Genetic Modification for Agriculture-Proposed Revision of GMO Regulation in Australia.

Genetic engineering (GM) of crops, modified with DNA transfer between species, has been highly regulated for over two decades. Now, genome editing (GE) enables a range of DNA alterations, from single base pair changes to precise gene insertion with site-directed nucleases (SDNs). Past regulations, established according to the precautionary principle of avoiding potential risks to human health and the environment, are predicated on fears fanned by well-funded and emotional anti-GM campaigns. These fears ignore the safety record of GM crops over the last 25 years and the benefits of GM to crop productivity, disease and pest resistance, and the environment. GE is now superseding GM, and public education is needed about its benefits and its potential to meet the challenges of climate change for crops. World population will exceed 9 billion by 2050, and world CO2 levels are now over 400 ppm in contrast with a pre-industrial 280 ppm, leading to a projected 1.5 °C global warming by 2050, with more stressful crop environments. The required abiotic and biotic stress tolerances can be introgressed from crop wild relatives (CWR) into domestic crops via GE. Restrictive regulations need to be lifted to facilitate GE technologies for sustainable agriculture in Australia and the world.

Enhanced Induction of Definitive Endoderm Differentiation of Mouse Embryonic Stem Cells in Simulated Microgravity.

Directed in vitro differentiation of pluripotent stem cells toward definitive endoderm (DE) offers great research and therapeutic potential since these cells can further differentiate into cells of the respiratory and gastrointestinal tracts, as well as associated organs such as pancreas, liver, and thyroid. We hypothesized that culturing mouse embryonic stem cells (mESCs) under simulated microgravity (SMG) conditions in rotary bioreactors (BRs) will enhance the induction of directed DE differentiation. To test our hypothesis, we cultured the cells for 6 days in two-dimensional monolayer colony cultures or as embryoid bodies (EBs) in either static conditions or, dynamically, in the rotary BRs. We used flow cytometry and quantitative polymerase chain reaction to analyze the expression of marker proteins and genes, respectively, for pluripotency (Oct3/4) and mesendodermal (Brachyury T), endodermal (FoxA2, Sox17, CxCr4), and mesodermal (Vimentin, Meox1) lineages. Culture in the form of EBs in maintenance media in the presence of leukemia inhibitory factor, in static or SMG conditions, induced expression of some of the differentiation markers, suggesting heterogeneity of the cells. This is in line with previous studies showing that differentiation is initiated as cells are aggregated into EBs even without supplementing differentiation factors to the media. Culturing EBs in static conditions in differentiation media (DM) in the presence of activin A reduced Oct3/4 expression and significantly increased Brachyury T and CxCr4 expression, but downregulated FoxA2 and Sox17. However, culturing in SMG BRs in DM upregulated Brachyury T and all of the DE markers and reduced Oct3/4 expression, indicating the advantage of dynamic cultures in BRs to specifically enhance directed DE differentiation. Given the potential discrepancies between the SMG conditions on earth and actual microgravity conditions, as observed in other studies, future experiments in space flight are required to validate the effects of reduced gravity on mESC differentiation.

Soil Fertility Map for Food Legumes Production Areas in China.

Given the limited resources of fossil energy, and the environmental risks of excess fertilizer on crops, it is time to reappraise the potential role of food legume biological nitrogen fixation (BNF) as sources of nitrogen for cropping systems in China. 150 soil samples across 17 provinces and 2 municipalities of China were collected and analyzed. A distribution map of the soil fertilities and their patterns of distribution was constructed. The pH results indicated that soils were neutral to slightly alkaline overall. The soil organic matter (SOM) and the available nitrogen (AN) content were relatively low, while the available phosphorus (AP) and available potassium (AK) contents were from moderate to high. Production areas of food legumes (faba bean, pea, adzuki bean, mung bean and common bean) were clearly separated into 4 soil fertility type clusters. In addition, regions with SOM, AN, AP and AK deficiency, high acidity and high alkalinity were listed as target areas for further soil improvement. The potential was considered for biological nitrogen fixation to substitute for the application of mineral nitrogen fertiliser.

Pediatric laryngotracheal reconstruction with tissue-engineered cartilage in a rabbit model.

OBJECTIVES/HYPOTHESIS: To develop an effective rabbit model of in vitro- and in vivo-derived tissue-engineered cartilage for laryngotracheal reconstruction (LTR). STUDY DESIGN: 1) Determination of the optimal scaffold 1% hyaluronic acid (HA), 2% HA, and polyglycolic acid (PGA) and in vitro culture time course using a pilot study of 4 by 4-mm in vitro-derived constructs analyzed on a static culture versus zero-gravity bioreactor for 4, 8, and 12 weeks, with determination of compressive modulus and histology as outcome measures. 2) Three-stage survival rabbit experiment utilizing autologous auricular chondrocytes seeded in scaffolds, either 1% HA or PGA. The constructs were cultured for the determined in vitro time period and then cultured in vivo for 12 weeks. Fifteen LTRs were performed using HA cartilage constructs, and one was performed with a PGA construct. All remaining specimens and the final reconstructed larynx underwent mechanical testing, histology, and glycosaminoglycan (GAG) content determination, and then were compared to cricoid control specimens (n = 13) and control LTR using autologous thyroid cartilage (n = 18). METHODS: 1) One rabbit underwent an auricular punch biopsy, and its chondrocytes were isolated and expanded and then encapsulated in eight 4 by 4-mm discs of 1% HA, 2% HA, PGA either in rotary bioreactor or static culture for 4, 8, and 12 weeks, respectively, with determination of compressive modulus, GAG content, and histology. 2) Sixteen rabbits underwent ear punch biopsy; chondrocytes were isolated and expanded. The cells were seeded in 13 by 5 by 2.25-mm UV photopolymerized 1% HA (w/w) or calcium alginate encapsulated synthetic PGA (13 × 5 × 2 mm); the constructs were then incubated in vitro for 12 weeks (the optimal time period determined above in paragraph 1) on a shaker. One HA and one PGA construct from each animal was tested mechanically and histologically, and the remaining eight (4 HA and 4 PGA) were implanted in the neck. After 12 weeks in vivo, the most optimal-appearing HA construct was used as a graft for LTR in 15 rabbits and PGA in one rabbit. The seven remaining specimens underwent hematoxylin and eosin, Safranin O, GAG content determination, and flexural modulus testing. At 12 weeks postoperative, the animals were euthanized and underwent endoscopy. The larynges underwent mechanical and histological testing. All animals that died underwent postmortem examination, including gross and microhistological analysis of the reconstructed airway. RESULTS: Thirteen of the 15 rabbits that underwent LTR with HA in vitro- and in vivo-derived tissue-engineered cartilage constructs survived. The 1% HA specimens had the highest modulus and GAG after 12 weeks in vitro. The HA constructs became well integrated in the airway, supported respiration for the 12 weeks, and were histologically and mechanically similar to autologous cartilage. CONCLUSIONS: The engineering of in vitro- and in vivo-derived cartilage with HA is a novel approach for laryngotracheal reconstruction. The data suggests that the in vitro- and in vivo-derived tissue-engineered approaches may offer a promising alternative to current strategies used in pediatric airway reconstruction, as well as other head and neck applications. LEVEL OF EVIDENCE: NA. Laryngoscope, 126:S5-S21, 2016.

Identification of Putative Candidate Genes for Water Stress Tolerance in Canola (Brassica napus).

Drought stress can directly inhibit seedling establishment in canola (Brassica napus), resulting in lower plant densities and reduced yields. To dissect this complex trait, 140 B. napus accessions were phenotyped under normal (0.0 MPa, S0) and water-stressed conditions simulated by polyethylene glycol (PEG) 6000 (-0.5 MPa, S5) in a hydroponic system. Phenotypic variation and heritability indicated that the root to shoot length ratio was a reliable indicator for water stress tolerance. Thereafter, 66 accessions (16 water stress tolerant, 34 moderate and 16 sensitive lines) were genotyped using 25,495 Brassica single nucleotide polymorphisms (SNPs). Genome-wide association studies (GWAS) identified 16 loci significantly associated with water stress response. Two B. napus accessions were used for RNA sequencing, with differentially-expressed genes under normal and water-stressed conditions examined. By combining differentially-expressed genes detected by RNA sequencing with significantly associated loci from GWAS, 79 candidate genes were identified, of which eight were putatively associated with drought tolerance based on gene ontology of Arabidopsis. Functional validation of these genes may confirm key drought-related genes for selection and breeding in B. napus. Our results provide insight into the genetic basis of water stress tolerance in canola.

Genetic diversity of grasspea and its relative species revealed by SSR markers.

The study of genetic diversity between Lathyrus sativus L. and its relative species may yield fundamental insights into evolutionary history and provide options to meet the challenge of climate changes. 30 SSR loci were employed to assess the genetic diversity and population structure of 283 individuals from wild and domesticated populations from Africa, Europe, Asia and ICARDA. The allele number per loci ranged from 3 to 14. The average gene diversity index and average polymorphism information content (PIC) was 0.5340 and 0.4817, respectively. A model based population structure analysis divided the germplasm resources into three subgroups: the relative species, the grasspea from Asia, and the grasspea from Europe and Africa. The UPGMA dendrogram and PCA cluster also demonstrated that Asian group was convincingly separated from the other group. The AMOVA result showed that the cultivated species was quite distinct from its relative species, however a low level of differentiation was revealed among their geographic origins. In all, these results provided a molecular basis for understanding genetic diversity of L. sativus and its relatives.

High-throughput genotyping for species identification and diversity assessment in germplasm collections.

Germplasm collections provide an extremely valuable resource for breeders and researchers. However, misclassification of accessions by species often hinders the effective use of these collections. We propose that use of high-throughput genotyping tools can provide a fast, efficient and cost-effective way of confirming species in germplasm collections, as well as providing valuable genetic diversity data. We genotyped 180 Brassicaceae samples sourced from the Australian Grains Genebank across the recently released Illumina Infinium Brassica 60K SNP array. Of these, 76 were provided on the basis of suspected misclassification and another 104 were sourced independently from the germplasm collection. Presence of the A- and C-genomes combined with principle components analysis clearly separated Brassica rapa, B. oleracea, B. napus, B. carinata and B. juncea samples into distinct species groups. Several lines were further validated using chromosome counts. Overall, 18% of samples (32/180) were misclassified on the basis of species. Within these 180 samples, 23/76 (30%) supplied on the basis of suspected misclassification were misclassified, and 9/105 (9%) of the samples randomly sourced from the Australian Grains Genebank were misclassified. Surprisingly, several individuals were also found to be the product of interspecific hybridization events. The SNP (single nucleotide polymorphism) array proved effective at confirming species, and provided useful information related to genetic diversity. As similar genomic resources become available for different crops, high-throughput molecular genotyping will offer an efficient and cost-effective method to screen germplasm collections worldwide, facilitating more effective use of these valuable resources by breeders and researchers.

Large-scale microsatellite development in grasspea (Lathyrus sativus L.), an orphan legume of the arid areas.

BACKGROUND: Grasspea (Lathyrus sativus L., 2n = 14), a member of the family Leguminosae, holds great agronomic potential as grain and forage legume crop in the arid areas for its superb resilience to abiotic stresses such as drought, flood and salinity. The crop could not make much progress through conventional breeding in the past, and there are hardly any detailed molecular biology studies due to paucity of reliable molecular markers representative of the entire genome. RESULTS: Using the 454 FLX Titanium pyrosequencing technique, 651,827 simple sequence repeat (SSR) loci were identified and 50,144 nonredundant primer pairs were successfully designed, of which 288 were randomly selected for validation among 23 L. sativus and one L. cicera accessions of diverse provenance. 74 were polymorphic, 70 monomorphic, and 144 with no PCR product. The number of observed alleles ranged from two to five, the observed heterozygosity from 0 to 0.9545, and Shannon's information index ranged from 0.1013 to 1.0980, respectively. The dendrogram constructed by using unweighted pair group method with arithmetic mean (UPGMA) based on Nei's genetic distance, showed obvious distinctions and understandable relationships among the 24 accessions. CONCLUSIONS: The large number of SSR primer pairs developed in this study would make a significant contribution to genomics enabled improvement of grasspea.

Geographical gradient of the eIF4E alleles conferring resistance to potyviruses in pea (Pisum) germplasm.

BACKGROUND: The eukaryotic translation initiation factor 4E was shown to be involved in resistance against several potyviruses in plants, including pea. We combined our knowledge of pea germplasm diversity with that of the eIF4E gene to identify novel genetic diversity. METHODOLOGY/PRINCIPAL FINDINGS: Germplasm of 2803 pea accessions was screened for eIF4E intron 3 length polymorphism, resulting in the detection of four eIF4E(A-B-C-S) variants, whose distribution was geographically structured. The eIF4E(A) variant conferring resistance to the P1 PSbMV pathotype was found in 53 accessions (1.9%), of which 15 were landraces from India, Afghanistan, Nepal, and 7 were from Ethiopia. A newly discovered variant, eIF4E(B), was present in 328 accessions (11.7%) from Ethiopia (29%), Afghanistan (23%), India (20%), Israel (25%) and China (39%). The eIF4E(C) variant was detected in 91 accessions (3.2% of total) from India (20%), Afghanistan (33%), the Iberian Peninsula (22%) and the Balkans (9.3%). The eIF4E(S) variant for susceptibility predominated as the wild type. Sequencing of 73 samples, identified 34 alleles at the whole gene, 26 at cDNA and 19 protein variants, respectively. Fifteen alleles were virologically tested and 9 alleles (eIF4E(A-1-2-3-4-5-6-7), eIF4E(B-1), eIF4E(C-2)) conferred resistance to the P1 PSbMV pathotype. CONCLUSIONS/SIGNIFICANCE: This work identified novel eIF4E alleles within geographically structured pea germplasm and indicated their independent evolution from the susceptible eIF4E(S1) allele. Despite high variation present in wild Pisum accessions, none of them possessed resistance alleles, supporting a hypothesis of distinct mode of evolution of resistance in wild as opposed to crop species. The Highlands of Central Asia, the northern regions of the Indian subcontinent, Eastern Africa and China were identified as important centers of pea diversity that correspond with the diversity of the pathogen. The series of alleles identified in this study provides the basis to study the co-evolution of potyviruses and the pea host.

Rotary bioreactor culture can discern specific behavior phenotypes in Trk-null and Trk-expressing neuroblastoma cell lines.

Neuroblastoma is characterized by biological and genetic heterogeneity that leads to diverse, often unpredictable, clinical behavior. Differential expression of the Trk family of neurotrophin receptors strongly correlates with clinical behavior; TrkA expression is associated with favorable outcome, whereas TrkB with unfavorable outcome. Neuroblastoma cells cultured in a microgravity rotary bioreactor spontaneously aggregate into tumor-like structures, called organoids. We wanted to determine if the clinical heterogeneity of TrkA- or TrkB-expressing neuroblastomas was reflected in aggregation kinetics and organoid morphology. Trk-null SY5Y cells were stably transfected to express either TrkA or TrkB. Short-term aggregation kinetics were determined by counting the number of single (non-aggregated) viable cells in the supernatant over time. Organoids were harvested after 8 d of bioreactor culture, stained, and analyzed morphometrically. SY5Y-TrkA cells aggregated significantly slower than SY5Y and SY5Y-TrkB cells, as quantified by several measures of aggregation. SY5Y and TrkB cell lines formed irregularly shaped organoids, featuring stellate projections. In contrast, TrkA cells formed smooth (non-stellate) organoids. SY5Y organoids were slightly smaller on average, but had significantly larger average perimeter than TrkA or TrkB organoids. TrkA expression alone is sufficient to dramatically alter the behavior of neuroblastoma cells in three-dimensional, in vitro rotary bioreactor culture. This pattern is consistent with both clinical behavior and in vivo tumorigenicity, in that SY5Y-TrkA represents a more differentiated, less aggressive phenotype. The microgravity bioreactor is a useful in vitro tool to rapidly investigate the biological characteristics of neuroblastoma and potentially to assess the effect of cytotoxic as well as biologically targeted drugs.

Evidence from genome-wide simple sequence repeat markers for a polyphyletic origin and secondary centers of genetic diversity of Brassica juncea in China and India.

The oilseed Brassica juncea is an important crop with a long history of cultivation in India and China. Previous studies have suggested a polyphyletic origin of B. juncea and more than one migration from the primary to secondary centers of diversity. We investigated molecular genetic diversity based on 99 simple sequence repeat markers in 119 oilseed B. juncea varieties from China, India, Europe, and Australia to test whether molecular differentiation follows Vavilov's proposal of secondary centers of diversity in India and China. Two distinct groups were identified by markers in the A genome, and the same two groups were confirmed by markers in the B genome. Group 1 included accessions from central and western India, in addition to those from eastern China. Group 2 included accessions from central and western China, as well as those from northern and eastern India. European and Australian accessions were found only in Group 2. Chinese accessions had higher allelic diversity per accession (Group 1) and more private alleles per accession (Groups 1 and 2) than those from India. The marker data and geographic distribution of Groups 1 and 2 were consistent with two independent migrations of B. juncea from its center of origin in the Middle East and neighboring regions along trade routes to western China and northern India, followed by regional adaptation. Group 1 migrated further south and west in India, and further east in China, than Group 2. Group 2 showed diverse agroecological adaptation, with yellow-seeded spring-sown types in central and western China and brown-seeded autumn-sown types in India.

Genetic diversity and relationship of global faba bean (Vicia faba L.) germplasm revealed by ISSR markers.

Genetic diversity and relationships of 802 faba bean (Vicia faba L.) landraces and varieties from different geographical locations of China and abroad were examined using ISSR markers. A total of 212 repeatable amplified bands were generated with 11 ISSR primers, of which 209 were polymorphic. Accessions from North China showed highest genetic diversity, while accessions from central China showed low level of diversity. Chinese spring faba bean germplasm was clearly separated from Chinese winter faba bean, based on principal component analysis and UPGMA clustering analysis. Winter accessions from Zhejiang (East China), Jiangxi (East China), Sichuan (Southwest China) and Guizhou (Southwest China) were quite distinct to that from other provinces in China. Great differentiation between Chinese accessions and those from rest of the world was shown with a UPGMA dendrogram. AMOVA analyses demonstrated large variation and differentiation within and among groups of accessions from China. As a continental geographic group, accessions from Europe were genetically closer to those from North Africa. Based on ISSR data, grouping results of accessions from Asia, Europe and Africa were obviously associated with their geographical origin. The overall results indicated that the genetic relationship of faba bean germplasm was closely associated with their geographical origin and their ecological habit.

Microgravity assay of neuroblastoma: in vitro aggregation kinetics and organoid morphology correlate with MYCN expression.

Neuroblastoma, the most common and deadly solid pediatric tumor, features genetic and biologic heterogeneity that defies simple risk assessments, drives diverse clinical behavior, and demands more extensive characterization. This research served to investigate the utility of a microgravity assay-rotary bioreactor culture-to evaluate and characterize the cell-specific, in vitro behavior of neuroblastoma cell lines: aggregation kinetics of single cells and the morphology of the formed structures, called organoids. Specifically, we examined the effect of amplification of the oncogene MYCN, a genetic factor that is strongly associated with poor clinical outcome. Three human neuroblastoma cell lines with varied MYCN expression (CHP-212 (unamplified), SK-N-AS (unamplified), IMR-32 (amplified)) were cultured in the microgravity rotary bioreactor. Simple aggregation kinetics were determined by periodically performing counts of non-aggregated single cells in the media. Organoids were harvested, stained with hematoxylin and eosin, and evaluated microscopically in terms of size and shape. The MYCN-amplified cell line (IMR32) aggregated much more rapidly than the unamplified cell lines, as indicated by a significantly lower area under its aggregation curve (single non-aggregated cells vs. time): IMR32=4.3, CHP-212 =12.4, SK-N-AS=9.8 (adhesion index ×10(5)). Further, the organoid morphology of the MYCN-amplified cell line was noticeably different compared to the unamplified lines. The CHP-212 and SK-N-AS cells formed spherical structures with average cross-sectional area 0.213 and 0.138 mm(2), respectively, and featured an outer viable zone of cells (average length of 0.175, 0.129 mm, respectively; the "diffusion distance"), surrounding an inner necrotic core. In contrast, the MYCN-amplified cell line formed a large single mass of cells but had a similar diffusion distance (0.175 mm). This microgravity assay provides a rapid, reproducible assessment of in vitro behavior of neuroblastoma, and the measured parameters, aggregation kinetics and organoid size and shape correlated with malignant potential in terms of MYCN amplification. This assay allows for the examination of cell-specific biologic and genetic factors that should provide valuable insight into the clinical behavior of neuroblastoma.

Molecular variation among Chinese and global winter faba bean germplasm.

A sample of winter faba bean germplasm from China was compared with germplasm from outside China, using AFLP analyses. Both sets of germplasm were obtained from the National Genebank of China, Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences, Beijing, China. A sample of 39 winter type accessions from outside of China and 204 Chinese landraces and varieties (201 winter types and 3 spring types) were characterized with 10 AFLP primers. These detected 266 polymorphic bands. The Chinese germplasm was clearly separated from the rest of the world in principal component analysis and clustering analysis, with the spring types from China showing the greatest separation. Yunnan germplasm, both landraces and commercial varieties, showed the greatest separation among the germplasm of Chinese winter faba bean provinces. The landraces/varieties from Anhui, Zhejiang, Sichuan, Jianxi, Guizhou and Fujian provinces clustered in a central group.

Analysis of a diverse global Pisum sp. collection and comparison to a Chinese local P. sativum collection with microsatellite markers.

Twenty-one informative microsatellite loci were used to assess and compare the genetic diversity among Pisum genotypes sourced from within and outside China. The Chinese germplasm comprised 1243 P. sativum genotypes from 28 provinces and this was compared to 774 P. sativum genotypes that represented a globally diverse germplasm collection, as well as 103 genotypes from related Pisum species. The Chinese P. sativum germplasm was found to contain genotypes genetically distinct from the global gene pool sourced outside China. The Chinese spring type genotypes were separate from the global gene pool and from the other main Chinese gene pool of winter types. The distinct Chinese spring gene pool comprised genotypes from Inner Mongolia and Sha'anxi provinces, with those from Sha'anxi showing the greatest diversity. The other main gene pool within China included both spring types from other northern provinces and winter types from central and southern China, plus some accessions from Inner Mongolia and Sha'anxi. A core collection of Chinese landraces chosen to represent molecular diversity was compared both to the wider Chinese collection and to a geographically diverse core collection of Chinese landraces. The average gene diversity and allelic richness per locus of both the micro-satellite based core and the wider collection were similar, and greater than the geographically diverse core. The genetic diversity of P. sativum within China appears to be quite different to that detected in the global gene pool, including the presence of several rare alleles, and may be a useful source of allelic variation for both major gene and quantitative traits.

Treatment of diabetic wounds with fetal murine mesenchymal stromal cells enhances wound closure.

Diabetes impairs multiple aspects of the wound-healing response. Delayed wound healing continues to be a significant healthcare problem for which effective therapies are lacking. We have hypothesized that local delivery of mesenchymal stromal cells (MSC) at a wound might correct many of the wound-healing impairments seen in diabetic lesions. We treated excisional wounds of genetically diabetic (Db-/Db-) mice and heterozygous controls with either MSC, CD45(+) cells, or vehicle. At 7 days, treatment with MSC resulted in a decrease in the epithelial gap from 3.2 +/- 0.5 mm in vehicle-treated wounds to 1.3 +/- 0.4 mm in MSC-treated wounds and an increase in granulation tissue from 0.8 +/- 0.3 mm(2) to 2.4 +/- 0.6 mm(2), respectively (mean +/- SD, P < 0.04). MSC-treated wounds also displayed a higher density of CD31(+) vessels and exhibited increases in the production of mRNA for epidermal growth factor, transforming growth factor beta 1, vascular endothelial growth factor, and stromal-derived growth factor 1-alpha. MSC also demonstrated greater contractile ability than fibroblast controls in a collagen gel contraction assay. The effects of locally applied MSC are thus sufficient to improve healing in diabetic mice. Possible mechanisms of this effect include augmented local growth-factor production, improved neovascularization, enhanced cellular recruitment to wounds, and improved wound contraction.

Structural and functional characterization of bladder smooth muscle in fetal rats with retinoic acid-induced myelomeningocele.

Myelomeningocele (MMC) is the most common cause of neurogenic bladder dysfunction (NBD). We recently developed a novel retinoic acid (RA)-induced MMC model in fetal rats. The objective of this study was to use this model to assess functional and structural characteristics of the detrusor muscle in MMC-associated NBD. Time-dated pregnant Sprague-Dawley rats were gavage fed 60 mg/kg RA dissolved in olive oil or olive oil alone [embryonic day 10 (E10)]. Bladder specimens from olive oil-exposed fetuses (OIL; n = 71), MMC (n = 79), and RA-exposed-no MMC (RA, n = 62) were randomly assigned for functional and histopathological evaluation and protein analysis. Contractility responses to field and agonist-mediated stimulation (KCl and bethanecol) were analyzed. The expression patterns of alpha-smooth muscle actin, myosin, desmin, vimentin, and collagen III and I were analyzed by immunohistochemistry and Western blotting. Spatial and temporal distribution of nerve fibers within the detrusor muscle was monitored by neurotubulin-beta-III throughout gestation. Neither OIL, MMC, nor RA detrusor responded to field stimulation. MMC bladder strips showed a significant decrease in contractility after KCl and bethanechol stimulation compared with OIL and RA bladders. Bladder detrusor morphology and expression patterns of smooth muscle markers were similar between groups. Detrusor muscles in OIL and RA fetuses were densely innervated, possessing abundant intramural ganglia and nerve trunks that branch to supply smooth muscle bundles. In MMC bladders, neurotubulin-beta-III-positive nerve fibers were markedly decreased with advancing gestational age and were almost completely absent at term (E22). We conclude that the biomechanical properties of fetal rat MMC bladders are analogous to that seen in humans with MMC-associated NBD. Decreased nerve density indicates loss of peripheral neural innervation throughout gestation. The early observation of decreased innervation and decreased contractility in the absence of morphologic abnormalities in muscle structure or extracellular matrix supports a pathophysiological hypothesis that denervation is the primary insult preceding the observed alterations in bladder muscle structure and function.

Complementary roles of microtubules and microfilaments in the lung fibroblast-mediated contraction of collagen gels: Dynamics and the influence of cell density.

Fibroblasts are important cellular components in wound healing, scar formation, and fibrotic disorders; and the fibroblast-populated collagen-gel (FPCG) model allows examination of fibroblast behavior in an in vitro three-dimensional environment similar to that in vivo. Contraction of free-floating FPCGs depends on an active and dynamic cytoskeleton, and the contraction dynamics are highly influenced by cell density. We investigated mechanistic differences between high- and low-cell density FPCG contraction by evaluating contraction dynamics in detail, using specific cytoskeletal disruptors. Collagen gels were seeded with human lung fibroblasts at either high (HD) or low (LD) density, and incubated with or without cytoskeletal disruptors colchicine (microtubules) or cytochalasin D (microfilaments). Gel area was measured daily. FPCG contraction curves were essentially sigmoidal, featuring an initial period of no contraction (lag phase), followed by a period of rapid contraction (log phase). Contraction curves of HD-FPCGs were distinct from those of LD-FPCGs. For example, HD-FPCGs had a negligible lag phase (compared with 3 d for LD-FPCGs) and exhibited a higher rate of log-phase contraction. Both colchicine and cytochalasin dose-dependently inhibited contraction but specifically affected different phases of contraction in HD- and LD-FPCGs; and colchicine inhibited LD-FPCGs much more than HD-FPCGs. The data indicate that LD- and HD-FPCGs contract through different primary mechanisms. Microtubules and microfilaments are both complementarily and dynamically involved in the contraction of FPCGs, and cell density influences primary cytoskeletal mechanisms. These results provide valuable information about fibroblast behavior in healing and fibrosis, and may suggest novel treatment options.

Collagen crosslinking and cell density have distinct effects on fibroblast-mediated contraction of collagen gels.

BACKGROUND/PURPOSE: Fibroblasts are actively and dynamically involved in wound healing (dermal regeneration, wound contraction, and scar contracture) and fibrosis. Fibroblast-seeded collagen gels provide an in vitro model for these processes. Over time, fibroblasts will contract the gels, but the mechanisms are not completely understood. This research investigated the influence of cell density and collagen crosslinking on the contraction of fibroblast-populated gels by varying seeding density and blocking the catalyzing enzyme lysyl oxidase, respectively. METHODS: Collagen gels were seeded with fibroblasts at either 3 x 104 or 1 x 105 cells/mL and incubated with or without the lathyrogen beta-aminoproprionitrile (BAPN) for 8 days. In all, four experimental groups were analyzed: low cell density control, high cell density control, low density plus BAPN, and high density plus BAPN. Digital images were taken daily and gel area was calculated. RESULTS: Contraction was dependent on cell concentration, with higher density gels being contracted to a greater extent. BAPN had no effect until after day 2 when it inhibited (high density) or almost completely blocked (low density) the gel contraction. BAPN also reduced total long-term contraction. CONCLUSION: The results demonstrated a bimodal nature to fibroblast-mediated gel contraction: a cell density-dependent component, most likely mediated through cellular forces, and a delayed collagen crosslinking component that could be blocked by BAPN. In the long-term, similar contraction rates among the four experimental groups, particularly between the two BAPN groups, implies that the collagen crosslinking effect is discrete and independent of cell density.