Landscape-scale predictions of future grassland conversion to cropland or development.

Grassland conservation planning often focuses on high-risk landscapes, but many grassland conversion models are not designed to optimize conservation planning because they lack multidimensional risk assessments and are misaligned with ecological and conservation delivery scales. To aid grassland conservation planning, we developed landscape-scale models at relevant scales that predict future (2021-2031) total and proportional loss of unprotected grassland to cropland or development. We developed models for 20 ecoregions across the contiguous United States by relating past conversion (2011-2021) to a suite of covariates in random forest regression models and applying the models to contemporary covariates to predict future loss. Overall, grassland loss models performed well, and explanatory power varied spatially across ecoregions (total loss model: weighted group mean R2 = 0.89 [range: 0.83-0.96], root mean squared error [RMSE] = 9.29 ha [range: 2.83-22.77 ha]; proportional loss model: weighted group mean R2 = 0.74 [range: 0.64-0.87], RMSE = 0.03 [range: 0.02-0.06]). Amount of crop in the landscape and distance to cities, ethanol plants, and concentrated animal feeding operations had high variable importance in both models. Total grass loss was greater when there were moderate amounts of grass, crop, or development (∼50%) in the landscape. Proportional grass loss was greater when there was less grass (∼<30%) and more crop or development (∼>50%). Some variables had a large effect on only a subset of ecoregions, for example, grass loss was greater when ∼>70% of the landscape was enrolled in the Conservation Reserve Program. Our methods provide a simple and flexible approach for developing risk layers well suited for conservation that can be extended globally. Our conversion models can support conservation planning by enabling prioritization as a function of risk that can be further optimized by incorporating biological value and cost.

Predicciones a escala de paisaje de la conversión futura de los pastizales a tierras de cultivo o desarrollo Resumen La planificación de la conservación de los pastizales a menudo se centra en paisajes de alto riesgo, pero muchos modelos de conversión de pastizales no están diseñados para optimizar la planificación de la conservación porque carecen de evaluaciones de riesgo multidimensionales y están mal alineados con las escalas ecológicas y de conservación. Para ayudar a la planificación de la conservación de los pastizales, desarrollamos modelos a escala de paisaje en escalas relevantes que predicen la pérdida futura (2021­2031) total y proporcional de pastizales no protegidos a tierras de cultivo o desarrollo. Desarrollamos modelos para 20 ecorregiones a lo largo de los Estados Unidos en relación con la conversión pasada (2011­2021) con un conjunto de covariables en modelos de regresión de bosque aleatorio y aplicando los modelos a covariables contemporáneas para predecir la pérdida futura. En general, los modelos de pérdida de pastizales funcionaron bien y el poder explicativo varió espacialmente entre las ecorregiones (modelo de pérdida total: media ponderada del grupoR2 = 0.89 [rango 0.83­0.96], error cuadrático medio [RMSE] = 9,29 ha [rango 2,83­22,77 ha]; modelo de pérdidas proporcionales: R2 medio ponderado del grupo = 0,74 [rango 0,64­0,87], RMSE = 0,03 [rango 0,02­0,06]). La cantidad de cultivos en el paisaje y la distancia a ciudades, plantas de etanol y operaciones concentradas de alimentación animal tuvieron una importancia variable alta en ambos modelos. La pérdida total de pastos fue mayor cuando había cantidades moderadas de pastos, cultivos o desarrollo (∼50%) en el paisaje. La pérdida proporcional de pastos fue mayor cuando había menos pastos (∼<30%) y más cultivos o desarrollo (∼>50%). Algunas variables tuvieron un gran efecto sólo en un subconjunto de ecorregiones, por ejemplo, la pérdida de pastos fue mayor cuando ∼>70% del paisaje estaba inscrito en el Programa de Reservas de Conservación. Nuestros métodos proporcionan un enfoque sencillo y flexible para desarrollar capas de riesgo adecuadas para la conservación que pueden extenderse globalmente. Nuestros modelos de conversión pueden apoyar la planificación de la conservación al permitir la priorización en función del riesgo, que puede optimizarse aún más si se incorporan el valor biológico y el costo.

Triplin: Functional Probing of Its Structure and the Dynamics of the Voltage-Gating Process.

Gram-negative bacteria have a large variety of channel-forming proteins in their outer membrane, generally referred to as porins. Some display weak voltage dependence. A similar trimeric channel former, named Triplin, displays very steep voltage dependence, rivaling that responsible for the electrical excitability of mammals, and high inter-subunit cooperativity. We report detailed insights into the molecular basis for these very unusual properties explored at the single-molecule level. By using chemical modification to reduce the charge on the voltage sensors, they were shown to be positively charged structures. Trypsin cleavage of the sensor eliminates voltage gating by cleaving the sensor. From asymmetrical addition of these reagents, the positively charged voltage sensors translocate across the membrane and are, thus, responsible energetically for the steep voltage dependence. A mechanism underlying the cooperativity was also identified. Theoretical calculations indicate that the charge on the voltage sensor can explain the rectification of the current flowing through the open pores if it is located near the pore mouth in the open state. All results support the hypothesis that one of the three subunits is oriented in a direction opposite to that of the other two. These properties make Triplin perhaps the most complex pore-forming molecular machine described to date.

DLG2 variants in patients with pubertal disorders.

PURPOSE: Impaired function of gonadotropin-releasing hormone (GnRH) neurons can cause a phenotypic spectrum ranging from delayed puberty to isolated hypogonadotropic hypogonadism (IHH). We sought to identify a new genetic etiology for these conditions. METHODS: Exome sequencing was performed in an extended family with autosomal dominant, markedly delayed puberty. The effects of the variant were studied in a GnRH neuronal cell line. Variants in the same gene were sought in a large cohort of individuals with IHH. RESULTS: We identified a rare missense variant (F900V) in DLG2 (which encodes PSD-93) that cosegregated with the delayed puberty. The variant decreased GnRH expression in vitro. PSD-93 is an anchoring protein of NMDA receptors, a type of glutamate receptor that has been implicated in the control of puberty in laboratory animals. The F900V variant impaired the interaction between PSD-93 and a known binding partner, Fyn, which phosphorylates NMDA receptors. Variants in DLG2 that also decreased GnRH expression were identified in three unrelated families with IHH. CONCLUSION: The findings indicate that variants in DLG2/PSD-93 cause autosomal dominant delayed puberty and may also contribute to IHH. The findings also suggest that the pathogenesis involves impaired NMDA receptor signaling and consequently decreased GnRH secretion.

Cartilage-Targeted IGF-1 Treatment to Promote Longitudinal Bone Growth.

Recombinant human growth hormone (GH) is commonly used to treat short stature in children. However, GH treatment has limited efficacy, particularly in severe, non-GH-deficient conditions such as chondrodysplasias, and potential off-target effects. Because short stature results from decreased growth plate chondrogenesis, we developed a cartilage-targeting single-chain human antibody fragment (CaAb) aiming to deliver therapeutic molecules to the growth plate, thereby increasing treatment efficacy while minimizing adverse effects on other tissues. To this end, we created fusion proteins of these CaAbs conjugated with insulin-like growth factor 1 (IGF-1), an endocrine and/or paracrine factor that positively regulates chondrogenesis. These CaAb-IGF-1 fusion proteins retained both cartilage binding and IGF-1 biological activity, and they were able to stimulate bone growth in an organ culture system. Using a GH-deficient (lit) mouse model, we found that subcutaneous injections of these CaAb-IGF-1 fusion proteins increased overall growth plate height without increasing proliferation in kidney cortical cells, suggesting on-target efficacy at the growth plate and less off-target effect on the kidney than IGF-1 alone. Alternate-day injections of these fusion proteins, unlike IGF-1 alone, were sufficient to produce a therapeutic effect. Our findings provide proof of principle that targeting therapeutics to growth plate cartilage can potentially improve treatment for childhood growth disorders.

Effect of Antiandrogen, Aromatase Inhibitor, and Gonadotropin-releasing Hormone Analog on Adult Height in Familial Male Precocious Puberty.

OBJECTIVE: Antiandrogen, aromatase inhibitor, and gonadotropin-releasing hormone analog (GnRHa) treatment normalizes growth rate and bone maturation and increases predicted adult height (AH) in boys with familial male-limited precocious puberty (FMPP). To evaluate the effect of long-term antiandrogen, aromatase inhibitor, and GnRHa on AH, boys with FMPP who were treated were followed to AH. STUDY DESIGN: Twenty-eight boys with FMPP, referred to the National Institutes of Health, were started on antiandrogen and aromatase inhibitor at 4.9 ± 1.5 years of age; GnRHa was added at 6.9 ± 1.5 years of age. Treatment was discontinued at 12.2 ± 0.5 years of age (bone age, 14.4 ± 1.3). AH was assessed at 16.4 ± 1.3 years of age (bone age, 18.5 ± 0.6). RESULTS: AH (mean ± SD) for all treated subjects was 173.6 ± 6.8 cm (-0.4 ± 1.0 SD relative to adult US males). For 25 subjects with pretreatment predicted AH, AH significantly exceeded predicted AH at treatment onset (173.8 ± 6.9 vs 164.9 ± 10.7 cm; P < .001), but fell short of predicted AH at treatment discontinuation (177.3 ± 9.0 cm; P < .001). For 11 subjects with maternal or sporadic inheritance, the mean AH was 3.1 cm (0.4 SD score) below sex-adjusted midparental height (175.4 ± 5.8 vs 178.5 ± 3.1 cm [midparental height]; P = .10). For 16 subjects with affected and untreated fathers, AH was significantly greater than fathers' AH (172.8 ± 7.4 vs 168.8 ± 7.2 cm; P < .05). CONCLUSIONS: Long-term treatment with antiandrogen, aromatase inhibitor, and GnRHa in boys with FMPP results in AH modestly below sex-adjusted midparental height and within the range for adult males in the general population.

CK2α regulates the transcription of BRP in Drosophila.

Development and plasticity of synapses are brought about by a complex interplay between various signaling pathways. Typically, either changing the number of synapses or strengthening an existing synapse can lead to changes during synaptic plasticity. Altering the machinery that governs the exocytosis of synaptic vesicles, which primarily fuse at specialized structures known as active zones on the presynaptic terminal, brings about these changes. Although signaling pathways that regulate the synaptic plasticity from the postsynaptic compartments are well defined, the pathways that control these changes presynaptically are poorly described. In a genetic screen for synapse development in Drosophila, we found that mutations in CK2α lead to an increase in the levels of Bruchpilot (BRP), a scaffolding protein associated with the active zones. Using a combination of genetic and biochemical approaches, we found that the increase in BRP in CK2α mutants is largely due to an increase in the transcription of BRP. Interestingly, the transcripts of other active zone proteins that are important for function of active zones were also increased, while the transcripts from some other synaptic proteins were unchanged. Thus, our data suggest that CK2α might be important in regulating synaptic plasticity by modulating the transcription of BRP. Hence, we propose that CK2α is a novel regulator of the active zone protein, BRP, in Drosophila.

Chemokines Kill Bacteria by Binding Anionic Phospholipids without Triggering Antimicrobial Resistance.

Classically, chemokines coordinate leukocyte trafficking during immune responses; however, many chemokines have also been reported to possess direct antibacterial activity in vitro. Yet, the bacterial killing mechanism of chemokines and the biochemical properties that define which members of the chemokine superfamily are antimicrobial remain poorly understood. Here we report that the antimicrobial activity of chemokines is defined by their ability to bind phosphatidylglycerol and cardiolipin, two anionic phospholipids commonly found in the bacterial plasma membrane. We show that only chemokines able to bind these two phospholipids kill Escherichia coli and Staphylococcus aureus and that they exert rapid bacteriostatic and bactericidal effects against E. coli with a higher potency than the antimicrobial peptide beta-defensin 3. Furthermore, our data support that bacterial membrane cardiolipin facilitates the antimicrobial action of chemokines. Both biochemical and genetic interference with the chemokine-cardiolipin interaction impaired microbial growth arrest, bacterial killing, and membrane disruption by chemokines. Moreover, unlike conventional antibiotics, E. coli failed to develop resistance when placed under increasing antimicrobial chemokine pressure in vitro. Thus, we have identified cardiolipin and phosphatidylglycerol as novel binding partners for chemokines responsible for chemokine antimicrobial action. Our results provide proof of principle for developing chemokines as novel antibiotics resistant to bacterial antimicrobial resistance mechanisms.

Loss-of-function variant in SPIN4 causes an X-linked overgrowth syndrome.

Overgrowth syndromes can be caused by pathogenic genetic variants in epigenetic writers, such as DNA and histone methyltransferases. However, no overgrowth disorder has previously been ascribed to variants in a gene that acts primarily as an epigenetic reader. Here, we studied a male individual with generalized overgrowth of prenatal onset. Exome sequencing identified a hemizygous frameshift variant in Spindlin 4 (SPIN4), with X-linked inheritance. We found evidence that SPIN4 binds specific histone modifications, promotes canonical WNT signaling, and inhibits cell proliferation in vitro and that the identified frameshift variant had lost all of these functions. Ablation of Spin4 in mice recapitulated the human phenotype with generalized overgrowth, including increased longitudinal bone growth. Growth plate analysis revealed increased cell proliferation in the proliferative zone and an increased number of progenitor chondrocytes in the resting zone. We also found evidence of decreased canonical Wnt signaling in growth plate chondrocytes, providing a potential explanation for the increased number of resting zone chondrocytes. Taken together, our findings provide strong evidence that SPIN4 is an epigenetic reader that negatively regulates mammalian body growth and that loss of SPIN4 causes an overgrowth syndrome in humans, expanding our knowledge of the epigenetic regulation of human growth.

A set of imprinted genes required for normal body growth also promotes growth of rhabdomyosarcoma cells.

INTRODUCTION: In many normal tissues, proliferation rates decline postnatally, causing somatic growth to slow. Previous evidence suggests that this decline is due, in part, to decline in the expression of growth-promoting imprinted genes including Mest, Plagl1, Peg3, Dlk1, and Igf2. Embryonal cancers are composed of cells that maintain embryonic characteristics and proliferate rapidly in childhood. We hypothesized that the abnormal persistent rapid proliferation in embryonal cancers occurs in part because of abnormal persistent high expression of growth-promoting imprinted genes. RESULTS: Analysis of microarray data showed elevated expression of MEST, PLAGL1, PEG3, DLK1, and IGF2 in various embryonal cancers, especially rhabdomyosarcoma, as compared to nonembryonal cancers and normal tissues. Similarly, mRNA expression, assessed by real-time PCR, of MEST, PEG3, and IGF2 in rhabdomyosarcoma cell lines was increased as compared to nonembryonal cancer cell lines. Furthermore, siRNA-mediated knockdown of MEST, PLAGL1, PEG3, and IGF2 expression inhibited proliferation in Rh30 rhabdomyosarcoma cells. DISCUSSION: These findings suggest that the normal postnatal downregulation of growth-promoting imprinted genes fails to occur in some embryonal cancers, particularly rhabdomyosarcoma, and contributes to the persistent rapid proliferation of rhabdomyosarcoma cells and, more generally, that failure of the mechanisms responsible for normal somatic growth deceleration can promote tumorigenesis.

The synovial microenvironment suppresses chondrocyte hypertrophy and promotes articular chondrocyte differentiation.

During the development of the appendicular skeleton, the cartilaginous templates undergo hypertrophic differentiation and remodels into bone, except for the cartilage most adjacent to joint cavities where hypertrophic differentiation and endochondral bone formation are prevented, and chondrocytes instead form articular cartilage. The mechanisms that prevent hypertrophic differentiation and endochondral bone formation of the articular cartilage have not been elucidated. To explore the role of the synovial microenvironment in chondrocyte differentiation, osteochondral allografts consisting of articular cartilage, epiphyseal bone, and growth plate cartilage from distal femoral epiphyses of inbred Lewis rats expressing enhanced green fluorescent protein from a ubiquitous promoter were transplanted either in inverted or original (control) orientation to matching sites in wildtype littermates, thereby allowing for tracing of transplanted cells and their progenies. We found that no hypertrophic differentiation occurred in the growth plate cartilage ectopically placed at the joint surface. Instead, the transplanted growth plate cartilage, with time, remodeled into articular cartilage. This finding suggests that the microenvironment at the articular surface inhibits hypertrophic differentiation and supports articular cartilage formation. To explore this hypothesis, rat chondrocyte pellets were cultured with and without synoviocyte-conditioned media. Consistent with the hypothesis, hypertrophic differentiation was inhibited and expression of the articular surface marker lubricin (Prg4) was dramatically induced when chondrocyte pellets were exposed to synovium- or synoviocyte-conditioned media, but not to chondrocyte- or osteoblast-conditioned media. Taken together, we present evidence for a novel mechanism by which synoviocytes, through the secretion of a factor or factors, act directly on chondrocytes to inhibit hypertrophic differentiation and endochondral bone formation and promote articular cartilage formation. This mechanism may have important implications for articular cartilage development, maintenance, and regeneration.

Coordinated postnatal down-regulation of multiple growth-promoting genes: evidence for a genetic program limiting organ growth.

Children grow, but adults do not. The cessation of growth in multiple organs is the end result of a progressive decline in cell proliferation beginning in early life. The mechanisms responsible for this growth deceleration are largely unknown. Using expression microarray and real-time PCR, we identified a common program of gene expression in lung, kidney, and liver during growth deceleration in juvenile rats. Gene ontology analyses and siRNA-mediated knockdown in vitro indicated that many of the down-regulated genes are growth promoting. Down-regulated genes in the program showed declining histone H3K4 trimethylation with age, implicating underlying epigenetic mechanisms. To investigate the physiological processes driving the genetic program, a tryptophan-deficient diet was used to temporarily inhibit juvenile growth in newborn rats for 4 wk. Afterward, microarray analysis showed that the genetic program had been delayed, implying that it is driven by body growth itself rather than age. Taken together, the findings suggest that growth in early life induces progressive down-regulation of a large set of proliferation-stimulating genes, causing organ growth to slow and eventually cease.

Persistent Sox9 expression in hypertrophic chondrocytes suppresses transdifferentiation into osteoblasts.

Longitudinal bone growth is driven by endochondral ossification, a process in which cartilage tissue is generated by growth plate chondrocytes and then remodeled into bone by osteoblasts. In the postnatal growth plate, as hypertrophic chondrocytes approach the chondro-osseous junction, they may undergo apoptosis, or directly transdifferentiate into osteoblasts. The molecular mechanisms governing this switch in cell lineage are poorly understood. Here we show that the physiological downregulation of Sox9 in hypertrophic chondrocyte is associated with upregulation of osteoblast-associated genes (such as Mmp13, Cola1, Ibsp) in hypertrophic chondrocytes, before they enter the metaphyseal bone. In transgenic mice that continued to express Sox9 in all cells derived from the chondrocytic lineage, upregulation of these osteoblast-associated genes in the hypertrophic zone failed to occur. Furthermore, lineage tracing experiments showed that, in transgenic mice expressing Sox9, the number of chondrocytes transdifferentiating into osteoblasts was markedly reduced. Collectively, our findings suggest that Sox9 downregulation in hypertrophic chondrocytes promotes expression of osteoblast-associated genes in hypertrophic chondrocytes and promotes the subsequent transdifferentiation of these cells into osteoblasts.

Catch-up growth after hypothyroidism is caused by delayed growth plate senescence.

Catch-up growth is defined as a linear growth rate greater than expected for age after a period of growth inhibition. We hypothesized that catch-up growth occurs because growth-inhibiting conditions conserve the limited proliferative capacity of growth plate chondrocytes, thus slowing the normal process of growth plate senescence. When the growth-inhibiting condition resolves, the growth plates are less senescent and therefore grow more rapidly than normal for age. To test this hypothesis, we administered propylthiouracil to newborn rats for 8 wk to induce hypothyroidism and then stopped the propylthiouracil to allow catch-up growth. In untreated controls, the growth plates underwent progressive, senescent changes in multiple functional and structural characteristics. We also identified genes that showed large changes in mRNA expression in growth plate and used these changes as molecular markers of senescence. In treated animals, after stopping propylthiouracil, these functional, structural, and molecular senescent changes were delayed, compared with controls. This delayed senescence included a delayed decline in longitudinal growth rate, resulting in catch-up growth. The findings demonstrate that growth inhibition due to hypothyroidism slows the developmental program of growth plate senescence, including the normal decline in the rate of longitudinal bone growth, thus accounting for catch-up growth.

Changes in cell-cycle kinetics responsible for limiting somatic growth in mice.

In mammals, the rate of somatic growth is rapid in early postnatal life but then slows with age, approaching zero as the animal approaches adult body size. To investigate the underlying changes in cell-cycle kinetics, [methyl-H]thymidine and 5'-bromo-2'deoxyuridine were used to double-label proliferating cells in 1-, 2-, and 3-wk-old mice for four weeks. Proliferation of renal tubular epithelial cells and hepatocytes decreased with age. The average cell-cycle time did not increase in liver and increased only 1.7 fold in kidney. The fraction of cells in S-phase that will divide again declined approximately 10 fold with age. Concurrently, average cell area increased approximately 2 fold. The findings suggest that somatic growth deceleration primarily results not from an increase in cell-cycle time but from a decrease in growth fraction (fraction of cells that continue to proliferate). During the deceleration phase, cells appear to reach a proliferative limit and undergo their final cell divisions, staggered over time. Concomitantly, cells enlarge to a greater volume, perhaps because they are relieved of the size constraint imposed by cell division. In conclusion, a decline in growth fraction with age causes somatic growth deceleration and thus sets a fundamental limit on adult body size.

Ezh2 Mutations Found in the Weaver Overgrowth Syndrome Cause a Partial Loss of H3K27 Histone Methyltransferase Activity.

Context: Weaver syndrome is characterized by tall stature, advanced bone age, characteristic facies, and variable intellectual disability. It is caused by heterozygous mutations in enhancer of zeste homolog 2 (EZH2), a histone methyltransferase responsible for histone H3 at lysine 27 (H3K27) trimethylation. However, no early truncating mutations have been identified, suggesting that null mutations do not cause Weaver syndrome. Objective: To test alternative hypotheses that EZH2 variants found in Weaver syndrome cause either a gain of function or a partial loss of function. Design: Exome sequencing was performed in a boy with tall stature, advanced bone age, and mild dysmorphic features. Mutant or wild-type EZH2 protein was expressed in mouse growth plate chondrocytes with or without endogenous EZH2, and enzymatic activity was measured. A mouse model was generated, and histone methylation was assessed in heterozygous and homozygous embryos. Results: A de novo missense EZH2 mutation [c.1876G>A (p.Val626Met)] was identified in the proband. When expressed in growth plate chondrocytes, the mutant protein showed decreased histone methyltransferase activity. A mouse model carrying this EZH2 mutation was generated using CRISPR/Cas9. Homozygotes showed perinatal lethality, whereas heterozygotes were viable, fertile, and showed mild overgrowth. Both homozygous and heterozygous embryos showed decreased H3K27 methylation. Conclusion: We generated a mouse model with the same mutation as our patient, found that it recapitulates the Weaver overgrowth phenotype, and demonstrated that EZH2 mutations found in Weaver syndrome cause a partial loss of function.

Wnt gene expression in the post-natal growth plate: regulation with chondrocyte differentiation.

Longitudinal growth of long bones occurs at the growth plate by endochondral ossification. In the embryonic mouse, this process is regulated by Wnt signaling. Little is known about which members of the Wnt family of secreted signaling proteins might be involved in the regulation of the postnatal growth plate. We used microdissection and real-time PCR to study mRNA expression of Wnt genes in the mouse growth plate. Of the 19 known members of the Wnt family, only six were expressed in postnatal growth plate. Of these, Wnts -2b, -4, and -10b signal through the canonical beta-catenin pathway and Wnts -5a, -5b, and -11 signal through the noncanonical calcium pathway. The spatial expression for these six Wnts was remarkably similar, showing low mRNA expression in the resting zone, increasing expression as the chondrocytes differentiated into the proliferative and prehypertrophic state and then (except Wnt-2b) decreasing expression as the chondrocytes underwent hypertrophic differentiation. This overall pattern is broadly consistent with previous studies of embryonic mouse growth cartilage suggesting that Wnt signaling modulates chondrocyte proliferation and hypertrophic differentiation. We also found that mRNA expression of these Wnt genes persisted at similar levels at 4 weeks, when longitudinal bone growth is waning. In conclusion, we have identified for the first time the specific Wnt genes that are expressed in the postnatal mammalian growth plate. The six identified Wnt genes showed a similar pattern of expression during chondrocyte differentiation, suggesting overlapping or interacting roles in postnatal endochondral bone formation.

Gradients in bone morphogenetic protein-related gene expression across the growth plate.

In the growth plate, stem-like cells in the resting zone differentiate into rapidly dividing chondrocytes of the proliferative zone and then terminally differentiate into the non-dividing chondrocytes of the hypertrophic zone. To explore the molecular switches responsible for this two-step differentiation program, we developed a microdissection method to isolate RNA from the resting (RZ), proliferative (PZ), and hypertrophic zones (HZ) of 7-day-old male rats. Expression of approximately 29,000 genes was analyzed by microarray and selected genes verified by real-time PCR. The analysis identified genes whose expression changed dramatically during the differentiation program, including multiple genes functionally related to bone morphogenetic proteins (BMPs). BMP-2 and BMP-6 were upregulated in HZ compared with RZ and PZ (30-fold each, P < 0.01 and 0.001 respectively). In contrast, BMP signaling inhibitors were expressed early in the differentiation pathway; BMP-3 and gremlin were differentially expressed in RZ (100- and 80-fold, compared with PZ, P < 0.001 and 0.005 respectively) and growth differentiation factor (GDF)-10 in PZ (160-fold compared with HZ, P < 0.001). Our findings suggest a BMP signaling gradient across the growth plate, which is established by differential expression of multiple BMPs and BMP inhibitors in specific zones. Since BMPs can stimulate both proliferation and hypertrophic differentiation of growth plate chondrocytes, these findings suggest that low levels of BMP signaling in the resting zone may help maintain these cells in a quiescent state. In the lower RZ, greater BMP signaling may help induce differentiation to proliferative chondrocytes. Farther down the growth plate, even greater BMP signaling may help induce hypertrophic differentiation. Thus, BMP signaling gradients may be a key mechanism responsible for spatial regulation of chondrocyte proliferation and differentiation in growth plate cartilage.

Establishing a reliable gait evaluation method for rodent studies.

BACKGROUND: CatWalk is one of the most popular tools for evaluating gait recovery in preclinical research, however, there is currently no consensus on which of the many gait parameters captured by CatWalk can reliably model recovery. There are conflicting interpretations of results, along with many common but seldom reported problems such as heel walking and poor compliance. NEW METHOD: We developed a systematic manual classification method that overcomes common problems such as heel walking and poor compliance. By correcting automation errors and removing inconsistent gait cycles, we isolated stretches of recordings that are more reliable for analysis. Recovery outcome was also assessed by quantitative histomorphometric analysis of myelinated axons. RESULTS: While 40-60% of runs were erroneously classified without manual intervention, we corrected all errors with our new method, and showed that Stand Time, Duty Cycle, and Swing Speed are able to track significant differences over time and between experimental groups (all p<0.05). The usability of print area and intensity parameters requires further validation beyond the capabilities of CatWalk. COMPARISON WITH EXISTING METHOD(S): There is currently no strategy that addresses problems such as heel walking and poor compliance, and therefore no standard set of parameters that researchers can rely on to report their findings. CONCLUSION: Manual classification is a crucial step to generate reliable CatWalk data, and Stand Time, Duty Cycle, and Swing Speed are suitable parameters for evaluating gait recovery. Static parameters such as print area and intensity should be used with extreme caution.

Depletion of resting zone chondrocytes during growth plate senescence.

With age, the growth plate undergoes senescent changes that cause linear bone growth to slow and finally cease. Based on previous indirect evidence, we hypothesized that this senescent decline occurs because growth plate stem-like cells, located in the resting zone, have a finite proliferative capacity that is gradually depleted. Consistent with this hypothesis, we found that the proliferation rate in rabbit resting zone chondrocytes (assessed by continuous 5-bromo-2'-deoxy-uridine labeling) decreases with age, as does the number of resting zone chondrocytes per area of growth plate. Glucocorticoid excess slows growth plate senescence. To explain this effect, we hypothesized that glucocorticoid inhibits resting zone chondrocyte proliferation, thus conserving their proliferative capacity. Consistent with this hypothesis, we found that dexamethasone treatment decreased the proliferation rate of rabbit resting zone chondrocytes and slowed the numerical depletion of these cells. Estrogen is known to accelerate growth plate senescence. However, we found that estradiol cypionate treatment slowed resting zone chondrocyte proliferation. Our findings support the hypotheses that growth plate senescence is caused by qualitative and quantitative depletion of stem-like cells in the resting zone and that growth-inhibiting conditions, such as glucocorticoid excess, slow senescence by slowing resting zone chondrocyte proliferation and slowing the numerical depletion of these cells, thereby conserving the proliferative capacity of the growth plate. We speculate that estrogen might accelerate senescence by a proliferation-independent mechanism, or by increasing the loss of proliferative capacity per cell cycle.

Synthesis and antimicrobial applications of 5,5'-ethylenebis[5-methyl-3-(3-triethoxysilylpropyl)hydantoin].

A novel, durable, long lasting, N-halamine siloxane monomer precursor, 5,5'-ethylenebis[5-methyl-3-(3-triethoxysilylpropyl)hydantoin] has been prepared and characterized by (1)H-NMR and FTIR for the purpose of functionalizing the surfaces of various materials. In this work, the precursor N-halamine moiety was attached by siloxane covalent bonding to surfaces of cotton fibers. Simulated laundering tests indicated that the chlorinated N-halamine structure could survive many repeated home launderings. The materials were rendered biocidal after exposure to oxidative halogen solutions, i.e. dilute household bleach. Once chlorinated, these materials were biocidal against Staphylococcus aureus and Escherichia coli. Upon loss of the halogen from either long-term use or consumption by the microbes on the surfaces, they could be simply recharged by further exposure to dilute bleach to regain biocidal activity.