A free customizable tool for easy integration of microfluidics and smartphones

The integration of smartphones and microfluidics is nowadays the best possible route to achieve effective point-of-need testing (PONT), a concept increasingly demanded in the fields of human health, agriculture, food safety, and environmental monitoring. Nevertheless, efforts are still required to integrally seize all the advantages of smartphones, as well as to share the developments in easily adoptable formats. For this purpose, here we present the free platform appuente that was designed for the easy integration of microfluidic chips, smartphones, and the cloud. It includes a mobile app for end users, which provides chip identification and tracking, guidance and control, processing, smart-imaging, result reporting and cloud and Internet of Things (IoT) integration. The platform also includes a web app for PONT developers, to easily customize their mobile apps and manage the data of administered tests. Three application examples were used to validate appuente: a dummy grayscale detector that mimics quantitative colorimetric tests, a root elongation assay for pesticide toxicity assessment, and a lateral flow immunoassay for leptospirosis detection. The platform openly offers fast prototyping of smartphone apps to the wide community of lab-on-a-chip developers, and also serves as a friendly framework for new techniques, IoT integration and further capabilities. Exploiting these advantages will certainly help to enlarge the use of PONT with real-time connectivity in the near future.Fil: Schaumburg, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Vidocevich, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Gerlero, Gabriel Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; ArgentinaFil: Pujato, Nazarena. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Macagno, Joana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Kler, Pablo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; ArgentinaFil: Berli, Claudio Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentin

FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours

A study of focal adhesion kinase in cancer using drosophila melanogaster

Cancer is a group of diseases that affects almost every organ of the human body. A normal cell transforms into a cancer cell as a consequence of cumulative failures that alter diverse cellular processes such as cell proliferation, adhesion, migration, and cell death. Focal Adhesion Kinase (FAK) is a ubiquitous protein that is involved in all these cellular processes. Therefore, it is not surprising that FAK plays important roles in cancer; in fact, it has been linked to tumour progression or regression depending on the cellular and genetic context. We used Drosophila melanogaster as a model organism to study FAK’s duality in cancer. In this thesis we describe two novel roles of Drosophila FAK (FAK56): as a tumour suppressor within receptor tyrosine kinases (RTKs)-driven contexts, and as a tumour promoter by inhibiting cell death in nervous tissues. We investigated how FAK56 regulates signalling resulting from the overexpression of RTKs RET and EGFR. Our data indicated that FAK is a suppressor of RTKs in fly epithelia. This was also observed in human cancer cell lines, suggesting an evolutionary conserved mechanism. On the other hand, we found FAK56 prevented caspase-dependent cell death and uncovered a novel link between FAK56 and Relish, the Drosophila homologue of human NF-κB: Relish mutants suppressed FAK56 loss-induced cell death in the larval central nervous system and eye imaginal discs. As supported by the results presented in this thesis, FAK may be a good therapeutic target in cancer biology; however, in some contexts it may also behave as a tumour suppressor. Therefore, we conclude it will be necessary to identify the context of FAK activity before designing therapeutic strategies against FAK-expressing tumours

AllR controls the expression of allantoin pathway genes in Streptomyces coelicolor

Streptomyces species are native inhabitants of soil, a natural environment where nutrients can be scarce and competition fierce. They have evolved ways to metabolize unusual nutrients, such as purines and its derivatives, which are highly abundant in soil. Catabolism of these uncommon carbon and nitrogen sources needs to be tightly regulated in response to nutrient availability and environmental stimulus. Recently, the allantoin degradation pathway was characterized in Streptomyces coelicolor. However, there are questions that remained unanswered, particularly regarding pathway regulation. Here, using a combination of proteomics and genetic approaches, we identified the negative regulator of the allantoin pathway, AllR. In vitro studies confirmed that AllR binds to the promoter regions of allantoin catabolic genes and determined the AllR DNA binding motif. In addition, effector studies showed that allantoic acid, and glyoxylate, to a lesser extent, inhibit the binding of AllR to the DNA. Inactivation of AllR repressor leads to the constitutive expression of the AllR regulated genes and intriguingly impairs actinorhodin and undecylprodigiosin production. Genetics and proteomics analysis revealed that among all genes from the allantoin pathway that are upregulated in the allR mutant, the hyi gene encoding a hydroxypyruvate isomerase (Hyi) is responsible of the impairment of antibiotic production.Fil: Navone, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Macagno, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Licona Cassani, Cuauhtémoc. The University Of Queensland; Australia. Laboratorio Nacional de Genómica para la Biodiversidad; MéxicoFil: Marcellin, Esteban. The University Of Queensland; AustraliaFil: Nielsen, Lars K.. The University Of Queensland; AustraliaFil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Rodriguez, Eduardo Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

FAK inhibits RTK signalling by impairing Ras/MAPK pathway.

<p>(A–D) Phosphorylated (active) MAPK immunostainings from wing discs with the indicated genotypes. (A–B) When dFAK was expressed in the <i>ptc</i>-compartment (green), pMAPK staining was unchanged compared to GFP-only expressing cells. (C–D) dRET^CA expression increased pMAPK staining in the <i>ptc</i> domain but co-expression with dFAK suppressed this dRET^CA-induced activation of MAPK. Scale bars, 50 µm. (E) Quantification of pMAPK immunostaining within the <i>ptc</i> stripe (see methods). Intensity of pMAPK signal is represented as relative values to the mean intensity of control tissues (A) (‘ns’: not statistically significant; **** = p<0.0001; n = 4–6 for each genotype).</p

FAK decreases EGFR at the plasma membrane via reduced recycling.

<p>(A–B) MDA-MB-231 cells transfected with non-targeting (NT) siRNA or FAK siRNA were immunostained with anti-EGFR antibody (green, A″ and B″), Rhodamine-phalloidin (red, A′ and B′) and DAPI (blue). Note the differential localisation of EGFR; while in siNT cells the receptor is distributed in plasma membrane and internal vesicles, FAK downregulation leads to an increase of EGFR levels at the cellular membrane. Scale bar, 10 µm. (C) Quantification of relative EGFR membrane levels, values are expressed as relative levels of the receptor against the mean value of siNT cells; four confocal fields for each condition were analysed: n = 347 cells (siNT), and n = 414 cells (siFAK). p<0.0286 in a Mann-Whitney test. (D) MDA-MB-231 cells were transfected with either non-targeting (siNT) or FAK-specific siRNA (siFAK) and deprived of serum prior to addition of 80 µM Dynasore. siNT-transfected cells showed an increased pERK1/2 level in response to both Dynasore treatment (80 µM, 30 minutes) and FAK knockdown. Total levels of EGFR and ERK were not changed and actin levels were probed as an additional loading control. (E) The internalization of membrane EGFR (top panel) and recycling of internalised EGFR (bottom panel) were determined in MDA-MB-231 cells transfected with non-targeting siRNA (siCTR) or FAK siRNA (siFAK). Values are means ± Standard Deviation (SD) of two independent experiments with four to eight replicates of each time point per genotype. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004262#s4" target="_blank">materials and methods</a> for more details. FAK knockdown did not affect receptor internalization but increased the recycling of the internalised EGFR pool. (F) A working model for the regulatory mechanism of FAK. Ectopic expression and/or hyperactivation of RTKs activate FAK and Ras among other signalling pathways. FAK mediates a negative regulation of receptor recycling; when FAK is reduced or absent, there are more RTKs molecules at the plasma membrane, thus enabling a higher flux of signalling through Ras/MAPK pathway. See the text for more details.</p

Moderate relative RET/FAK levels lead to inhibition of programmed cell death.

<p>(A–C) Armadillo immunostaining revealed cell outlines of <i>wild type</i> (A), <i>dFAK^CG1</i> (B), and <i>GMR-dRET^WT</i> (C) retinas at 42 hs after puparium formation (APF). The boxed areas were traced to highlight their cellular composition (A′–C′). Each ommatidium is composed of 4 cone cells (red), 2 primary pigments cells (yellow), 6 secondary and three tertiary cells (white), and three-bristle cells (green) make the hexagonal lattice. Note that <i>dFAK^CG1</i> eyes display normal patterning (B′) while <i>GMR-dRET^WT</i> retinas displayed normal ommatidial cores but additional interommatidial cells (white cells in C′). Scale bars, 10 µm. (D–G) TUNEL labelling of retinas at 28 h APF. Note that the developmental programmed cell death observed in <i>wild type</i> and <i>dFAK^CG1</i> retinas were suppressed in <i>GMR-dRET^WT</i> retinas. Co-expression of dFAK rescued this inhibition of cell death (G). Scale bar, 50 µm. (H–K) Hid overexpression (<i>GMR-hid</i>) gave a small eye phenotype, which was suppressed by dRET^WT co-expression (J). This dRET-dependent inhibition was also suppressed by dFAK co-expression (K) while dFAK itself did not suppress Hid-mediated effects in the eye (I). Scale bar, 100 µm. (L) Eye size quantification of the indicated genotypes (as depicted in panels H–K) represented as relative values to the <i>wild type</i> mean value (‘ns’: not statistically significant; **** = p<0.0001; n = 8–10 for each genotype).</p

Requirement of the N-terminal FAK FERM domain.

<p>(A) Linear representation of <i>dFAK</i> mRNA, its derivatives UAS-transgenes and their resultant protein isoforms: a full-length dFAK isoform; an N-terminal deletion mutant that lacks the first 400 amino acids residues of dFAK including its FERM domain; and a point mutant isoform that bears a replacement of the Tyrosine⁴³⁰ residue for a Phenylalanine residue, which impairs the auto-phosphorylation site and consequently the kinase activity of dFAK. (B) Expression profiles of each <i>UAS-dFAK</i> transgene in the eye (driven by <i>GMR-gal4</i>) as determined by quantitative (q) PCR of RNA samples (see methods). We used a pair of primers (3F and 3L) flanking a 200 bp region corresponding to the C-terminal domain (FAT: Focal adhesion targeting domain), which is a common region to all the isoforms. (C) Eye size quantification of the indicated genotypes, shown in D-G. Eye sizes on the Y-axis are represented as relative values to the mean of <i>GMR>dRET^CA</i> (‘ns’: not statistically significant; **** = p<0.0001; n = 8–10 for each genotype). (D–G) Eye micrographs correspond to the indicated genotypes. Note that while the auto-phosphorylation mutant version of dFAK was expressed at lower levels than the N-terminal mutant isoform (B), it was still able to rescue the size of dRET^CA-expressing eyes (G), to a similar extent as the full-length dFAK isoform (E). However, the N-terminal deletion mutant isoform did not suppress the small eye size of dRET^CA animals (C, D and F). Scale bar, 100 µm.</p

FAK suppression of RTK signalling is conserved.

<p>(A–B) Adult eyes images of animals expressing <i>Drosophila</i> EGFR (dEGFR) alone or in combination with dFAK. Note that dFAK expression suppressed the rough, small eye phenotype driven by dEGFR. Scale bar, 100 µm. (C–C′) Expression of dEGFR within the <i>ptc</i> domain resulted in increased MAPK phosphorylation, and co-expression of dFAK rescued the ectopic pMAPK staining within the <i>ptc</i> stripe (D–D′). Scale bar, 50 µm. (E) Quantification of pMAPK immunostaining within the <i>ptc</i> stripe (see methods). Intensity of pMAPK signal is represented as relative values to the mean intensity of control tissues (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004262#pgen-1004262-g006" target="_blank">Figure 6A</a>; **** = p<0.0001; n = 4–6 for each genotype). (F) Quantification of the penetrance on adult eclosion for the indicated genotypes. Note that dFAK co-expression significantly rescued the developmental lethality associated to <i>ptc</i>-driven dEGFR expression (* = p<0.05). (G) Western blots from protein extracts from MDA-MB-231 cells after 48 or 72 h transfection with FAK siRNA. FAK protein levels were effectively knocked down. While total levels of EGFR and ERK were not changed at 48 h, there was a marked upregulation in phosphorylated ERK1/2 upon FAK knockdown, which was more apparent at 48 h after siRNA transfection. Actin levels were probed as an additional loading control. (H) MDA-MB-231 cells were transfected with either non-targeting (siNT) or FAK-specific siRNA (siFAK) and serum starved prior to addition of EGF. Note that FAK knockdown resulted in increased phosphorylation of ERK1/2 in response to EGF treatment (30 µM, 15 minutes).</p

High relative levels between RET and FAK induce ectopic cone cell differentiation in the eye.

<p>We examined the cellular patterning of the pupal retinas in correspondence to the adult eye phenotypes shown in panels A–D. Scale bars, 100 µm. (E–H) Merged images of stainings for nuclei (DAPI, blue), Dlg (cell outlines, red) and Cut (cone cells, green), from retinas at 42 h APF. Bottom panels show Dlg (E′–H′) and Cut (E″–H″) immunostainings individually. (E) Note the symmetric hexagonal array, and four Cut⁺ cone cells per ommatidium (white arrows) in control retinas. (F and G) Note the change in cellular composition of these retinas with high RET/FAK ratios, primarily composed of Cut⁺ cone-like cells. (H) dFAK expression within a <i>2X GMR-dRET^CA</i> background suppressed this phenotype (also see S1D); some normal four-cone cell clusters (white arrows) can be identified and interommatidial cells reappeared (yellow arrows). Scale bars, 10 µm.</p