RHAMM regulates MMTV-PyMT-induced lung metastasis by connecting STING-dependent DNA damage sensing to interferon/STAT1 pro-apoptosis signaling.

BACKGROUND: RHAMM is a multifunctional protein that is upregulated in breast tumors, and the presence of strongly RHAMM+ve cancer cell subsets associates with elevated risk of peripheral metastasis. Experimentally, RHAMM impacts cell cycle progression and cell migration. However, the RHAMM functions that contribute to breast cancer metastasis are poorly understood. METHODS: We interrogated the metastatic functions of RHAMM using a loss-of-function approach by crossing the MMTV-PyMT mouse model of breast cancer susceptibility with Rhamm-/- mice. In vitro analyses of known RHAMM functions were performed using primary tumor cell cultures and MMTV-PyMT cell lines. Somatic mutations were identified using a mouse genotyping array. RNA-seq was performed to identify transcriptome changes resulting from Rhamm-loss, and SiRNA and CRISPR/Cas9 gene editing was used to establish cause and effect of survival mechanisms in vitro. RESULTS: Rhamm-loss does not alter initiation or growth of MMTV-PyMT-induced primary tumors but unexpectedly increases lung metastasis. Increased metastatic propensity with Rhamm-loss is not associated with obvious alterations in proliferation, epithelial plasticity, migration, invasion or genomic stability. SNV analyses identify positive selection of Rhamm-/- primary tumor clones that are enriched in lung metastases. Rhamm-/- tumor clones are characterized by an increased ability to survive with ROS-mediated DNA damage, which associates with blunted expression of interferon pathway and target genes, particularly those implicated in DNA damage-resistance. Mechanistic analyses show that ablating RHAMM expression in breast tumor cells by siRNA knockdown or CRISPR-Cas9 gene editing blunts interferon signaling activation by STING agonists and reduces STING agonist-induced apoptosis. The metastasis-specific effect of RHAMM expression-loss is linked to microenvironmental factors unique to tumor-bearing lung tissue, notably high ROS and TGFB levels. These factors promote STING-induced apoptosis of RHAMM+ve tumor cells to a significantly greater extent than RHAMM-ve comparators. As predicted by these results, colony size of Wildtype lung metastases is inversely related to RHAMM expression. CONCLUSION: RHAMM expression-loss blunts STING-IFN signaling, which offers growth advantages under specific microenvironmental conditions of lung tissue. These results provide mechanistic insight into factors controlling clonal survival/expansion of metastatic colonies and has translational potential for RHAMM expression as a marker of sensitivity to interferon therapy.

Soil bacterial community dynamics in plots managed with cover crops and no-till farming in the Lower Mississippi Alluvial Valley, USA.

AIMS: Assess bacterial community changes over time in soybean (Glycine max) crop fields following cover crop (CC) and no-till (NT) implementation under natural abiotic stressors. METHOD AND RESULTS: Soil bacterial community composition was obtained by amplifying, sequencing, and analysing the V4 region of the 16S rRNA gene. Generalized linear mixed models were used to assess the effects of tillage, CC, and time on bacterial community response. The most abundant phyla present were Acidobacteria, Actinobacteria, Bacteroidetes, and Verrucomicrobia. Bacterial diversity increased in periods with abundant water. Reduced tillage (RT) increased overall bacterial diversity, but NT with a CC was not significantly different than RT treatments under drought conditions. CCs shifted abundances of Firmicutes and Bacteroidetes depending on abiotic conditions. CONCLUSIONS: In the Lower Mississippi Alluvial Valley (LMAV), USA, NT practices lower diversity and influence long-term community changes while cover crops enact a seasonal response to environmental conditions. NT and RT management affect soil bacterial communities differently than found in other regions of the country.

Can Pesticides Dissolved in Runoff and Exposed to Maturing Rice (Oryza sativa) Plants be Transferred to Seeds?

Agriculture's global challenge to feed an estimated 7.7 billion people is further exacerbated by less available cropland for production and rapidly changing climate patterns. Pesticides are often utilized to minimize crop losses due to pest infestations; however, problems arise when these chemicals are transported off production acreage, either by storm or irrigation events, and into nearby water bodies. Innovative management practices are needed to not only reduce the volume of runoff, but also to mitigate various pollutants, such as pesticides, within the runoff. One such practice being evaluated involves using rice (Oryza sativa) as a pesticide mitigation tool. While rice plants may serve as a mechanism for phytoremediation, whether the seeds harvested from exposed plants could then be utilized as a human food source is an unanswered question. Thirty round mesocosms (55 L volume; 56 cm diameter; six replicates per treatment) were established with rice and exposed to aqueous concentrations of the pesticides clomazone, propanil, or cyfluthrin, as well as a mixture of the three pesticides. Six replicates with rice and no pesticide exposure served as controls. Initial pesticide exposure took place 8 weeks post-planting and continued once a week for 5 weeks. Rice plants, unmilled seeds, and mesocosm sediment were collected from each mesocosm 2 weeks after seed formation began and analyzed for pesticide concentrations using gas chromatography. Concentrations of pesticides in unmilled seed were below detection for individual exposures of clomazone, propanil, and cyfluthrin. When rice was exposed to the pesticide mixture, the mean ± SE unmilled seed cyfluthrin concentration was 14.8 ± 1.25 µg kg-1. These small-scale, preliminary studies offer insight into the possibility of using immature rice plants as a phytoremediation tool, while harvesting its grain after plant maturation for human consumption. Further research is needed to address this question on a larger scale and with multiple pesticide mixtures.

Advancing the Sustainability of US Agriculture through Long-Term Research.

Agriculture in the United States must respond to escalating demands for productivity and efficiency, as well as pressures to improve its stewardship of natural resources. Growing global population and changing diets, combined with a greater societal awareness of agriculture's role in delivering ecosystem services beyond food, feed, fiber, and energy production, require a comprehensive perspective on where and how US agriculture can be sustainably intensified, that is, made more productive without exacerbating local and off-site environmental concerns. The USDA's Long-Term Agroecosystem Research (LTAR) network is composed of 18 locations distributed across the contiguous United States working together to integrate national and local agricultural priorities and advance the sustainable intensification of US agriculture. We explore here the concept of sustainable intensification as a framework for defining strategies to enhance production, environmental, and rural prosperity outcomes from agricultural systems. We also elucidate the diversity of factors that have shaped the past and present conditions of cropland, rangeland, and pastureland agroecosystems represented by the LTAR network and identify priorities for research in the areas of production, resource conservation and environmental quality, and rural prosperity. Ultimately, integrated long-term research on sustainable intensification at the national scale is critical to developing practices and programs that can anticipate and address challenges before they become crises.

Can Rice (Oryza sativa) Mitigate Pesticides and Nutrients in Agricultural Runoff?

Phytoremediation of nutrients and pesticides in runoff is a growing conservation effort, particularly in agriculturally intensive areas such as the lower Mississippi River Valley. In the current study, rice (Oryza sativa) was examined for its mitigation capacity of nitrogen, phosphorus, diazinon, and permethrin. Twenty-two high density polyethylene circular containers (56 cm x 45 cm) were used as mesocosms, with 12 mesocosms planted with rice and 10 mesocosms remaining unvegetated. Mesocosms were hydraulically connected and arranged in a series of two, with each system providing a 4 h hydraulic retention time (HRT) for a total system retention time of 8 h. Two treatments (RICE/RICE and RICE/BARE) of four replicates each were utilized, with three replicates of controls (BARE/BARE). Systems with RICE/RICE (8 h HRT) significantly reduced diazinon (p = 0.0126), cis-permethrin (p = 0.0442), filtered orthophosphate (p = 0.0058), and total orthophosphate (p = 0.0123) compared to control systems. No significant differences were noted for trans-permethrin, nitrate, or ammonium. Results indicate promise in phytoremediation of agricultural runoff by rice. If further studies reveal contaminants are not transferred into seeds, then rice could potentially serve as both a remediation tool and food source in countries facing agricultural pollution challenges.

Application of Polyacrylamide (PAM) through Lay-Flat Polyethylene Tubing: Effects on Infiltration, Erosion, N and P Transport, and Corn Yield.

Polyacrylamides (PAMs), when applied as a soil amendment, purportedly improve soil infiltration, decrease erosion, and reduce offsite agrochemical transport. The effect of PAM on infiltration, erosion, agrochemical transport, and crop yield when applied in furrow to mid-southern US production systems has not been evaluated. The objective of this study was to assess PAM effects on infiltration, erosion, corn ( L.) grain yield, and nitrogen (N) and phosphorus (P) transport when applied at 10 mg L through lay-flat polyethylene tubing. A 2-yr field study was conducted at the Mississippi State Delta Research and Extension Center in Stoneville, MS, on a Dundee silt loam and a Forestdale silty clay loam. The experimental design was a randomized complete block with four replications of each treatment: irrigated plus no PAM (control) and irrigated plus PAM at 10 mg L. Each irrigation event delivered 102 mm of water at 18.9 L m per furrow, and runoff was captured in a holding tank on the lower end of each plot. Pooled over year and soil texture, PAM increased infiltration and corn grain yield by 6% ( ≤ 0.0398). Polyacrylamide effects on the offsite transport of sediment and N and P were inconsistent, varying across year and soil texture. Results indicate that PAM improves infiltration and corn grain yield on silt loam and silty clay loam textured soils; however, further research is required before PAM can be recommended as a best management practice for mitigating erosion and offsite agrochemical transport in mid-southern production systems.

Drying and Storage Methods Affect Cyfluthrin Concentrations in Exposed Plant Samples.

Standard procedures do not exist for drying and storage of plant samples prior to chemical analyses. Since immediate analysis is not always possible, current research examined which plant drying and storage method yielded the highest cyfluthrin recovery rates compared to traditional mechanical freeze-drying methods. Fifteen mesocosms were planted with rice. Cyfluthrin (5 mg L(-1)) was amended into the water column of individual mesocosms. 48 h later, plant material in the water column was collected from each mesocosm. Control (mechanical freeze drying) recovery was significantly greater (p < 0.001) than all 14 combinations of drying and storage. Significant differences also existed between all 14 different combinations. Greatest cyfluthrin recoveries in non-control plants were from the freezer-greenhouse-freezer drying and storage method. Results offer evidence for the efficient plant drying and storage methods prior to cyfluthrin analysis. Future studies should perform comparable analyses on various pesticide classes to determine possible relationships.

Genomic copy number variation in Mus musculus.

BACKGROUND: Copy number variation is an important dimension of genetic diversity and has implications in development and disease. As an important model organism, the mouse is a prime candidate for copy number variant (CNV) characterization, but this has yet to be completed for a large sample size. Here we report CNV analysis of publicly available, high-density microarray data files for 351 mouse tail samples, including 290 mice that had not been characterized for CNVs previously. RESULTS: We found 9634 putative autosomal CNVs across the samples affecting 6.87% of the mouse reference genome. We find significant differences in the degree of CNV uniqueness (single sample occurrence) and the nature of CNV-gene overlap between wild-caught mice and classical laboratory strains. CNV-gene overlap was associated with lipid metabolism, pheromone response and olfaction compared to immunity, carbohydrate metabolism and amino-acid metabolism for wild-caught mice and classical laboratory strains, respectively. Using two subspecies of wild-caught Mus musculus, we identified putative CNVs unique to those subspecies and show this diversity is better captured by wild-derived laboratory strains than by the classical laboratory strains. A total of 9 genic copy number variable regions (CNVRs) were selected for experimental confirmation by droplet digital PCR (ddPCR). CONCLUSION: The analysis we present is a comprehensive, genome-wide analysis of CNVs in Mus musculus, which increases the number of known variants in the species and will accelerate the identification of novel variants in future studies.

Biological relevance of CNV calling methods using familial relatedness including monozygotic twins.

BACKGROUND: Studies involving the analysis of structural variation including Copy Number Variation (CNV) have recently exploded in the literature. Furthermore, CNVs have been associated with a number of complex diseases and neurodevelopmental disorders. Common methods for CNV detection use SNP, CNV, or CGH arrays, where the signal intensities of consecutive probes are used to define the number of copies associated with a given genomic region. These practices pose a number of challenges that interfere with the ability of available methods to accurately call CNVs. It has, therefore, become necessary to develop experimental protocols to test the reliability of CNV calling methods from microarray data so that researchers can properly discriminate biologically relevant data from noise. RESULTS: We have developed a workflow for the integration of data from multiple CNV calling algorithms using the same array results. It uses four CNV calling programs: PennCNV (PC), Affymetrix® Genotyping Console™ (AGC), Partek® Genomics Suite™ (PGS) and Golden Helix SVS™ (GH) to analyze CEL files from the Affymetrix® Human SNP 6.0 Array™. To assess the relative suitability of each program, we used individuals of known genetic relationships. We found significant differences in CNV calls obtained by different CNV calling programs. CONCLUSIONS: Although the programs showed variable patterns of CNVs in the same individuals, their distribution in individuals of different degrees of genetic relatedness has allowed us to offer two suggestions. The first involves the use of multiple algorithms for the detection of the largest possible number of CNVs, and the second suggests the use of PennCNV over all other methods when the use of only one software program is desirable.

Diazinon and permethrin mitigation across a grass-wetland buffer.

Vegetated buffers of different designs are often used as edge-of-field treatment practices to remove pesticides that may be entrained in agricultural runoff. However, buffer system efficacy in pesticide runoff mitigation varies widely due to a multitude of factors including, but not limited to, pesticide chemistry, vegetation composition, and hydrology. Two experimental systems, a control (no vegetation) and a grass-wetland buffer system, were evaluated for their ability to retain diazinon and permethrin associated with a simulated storm runoff. The two systems were equally inefficient at retaining diazinon (mean 9.6 % retention for control and buffer). Grass-wetland buffers retained 83 % and 85 % of cis- and trans-permethrin masses, respectively, while the control only retained 39 % and 44 % of cis- and trans-permethrin masses, respectively. Half-distances (the distance required to decrease pesticide concentration by one-half) for both permethrin isomers were 26 %-30 % shorter in grass buffers (22-23 m) than in the control (32 m). The current study demonstrates treatment efficacy was a function of pesticide properties with the more strongly sorbing permethrin retained to a greater degree. The study also demonstrates challenges in remediating multiple pesticides with a single management practice. By using suites of management practices, especially those employing vegetation, better mitigation of pesticide impacts may be accomplished.

Effect of storage method and associated holding time on nitrogen and phosphorus concentrations in surface water samples.

Assessments were conducted to determine the effect of sample storage method and associated holding time on surface water nutrient concentrations from field sites. Six surface water sites and two nutrient spiked, laboratory water samples were evaluated for nitrate, nitrite, ammonium, filtered orthophosphorus, and total orthophosphorus concentrations on four separate days throughout the period of 1 year. Samples stored at ambient temperature (23°C) for 24 h prior to nutrient analyses resulted in 18 % ± 2 % of results being significantly different from controls (which were analyzed immediately upon collection). Samples placed in the cooler (4°C) for 7 days prior to nutrient analyses resulted in 30 % ± 1 % of values being significantly different from controls. Samples placed in the freezer (-20°C) for 7 days prior to analyses resulted in 34 % ± 12 %, 44 % ± 10 %, and 28 % ± 5.7 % of ammonium, filtered orthophosphate, and total orthophosphate, respectively, values being significantly different from controls. This study highlights the challenges facing researchers in efficient collection, storage and nutrient analysis of samples, especially when sites are remote and difficult to access .

Phytotoxicity of atrazine, S-metolachlor, and permethrin to Typha latifolia (Linneaus) germination and seedling growth.

Phytotoxicity assessments were performed to compare responses of Typha latifolia (L.) seeds to atrazine (only) and atrazine + S-metolachlor exposure concentrations of 0.03, 0.3, 3, and 30 mg L(-1), as well as permethrin exposure concentrations of 0.008, 0.08, 0.8, and 8 mg L(-1). All atrazine + S-metolachlor exposures resulted in significantly reduced radicle development (p < 0.001). A stimulatory effect for coleoptile development was noted in the three highest atrazine (only) exposures (p = 0.0030, 0.0181, and 0.0016, respectively). This research provides data concerning the relative sensitivity of T. latifolia seeds to pesticides commonly encountered in agricultural settings, as well as critical understanding and development of using T. latifolia in phytoremediation efforts for pesticide exposures.

The challenge of documenting water quality benefits of conservation practices: a review of USDA-ARS's conservation effects assessment project watershed studies.

The Conservation Effects Assessment Project was established to quantify water quality benefits of conservation practices supported by the U.S. Department of Agriculture (USDA). In 2004, watershed assessment studies were begun in fourteen agricultural watersheds with varying cropping systems, landscapes, climate, and water quality concerns. This paper reviews USDA Agricultural Research Service 'Benchmark' watershed studies and the challenge of identifying water quality benefits in watersheds. Study goals included modeling and field research to assess practices, and evaluation of practice placement in watersheds. Not all goals were met within five years but important lessons were learned. While practices improved water quality, problems persisted in larger watersheds. This dissociation between practice-focused and watershed-scale assessments occurred because: (1) Conservation practices were not targeted at critical sources/pathways of contaminants; (2) Sediment in streams originated more from channel and bank erosion than from soil erosion; (3) Timing lags, historical legacies, and shifting climate combined to mask effects of practice implementation; and (4) Water quality management strategies addressed single contaminants with little regard for trade-offs among contaminants. These lessons could help improve conservation strategies and set water quality goals with realistic timelines. Continued research on agricultural water quality could better integrate modeling and monitoring capabilities, and address ecosystem services.

Protein uptake into multivesicular bodies and storage granules in the fat body of an insect.

The fat body in Calpodes ethlius (Lepidoptera, Hesperiidae) takes up protein from the blood throughout the larval stage before pupation. Depending upon the phase of development, the protein appears in multivesicular bodies, in large storage granules, and in structures of intermediate form. There are three phases in the 8 days of the last larval stage; the first devoted to growth (molting to 66 hr), the second to synthesis for storage or export (M + 66 to M + 156 hr), and the third to preparation for pupation (M + 156 to pupation at M + 192 hr). From M + O to M + 156 and from M + 180 to M + 188 hr, protein is taken up into multivesicular bodies. Larger MVB's form a continuous series with the protein granules formed from M + 162 to M + 180 hr. Blood proteins increase in concentration and amount from M + 66 to M + 156 hr at the same time as the fat body cells have a high rate of incorporation of amino acids and a structure appropriate for protein synthesis. During granule formation, both amino acid incorporation and blood protein concentration decrease. Since foreign proteins injected into the blood appear in the granules, they are probably made mainly from sequestered blood. Protein uptake involves two stages: concentration between the cells, and ingestion in pinocytotic vesicles. The vesicles fuse to become MVB's or storage granules, depending upon their rates of growth and the addition of lytic enzymes. Since MVB's do not accumulate in the fat body and since many of them contain acid phosphatase and appear empty, they are presumed to be concerned in protein turnover.

The origin and fate of microbodies in the fat body of an insect.

The structure and life history of insect microbodies are described during the development of the fat body from the 4th to 5th larval molt through the 5th to pupal molt. The mature microbodies are flattened spheres about 1.1 x 0.9 micro, with a depression on one side where a dense mass connects the limiting membrane to the core of coiled tubules. They contain catalase and urate oxidase. The precise synchrony of development of insect cells during the molt/intermolt cycle makes it easy to study the life history of particular organelles. Phases of growth are correlated with the hormonal milieu. Mature 4th stage microbodies decrease in size before ecdysis to the 5th stage when they atrophy at the same time as the new 5th stage generation arises. The 5th stage microbodies form as diverticula of the RER and, grow while confronted by RER cisternae. The mature microbodies decrease in size when the fat body engages in massive larval syntheses. At the end of the 5th larval stage, the microbodies are invested by isolation membranes and destroyed before pupation. There are thus two mechanisms for microbody destruction: atrophy of the 4th stage organelles and isolation with autophagy at the end of the 5th stage.

The beads in the Golgi complex-endoplasmic reticulum region.

The region between the rough endoplasmic reticulum (ER) and the Golgi complex has been studied in a variety of insect cell types in an attempt to find a marker for the exit gate or gates from the ER. We have found that the smooth surface of the rough endoplasmic reticulum near Golgi complex transitional elements has beadlike structures arranged in rings at the base of transition vesicles. They occur in all insect cell types and a variety of other organisms. The beads can be seen only after staining in bismuth salts. They are 10-12 nm in diameter and are separated from the membrane and one another by a clear halo giving them a center to center spacing of about 27 nm. The beads are not sensitive to nucleases under conditions which disrupt ribosomes or remove all Feulgen staining material from the nucleus. Under conditions similar to those used to stain tissue, bismuth does not react in vitro with nucleic acids. The component of the beads that stains preferentially with bismuth is therefore probably not nucleic acid.

Protein uptake in multivesicular bodies in the molt-intermolt cycle of an insect.

Plant peroxidase injected into the hemocoel is taken up in granules by almost all tissues. These granules may become multivesicular bodies or isolation bodies which later breakdown. There is most uptake and breakdown at times in the molt-intermolt cycle when cells are engaged in active syntheses.

Golgi complex--endoplasmic reticulum transition region has rings of beads.

The smooth surface of the rough endoplasmic reticulum that makes the forming face of the Golgi complex has beadlike structures arranged in rings at the base of transition vesicles. The beads can only be seen easily after staining in bismuth salts. They are 10 to 12 nanometers in diameter and occur in a variety of cell types and organisms.

Increased urokinase and consumption of α2 -antiplasmin as an explanation for the loss of benefit of tranexamic acid after treatment delay.

Essentials Delayed treatment with tranexamic acid results in loss of efficacy and poor outcomes. Increasing urokinase activity may account for adverse effects of late tranexamic acid treatment. Urokinase + tranexamic acid produces plasmin in plasma or blood and disrupts clotting. α2 -Antiplasmin consumption with ongoing fibrinolysis increases plasmin-induced coagulopathy. SUMMARY: Background Tranexamic acid (TXA) is an effective antifibrinolytic agent with a proven safety record. However, large clinical trials show TXA becomes ineffective or harmful if treatment is delayed beyond 3 h. The mechanism is unknown but urokinase plasminogen activator (uPA) has been implicated. Methods Inhibitory mechanisms of TXA were explored in a variety of clot lysis systems using plasma and whole blood. Lysis by tissue plasminogen activator (tPA), uPA and plasmin were investigated. Coagulopathy was investigated using ROTEM and activated partial thromboplastin time (APTT). Results IC50 values for antifibrinolytic activity of TXA varied from < 10 to > 1000 µmol L-1 depending on the system, but good fibrin protection was observed in the presence of tPA, uPA and plasmin. However, in plasma or blood, active plasmin was generated by TXA + uPA (but not tPA) and coagulopathy developed leading to no or poor clot formation. The extent of coagulopathy was sensitive to available α2 -antiplasmin. No clot formed with plasma containing 40% normal α2 -antiplasmin after short incubation with TXA + uPA. Adding purified α2 -antiplasmin progressively restored clotting. Plasmin could be inhibited by aprotinin, IC50 = 530 nmol L-1 , in plasma. Conclusions Tranexamic acid protects fibrin but stimulates uPA activity and slows inhibition of plasmin by α2 -antiplasmin. Plasmin proteolytic activity digests fibrinogen and disrupts coagulation, exacerbated when α2 -antiplasmin is consumed by ongoing fibrinolysis. Additional direct inhibition of plasmin by aprotinin may prevent development of coagulopathy and extend the useful time window of TXA treatment.

Modulation of gingival epithelial phenotypes by interactions with regionally defined populations of fibroblasts.

BACKGROUND AND OBJECTIVE: The unusual structure and functions of junctional epithelium, together with its pattern of migration in periodontal disease, raise interesting questions about the factors associated with the maintenance of its unique phenotype. To explore the effects of regionally differing fibroblast populations on the growth and patterns of differentiation of oral epithelia, this study used an organotypical in vitro model in an attempt to detect interactions occurring between populations of human oral fibroblasts and keratinocytes. MATERIAL AND METHODS: Keratinocytes and fibroblasts, isolated from the gingival region and periodontal ligament, were characterized by their patterns of growth and by their expression of known differentiation markers. Changes in cell behaviour and phenotypic marker expression were examined during in vitro passage as an indication of the maintenance of in vivo phenotypic traits. Using early passage cells, organotypical cultures were generated and patterns of epithelial growth and expression of phenotypic markers were examined. RESULTS: Phenotypically different populations of junctional and oral-gingival keratinocytes, and of oral-gingival and periodontal ligament fibroblasts, were successfully isolated, cultured and characterized. In the organotypic culture system, oral-gingival fibroblasts were found to have a markedly greater ability than periodontal ligament fibroblasts to support and maintain the growth of either type of epithelium. Shifts of epithelial phenotype were induced by different fibroblasts. CONCLUSION: Periodontal and gingival fibroblast subpopulations have differential effects on the growth and patterns of differentiation of oral and junctional epithelia. By modulating the epithelial phenotype, regionally differing fibroblasts can influence the stability and behaviour of the gingival attachment apparatus in health and disease.