AgAnimalGenomes: browsers for viewing and manually annotating farm animal genomes.

Current genome sequencing technologies have made it possible to generate highly contiguous genome assemblies for non-model animal species. Despite advances in genome assembly methods, there is still room for improvement in the delineation of specific gene features in the genomes. Here we present genome visualization and annotation tools to support seven livestock species (bovine, chicken, goat, horse, pig, sheep, and water buffalo), available in a new resource called AgAnimalGenomes. In addition to supporting the manual refinement of gene models, these browsers provide visualization tracks for hundreds of RNAseq experiments, as well as data generated by the Functional Annotation of Animal Genomes (FAANG) Consortium. For species with predicted gene sets from both Ensembl and RefSeq, the browsers provide special tracks showing the thousands of protein-coding genes that disagree across the two gene sources, serving as a valuable resource to alert researchers to gene model issues that may affect data interpretation. We describe the data and search methods available in the new genome browsers and how to use the provided tools to edit and create new gene models.

A quantitative comparison of points of departure between 28-day and 90-day repeated dose studies with a proposed extrapolation factor.

The influence of exposure duration on chemical toxicity has important implications for risk assessment. Although a default 10-fold extrapolation factor is commonly applied when the toxicological dataset includes a subchronic (90-day) study but lacks studies of chronic duration, little consensus has been reached on an appropriate extrapolation factor to apply when the dataset includes a 28-day study but lacks studies of longer durations. The goal of the present assessment was to identify a 28-day to 90-day extrapolation factor by analyzing distributions of ratios of No-Observed-Adverse-Effect Levels (NOAELs) and Benchmark Doses (BMDs) derived from 28-day and 90-day studies. The results of this analysis suggest that a default 10-fold extrapolation factor in chemical risk assessment applications is sufficient to account for the uncertainty associated with evaluating human health risk based on results from a 28-day study in the absence of results from a 90-day study. This analysis adds significantly to the growing body of literature interpreting the influence of exposure duration on chemical toxicity that will likewise facilitate discussions on the future state of testing requirements in the international regulatory community.

Biodistribution and Clearance of Stable Superparamagnetic Maghemite Iron Oxide Nanoparticles in Mice Following Intraperitoneal Administration.

Nanomedicine is an emerging field with great potential in disease theranostics. We generated sterically stabilized superparamagnetic iron oxide nanoparticles (s-SPIONs) with average core diameters of 10 and 25 nm and determined the in vivo biodistribution and clearance profiles. Healthy nude mice underwent an intraperitoneal injection of these s-SPIONs at a dose of 90 mg Fe/kg body weight. Tissue iron biodistribution was monitored by atomic absorption spectroscopy and Prussian blue staining. Histopathological examination was performed to assess tissue toxicity. The 10 nm s-SPIONs resulted in higher tissue-iron levels, whereas the 25 nm s-SPIONs peaked earlier and cleared faster. Increased iron levels were detected in all organs and body fluids tested except for the brain, with notable increases in the liver, spleen, and the omentum. The tissue-iron returned to control or near control levels within 7 days post-injection, except in the omentum, which had the largest and most variable accumulation of s-SPIONs. No obvious tissue changes were noted although an influx of macrophages was observed in several tissues suggesting their involvement in s-SPION sequestration and clearance. These results demonstrate that the s-SPIONs do not degrade or aggregate in vivo and intraperitoneal administration is well tolerated, with a broad and transient biodistribution. In an ovarian tumor model, s-SPIONs were shown to accumulate in the tumors, highlighting their potential use as a chemotherapy delivery agent.

The biology of meniscal pathology in osteoarthritis and its contribution to joint disease: beyond simple mechanics.

The meniscal cartilages in the knee function to improve congruity of the medial and lateral femoro-tibial joints and play critical roles in load distribution and joint stability. Meniscal tears of various configurations are one of the most common conditions of the knee and are associated with an increased risk of developing osteoarthritis (OA). While this risk has been largely attributed to loss of the biomechanical functions of the menisci, there is accumulating evidence suggesting that other aspects of meniscal biology may play a role in determining the long-term consequences of meniscal damage for joint health. In this narrative review, we examine the existing literature and present some new data implicating synthesis and secretion of enzymes and other pro-catabolic mediators by injured and degenerate menisci, contributing to the pathological change in other knee joint tissues in OA.

Interleukin-1α induces focal degradation of biglycan and tissue degeneration in an in-vitro ovine meniscal model.

We have developed an ovine meniscal explant model where the focal degradative events leading to characteristic fragmentation patterns of biglycan in human OA of the knee and hip, and evident in animal models of knee OA and IVD degeneration are reproduced in culture. Lateral and medial menisci were dissected into outer, mid and inner zones and established in explant culture±IL-1 (10ng/ml). The biglycan species present in conditioned media samples and in GuHCl extracts of tissues were examined by Western blotting using two C-terminal antibodies PR-85 and EF-Bgn. Clear differences were evident in the biglycan species in each meniscal tissue zone with the medial outer meniscus having lower biglycan levels and major fragments of 20, 28, 33 and 36, 39kDa. Similar fragmentation was detected in articular cartilage samples, 42-45kDa core protein species were also detected. Biglycan fragmentation was not as extensive in the IL-1 stimulated meniscal cultures with 36, 39, 42 and 45kDa biglycan species evident. Thus the medial meniscus outer zone displayed the highest levels of biglycan processing in this model and correlated with a major zone of meniscal remodelling in OA in man. Significantly, enzymatic digests of meniscal tissues with MMP-13, ADAMTS-4 and ADAMTS-5 have also generated similar biglycan species in-vitro. Zymography confirmed that the medial outer zone was the region of maximal MMP activity. This model represents a convenient system to recapitulate matrix remodelling events driven by IL-1 in pathological cartilages and in animal models of joint degeneration.

Centromeric transposable elements and epigenetic status drive karyotypic variation in the eastern hoolock gibbon.

Great apes have maintained a stable karyotype with few large-scale rearrangements; in contrast, gibbons have undergone a high rate of chromosomal rearrangements coincident with rapid centromere turnover. Here we characterize assembled centromeres in the Eastern hoolock gibbon, Hoolock leuconedys (HLE), finding a diverse group of transposable elements (TEs) that differ from the canonical alpha satellites found across centromeres of other apes. We find that HLE centromeres contain a CpG methylation centromere dip region, providing evidence this epigenetic feature is conserved in the absence of satellite arrays; nevertheless, we report a variety of atypical centromeric features, including protein-coding genes and mismatched replication timing. Further, large structural variations define HLE centromeres and distinguish them from other gibbons. Combined with differentially methylated TEs, topologically associated domain boundaries, and segmental duplications at chromosomal breakpoints, we propose that a "perfect storm" of multiple genomic attributes with propensities for chromosome instability shaped gibbon centromere evolution.

A fluorescent colorimetric pH sensor and the influences of matrices on sensing performances.

A fluorescent colorimetric pH sensor was developed by a polymerization of a monomeric fluorescein based green emitter (SM1) with a monomeric 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran derived red emitter (SM2) in poly(2-hydroxyethyl methacrylate)-co-polyacrylamide (PHEMA-co-PAM) matrices. Polymerized SM1 (PSM1) in the polymer matrices showed bright emissions at basic conditions and weak emissions at acidic conditions. Polymerized SM2 (PSM2) in the polymer matrices exhibited a vastly different response when compared to PSM1. The emissions of PSM2 are stronger under acidic conditions than those under basic conditions. When SM1 and SM2 were polymerized in the same polymer matrix, a dual emission sensor acting as a ratiometric pH sensor (PSM1,2) was successfully developed. Because the PSM1 and PSM2 exhibited different pH responses and separated emission windows, the changes in the emission colors were clearly observed in their dual color sensor of PSM1,2, which changed emission colors dramatically from green at pH 7 to red at pH 4, which was detected visually and/or by using a color camera under an excitation of 488 nm. In addition to the development of the dual color ratiometric pH sensor, we also studied the effects of different matrix compositions, crosslinkers, and charges on the reporting capabilities of the sensors (sensitivity and pKa).

Predictors of tissue infarction from distal emboli after mechanical thrombectomy.

BACKGROUND: Distal embolization after endovascular thrombectomy (EVT) is common. We aimed to determine factors associated with tissue infarction in the territories of distal emboli. METHODS: This is a retrospective cohort study of consecutive patients with anterior circulation large vessel occlusions who underwent EVT from 2015 to 2021. Patients with Thrombolysis In Cerebral Infarction (TICI) 2b reperfusion and follow-up imaging were identified. Baseline characteristics, procedural details, and imaging findings were reviewed. Primary outcome was categorized according to the occurrence of infarction at the territory of distal embolus on follow-up diffusion-weighted imaging MRI. RESULTS: Of 156 subjects, 97 (62%) had at least one infarction in the territories at risk. Hypertension was significantly more prevalent in the infarct group (83% vs 53%, P=0.001). General anesthesia was more commonly used in the infarct group (60% vs 43%, P=0.037). The median number of distal emboli and diameter of the occluded vessel were similar. After adjusting for confounders, hypertension (aOR 4.73, 95% CI 1.81 to 13.25, P=0.002), higher blood glucose (aOR 1.01, 95% CI 1.00 to 1.03, P=0.023), and general anesthesia (aOR 2.75, 95% CI 1.15 to 6.84, P=0.025) were independently associated with infarction. The presence of angiographic leptomeningeal collaterals predicted tissue survival (aOR 0.13, 95% CI 0.05 to 0.33, P<0.001). 90-day modified Rankin scale (mRS) scores were worse for the infarction patients (mRS 0-2: infarct, 39% vs 55%, P=0.046). CONCLUSIONS: Nearly 40% of patients with TICI 2b had no tissue infarction in the territory of a distal embolus. The association of infarction with hypertension and general anesthesia suggests late or post-procedural blood pressure management could be a modifiable factor. Patients with poor leptomeningeal collaterals or hyperglycemia may benefit from further attempts at revascularization.

Epivolve: A Protocol for Site-Directed Antibodies.

Researchers can often successfully generate antibodies to predicted epitopes. Especially when the epitopes are on the surface of a protein or in a hydrophilic loop. But it is difficult to direct recombinant antibodies to bind either to- or near a specific amino acid on a protein or peptide. We have developed a unique immune-targeting strategy, that we call "Epivolve," that enables us to make site-specific antibodies (Abs). Epivolve technology leverages a highly immunogenic modified amino acid that acts as a "pseudo-hapten" immuno-target and takes advantage of Ab affinity maturation technologies to make high-affinity site-specific antibodies. Epivolve functions by the evolution of an Ab paratope to either synonymous or especially non-synonymous amino acid (aa) binding. Here we describe the use of Epivolve technology in phage display and the protocols for developing site-specific antibodies.

1H NMR spectroscopy of serum reveals unique metabolic fingerprints associated with subtypes of surgically induced osteoarthritis in sheep.

Osteoarthritis (OA) is a highly prevalent joint disease. Its slow progressive nature and the correlation between pathological changes and clinical symptoms mean that OA is often well advanced by the time of diagnosis. In the absence of any specific pharmacological treatments, there is a pressing need to develop robust biomarkers for OA. We have adopted a nuclear magnetic resonance (NMR)-based metabolomic strategy to identify molecular responses to surgically induced OA in an animal model. Sheep underwent one of three types of surgical procedure (sham (control), meniscal destabilization, MD or anterior cruciate ligament transaction, ACLT), and for every animal a serum sample was collected both pre- and postoperatively, thus, affording two types of "control" data for comparison. 1D 1H NMR spectra were acquired from each sample at 800 MHz and the digitized spectral data were analyzed using principal components analysis and partial least-squares regression discriminant analysis. Our approach, combined with the study design, allowed us to separate the metabolic responses to surgical intervention from those associated with OA. We were able to identify dimethyl sulfone (DMSO2) as being increased in MD after 4 weeks, while ACLT-induced OA exhibited increased 3-methylhistidine and decreased branched chain amino acids (BCAAs). The findings are discussed in the context of interpretation of metabolomic results in studies of human disease, and the selection of appropriate "control" data sets.

Derivation of splice junction-specific antibodies using a unique hapten targeting strategy and directed evolution.

Alternative splicing of RNA occurs frequently in eukaryotic cells and can result in multiple protein isoforms that are nearly identical in amino acid sequence, but have unique biological roles. Moreover, the relative abundance of these unique isoforms can be correlative with diseased states and potentially used as biomarkers or therapeutic targets. However, due to high sequence similarities among isoforms, current proteomic methods are incapable of differentiating native protein isoforms derived from most alternative splicing events. Herein, a strategy employing a nonsynonymous, non-native amino acid (nnAA) pseudo-hapten (i.e. an amino acid or amino acid derivative that is different from the native amino acid at a particular position) as a targeting epitope in splice junction-spanning peptides was successful in directed antibody derivation. After isolating nnAA-specific antibodies, directed evolution reduced the antibody's binding dependence on the nnAA pseudo-hapten and improved binding to the native splice junction epitope. The resulting antibodies demonstrated codependent binding affinity to each exon of the splice junction and thus are splice junction- and isoform-specific. Furthermore, epitope scanning demonstrated that positioning of the nnAA pseudo-hapten within a peptide antigen can be exploited to predetermine the isolated antibody's specificity at, or near, amino acid resolution. Thus, this nnAA targeting strategy has the potential to robustly derive splice junction- and site-specific antibodies that can be used in a wide variety of research endeavors to unambiguously differentiate native protein isoforms.

MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot.

Antibody (Ab) validation is the procedure in which an Ab is thoroughly assayed for sensitivity and specificity in a given application. Validation of Abs against post-translationally modified (PTM) targets is particularly challenging because it requires specifically prepared antigen. Here we describe a novel validation method using surrogate proteins in a Western blot. The surrogate protein, which we termed 'MILKSHAKE,' is a modified maltose binding protein enzymatically conjugated to a peptide from the chosen target that is either modified or nonmodified at the residue of interest. The certainty of the residue's modification status can be used to confirm Ab specificity. This method also allows for Ab validation even in the absence or limited availability of treated cell lysates.

A cross-sectional study of social inequities in medical crowdfunding campaigns in the United States.

Americans are increasingly relying on crowdfunding to pay for the costs of healthcare. In medical crowdfunding (MCF), online platforms allow individuals to appeal to social networks to request donations for health and medical needs. Users are often told that success depends on how they organize and share their campaigns to increase social network engagement. However, experts have cautioned that MCF could exacerbate health and social disparities by amplifying the choices (and biases) of the crowd and leveraging these to determine who has access to financial support for healthcare. To date, research on potential axes of disparity in MCF, and their impacts on fundraising outcomes, has been limited. To answer these questions, this paper presents an exploratory cross-sectional study of a randomized sample of 637 MCF campaigns on the popular platform GoFundMe, for which the race, gender, age, and relationships of campaigners and campaign recipients were categorized alongside campaign characteristics and outcomes. Using both descriptive and inferential statistics, the analysis examines race, gender, and age disparities in MCF use, and tests how these are associated with differential campaign outcomes. The results show systemic disparities in MCF use and outcomes: people of color (and black women in particular) are under-represented; there is significant evidence of an additional digital care labor burden on women organizers of campaigns; and marginalized race and gender groups are associated with poorer fundraising outcomes. Outcomes are only minimally associated with campaign characteristics under users' control, such as photos, videos, and updates. These results corroborate widespread concerns with how technology fuels health inequities, and how crowdfunding may be creating an unequal and biased marketplace for those seeking financial support to access healthcare. Further research and better data access are needed to explore these dynamics more deeply and inform policy for this largely unregulated industry.

Acticoat versus biobrane: a retrospective review on the treatment of paediatric mid-dermal torso burns.

OBJECTIVES: Scalds involving toddlers commonly involve the torso and are frequently mid-dermal in depth. Initial management of a mid-dermal burn is conservative, progressing to grafting if healing has not been achieved in 10-14 days. Historically BiobraneTM (UDL Laboratories, Inc., Sugar Land, TX) is thought to have more favourable clinical outcomes compared to Acticoat TM (Smith and Nephew, St. Petersburg, Fl, USA). The Burns Unit at The Children's Hospital at Westmead (CHW) uses both dressings on a regular basis, providing the opportunity to compare the results of the dressings in a cohort of patients with mid-dermal torso burns. METHOD: A retrospective review was undertaken of all paediatric mid-dermal torso burns admitted to CHW between 2015 and 2017. The primary outcomes analysed were: time to complete healing and the need for grafting. Secondary outcomes included: operating theatre time, clinic visits, length of stay in hospital and positive wound swab colonisation. RESULTS: 78 children met the study criteria, 64 (82%) in the Acticoat group and 14 (18%) in the Biobrane group. 36 out of 78 children (56%) in the Acticoat group had their burns spontaneously healed without the need of skin graft surgery, compared with 10 out of 14 children (71%) in the Biobrane group. The days to complete healing were quicker in the Acticoat group (13 days) compared to the Biobrane group (17 days), although this was not statistically significant (P = 0.3). Overall patients managed with the Biobrane dressing required more operative sessions under general anaesthesia, a longer hospital stay, more clinic visits and a higher number of positive wound swab colonisation with heavy growth compared to the Acticoat group. CONCLUSION: This study suggests that the use of the Biobrane dressing does not significantly improve the clinical outcomes of mid-dermal torso burns in children compared to the Acticoat dressing. Acticoat reduced healing time, decreased the requirements for a general anaesthesia, reduced inpatient hospital stay and risk of infection.

Hyaluronan oligosaccharides stimulate matrix metalloproteinase and anabolic gene expression in vitro by intervertebral disc cells and annular repair in vivo.

The role of hyaluronan (HA) oligosaccharides in disc cell-mediated matrix metalloproteinase (MMP) and anabolic gene expression in vitro and annular repair in vivo were examined. Monolayer and alginate bead cultures of ovine intervertebral disc cells were stimulated with 10-12 mer hyaluronan oligosaccharides (HA-oligos). Annulus fibrosus (AF) monolayers were poorly responsive to the HA-oligos, proMMP-2 levels were marginally elevated and levels were MMP-9 unaffected. ProMMP-2 displayed a strong dose-dependent increase in the nucleus pulposus (NP) monolayers. In AF alginate bead cultures, proMMP-2 and active MMP-9 increased up to day 10, in NP cultures proMMP-2 was progressively converted to active MMP-2 over days 7-10 and active MMP-9 levels were elevated on day 10. A steady decline in MMP-2 and MMP-9 activity was evident over days 2-10 in the non-stimulated NP cultures. Disc cell viabilities were ≥92 ± 5% in all cultures indicating that the HA-oligo was not cytotoxic. Reverse-transcription polymerase chain reaction demonstrated an upregulation in MMP1, MMP113 and ADAMTS1 and the anabolic matrix repair genes ACAN, COL1A1 and COL2A1 in the NP by HA-oligos, whereas AF MMP13, ADAMTS1, ADAMTS4 and ADAMTS5, ACAN and COL2A1 were down-regulated; this differential regulation is expected to promote clearance of granulation/scar tissue from AF defects and matrix replenishment. The AF defect sites contained enlarged annular lamellae in vivo in response to the HA oligos, which is consistent with an active repair response. Masson trichrome and PicroSirius red histology and immunolocalization of type I collagen supported active remodelling in the outer lesion zone by the HA-oligo treatment but not the inner lesion. Copyright © 2016 John Wiley & Sons, Ltd.

Biglycan and fibromodulin fragmentation correlates with temporal and spatial annular remodelling in experimentally injured ovine intervertebral discs.

This study evaluated spatial and temporal extracellular matrix changes, induced by controlled surgical defects in the outer third of the annulus fibrosus (AF) of ovine intervertebral discs (IVDs). Thirty-two 4 year old sheep received a 4 mm deep x 10 mm wide standard annular surgical incision in the L1L2 and L3L4 IVDs (lesion group), 32 sheep were also subjected to the same surgical approach but the AF was not incised (sham-operated controls). Remodeling of the IVD matrix in the lesion and sham discs was assessed histochemically at 3, 6,12 and 26 month post operation (PO). Discs were also dissected into annular lesion site and contra-lateral AF and NP and equivalent zones in the sham sheep group, extracted with GuHCl, dialysed, freeze dried, digested with chondroitinase ABC/keratanase-I and aliquots examined for small leucine repeat proteoglycan (SLRP) core protein species by Western blotting using C-terminal antibodies to decorin, biglycan, lumican and fibromodulin and monoclonal antibody (Mab) 2B6 to unsaturated stub epitopes on chondroitin-4-sulphate generated by chondroitinase ABC. Masson Trichrome and Picrosirius red staining demonstrated re-organisation of the outermost collagenous lamellae in the incised discs 3-6 month PO. Toluidine blue staining also demonstrated a focal loss of anionic proteoglycan (PG) from the annular lesion 3-6 month PO with partial recovery of PG levels by 26 month. Specific fragments of biglycan and fibromodulin were associated with remodeling of the AF 12-26 month PO in the lesion IVDs but were absent from the NP of the lesion discs or all tissue zones in the sham animal group. Fragments of decorin were also observed in lesion zone extracts from 3 to 6 months but diminished after this. Isolation and characterization of the biglycan/fibromodulin fragments may identify them as prospective biomarkers of annular remodeling and characterization of the enzyme systems responsible for their generation may identify therapeutic target molecules.

Implementation of Epic Beaker Anatomic Pathology at an Academic Medical Center.

BACKGROUND: Beaker is a relatively new laboratory information system (LIS) offered by Epic Systems Corporation as part of its suite of health-care software and bundled with its electronic medical record, EpicCare. It is divided into two modules, Beaker anatomic pathology (Beaker AP) and Beaker Clinical Pathology. In this report, we describe our experience implementing Beaker AP version 2014 at an academic medical center with a go-live date of October 2015. METHODS: This report covers preimplementation preparations and challenges beginning in September 2014, issues discovered soon after go-live in October 2015, and some post go-live optimizations using data from meetings, debriefings, and the project closure document. RESULTS: We share specific issues that we encountered during implementation, including difficulties with the proposed frozen section workflow, developing a shared specimen source dictionary, and implementation of the standard Beaker workflow in large institution with trainees. We share specific strategies that we used to overcome these issues for a successful Beaker AP implementation. Several areas of the laboratory-required adaptation of the default Beaker build parameters to meet the needs of the workflow in a busy academic medical center. In a few areas, our laboratory was unable to use the Beaker functionality to support our workflow, and we have continued to use paper or have altered our workflow. In spite of several difficulties that required creative solutions before go-live, the implementation has been successful based on satisfaction surveys completed by pathologists and others who use the software. However, optimization of Beaker workflows has continued to be an ongoing process after go-live to the present time. CONCLUSIONS: The Beaker AP LIS can be successfully implemented at an academic medical center but requires significant forethought, creative adaptation, and continued shared management of the ongoing product by institutional and departmental information technology staff as well as laboratory managers to meet the needs of the laboratory.

Low-cost workflow improvement reduces gastrointestinal block use 17% by altering classic histotechnology testing.

OBJECTIVES: Gastrointestinal (GI) biopsy specimens were previously limited to four per cassette to facilitate established internal technical work practices and histotechnology best practice guidelines. We evaluated the workflow of these biopsy specimens. METHODS: We implemented three specific changes: (1) up to 10 GI biopsy specimens could be placed in each cassette, (2) histotechnologists would no longer orient GI biopsy specimens, and (3) embedding would be in a straight line rather than diagonal. We evaluated the effects of these changes on total block numbers, quality of slides, and perceptions of staff. RESULTS: The mean number of cassettes used was reduced 17% for GI biopsy cases, or an overall decrease of 3% of total blocks processed by our histopathology laboratory. Slide quality was unchanged. Staff reported increased job satisfaction. CONCLUSIONS: This simple, low-cost, low-effort process change yielded immediate and significant time savings for grossing and histology staff, increased job satisfaction, and challenges conventional histotechnology teaching.

Granulosa Cell-Specific Brca1 Loss Alone or Combined with Trp53 Haploinsufficiency and Transgenic FSH Expression Fails to Induce Ovarian Tumors.

BRCA1 mutations are associated with ovarian cancer. Previous studies reported that murine granulosa cell (GC) Brca1 loss caused ovarian-uterine tumors resembling serous cystadenomas, but the pathogenesis of these tumors may have been confounded by ectopic Brca1 expression and altered estrous cycling. We have used Tg.AMH.Cre conferring proven ovarian and GC-specific Cre activity to selectively target Brca1 disruption, denoted Brca1(GC-/-). Furthermore, ovary-specific Brca1(GC-/-) was combined with global Trp53 haploinsufficiency (Trp53(+/-)) and transgenic follicle-stimulating hormone (Tg.FSH) overexpression as a multi-hit strategy to investigate additional genetic and hormonal ovarian tumorigenesis mechanisms. However, 12-month-old Brca1(GC-/-) mice had no detectable ovarian or uterine tumors. Brca1(GC-/-) mice had significantly increased ovary weights, follicles exhibiting more pyknotic granulosa cells, and fewer corpora lutea with regular estrous cycling compared to controls. Isolated Brca1(GC-/-) mutation lengthened the estrous cycle and proestrus stage; however, ovarian cystadenomas were not observed, even when Brca1(GC-/-) was combined with Trp53(+/-) and overexpressed Tg.FSH. Our Brca1(GC-/-) models reveal that specific intra-follicular Brca1 loss alone, or combined with cancer-promoting genetic (Trp53 loss) and endocrine (high serum FSH) changes, was not sufficient to cause ovarian tumors. Our findings show that the ovary is remarkably resistant to oncogenesis, and support the emerging view of an extragonadal, multi-hit origin for ovarian tumorigenesis.

Culture models to define key mediators of cancer matrix remodeling.

High grade serous epithelial ovarian cancer (HG-SOC) is one of the most devastating gynecological cancers affecting women worldwide, with a poor survival rate despite clinical treatment advances. HG-SOC commonly metastasizes within the peritoneal cavity, primarily to the mesothelial cells of the omentum, which regulate an extracellular matrix rich in collagens type I, III, and IV along with laminin, vitronectin, and fibronectin. Cancer cells depend on their ability to penetrate and invade secondary tissue sites to spread, however a detailed understanding of the molecular mechanisms underlying these processes remain largely unknown. Given the high metastatic potential of HG-SOC and the associated poor clinical outcome, it is extremely important to identify the pathways and the components of which that are responsible for the progression of this disease. In vitro methods of recapitulating human disease processes are the critical first step in such investigations. In this context, establishment of an in vitro "tumor-like" micro-environment, such as 3D culture, to study early disease and metastasis of human HG-SOC is an important and highly insightful method. In recent years, many such methods have been established to investigate the adhesion and invasion of human ovarian cancer cell lines. The aim of this review is to summarize recent developments in ovarian cancer culture systems and their use to investigate clinically relevant findings concerning the key players in driving human HG-SOC.