while utilising prior anatomical knowledge derived from only 10-20 training samples. Requiring few training samples allows emphasis to be placed on producing high quality, neuro-anatomically accurate training data, and enables rapid adaptation to new tracts of interest. Avoiding streamline tractography at the point of application reduces computational time, false positives and vulnerabilities to pathology such as tumour deformations or oedema. Carefully filtered training streamlines and track orientation distribution mapping are used to construct tract specific orientation and spatial probability atlases in standard space. Atlases are then transformed to target subject space using affine registration and compared with the subject's voxel-wise fibre orientation distribution data using a mathematical measure of distribution overlap, resulting in a map of the tract's likely spatial distribution. This work includes extensive performance evaluation and comparison with benchmark techniques, including streamline tractography and the deep-learning method TractSeg, in two publicly available healthy diffusion MRI datasets (from TractoInferno and the Human Connectome Project) in addition to a clinical dataset comprising paediatric and adult brain tumour scans. Tract segmentation results display high agreement with established techniques while requiring less than 3 min on average when applied to a new subject. Results also display higher robustness than compared methods when faced with clinical scans featuring brain tumours and resections. As well as describing and evaluating a novel proposed tract delineation technique, this work continues the discussion on the challenges surrounding the white matter segmentation task, including issues of anatomical definitions and the use of quantitative segmentation comparison metrics.

Atezolizumab plus Magrolimab, Niraparib, or Tocilizumab versus Atezolizumab Monotherapy in Platinum-Refractory Metastatic Urothelial Carcinoma: A Phase Ib/II Open-Label, Multicenter, Randomized Umbrella Study (MORPHEUS Urothelial Carcinoma).

PURPOSE: The MORPHEUS platform was designed to identify early efficacy signals and evaluate the safety of novel immunotherapy combinations across cancer types. The phase Ib/II MORPHEUS-UC trial (NCT03869190) is evaluating atezolizumab plus magrolimab, niraparib, or tocilizumab in platinum-refractory locally advanced or metastatic urothelial carcinoma (mUC). Additional treatment combinations were evaluated and will be reported separately. PATIENTS AND METHODS: Patients had locally advanced or mUC that progressed during or following treatment with a platinum-containing regimen. The primary efficacy endpoint was investigator-assessed objective response rate (ORR). Key secondary endpoints included investigator-assessed progression-free survival (PFS) and overall survival (OS). Safety and exploratory biomarker analyses were also conducted. RESULTS: Seventy-six patients were randomized to receive either atezolizumab plus magrolimab (n = 16), atezolizumab plus niraparib (n = 15), atezolizumab plus tocilizumab (n = 15), or atezolizumab monotherapy (control; n = 30). No additive benefit in ORR, PFS, or OS was seen in the treatment arms versus the control. The best confirmed ORR was 26.7% with atezolizumab plus magrolimab, 6.7% with atezolizumab plus niraparib, 20.0% with atezolizumab plus tocilizumab, and 27.6% with atezolizumab monotherapy. Overall, the treatment combinations were tolerable, and adverse events were consistent with each agent's known safety profile. Trends were observed for shrinkage of programmed death-ligand 1-positive tumors (atezolizumab, atezolizumab plus magrolimab, atezolizumab plus tocilizumab), inflamed tumors, or tumors with high mutational burden (atezolizumab), and immune excluded tumors (atezolizumab plus magrolimab). CONCLUSIONS: The evaluated regimens in MORPHEUS-UC were tolerable. However, response rates for the combinations did not meet the criteria for further development in platinum-experienced locally advanced or mUC.

Fibre tract segmentation for intraoperative diffusion MRI in neurosurgical patients using tract-specific orientation atlas and tumour deformation modelling.

PURPOSE: Intraoperative diffusion MRI could provide a means of visualising brain fibre tracts near a neurosurgical target after preoperative images have been invalidated by brain shift. We propose an atlas-based intraoperative tract segmentation method, as the standard preoperative method, streamline tractography, is unsuitable for intraoperative implementation. METHODS: A tract-specific voxel-wise fibre orientation atlas is constructed from healthy training data. After registration with a target image, a radial tumour deformation model is applied to the orientation atlas to account for displacement caused by lesions. The final tract map is obtained from the inner product of the atlas and target image fibre orientation data derived from intraoperative diffusion MRI. RESULTS: The simple tumour model takes only seconds to effectively deform the atlas into alignment with the target image. With minimal processing time and operator effort, maps of surgically relevant tracts can be achieved that are visually and qualitatively comparable with results obtained from streamline tractography. CONCLUSION: Preliminary results demonstrate feasibility of intraoperative streamline-free tract segmentation in challenging neurosurgical cases. Demonstrated results in a small number of representative sample subjects are realistic despite the simplicity of the tumour deformation model employed. Following this proof of concept, future studies will focus on achieving robustness in a wide range of tumour types and clinical scenarios, as well as quantitative validation of segmentations.

The regression of the bovine tuberculin reaction: Results from the Reactor Quality Assurance study in Northern Ireland.

Tuberculin skin tests remain widely used in the control of bovine tuberculosis (bTB) in cattle. Little is known about the rate of regression of tuberculin reactions after the comparative intradermal cervical test (CICT) in cattle. This study aimed to collect data to describe tuberculin regression in reactors following the CICT at 72 ± 4 h post injection. Reactors were also tested using the interferon gamma (IFN-γ) assay to establish if any pattern existed between these results and the CICT reaction regression. The data were derived from 108 herds, 112 herd-level CICTs and 1008 animals. A multivariable linear mixed model was built to explore the regression of the bovine tuberculin reaction over time and the influence of potential predictors. The results confirmed a proportional decline in the bovine tuberculin reaction occurred over time. The predictors in the final model demonstrated that regression of the tuberculin reaction differed between reactors according to their IFN-γ test results and whether visible lesions were present at slaughter. Follow-up measurement of tuberculin reactions and the serial use of the IFN-γ assay in large breakdowns has the potential to provide both a mechanism for quality assurance of the current CICT bTB surveillance and the identification of atypical breakdowns or reactors requiring further investigation.

Serological test performance for bovine tuberculosis in cattle from herds with evidence of on-going infection in Northern Ireland.

The ability to accurately identify infected hosts is the cornerstone of effective disease control and eradication programs. In the case of bovine tuberculosis, accurately identifying infected individual animals has been challenging as all available tests exhibit limited discriminatory ability. Here we assess the utility of two serological tests (IDEXX Mycobacterium bovis Ab test and Enfer multiplex antibody assay) and assess their performance relative to skin test (Single Intradermal Comparative Cervical Tuberculin; SICCT), gamma-interferon (IFNγ) and post-mortem results in a Northern Ireland setting. Furthermore, we describe a case-study where one test was used in conjunction with statutory testing. Serological tests using samples taken prior to SICCT disclosed low proportions of animals as test positive (mean 3% positive), despite the cohort having high proportions with positive SICCT test under standard interpretation (121/921; 13%) or IFNγ (365/922; 40%) results. Furthermore, for animals with a post-mortem record (n = 286), there was a high proportion with TB visible lesions (27%) or with laboratory confirmed infection (25%). As a result, apparent sensitivities within this cohort was very low (≤15%), however the tests succeeded in achieving very high specificities (96-100%). During the case-study, 7/670 (1.04%) samples from SICCT negative animals from a large chronically infected herd were serology positive, with a further 17 animals being borderline positive (17/670; 2.54%). Nine of the borderline animals were voluntarily removed, none of which were found to be infected post-mortem (no lesions/bacteriology negative). One serology test negative animal was subsequently found to have lesions at slaughter with M. bovis confirmed in the laboratory.

The nanophthalmos protein TMEM98 inhibits MYRF self-cleavage and is required for eye size specification.

The precise control of eye size is essential for normal vision. TMEM98 is a highly conserved and widely expressed gene which appears to be involved in eye size regulation. Mutations in human TMEM98 are found in patients with nanophthalmos (very small eyes) and variants near the gene are associated in population studies with myopia and increased eye size. As complete loss of function mutations in mouse Tmem98 result in perinatal lethality, we produced mice deficient for Tmem98 in the retinal pigment epithelium (RPE), where Tmem98 is highly expressed. These mice have greatly enlarged eyes that are very fragile with very thin retinas, compressed choroid and thin sclera. To gain insight into the mechanism of action we used a proximity labelling approach to discover interacting proteins and identified MYRF as an interacting partner. Mutations of MYRF are also associated with nanophthalmos. The protein is an endoplasmic reticulum-tethered transcription factor which undergoes autoproteolytic cleavage to liberate the N-terminal part which then translocates to the nucleus where it acts as a transcription factor. We find that TMEM98 inhibits the self-cleavage of MYRF, in a novel regulatory mechanism. In RPE lacking TMEM98, MYRF is ectopically activated and abnormally localised to the nuclei. Our findings highlight the importance of the interplay between TMEM98 and MYRF in determining the size of the eye.

Gamma Tocopherol Reduced Chemotherapeutic-Induced ROS in an Ovarian Granulosa Cell Line, But Not in Breast Cancer Cell Lines In Vitro.

Doxorubicin and cyclophosphamide are used to treat breast cancer, but they also cause infertility through off-target cytotoxicity towards proliferating granulosa cells that surround eggs. Each chemotherapeutic generates reactive oxygen species (ROS) but the effects of the combination, or the antioxidants alpha (αToc) and gamma tocopherol (γToc) on ROS in breast cancer or ovarian cells are unknown. Human breast cancer (MCF7, T47D) and ovarian cancer (OVCAR, COV434) cells were loaded with DCDFA and exposed (1, 2, 3, 24 h) to the MCF7-derived EC25 values of individual agents, or to combinations of these. ROS were quantified and viable cells enumerated using crystal violet or DAPI. Each chemotherapeutic killed ~25% of MCF7, T47D and OVCAR cells, but 57 ± 2% (doxorubicin) and 66 ± 2% (cyclophosphamide) of the COV434 granulosa cells. The combined chemotherapeutics decreased COV434 cell viability to 34 ± 5% of control whereas doxorubicin + cyclophosphamide + γToc reduced ROS within 3 h (p < 0.01) and reduced cytotoxicity to 54 ± 4% (p < 0.05). αToc was not cytotoxic, whereas γToc killed ~25% of the breast cancer but none of the ovarian cells. Adding γToc to the combined chemotherapeutics did not change ROS or cytotoxicity in MCF7, T47D or OVCAR cells. The protection γToc afforded COV434 granulosa cells against chemotherapy-induced ROS and cytotoxicity suggests potential for fertility preservation.

Effect of Chemotherapeutics and Tocopherols on MCF-7 Breast Adenocarcinoma and KGN Ovarian Carcinoma Cell Lines In Vitro.

The combination of doxorubicin and cyclophosphamide commonly used to treat breast cancer can cause premature ovarian failure and infertility. α-Tocopherol is a potent antioxidant whereas γ-tocopherol causes apoptosis in a variety of cancer models in vitro including breast cancer. We hypothesised that the combination of doxorubicin (Dox) and 4-hydroperoxycyclophosphamide (4-Cyc) would be more cytotoxic in vitro than each agent alone, and that α-tocopherol would reduce and γ-tocopherol would augment the cytotoxicity of the combined chemotherapeutics. Human MCF-7 breast cancer and KGN ovarian cells were exposed to Dox, 4-Cyc, combined Dox and 4-Cyc, α-tocopherol, γ-tocopherol, or a combination of Dox and 4-Cyc with α-tocopherol or γ-tocopherol. Cell viability was assessed using a crystal violet assay according to four schedules: 24h exposure, 24h exposure + 24h culture in medium, 24h exposure + 48h culture in medium, or 72h continuous exposure. Supernatants from each separate KGN culture experiment (n=3) were examined using an estradiol ELISA. Dox was cytotoxic to both MCF-7 and KGN cells, but 4-Cyc only killed MCF-7 cells. γ-Tocopherol significantly decreased MCF-7 but not KGN cell viability. The combined chemotherapeutics and γ-tocopherol were more cytotoxic to MCF-7 than KGN cells, and α-tocopherol reduced the cytotoxicity of the combined chemotherapeutics towards KGN ovarian cells, but not MCF-7 cells. The addition of both γ-tocopherol and α-tocopherol to the chemotherapeutic combination of Dox and cyclophosphamide has the potential to increase in vitro chemotherapeutic efficacy against breast cancer cells whilst decreasing cytotoxicity towards ovarian granulosa cells.

Real time monitoring and quantification of reactive oxygen species in breast cancer cell line MCF-7 by 2',7'-dichlorofluorescin diacetate (DCFDA) assay.

The detection of reactive oxygen species (ROS) using 2',7'-dichlorofluorescin diacetate (DCFDA) is commonly performed by a single measurement of fluorescence but this fails to capture a profile of ROS generation over time. This study aimed to develop a real-time monitoring method to increase the utility of the assay, to incorporate cytotoxicity screening and to describe the combined effects of DCFDA and the ROS generator, Ter-butyl hydrogen peroxide (TBHP). Breast cancer MCF-7 cells were loaded with DCFDA (0-50 µM) for 45 min, and then exposed to TBHP (0-50 µM). Fluorescence was recorded according to three different schedules: every hour for 6 h, or once after 6 h or 24 h. Viability was assessed in a crystal violet assay and cell morphology was examined by microscopy. TBHP caused a time and dose-dependent increase in ROS and the magnitude of the fluorescent signal was affected by the loading concentration of DCFDA. Reading the fluorescence every hour for 6 h did not diminish the emission signal. The most sensitive and reliable combination for this ROS assay was 10 µM DCFDA with 25 µM TBHP; since higher concentrations of DCFDA compromised cell viability. In conclusion we adapted a single point ROS assay to enable production of a profile of ROS generation over an extended 6 h period, and related this to cell viability and morphology.

Effects of ovarian disaggregation on adult murine follicle yield and viability.

Follicles are isolated from ovaries for numerous reasons, including IVM, but adult murine yields are <2 folliclesmg-1. The aim of the present study was to optimise ovarian disaggregation and develop methods applicable to the rapid screening of follicle viability. Ovaries from adult mice (n=7) were halved and disaggregated mechanically, or by using collagenase IV (Col-IV; 590UmL-1) or animal origin-free collagenase IV (AOF) at 590 or 1180UmL-1. Isolated follicles were stained with 4',6'-diamidino-2-phenylindole (DAPI; nuclei), chloromethyl-X-rosamine (CMXRos; mitochondria) or fluorescein isothiocyanate-conjugated anti-α-tubulin antibody. Follicle diameters and staining were measured and analysed using ImageJ, and data analysed using GraphPad Prism. Col-IV disaggregation yielded the highest number of follicles (17±10 folliclesmg-1 ovarian tissue). All disaggregation methods released more secondary follicles (86±20 per ovary; P<0.05) than any other size cohort. Mechanical and Col-IV disaggregation yielded similar numbers of morphologically intact follicles, whereas AOF disaggregation caused more damage (P<0.01). As the morphological disruption increased, DAPI and CMXRos staining decreased (P<0.05), and tubulin localisation became more heterogeneous. Col-IV disaggregation gave the best yield of morphologically intact follicles containing viable granulosa cells. In conclusion, we improved adult murine follicle yields and applied molecular markers to assess follicle morphology, cellular cytoskeleton and mitochondrial function.

Angiomyolipoma with Epithelial Cysts Masquerading as a Cystic Renal Cell Carcinoma.

Angiomyolipoma with epithelial cysts (AMLEC) is a very uncommon renal tumor. AMLEC has a characteristic histological appearance and immunohistochemical staining pattern, knowledge of which should preclude misdiagnosis by pathologists. We present a rare case of an AMLEC which was suspected to be a cystic renal cell carcinoma radiologically. We describe the characteristic immunological staining pattern and ultrastructural features of this lesion and discuss the potential differential diagnoses.

Sclerosing odontogenic carcinoma presenting on the hard palate of a 43-year-old female: a case report.

We present a case of sclerosing odontogenic carcinoma (SOC) occurring on the hard palate of a 43-year-old female. The tumor presented as an asymptomatic firm swelling and histopathologically was characterized by widely dispersed nests and cords of bland cells infiltrating between hyalinized collagen fibers. Prominent perineural and intraneural invasion and erosion of bone was noted. The tumor cells showed staining with antibodies to pan-cytokeratin (PanCK), cytokeratin 19 (CK19), cytokeratin 5/6 (CK5/6), cytokeratin 14 (CK14), p63 and E-cadherin, but no staining with antibodies to carcinoembryonic antigen (CEA), cytokeratin 7 (CK7), cytokeratin 20 (CK20), estrogen receptor (ER), progesterone receptor (PR) or S100. Staining for mucin with alcian blue/periodic acid-Schiff with diastase was equivocal, with no definite evidence of mucin or muciphages. An initial diagnosis of adenocarcinoma NOS was made upon incisional biopsy, with the prominent filing pattern and cytoplasmic vacuolization prompting consideration of metastatic breast cancer in the first instance. The true nature of the tumor became clear after staging investigations and surgical resection. The patient was treated by surgery alone and is disease-free after 17 months.

Undifferentiated murine embryonic stem cells used to model the effects of the blue-green algal toxin cylindrospermopsin on preimplantation embryonic cell proliferation.

Undifferentiated mouse embryonic stem cell (mES) proliferation in vitro resembles aspects of in vivo pre-implantation embryonic development. mES were used to assess the embryo-toxicity of cylindrospermopsin (CYN), a water contaminant with an Australian Drinking Water Guideline (ADWG) of 1 µg/L. mES exposed to 0-1 µg/mL CYN for 24-168 h were subjected to an optimised crystal violet viability assay. mES exposed to retinoic acid ± 1 µg/L CYN differentiated into neural-like cells confirmed by morphological examination and RT-PCR for Oct4, Brachyury and Nestin. The CYN No Observed Effect Concentration (OEC) was 0.5 µg/mL, the Lowest OEC was 1 µg/mL (p < 0.001, n = 3), and the IC50 was 0.86 µg/mL after 24 h. The ADWG 1 µg/L CYN did not affect differentiation or proliferation after 72 h, but decreased proliferation after 168 h (p < 0.05). We conclude that higher algal bloom-associated CYN concentrations have the potential to impair in vivo pre-implantation development, and the mES crystal violet assay has broad application to screening environmental toxins.

Are the Traditional Medical Uses of Muricidae Molluscs Substantiated by Their Pharmacological Properties and Bioactive Compounds?

Marine molluscs from the family Muricidae hold great potential for development as a source of therapeutically useful compounds. Traditionally known for the production of the ancient dye Tyrian purple, these molluscs also form the basis of some rare traditional medicines that have been used for thousands of years. Whilst these traditional and alternative medicines have not been chemically analysed or tested for efficacy in controlled clinical trials, a significant amount of independent research has documented the biological activity of extracts and compounds from these snails. In particular, Muricidae produce a suite of brominated indoles with anti-inflammatory, anti-cancer and steroidogenic activity, as well as choline esters with muscle-relaxing and pain relieving properties. These compounds could explain some of the traditional uses in wound healing, stomach pain and menstrual problems. However, the principle source of bioactive compounds is from the hypobranchial gland, whilst the shell and operculum are the main source used in most traditional remedies. Thus further research is required to understand this discrepancy and to optimise a quality controlled natural medicine from Muricidae.

The juxtamembrane region of synaptotagmin 1 interacts with dynamin 1 and regulates vesicle fission during compensatory endocytosis in endocrine cells.

Synaptotagmin 1 (Syt1) is a synaptic vesicle protein that is important for the kinetics of both exocytosis and endocytosis, and is thus a candidate molecule to link these two processes. Although the tandem Ca(2+)-binding C2 domains of Syt1 have important roles in exocytosis and endocytosis, the function of the conserved juxtamembrane (jxm) linker region has yet to be determined. We now demonstrate that the jxm region of Syt1 interacts directly with the pleckstrin homology (PH) domain of the endocytic protein dynamin 1. By using cell-attached capacitance recordings with millisecond time resolution to monitor clathrin-mediated endocytosis of single vesicles in neuroendocrine chromaffin cells, we find that loss of this interaction prolongs the lifetime of the fission pore leading to defects in the dynamics of vesicle fission. These results indicate a previously undescribed interaction between two major regulatory proteins in the secretory vesicle cycle and that this interaction regulates endocytosis.

A dominant-negative mutation of mouse Lmx1b causes glaucoma and is semi-lethal via LDB1-mediated dimerization [corrected].

Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice.

An in vitro high-throughput assay for screening reproductive and toxic effects of anticancer compounds.

An in vitro assay was developed that simultaneously tested the effects of anticancer drug candidates on cytotoxicity, hormone synthesis, and gonadotrophin responsiveness using the choriocarcinoma JAr cell line. JAr culture conditions were optimized and then cells were exposed to a marine mollusc extract in the presence and absence of hCG. The intra- and interassay coefficients of variation of the optimized 1 H thiazolyl blue tetrazolium bromide assay were 11.3% and 10.9%, respectively. hCG (1,000 mIU/mL) increased progesterone (P4) synthesis after 24 H (P<0.05). The mollusc extract significantly decreased cell viability, with the IC50 affected by incubation time, but not hCG. P4 synthesis was inhibited at low concentrations of the anticancer extract, but stimulated at the highest concentration, and complex interactions of P4 were also found with hCG. In conclusion, the optimized assay is useful to characterize the effects of novel drugs on cytotoxicity, basal, and gonadotrophin-stimulated P4 synthesis in vitro, and can be used to inform subsequent in vivo studies.