Himalayan lichen biomass for green synthesis of silver nanocolloids: growth kinetics, effect of pH and metal sensing.

Lichen is one of the most abundant non-vascular biomasses; however, a systematic study on the application of biomass in nanomaterial synthesis is very limited. In this study, an aqueous lichen extract was obtained from Hypotrachyna cirrhata, one of the most abundant Himalayan lichen biomasses, using a simple cold percolation method. The effects of extract-to-silver nitrate mixing ratio, pH and waiting time on the growth and stability of nanoparticles were systematically explored. The rate constant for bio-reduction was found to be 5.3 × 10-3 min-1. Transmission electron microscopy showed a narrow particle size distribution with a mean particle size of 11.1 ± 3.6 nm (n = 200). The X-ray diffraction and selected area electron diffraction techniques confirmed the formation of cubic crystals. The synthesized colloidal solution showed excellent response to Hg2+ and Cu2+ ions in spiked water samples. The limit of detection and calibration sensitivity for Hg2+ and Cu2+ ions were found to be 1 and 5 mg l-1 and 2.9 × 10-3 and 1.6 × 10-3 units ppm-1, respectively. These findings suggested that spherical silver nanoparticles with a narrow particle size distribution can be synthesized on a laboratory scale using an aqueous H. cirrhata lichen extract, and the colloidal solution can be used for the detection of selected heavy metals in water samples.

Stigma, depression and quality of life among people affected by neglected tropical diseases in Nepal.

BACKGROUND: Stigma and poor mental health are important factors influencing the quality of life (QOL) of people with neglected tropical diseases (NTDs). This study examines the relationship between stigma, depression and QOL among people affected by leprosy and lymphatic filariasis (LF) in Nepal. METHODS: A cross-sectional community-based survey was conducted among 102 NTD-affected persons (70 leprosy and 32 LF) using interview-administered questionnaires measuring the level of stigma (5-QSI-AP), symptoms of depression (PHQ-9) and QOL (WHOQOL-8). Three different regression models were developed, each using the ordinary least squares and Poisson regression to evaluate the association between stigma and QOL, depression and QOL, and stigma and depression. RESULTS: The mean scores were 21.8±4.4 for QOL, 6.6±5.6 for depression and 3.0±2.8 for stigma. Almost 17% reported the prevalence of depression symptoms. Both stigma (ß=-0.65, p<0.001) and depression (ß=-0.32, p<0.001) were associated with lower scores for QOL, while there was a significant positive relationship between stigma and depression (ß=0.92, p<0.001). Similar results were obtained from the Poisson regression models. CONCLUSIONS: The study showed a considerable burden of depression, stigma and poor QOL among study participants with leprosy and LF in Nepal. A holistic package of care that addresses the physical, mental and psychological well-being of people with NTD is required. CONTEXTE: La stigmatisation et la mauvaise santé mentale sont des facteurs importants qui influencent la qualité de vie des personnes atteintes de maladies tropicales négligées (MTN). Cette étude examine la relation entre la stigmatisation, la dépression et la qualité de vie chez les personnes atteintes de lèpre et de filariose lymphatique au Népal. MÉTHODES UTILISÉES: Une enquête communautaire transversale a été menée auprès de 102 personnes atteintes de MTN (70 de la lèpre et 32 de la filariose lymphatique) à l'aide de questionnaires administrés par entretien mesurant le niveau de stigmatisation (5-QSI-AP), les symptômes de dépression (PHQ-9) et la qualité de vie (WHOQOL-8). Trois modèles de régression différents ont été développés, chacun utilisant les moindres carrés ordinaires et la régression de Poisson pour évaluer l'association entre : (i) la stigmatisation et la QV; (ii) la dépression et la QV; et (iii) la stigmatisation et la dépression. RÉSULTATS: Les scores moyens étaient de 21,8±4,4 pour la QV, 6,6±5,6 pour la dépression, et 3,0±2,8 pour la stigmatisation. Près de 17% des personnes interrogées ont signalé la prévalence de symptômes dépressifs. La stigmatisation (ß = -0,65, p<0 001) et la dépression (ß = -0,32, p<0 001) ont été associées à des scores plus faibles pour la qualité de vie, tandis qu'il existait une relation positive significative entre la stigmatisation et la dépression (ß = 0,92, p<0 001). Des résultats similaires ont été obtenus à partir des modèles de régression de Poisson. CONCLUSIONS: L'étude a montré une incidence importante de dépression, de stigmatisation et d'une mauvaise qualité de vie parmi les participants atteints de lèpre et de FL au Népal. Il convient donc de mettre en place un ensemble de soins holistiques pour ces patients qui prendrait en compte le bien-être physique, mental et psychologique des personnes atteintes de MTN. ANTECEDENTES: La estigmatización y la mala salud mental son factores importantes que influyen en la calidad de vida de las personas con enfermedades tropicales desatendidas. las personas con enfermedades tropicales desatendidas (ETD). Este estudio examina la relación entre el estigma, la depresión y la CdV entre las personas afectadas por lepra y lepra y la filariasis linfática en Nepal. MÉTODOS: Se realizó una encuesta comunitaria transversal entre 102 personas afectadas por ETD (70 de lepra y 32 de filariasis linfática) utilizando cuestionarios entrevistas para medir el nivel de estigma (5-QSI-AP), los síntomas de depresión (PHQ- 9) y la CdV (OMS- 9). 9) y la calidad de vida (WHOQOL-8). Se desarrollaron tres modelos de regresión diferentes regresión de Poisson para evaluar la asociación entre: (i) el estigma y (ii) la depresión. entre: (i) estigma y CdV; (ii) depresión y CdV; y (iii) estigma y depresión. RESULTADOS: Las puntuaciones medias fueron 21,8±4,4 para la CdV, 6,6±5,6 para la depresión y 3,0±2,8 para el estigma. Casi el 17% informó de la prevalencia de síntomas de depresión. Tanto el estigma (ß = -0,65, p<0 001), como la depresión (ß = -0,32, p<0 001) se asociaron con puntuaciones más bajas para la CdV, mientras que hubo una relación positiva significativa entre el estigma y la depresión (ß = 0,92, p<0 001). Se obtuvieron resultados similares en los modelos de regresión de Poisson. CONCLUSIONES: El estudio mostró una carga considerable de depresión, estigma y mala CdV entre los participantes del estudio con lepra y FL en Nepal. Se requiere un paquete holístico de atención que aborde el bienestar físico, mental y psicológico de las personas con ETD.

Polyhydroxybutyrate synthesis in Camelina: Towards coproduction of renewable feedstocks for bioplastics and fuels.

Plant-based co-production of polyhydroxyalkanoates (PHAs) and seed oil has the potential to create a viable domestic source of feedstocks for renewable fuels and plastics. PHAs, a class of biodegradable polyesters, can replace conventional plastics in many applications while providing full degradation in all biologically active environments. Here we report the production of the PHA poly[(R)-3-hydroxybutyrate] (PHB) in the seed cytosol of the emerging bioenergy crop Camelina sativa engineered with a bacterial PHB biosynthetic pathway. Two approaches were used: cytosolic localization of all three enzymes of the PHB pathway in the seed, or localization of the first two enzymes of the pathway in the cytosol and anchoring of the third enzyme required for polymerization to the cytosolic face of the endoplasmic reticulum (ER). The ER-targeted approach was found to provide more stable polymer production with PHB levels up to 10.2% of the mature seed weight achieved in seeds with good viability. These results mark a significant step forward towards engineering lines for commercial use. Plant-based PHA production would enable a direct link between low-cost large-scale agricultural production of biodegradable polymers and seed oil with the global plastics and renewable fuels markets.

Fgf8 promotes survival of nephron progenitors by regulating BAX/BAK-mediated apoptosis.

Fibroblast growth factors (Fgfs) have long been implicated in processes critical to embryonic development, such as cell survival, migration, and differentiation. Several mouse models of organ development ascribe a prosurvival requirement specifically to FGF8. Here, we explore the potential role of prosurvival FGF8 signaling in kidney development. We have previously demonstrated that conditional deletion of Fgf8 in the mesodermal progenitors that give rise to the kidney leads to renal aplasia in the mutant neonate. Deleterious consequences caused by loss of FGF8 begin to manifest by E14.5 when massive aberrant cell death occurs in the cortical nephrogenic zone in the rudimentary kidney as well as in the renal vesicles that give rise to the nephrons. To rescue cell death in the Fgf8 mutant kidney, we inactivate the genes encoding the pro-apoptotic factors BAK and BAX. In a wild-type background, the loss of Bak and Bax abrogates normal cell death and has minimal effect on renal development. However, in Fgf8 mutants, the combined loss of Bak and Bax rescues aberrant cell death in the kidneys and restores some measure of kidney development: 1) the nephron progenitor population is greatly increased; 2) some glomeruli form, which are rarely observed in Fgf8 mutants; and 3) kidney size is rescued by about 50% at E18.5. The development of functional nephrons, however, is not rescued. Thus, FGF8 signaling is required for nephron progenitor survival by regulating BAK/BAX and for subsequent steps involving, as yet, undefined roles in kidney development.

Development of a High-throughput NanoBRET Screening Platform to Identify Modulators of the RAS/RAF Interaction.

Activating mutations in RAS are found in approximately 30% of human cancers, resulting in the delivery of a persistent signal to critical downstream effectors that drive tumorigenesis. RAS-driven malignancies respond poorly to conventional cancer treatments and inhibitors that target RAS directly are limited; therefore, the identification of new strategies and/or drugs to disrupt RAS signaling in tumor cells remains a pressing therapeutic need. Taking advantage of the live-cell bioluminescence resonance energy transfer (BRET) methodology, we describe the development of a NanoBRET screening platform to identify compounds that modulate binding between activated KRAS and the CRAF kinase, an essential effector of RAS that initiates ERK cascade signaling. Using this strategy, libraries containing synthetic compounds, targeted inhibitors, purified natural products, and natural product extracts were evaluated. These efforts resulted in the identification of compounds that inhibit RAS/RAF binding and in turn suppress RAS-driven ERK activation, but also compounds that have the deleterious effect of enhancing the interaction to upregulate pathway signaling. Among the inhibitor hits identified, the majority were compounds derived from natural products, including ones reported to alter KRAS nanoclustering (ophiobolin A), to impact RAF function (HSP90 inhibitors and ROS inducers) as well as some with unknown targets and activities. These findings demonstrate the potential for this screening platform in natural product drug discovery and in the development of new therapeutic agents to target dysregulated RAS signaling in human disease states such as cancer.

An Effective Solution for Large Scale Single Machine Total Weighted Tardiness Problem using Lunar Cycle Inspired Artificial Bee Colony Algorithm.

Single machine total weighted tardiness problem (SMTWTP) is one of the fundamental combinatorial optimization problems. The problem consists of a set of independent jobs with distinct processing times, weights, and due dates to be scheduled on a single machine. The goal of the problem is to minimize the total weighted tardiness. Several swarm intelligence (SI) motivated techniques have been proposed to solve SMTWTP. Still, the solution for large scale SMTWTP instances within a reasonable amount of time is a challenging task. Artificial bee colony (ABC) algorithm is one of the efficient SI based techniques to solve real world optimization problems. This article presents an effective amended ABC based strategy to solve SMTWTP. A local search (LS) approach, influenced from the lunar cycle is proposed and hybridized with ABC to escalate the exploitation capacity of the algorithm. The proposed LS approach is titled as the lunar inspired LS (LLS) approach and the proposed hybridized strategy is known as lunar inspired ABC (LuABC) algorithm. The proposed LuABC algorithm has been applied on 25 large SMTWTP instances of job size 1000. The obtained outcomes prove that the proposed algorithm obtains the optimum solutions for all the considered instances within a reasonable amount of time.

Camelina sativa, an oilseed at the nexus between model system and commercial crop.

The rapid assessment of metabolic engineering strategies in plants is aided by crops that provide simple, high throughput transformation systems, a sequenced genome, and the ability to evaluate the resulting plants in field trials. Camelina sativa provides all of these attributes in a robust oilseed platform. The ability to perform field evaluation of Camelina is a useful, and in some studies essential benefit that allows researchers to evaluate how traits perform outside the strictly controlled conditions of a greenhouse. In the field the plants are subjected to higher light intensities, seasonal diurnal variations in temperature and light, competition for nutrients, and watering regimes dictated by natural weather patterns, all which may affect trait performance. There are difficulties associated with the use of Camelina. The current genetic resources available for Camelina pale in comparison to those developed for the model plant Arabidopsis thaliana; however, the sequence similarity of the Arabidopsis and Camelina genomes often allows the use of Arabidopsis as a reference when additional information is needed. Camelina's genome, an allohexaploid, is more complex than other model crops, but the diploid inheritance of its three subgenomes is straightforward. The need to navigate three copies of each gene in genome editing or mutagenesis experiments adds some complexity but also provides advantages for gene dosage experiments. The ability to quickly engineer Camelina with novel traits, advance generations, and bulk up homozygous lines for small-scale field tests in less than a year, in our opinion, far outweighs the complexities associated with the crop.

Selective In Vitro Propagation of Nephron Progenitors Derived from Embryos and Pluripotent Stem Cells.

Nephron progenitors in the embryonic kidney propagate while generating differentiated nephrons. However, in mice, the progenitors terminally differentiate shortly after birth. Here, we report a method for selectively expanding nephron progenitors in vitro in an undifferentiated state. Combinatorial and concentration-dependent stimulation with LIF, FGF2/9, BMP7, and a WNT agonist is critical for expansion. The purified progenitors proliferated beyond the physiological limits observed in vivo, both for cell numbers and lifespan. Neonatal progenitors were maintained for a week, while progenitors from embryonic day 11.5 expanded 1,800-fold for nearly 20 days and still reconstituted 3D nephrons containing glomeruli and renal tubules. Furthermore, progenitors generated from mouse embryonic stem cells and human induced pluripotent cells could be expanded with retained nephron-forming potential. Thus, we have established in vitro conditions for promoting the propagation of nephron progenitors, which will be essential for dissecting the mechanisms of kidney organogenesis and for regenerative medicine.

Preferential Propagation of Competent SIX2+ Nephronic Progenitors by LIF/ROCKi Treatment of the Metanephric Mesenchyme.

Understanding the mechanisms responsible for nephrogenic stem cell preservation and commitment is fundamental to harnessing the potential of the metanephric mesenchyme (MM) for nephron regeneration. Accordingly, we established a culture model that preferentially expands the MM SIX2+ progenitor pool using leukemia inhibitory factor (LIF), a Rho kinase inhibitor (ROCKi), and extracellular matrix. Passaged MM cells express the key stem cell regulators Six2 and Pax2 and remain competent to respond to WNT4 induction and form mature tubular epithelia and glomeruli. Mechanistically, LIF activates STAT, which binds to a Stat consensus sequence in the Six2 proximal promoter and sustains SIX2 levels. ROCKi, on the other hand, attenuates the LIF-induced differentiation activity of JNK. Concomitantly, the combination of LIF/ROCKi upregulates Slug expression and activates YAP, which maintains SIX2, PAX2, and SALL1. Using this novel model, our study underscores the pivotal roles of SIX2 and YAP in MM stem cell stability.

Cause of Death in Women of Reproductive Age in Rural Nepal Obtained Through Community-Based Surveillance: Is Reducing Maternal Mortality the Right Priority for Women's Health Programs?

We used a community surveillance system to gather information regarding pregnancy outcomes and the cause of death for women of reproductive age (WRA) in Kanchanpur, Nepal. A total of 784 mother groups participated in the collection of pregnancy outcomes and mortality data. Of the 273 deaths among WRA, the leading causes of death reported were chronic diseases (94, 34.4%) poisoning, snake bites, and suicide (grouped together; 55, 20.1%), and accidents (29, 10.6%), while maternal mortality accounted for 7%. Nevertheless, the calculated maternal mortality ratio was quite high (259.3 per 100,000 live births).

Production of high levels of poly-3-hydroxybutyrate in plastids of Camelina sativa seeds.

Poly-3-hydroxybutyrate (PHB) production in plastids of Camelina sativa seeds was investigated by comparing levels of polymer produced upon transformation of plants with five different binary vectors containing combinations of five seed-specific promoters for expression of transgenes. Genes encoding PHB biosynthetic enzymes were modified at the N-terminus to encode a plastid targeting signal. PHB levels of up to 15% of the mature seed weight were measured in single sacrificed T1 seeds with a genetic construct containing the oleosin and glycinin promoters. A more detailed analysis of the PHB production potential of two of the best performing binary vectors in a Camelina line bred for larger seed size yielded lines containing up to 15% polymer in mature T2 seeds. Transmission electron microscopy showed the presence of distinct granules of PHB in the seeds. PHB production had varying effects on germination, emergence and survival of seedlings. Once true leaves formed, plants grew normally and were able to set seeds. PHB synthesis lowered the total oil but not the protein content of engineered seeds. A change in the oil fatty acid profile was also observed. High molecular weight polymer was produced with weight-averaged molecular weights varying between 600 000 and 1 500 000, depending on the line. Select lines were advanced to later generations yielding a line with 13.7% PHB in T4 seeds. The levels of polymer produced in this study are the highest reported to date in a seed and are an important step forward for commercializing an oilseed-based platform for PHB production.

Non-canonical Wnt5a/Ror2 signaling regulates kidney morphogenesis by controlling intermediate mesoderm extension.

Congenital anomalies of the kidney and urinary tract (CAKUT) affect about 1 in 500 births and are a major cause of morbidity in infants. Duplex collecting systems rank among the most common abnormalities of CAKUT, but the molecular basis for this defect is poorly understood. In mice, conditional deletion of Wnt5a in mesoderm results in bilateral duplex kidney and ureter formation. The ureteric buds (UBs) in mutants emerge as doublets from the intermediate mesoderm (IM)-derived nephric duct (ND) without anterior expansion of the glial cell line-derived neurotrophic factor (Gdnf) expression domain in the surrounding mesenchyme. Wnt5a is normally expressed in a graded manner at the posterior end of the IM, but its expression is down-regulated prior to UB outgrowth at E10.5. Furthermore, ablation of Wnt5a in the mesoderm with an inducible Cre at E7.5 results in duplex UBs, whereas ablation at E8.5 yields normal UB outgrowth, demonstrating that Wnt5a functions in IM development well before the formation of the metanephros. In mutants, the posterior ND is duplicated and surrounding Pax2-positive mesenchymal cells persist in the nephric cord, suggesting that disruption of normal ND patterning prompts the formation of duplex ureters and kidneys. Ror2 homozygous mutants, which infrequently yield duplex collecting systems, show a dramatic increase in incidence with the additional deletion of one copy of Wnt5a, implicating this receptor in non-canonical Wnt5a signaling during IM development. This work provides the first evidence of a role of Wnt5a/Ror2 signaling in IM extension and offers new insights into the etiology of CAKUT and possible involvement of Wnt5a/Ror2 mutations.

Protein/peptide transduction in metanephric explant culture.

While gene targeting methods have largely supplanted cell/explant culture models for studying developmental processes, they have not eliminated the need for or value of such approaches in the investigator's technical arsenal. Explant culture models, such as those devised for the metanephric kidney and its progenitors, remain invaluable as tools for screening regulatory factors involved in tissue induction or in the inhibition of progenitor specification. Thus, some factors capable of inducing tissue condensations or nephronic tubule formation in explants of metanephric mesenchyme have been identified through direct treatment of cultures rather than lengthy genetic engineering in animals. Unfortunately, renal progenitors are largely refractory to most contemporary methods for gene manipulation, including transfection and viral transduction, so the applications of explant culture have been rather limited. However, methods for protein or peptide transduction offer greatly improved efficiencies for uptake and expression/regulation of proteins within cells and tissues. Biologically active TAT- or penetratin-fusion proteins/peptides are readily taken up by most cells in metanephric explants or monolayer cultured cells (Plisov et al., J Am Soc Nephrol 16:1632-1644, 2005; Osafune et al., Development 133:151-161, 2006; Wang et al., Cell Signal 22:1717-1726, 2010; Tanigawa, Dev Biol 352:58-69, 2011), allowing a direct functional evaluation of theoretically any protein, including biologically active enzymes and transcription factors, or any targeted interactive domain within a protein.

High-level expression of sugar inducible gene2 (HSI2) is a negative regulator of drought stress tolerance in Arabidopsis.

BACKGROUND: HIGH-LEVEL EXPRESSION OF SUGAR INDUCIBLE GENE2 (HSI2), also known as VAL1, is a B3 domain transcriptional repressor that acts redundantly with its closest relative, HSI2-LIKE1 (HSL1), to suppress the seed maturation program following germination. Mutant hsi2 hsl1 seedlings are arrested early in development and differentially express a number of abiotic stress-related genes. To test the potential requirement for HSI2 during abiotic stress, hsi2 single mutants and plants overexpressing HSI2 were subjected to simulated drought stress by withholding watering, and characterized through physiological, metabolic and gene expression studies. RESULTS: The hsi2 mutants demonstrated reduced wilting and maintained higher relative water content than wild-type after withholding watering, while the overexpressing lines displayed the opposite phenotype. The hsi2 mutant displayed lower constitutive and ABA-induced stomatal conductance than wild-type and accumulated lower levels of ABA metabolites and several osmolytes and osmoprotectants following water withdrawal. Microarray comparisons between wild-type and the hsi2 mutant revealed that steady-state levels of numerous stress-induced genes were up-regulated in the mutant in the absence of stress but down-regulated at visible wilting. Plants with altered levels of HSI2 responded to exogenous application of ABA and a long-lived ABA analog, but the hsi2 mutant did not show altered expression of several ABA-responsive or ABA signalling genes 4 hr after application. CONCLUSIONS: These results implicate HSI2 as a negative regulator of drought stress response in Arabidopsis, acting, at least in part, by regulating transpirational water loss. Metabolic and global transcript profiling comparisons of the hsi2 mutant and wild-type plants do not support a model whereby the greater drought tolerance observed in the hsi2 mutant is conferred by the accumulation of known osmolytes and osmoprotectants. Instead, data are consistent with mutants experiencing a relatively milder dehydration stress following water withdrawal.

FGF8 is essential for formation of the ductal system in the male reproductive tract.

During development of the urogenital tract, fibroblast growth factor 8 (Fgf8) is expressed in mesonephric tubules, but its role in this tissue remains undefined. An evaluation of previously generated T-Cre-mediated Fgf8-deficient mice (T-Cre; Fgf8(flox/Δ2,3) mice), which lack Fgf8 expression in the mesoderm, revealed that the cranial region of the Wolffian duct degenerated prematurely and the cranial mesonephric tubules were missing. As a result, the epididymis, vas deferens and efferent ductules were largely absent in mutant mice. Rarb2-Cre was used to eliminate FGF8 from the mesonephric tubules but to allow expression in the adjacent somites. These mutants retained the cranial end of the Wolffian duct and formed the epididymis and vas deferens, but failed to elaborate the efferent ductules, indicating that Fgf8 expression by the mesonephric tubules is required specifically for the formation of the ductules. Ret knockout mice do not form the ureteric bud, a caudal outgrowth of the Wolffian duct and progenitor for the collecting duct network in the kidney, but they do develop the cranial end normally. This indicates that Fgf8, but not Ret, expression is essential to the outgrowth of the cranial mesonephric tubules from the Wolffian duct and to the development of major portions of the sex accessory tissues in the male reproductive tract. Mechanistically, FGF8 functions upstream of Lhx1 expression in forming the nephron, and analysis of Fgf8 mutants similarly shows deficient Lhx1 expression in the mesonephric tubules. These results demonstrate a multifocal requirement for FGF8 in establishing the male reproductive tract ducts and implicate Lhx1 signaling in tubule elongation.

Wnt4 induces nephronic tubules in metanephric mesenchyme by a non-canonical mechanism.

Wnt4 and ß-catenin are both required for nephrogenesis, but studies using TCF-reporter mice suggest that canonical Wnt signaling is not activated in metanephric mesenchyme (MM) during its conversion to the epithelia of the nephron. To better define the role of Wnt signaling, we treated rat metanephric mesenchymal progenitors directly with recombinant Wnt proteins. These studies revealed that Wnt4 protein, which is required for nephron formation, induces tubule formation and differentiation markers Lim1 and E-cadherin in MM cells, but does not activate a TCF reporter or up regulate expression of canonical Wnt target gene Axin-2 and has little effect on the stabilization of ß-catenin or phosphorylation of disheveled-2. Furthermore, Wnt4 causes membrane localization of ZO-1 and occludin in tight junctions. To directly examine the role of ß-catenin/TCF-dependent transcription, we developed synthetic cell-permeable analogs of ß-catenin's helix C, which is required for transcriptional activation, in efforts to specifically inhibit canonical Wnt signaling. One inhibitor blocked TCF-dependent transcription and induced degradation of ß-catenin but did not affect tubule formation and stimulated the expression of Lim1 and E-cadherin. Since a canonical mechanism appears not to be operative in tubule formation, we assessed the involvement of the non-canonical Ca(2+)-dependent pathway. Treatment of MM cells with Wnt4 induced an influx of Ca(2+) and caused phosphorylation of CaMKII. Moreover, Ionomycin, a Ca(2+)-dependent pathway activator, stimulated tubule formation. These results demonstrate that the canonical Wnt pathway is not responsible for mesenchymal-epithelial transition (MET) in nephron formation and suggest that the non-canonical calcium/Wnt pathway mediates Wnt4-induced tubulogenesis in the kidney.

STAT1 activation regulates proliferation and differentiation of renal progenitors.

We have shown previously that activation of STAT1 contributes to the pathogenesis of Wilms tumor. This neoplasm caricatures metanephric development and is believed to originate from embryonic renal mesenchymal progenitors that lose their ability to undergo mesenchymal-epithelial transition (MET). Therefore, we hypothesized that STAT1 is also activated and functional during metanephric development. Here we have demonstrated that both STAT1 and STAT3 are activated during normal development of the embryonic kidney. Furthermore, activation of STAT1 stimulated the proliferation of metanephric mesenchymal cells, but it prevented MET and tubulogenesis induced by leukemia inhibitory factor, which preferentially activates STAT3. Consistent with its negative regulation of metanephric mesenchymal differentiation, inhibition of STAT1 activation with protein kinase CK2 inhibitor TBB or RNAi-mediated knockdown of STAT1 promoted differentiation of metanephric progenitors and abolished the effect of cytokine-induced STAT1 activation in these cells. Additionally, a cell-permeable peptide that inhibits STAT1-mediated transactivation by targeting the STAT1 N-domain also blocked cytokine-induced STAT1-dependent proliferation in metanephric progenitors and promoted LIF-induced MET and tubulogenesis. Finally, the STAT1 peptide inhibitor caused the down regulation of survival/anti-apoptotic factors, Mcl-1 and Hsp-27, and induced apoptosis in renal tumor cells with constitutively active STAT1, indicating that STAT1 is required for these cells to survive. These findings show that both metanephric progenitors and renal tumor cells utilize a STAT1-dependent mechanism for growth or survival.

Inheritance of susceptibility to induction of nephroblastomas in the Noble rat.

Noble (Nb) strain rats are susceptible to nephroblastoma induction with transplacental exposure to direct-acting alkylating agent N-nitrosoethylurea (ENU), while F344 strain rats are highly resistant. To study the inheritance of susceptibility to induction of these embryonal renal tumors, fetal Nb and F344 rats and F1, F2 and reciprocal backcross hybrids were exposed transplacentally to ENU once on day 18 of gestation. Nephroblastomas developed in 53% of Nb offspring with no apparent gender difference, while no nephroblastomas developed in inbred F344 offspring. F1 and F2 hybrid offspring had intermediate responses, 28% and 30%, respectively. Nephroblastoma incidence in the offspring of F1 hybrids backcrossed to the susceptible strain Nb was 46%, while that in F1 hybrids backcrossed to resistant strain F344 was much lower (16%). Carcinogenic susceptibility is therefore consistent with the involvement of one major autosomal locus; the operation of a gene dosage effect; and a lack of simple Mendelian dominance for either susceptibility or resistance. Since established Wilms tumor-associated suppressor genes, Wt1 and Wtx, were not mutated in normal or neoplastic tissues, genomic profiling was performed on isolated Nb and F344 metanephric progenitors to identify possible predisposing factors to nephroblastoma induction. Genes preferentially elevated in expression in Nb rat progenitors included Wnt target genes Epidermal growth factor receptor, Inhibitor of DNA binding 2, and Jagged1, which were further increased in nephroblastomas. These studies demonstrate the value of this model for genetic analysis of nephroblastoma development and implicate both the Wnt and Notch pathways in its pathogenesis.

Transgenic increases in seed oil content are associated with the differential expression of novel Brassica-specific transcripts.

BACKGROUND: Seed oil accumulates primarily as triacylglycerol (TAG). While the biochemical pathway for TAG biosynthesis is known, its regulation remains unclear. Previous research identified microsomal diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) as controlling a rate-limiting step in the TAG biosynthesis pathway. Of note, overexpression of DGAT1 results in substantial increases in oil content and seed size. To further analyze the global consequences of manipulating DGAT1 levels during seed development, a concerted transcriptome and metabolome analysis of transgenic B. napus prototypes was performed. RESULTS: Using a targeted Brassica cDNA microarray, about 200 genes were differentially expressed in two independent transgenic lines analyzed. Interestingly, 24-33% of the targets showing significant changes have no matching gene in Arabidopsis although these represent only 5% of the targets on the microarray. Further analysis of some of these novel transcripts indicated that several are inducible by ABA in microspore-derived embryos. Of the 200 Arabidopsis genes implicated in lipid biology present on the microarray, 36 were found to be differentially regulated in DGAT transgenic lines. Furthermore, kinetic reverse transcriptase Polymerase Chain Reaction (k-PCR) analysis revealed up-regulation of genes encoding enzymes of the Kennedy pathway involved in assembly of TAGs. Hormone profiling indicated that levels of auxins and cytokinins varied between transgenic lines and untransformed controls, while differences in the pool sizes of ABA and catabolites were only observed at later stages of development. CONCLUSION: Our results indicate that the increased TAG accumulation observed in transgenic DGAT1 plants is associated with modest transcriptional and hormonal changes during seed development that are not limited to the TAG biosynthesis pathway. These might be associated with feedback or feed-forward effects due to altered levels of DGAT1 activity. The fact that a large fraction of significant amplicons have no matching genes in Arabidopsis compromised our ability to draw concrete inferences from the data at this stage, but has led to the identification of novel genes of potential interest.

Hormonal regulation of oil accumulation in Brassica seeds: metabolism and biological activity of ABA, 7'-, 8'- and 9'-hydroxy ABA in microspore derived embryos of B. napus.

Developing seeds of Brassica napus contain significant levels of ABA and products of oxidation at the 7'- and 9'-methyl groups of ABA, 7'- and 9'-hydroxy ABA, as well stable products of oxidation of the 8'-methyl group, phaseic acid and dihydrophaseic acid. To probe the biological roles of the initially formed hydroxylated compounds, we have compared the effects of supplied ABA and the hydroxylated metabolites in regulating oil synthesis in microspore-derived embryos of B. napus, cv Hero that accumulate long chain fatty acids. Uptake into the embryos and metabolism of each of the hormone metabolites was studied by using deuterium labeled analogs. Supplied ABA, which was rapidly metabolized, induced expression of oleosin and fatty acid elongase genes and increased the accumulation of triacylglycerols and very long chain fatty acids. The metabolites 7'- and 9'-hydroxy ABA had similar effects, with the 9'-hydroxy ABA having even greater activity than ABA. The principal catabolite of ABA, 8'-hydroxy ABA, also had hormonal activity and led to increased oil synthesis but induced the genes weakly. These results indicate that all compounds tested could be involved in lipid synthesis in B. napus, and may have hormonal roles in other ABA-regulated processes.