Developmental mechanisms understood quantitatively.

Across developmental systems, quantitative and imaging-based approaches have provided unprecedented resolution of dynamic changes in gene regulation and cell fate specification, along with complex changes in tissue morphology. This has set the stage for a wealth of comprehensive theoretical models, parameterised by experimental data, able to reproduce key aspects of biological behaviour and jointly enabling a higher level of abstraction, going from the identification of the molecular components to understanding complex functional relationships between these components. Despite these successes, gaining a cross-scale understanding of developmental systems will require further collaboration between disciplines, from developmental biology to bioengineering, systems biology and biophysics. We highlight the exciting multi-disciplinary research discussed at The Company of Biologists workshop 'Fostering quantitative modelling and experimentation in Developmental Biology'.

Transmission of cytokinesis forces via E-cadherin dilution and actomyosin flows.

During epithelial cytokinesis, the remodelling of adhesive cell-cell contacts between the dividing cell and its neighbours has profound implications for the integrity, arrangement and morphogenesis of proliferative tissues. In both vertebrates and invertebrates, this remodelling requires the activity of non-muscle myosin II (MyoII) in the interphasic cells neighbouring the dividing cell. However, the mechanisms that coordinate cytokinesis and MyoII activity in the neighbours are unknown. Here we show that in the Drosophila notum epithelium, each cell division is associated with a mechanosensing and transmission event that controls MyoII dynamics in neighbouring cells. We find that the ring pulling forces promote local junction elongation, which results in local E-cadherin dilution at the ingressing adherens junction. In turn, the reduction in E-cadherin concentration and the contractility of the neighbouring cells promote self-organized actomyosin flows, ultimately leading to accumulation of MyoII at the base of the ingressing junction. Although force transduction has been extensively studied in the context of adherens junction reinforcement to stabilize adhesive cell-cell contacts, we propose an alternative mechanosensing mechanism that coordinates actomyosin dynamics between epithelial cells and sustains the remodelling of the adherens junction in response to mechanical forces.

In vitro anti-inflammatory activity and ameliorative effects on gastric ulcers of Licania rigida benth seed extract.

Licania rigida Benth., a Brazilian endemic plant, has been traditionally used for treating inflammation and stomach pain. This work investigates the anti-inflammatory and gastroprotective activities of the ethanolic extract from L. rigida seeds (EELr) by in vitro and in vivo methods. The phytochemical profile was determined and the in vitro antioxidant activity was investigated by radical scavenging and thiobarbituric acid reactive substances methods. The ovalbumin denaturation method was used with sodium diclofenac as standard for the in vitro anti-inflammatory activity assessment. Acetylsalicylic acid was used to induce gastric ulcers in male mice and then to evaluate the preventive and therapeutic gastroprotective effect of EELr, using omeprazole as the reference drug. The extract exhibited relevant amount of phenolic compounds and flavonoids, in particular, demonstrating in vitro antioxidant capacity. EELr was able to inhibit almost 60% of ovalbumin denaturation at a concentration considered low. It also prevented the decrease of biochemical markers for oxidative stress such as superoxide dismutase (SOD) and reduced glutathione (GSH) in the stomach and SOD and catalase (CAT) in the liver. EELr also significantly decreased the number of lesions as well as reduced the ulcerated area when used as therapy. The observed effect may be due to its phenolic compounds, such as chlorogenic acid, caffeic acid and tannins, as previously reported. EELr is a potential source of compounds with anti-inflammatory activity, protects the liver from oxidative damage and improves healing of aspirin-induced ulcers. This work contributes to the knowledge of L. rigida species.

Drosophila Polo regulates the spindle assembly checkpoint through Mps1-dependent BubR1 phosphorylation.

Maintenance of genomic stability during eukaryotic cell division relies on the spindle assembly checkpoint (SAC) that prevents mitotic exit until all chromosomes are properly attached to the spindle. Polo is a mitotic kinase proposed to be involved in SAC function, but its role has remained elusive. We demonstrate that Polo and Aurora B functional interdependency comprises a positive feedback loop that promotes Mps1 kinetochore localization and activity. Expression of constitutively active Polo restores normal Mps1 kinetochore levels even after Aurora B inhibition, highlighting a role for Polo in Mps1 recruitment to unattached kinetochores downstream of Aurora B. We also show that Mps1 kinetochore localization is required for BubR1 hyperphosphorylation and formation of the 3F3/2 phosphoepitope. This is essential to allow recruitment of Cdc20 to unattached kinetochores and the assembly of anaphase-promoting complex/cyclosome-inhibitory complexes to levels that ensure long-term SAC activity. We propose a model in which Polo controls Mps1-dependent BubR1 phosphorylation to promote Cdc20 kinetochore recruitment and sustained SAC function.

Selective tracking of template DNA strands after induction of mitosis with unreplicated genomes (MUGs) in Drosophila S2 cells.

According to the "immortal" DNA strand hypothesis (Cairns Nature 255:197-200, 1975), stem cells would keep their template strands in order to prevent the accumulation of mutations, which could occur during DNA replication. Despite the growing number of studies that attempt to test this hypothesis, the conclusions remain highly controversial. In the base of this controversy lie the current limitations of available methodology to selectively and faithfully track the fate of template DNA strands throughout and upon cell division. Here, we developed a method that allows the unequivocal tracking of single chromatids containing template DNA strands in Drosophila S2 cells in culture. This method consists in the induction of mitosis with unreplicated genomes (MUGs) in which cells are allowed to enter mitosis without prior DNA replication. This is achieved by RNAi-mediated knockdown of Double parked, a conserved protein required for the initiation of DNA replication and post-replication checkpoint response. The advantages of this system when compared with MUGs generated in mammalian cells is the preservation of chromatid morphology, the ease of loss-of-function studies and the possibility of in vivo applications. Altogether, this approach allows for the readily visualization and tracking of template DNA strands by simply monitoring cells stably expressing GFP-fusions with either Histone H2B or the centromeric Histone variant CID/CENP-A by time-lapse fluorescence microscopy. This might be useful for the dissection of the molecular mechanism behind asymmetric DNA strand segregation.

Pulling the strings on solid-to-liquid phase transitions in cell collectives.

Cell collectives must dynamically adapt to different biological contexts. For instance, in homeostatic conditions, epithelia must establish a barrier between body compartments and resist external stresses, while during development, wound healing or cancer invasion, these tissues undergo extensive remodeling. Using analogies from inert, passive materials, changes in cellular density, shape, rearrangements and/or migration were shown to result in collective transitions between solid and fluid states. However, what biological mechanisms govern these transitions remains an open question. In particular, the upstream signaling pathways and molecular effectors controlling the key physical axes determining tissue rheology and dynamics remain poorly understood. In this perspective, we focus on emerging evidence identifying the first biological signals determining the collective state of living tissues, with an emphasis on how these mechanisms are exploited for functionality across biological contexts.

Dynamics of recombinant hG-CSF in transgenic goat: preliminary study in the founder during hormonally induced lactation.

This study aimed to characterize the dynamic of human granulocyte colony-stimulating factor (hG-CSF) during artificial lactation in a transgenic founder goat and to assess its potential ectopic expression and health. The female secreted 93.9 to 1,474.6 µg hG-CSF per mL of milk. Two peaks of serum hG-CSF (3,470 and 7,390 pg/mL) were detected in the first half of the lactation. Outside of the lactation, hG-CSF was absent from serum, indicating no ectopic expression. During the treatment to induce lactation, transgenic female presented increased neutrophil and lymphocyte blood counts when compared to nontransgenic female. Despite transient neutrophilia, serum biochemistry profiles indicated normal liver and renal functions. Thus, transgenic goat expressed hG-CSF in quantities sufficient for a commercial bioreactor and remained clinically healthy.

A hydraulic feedback loop between mesendoderm cell migration and interstitial fluid relocalization promotes embryonic axis formation in zebrafish.

Interstitial fluid (IF) accumulation between embryonic cells is thought to be important for embryo patterning and morphogenesis. Here, we identify a positive mechanical feedback loop between cell migration and IF relocalization and find that it promotes embryonic axis formation during zebrafish gastrulation. We show that anterior axial mesendoderm (prechordal plate [ppl]) cells, moving in between the yolk cell and deep cell tissue to extend the embryonic axis, compress the overlying deep cell layer, thereby causing IF to flow from the deep cell layer to the boundary between the yolk cell and the deep cell layer, directly ahead of the advancing ppl. This IF relocalization, in turn, facilitates ppl cell protrusion formation and migration by opening up the space into which the ppl moves and, thereby, the ability of the ppl to trigger IF relocalization by pushing against the overlying deep cell layer. Thus, embryonic axis formation relies on a hydraulic feedback loop between cell migration and IF relocalization.

In vitro long-term culture of isolated ovine preantral follicles: Influence of ethanol on steroid production, oocyte meiotic resumption, and metabolomic profile.

Ethanol is used routinely to dilute cell culture media supplements with little or no water solubility. This study evaluates the effect of low concentration of ethanol on the follicular development, oocyte maturation, hormone production, gene expression, and metabolomics profile of spent culture medium after long-term culture of isolated ovine preantral follicles. For this, follicles were cultured for 18 days in α-Minimum Essential Medium+ alone (control treatment) or supplemented with 100 ng/mL recombinant bovine FSH (rbFSH treatment) or with 0.2%-v/v ethanol (ethanol treatment). Ethanol treatment increased the percentage of degenerated follicles and oocytes significantly, however, it showed the highest estradiol secretion. Also, the rate of meiosis resumption was higher in ethanol treatment than Control treatment. Ethanol treatment decreased the mRNA levels of B-cell lymphoma 2 (BCL2), BCL2 associated X, Aquaporin 3, Connexin 43, Inhibin Subunit Beta A, kit ligand, Heat Shock Protein (HSP A1A) significantly when compared to the Control treatment. However, mRNA levels of cytochrome P450 family 19, and FSH receptors were significantly higher in ethanol treatment than in the Control treatment. The levels of some metabolites, which are likely amino acids, lipids, an analog of Cyclic guanosine monophosphate, and a derivative of phosphoinositol phosphate metabolism, had higher relative concentrations in ethanol and rbFSH treatments than the Control treatment. In conclusion, ethanol addition augmented the follicular and oocyte degeneration rates but increased the estradiol production and the meiotic resumption. Furthermore, the follicular metabolomic profile was similar between ethanol and rbFSH treatments being both treatments; however, different from the Control treatment.

Zebrafish gastrulation: Putting fate in motion.

Gastrulation entails specification and formation of three embryonic germ layers-ectoderm, mesoderm and endoderm-thereby establishing the basis for the future body plan. In zebrafish embryos, germ layer specification occurs during blastula and early gastrula stages (Ho & Kimmel, 1993), a period when the main morphogenetic movements underlying gastrulation are initiated. Hence, the signals driving progenitor cell fate specification, such as Nodal ligands from the TGF-ß family, also play key roles in regulating germ layer progenitor cell segregation (Carmany-Rampey & Schier, 2001; David & Rosa, 2001; Feldman et al., 2000; Gritsman et al., 1999; Keller et al., 2008). In this review, we summarize and discuss the main signaling pathways involved in germ layer progenitor cell fate specification and segregation, specifically focusing on recent advances in understanding the interplay between mesoderm and endoderm specification and the internalization movements at the onset of zebrafish gastrulation.

Zebrafish embryonic explants undergo genetically encoded self-assembly.

Embryonic stem cell cultures are thought to self-organize into embryoid bodies, able to undergo symmetry-breaking, germ layer specification and even morphogenesis. Yet, it is unclear how to reconcile this remarkable self-organization capacity with classical experiments demonstrating key roles for extrinsic biases by maternal factors and/or extraembryonic tissues in embryogenesis. Here, we show that zebrafish embryonic tissue explants, prepared prior to germ layer induction and lacking extraembryonic tissues, can specify all germ layers and form a seemingly complete mesendoderm anlage. Importantly, explant organization requires polarized inheritance of maternal factors from dorsal-marginal regions of the blastoderm. Moreover, induction of endoderm and head-mesoderm, which require peak Nodal-signaling levels, is highly variable in explants, reminiscent of embryos with reduced Nodal signals from the extraembryonic tissues. Together, these data suggest that zebrafish explants do not undergo bona fide self-organization, but rather display features of genetically encoded self-assembly, where intrinsic genetic programs control the emergence of order.

Toxicological effects of the rare earth element neodymium in Mytilus galloprovincialis.

The wide range of applications of rare earth elements (REE) is leading to their occurrence in worldwide aquatic environments. Among the most popular REE is Neodymium (Nd), being widely used in permanent magnets, lasers, and glass additives. Neodymium-iron-boron (NdFeB) magnets is the main application of Nd since they are used in electric motors, hard disk drives, speakers and generators for wind turbines. Recent studies have already evaluated the toxic potential of different REE, but no information is available on the effects of Nd towards marine bivalves. Thus, the present study evaluated the biochemical alterations caused by Nd in the mussel Mytilus galloprovincialis exposed to this element for 28 days. The results obtained clearly demonstrated that Nd was accumulated by mussels, leading to mussel's metabolic capacity increase and GLY expenditure, in an attempt to fuel up defense mechanisms. Antioxidant and biotransformation defenses were insufficient in the elimination of ROS excess, resulting from the presence of Nd and increased electron transport system activity, which caused cellular damages (measured by lipid peroxidation) and loss of redox balance (assessed by the ratio between reduced and oxidized glutathione). The results obtained clearly highlight the potential toxicity of REEs and, in particular of Nd, with impacts at cellular level, which may have consequences in mussel's survival, growth and reproduction, affecting mussel's population.

Clinical outcomes of 28 cats 12-24 months after urethrostomy.

OBJECTIVES: The aim of this study was to evaluate and compare the long-term clinical outcomes and quality of life of cats having undergone perineal urethrostomy (PU) or prepubic urethrostomy (PPU). METHODS: This clinical study followed 28 cats (PU, n = 22; PPU, n = 6) that underwent a urethrostomy, with a minimum of 1 year postoperative follow-up. Medical records, pet owner surveys and urologic laboratory tests were used for assessment. Urologic laboratory tests included serum symmetric dimethylarginine (SDMA), serum creatinine, urinalysis, urine specific gravity (USG), urine protein:creatinine (UPC) ratio and urine culture. RESULTS: The main indications for urethrostomy were multiple catheterizations and PU stricture. The overall complication rates of PU and PPU were 31.8% and 83.3%, respectively. Recurrent urinary tract infection (UTI) and urine scald dermatitis were less frequent in PU than in PPU cats (UTI 22.7% vs 66.6%; dermatitis 4.5% vs 83.3%). Bacteriuria was present in 77.2% and 100% of PU and PPU cats, respectively. Owner satisfaction rates were excellent in 81.8% of PU and 33.3% of PPU cases. CONCLUSIONS AND RELEVANCE: A proportion of cats that underwent urethrostomy showed bacteriuria, recurrent UTIs and increased levels of SDMA. PPU is important as a salvage procedure; however, it should be limited to cases in which standard techniques for PU cannot be performed, owing to the potential for recurrent complications and lower owner satisfaction.

Comparative study of cystatin C and serum creatinine in the estimative of glomerular filtration rate in children.

BACKGROUND: To compare estimated glomerular filtration rate (GFR) by Schwartz formula and cystatin C-derived formula in a large population of children with a large spectrum of renal disease. METHODS: Serum creatinine, cystatin C and estimated GFR were determined in 273 children, 254 with renal disease, and a mean age of 10.0+/-4.4 y. Nineteen children were used as control, with a mean age of 8.5+/-4.2 y. RESULTS: The children had nephrotic syndrome (16.5%), glomerulonephritis (11.4%), neurogenic bladder (11.4%), hydronephrosis (9.8%), asymptomatic hematuria (11%), chronic renal disease (5.9%) and other diseases (11%). Cystatin C, creatinine, Schwartz estimated GFR and cystatin C estimated GFR (mean+/-SD) were 1.30+/-1.03 mg/dl, 0.82+/-1.20 mg/l, 143+/-72 ml/min/1.73 m(2) and 88+/-36 ml/min/1.73 m(2), respectively. Although GFR estimated by creatinine and cystatin C had a significant correlation, the Bland-Altman analysis showed greater differences between GFR estimated by the 2 methods, with a mean difference of 50 ml/min. Besides, >50% of the patients with a reduced cystatin C estimated GFR had a normal GFR when analyzed by the Schwartz formula. CONCLUSIONS: Our data shows that cystatin C-based GFR is more sensitive than previous study had demonstrated. It is important to perform studies in specific populations to determine the variability in GFR measurements.

Mechanical Force-Driven Adherens Junction Remodeling and Epithelial Dynamics.

During epithelial tissue development, repair, and homeostasis, adherens junctions (AJs) ensure intercellular adhesion and tissue integrity while allowing for cell and tissue dynamics. Mechanical forces play critical roles in AJs' composition and dynamics. Recent findings highlight that beyond a well-established role in reinforcing cell-cell adhesion, AJ mechanosensitivity promotes junctional remodeling and polarization, thereby regulating critical processes such as cell intercalation, division, and collective migration. Here, we provide an integrated view of mechanosensing mechanisms that regulate cell-cell contact composition, geometry, and integrity under tension and highlight pivotal roles for mechanosensitive AJ remodeling in preserving epithelial integrity and sustaining tissue dynamics.

Himatanthus drasticus (Apocynaceae) latex reduces oxidative stress and modulates CD4+, CD8+, FoxP3+ and HSP-60+ expressions in Sarcoma 180-bearing mice.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil, latex of Himatanthus drasticus is used to treat inflammation, wound healing and cancer. The present study evaluated the antitumoral potential of H. drasticus latex (HdCL) in Sarcoma 180-bearing mice (S180). MATERIALS AND METHODS: HdCL was obtained in Crato-CE, Brazil. Qualitative phytochemicals assays, nuclear magnetic resonance (NMR) and microbiological analyzes were performed. Swiss mice were divided into six groups, according to tumor forms: 1) ascitic model, GI (Control; 0.9% saline), GII (S180asc) and GIII (S180asc/HdCL/14 days); 2) solid model, GIV (Control; 0.9% saline), GV (S180sol) and GVI (S180sol/HdCL/10 days). HdCL and 0.9% saline were administered at 0.2 mL, SID, by gavage, for 10 or 14 days. For ascitic model, 0.5 mL of S180 suspension (4×106 cells/mL) was inoculated intraperitoneally and for solid model, cells were inoculated subcutaneously (25 µL) on the right hind paw of mice. Blood samples were collected for hematological and oxidative stress evaluation. Thickness, volume and weight of paws were measured in solid model. After euthanasia, spleen, liver and kidney were collected in order to assess the relative organ weight. Tissue fragments of paws and popliteal lymph nodes (PLN) were analyzed by H&E and CD4+, CD8+, HSP-60+ and Foxp3+ immunohistochemistry. RESULTS: HdCL presented milky aspect and pinkish supernatant. Phenols, flavonols, flavanones, free steroids and cinnamoyl derivatives of lupeol, α-amyrin and ß-amyrin were detected at the phytochemistry analysis. HdCL did not alter the relative weight of organs, hematological parameters and volume of ascitic fluid recovered. In solid model, HdCL reduced (P < 0.05) paw volume, but did not altered thickness, paw weight and histological parameters. S180sol induced necrosis, metastasis and destruction of bone, cartilage and muscles. Bleeding, vessel congestion and oncocytes were observed in PLN. In paw, HdCL did not alter FoxP3+ and HSP-60+ expressions but reduced the CD4+ and CD8+ expressions, while at PLN, HdCL reduced the expressions of all markers. HdCL decreased (P < 0.05) serum levels of malondialdehyde in ascitic model. CONCLUSIONS: Treatment with HdCL reduced oxidative damage and modulated the expressions of CD4+, CD8+, FoxP3+and HSP-60+ in S180 solid tumor model, which can be associated to the presence of triterpenes, such as α-amyrin, ß-amyrin and lupeol cinnamate. Present data emphasizes the importance of immune system in cancer and highlights the evaluation of the pharmacological properties of plants used by population as phytoterapics.

Phylogenetic analysis of foot-and-mouth disease virus type O re-emerging in free areas of South America.

The nucleotide sequences of the complete VP(1)-coding region of foot-and-mouth disease viruses (FMDV), type O, isolated during the recent emergencies of the disease in free areas of South America (Mato Grosso do Sul, Brazil, October 2005, and Corrientes, Argentina, February 2006), were determined. Also established were the complete VP(1)-coding sequences of viruses occurring in neighbouring locations between the years 2000 and 2003. A phylogenetic analysis was performed based on comparison with continental relevant field and vaccine strains, as well as with extra-continental representative viruses. The results show that the emergencies in Argentina and Brazil were caused by viruses presenting 93% genetic relatedness. Both variants are endogenous to South America, as they were placed within the Europe-South America topotype. When compared with the continental viruses available for the phylogenetic studies, they show the closest relationship with viruses responsible for previous emergencies in neighbouring free areas, or for sporadic outbreaks in the adjacent places with advanced eradication stages, presenting similarity values of at least 90% among them, and clustering together in a unique lineage. This lineage represents the only one sporadically appearing in the Southern Cone and differs from those including viruses presently circulating in the Andean region, reflecting the different livestock circuits and epidemiological scenarios.

Topical anti-inflammatory, gastroprotective and antioxidant effects of the essential oil of Lippia sidoides Cham. leaves.

Lippia sidoides in Northeastern Brazil is widely used in the social medicine program named "Live Pharmacies" run by the municipal governments of country towns, to help poor people with phytotherapy, performed with local plants that are inexpensive but very effective. This plant is mainly used as a general antiseptic due to its strong action against many microorganisms. In order to evaluate the action spectrum of this plant, pharmacological studies were performed on acute toxicity, topical inflammation and ethanol-induced gastric lesions in mice, using the leaf essential oil (EO) of Lippia sidoides. The topical application of EO at doses of 1 and 10mg/ear, respectively, significantly reduced (P<0.05) in 45.93 and 35.26% the acute ear edema induced by 12-otetradecanoylphorbol 13-acetate (TPA). The gastroprotective effect was demonstrated by oral pretreatment with EO at doses of 10, 50 and 100mg/kg, which, respectively, significantly inhibited (P<0.05) by 58.33, 45.83 and 41.66% the damage produced by ethanol, but altered neither the weight nor the protein gastric mucus induced by ethanol administration. This study confirmed the great potential of this plant for medicinal proposals.

Renal biochemistry variables and ultrasonographic imaging in healthy Squirrel monkeys (Saimiri collinsi).

BACKGROUND: The combined use of renal biochemistry and ultrasonographic imaging may improve the correct management of renal disease. Although renal disease is frequently observed in nonhuman primates, renal function markers have not yet been studied in Squirrel monkeys (Saimiri collinsi). OBJECTIVES: The aim of this study was to establish normal renal biochemistry variables and ultrasonographic features in Squirrel monkeys. MATERIAL AND METHODS: Renal biochemistry variables and ultrasonographic images were documented in 29 healthy Squirrel monkeys (15 males and 14 females). Urea, serum creatinine (SCr), and uric acid (UA) concentrations were measured by kinetic assay. Cystatin C (CysC) was analyzed by immunonephelometry. A multiple frequency linear array probe (5-12 MHz) was used for ultrasonographic imaging. The studied indicators of renal function were related to sex, age, and body mass. RESULTS: Serum creatinine was influenced by sex and body mass. Serum concentration of urea, UA, and CysC were not influenced by sex, age, and body mass. Ultrasonographic images provided accurate and comprehensive data for making clinical decisions for Squirrel monkeys. The total renal volume was only influenced by the body mass nested in sex and was positively correlated to body mass. Right renal volume was bigger than the left one. CONCLUSION: Normative standards for the renal evaluation, including biochemistry and ultrasonography, in the Squirrel monkey have been established correlated to age, sex, and body mass.