Manuel G. Forero

Identification of tuberculosis bacteria based on shape and color

Tuberculosis and other mycobacteriosis are serious illnesses which control is based on early diagnosis. A technique commonly used consists of analyzing sputum images for detecting bacilli. However, the analysis of sputum is time consuming and requires highly trained personnel to avoid high errors. Image-processing techniques provide a good tool for improving the manual screening of samples. In this paper, a new autofocus algorithm and a new bacilli detection technique is presented with the aim to attain a high specificity rate and reduce the time consumed to analyze such sputum samples. This technique is based on the combined use of some invariant shape features together with a simple thresholding operation on the chromatic channels. Some feature descriptors have been extracted from bacilli shape using an edited dataset of samples. A k-means clustering technique was applied for classification purposes and the sensitivity vs specificity results were evaluated using a standard ROC analysis procedure.

Drosophila Neurotrophins Reveal a Common Mechanism for Nervous System Formation

Neurotrophic interactions occur in Drosophila, but to date, no neurotrophic factor had been found. Neurotrophins are the main vertebrate secreted signalling molecules that link nervous system structure and function: they regulate neuronal survival, targeting, synaptic plasticity, memory and cognition. We have identified a neurotrophic factor in flies, Drosophila Neurotrophin (DNT1), structurally related to all known neurotrophins and highly conserved in insects. By investigating with genetics the consequences of removing DNT1 or adding it in excess, we show that DNT1 maintains neuronal survival, as more neurons die in DNT1 mutants and expression of DNT1 rescues naturally occurring cell death, and it enables targeting by motor neurons. We show that Spätzle and a further fly neurotrophin superfamily member, DNT2, also have neurotrophic functions in flies. Our findings imply that most likely a neurotrophin was present in the common ancestor of all bilateral organisms, giving rise to invertebrate and vertebrate neurotrophins through gene or whole-genome duplications. This work provides a missing link between aspects of neuronal function in flies and vertebrates, and it opens the opportunity to use Drosophila to investigate further aspects of neurotrophin function and to model related diseases.

Automatic identification of Mycobacterium tuberculosis by Gaussian mixture models

Tuberculosis and other kinds of mycobacteriosis are serious illnesses for which early diagnosis is critical for disease control. Sputum sample analysis is a common manual technique employed for bacillus detection but current sample‐analysis techniques are time‐consuming, very tedious, subject to poor specificity and require highly trained personnel. Image‐processing and pattern‐recognition techniques are appropriate tools for improving the manual screening of samples. Here we present a new technique for sputum image analysis that combines invariant shape features and chromatic channel thresholding. Some feature descriptors were extracted from an edited bacillus data set to characterize their shape. They were statistically represented by using a Gaussian mixture model representation and a minimal error Bayesian classification procedure was employed for the last identification stage. This technique constitutes a step towards automating the process and providing a high specificity.

Automatic identification techniques of tuberculosis bacteria

Tuberculosis is a serious illness which control is mainly based on presumptive diagnosis. A technique commonly used consists of analyzing sputum images for detecting bacilli. However, the analysis of sputum is quite expensive, time consuming and requires highly trained personnel to avoid high errors. Image processing techniques provide a good tool for improving the manual screening of samples. In this paper we present a new bacilli detection technique with the aim to attain a high specificity rate and therefore for reducing the time required to analyze such sputum samples. This technique is based on the neuristic acknowlege extracted from the bacilli shape contour. It uses also the color information for image segmentation and finally a classification tree is used to categorize if a sample is positive or negative.

Gliatrophic and gliatropic roles of PVF/PVR signaling during axon guidance.

Evidence of molecular and functional homology between vertebrate and Drosophila glia is limited, restricting the power of Drosophila as a model system to unravel the molecular basis of glial function. Like in vertebrates, in the Drosophila central nervous system glial cells are produced in excess and surplus glia are eliminated by apoptosis adjusting final glial number to axons. The underlying molecular mechanisms are largely unknown, as the only gliatrophic pathway known to date in flies is the EGFR and its ligands. The PDGFR signaling pathway plays a major role in regulating oligodendrocyte migration and number in vertebrates. Here, we show that the Drosophila PDGFR/VEGFR homologue PVR is required in midline glia during axon guidance for glial survival and migration, ultimately enabling axonal enwrapment. The midline glia migrate aided by the VUM and the MP1 midline neurons—sources of PVF ligands—and concomitantly interactions with neurons maintain midline glia survival. Upon loss of function for PVF/PVR signaling midline glia apoptosis increases, and gain of function induces supernumerary midline glia. Midline glial cells are displaced towards ectopic sources of PVF ligands. PVR signaling promotes midline glia survival through AKT and ERK pathways. This work shows that the PVR/PDGFR pathway plays conserved gliatrophic and gliatropic roles in subsets of glial cells in flies and vertebrates.

The Glial Regenerative Response to Central Nervous System Injury Is Enabled by Pros-Notch and Pros-NFκB Feedback

A gene network involving Notch and Pros underlies the glial regenerative response to injury in the Drosophila central nervous system.

Neuron-Type Specific Functions of DNT1, DNT2 and Spz at the Drosophila Neuromuscular Junction

Retrograde growth factors regulating synaptic plasticity at the neuromuscular junction (NMJ) in Drosophila have long been predicted but their discovery has been scarce. In vertebrates, such retrograde factors produced by the muscle include GDNF and the neurotrophins (NT: NGF, BDNF, NT3 and NT4). NT superfamily members have been identified throughout the invertebrates, but so far no functional in vivo analysis has been carried out at the NMJ in invertebrates. The NT family of proteins in Drosophila is formed of DNT1, DNT2 and Spätzle (Spz), with sequence, structural and functional conservation relative to mammalian NTs. Here, we investigate the functions of Drosophila NTs (DNTs) at the larval NMJ. All three DNTs are expressed in larval body wall muscles, targets for motor-neurons. Over-expression of DNTs in neurons, or the activated form of the Spz receptor, Toll 10b, in neurons only, rescued the semi-lethality of spz 2 and DNT1 41 , DNT2 e03444 double mutants, indicating retrograde functions in neurons. In spz 2 mutants, DNT1 41 , DNT2 e03444 double mutants, and upon over-expression of the DNTs, NMJ size and bouton number increased. Boutons were morphologically abnormal. Mutations in spz and DNT1,DNT2 resulted in decreased number of active zones per bouton and decreased active zone density per terminal. Alterations in DNT function induced ghost boutons and synaptic debris. Evoked junction potentials were normal in spz 2 mutants and DNT1 41 , DNT2 e03444 double mutants, but frequency and amplitude of spontaneous events were reduced in spz 2 mutants suggesting defective neurotransmission. Our data indicate that DNTs are produced in muscle and are required in neurons for synaptogenesis. Most likely alterations in DNT function and synapse formation induce NMJ plasticity leading to homeostatic adjustments that increase terminal size restoring overall synaptic transmission. Data suggest that Spz functions with neuron-type specificity at the muscle 4 NMJ, and DNT1 and DNT2 function together at the muscles 6,7 NMJ.

DeadEasy Caspase: Automatic Counting of Apoptotic Cells in Drosophila

Development, cancer, neurodegenerative and demyelinating diseases, injury, and stem cell manipulations are characterised by alterations in cell number. Research into development, disease, and the effects of drugs require cell number counts. These are generally indirect estimates, because counting cells in an animal or organ is paradoxically difficult, as well as being tedious and unmanageable. Drosophila is a powerful model organism used to investigate the genetic bases of development and disease. There are Drosophila models for multiple neurodegenerative diseases, characterised by an increase in cell death. However, a fast, reliable, and accurate way to count the number of dying cells in vivo is not available. Here, we present a method based on image filtering and mathematical morphology techniques, to count automatically the number of dying cells in intact fruit-fly embryos. We call the resulting programme DeadEasy Caspase. It has been validated for Drosophila and we present examples of its power to address biological questions. Quantification is automatic, accurate, objective, and very fast. DeadEasy Caspase will be freely available as an ImageJ plug-in, and it can be modified for use in other sample types. It is of interest to the Drosophila and wider biomedical communities. DeadEasy Caspase is a powerful tool for the analysis of cell survival and cell death in development and in disease, such as neurodegenerative diseases and ageing. Combined with the power of Drosophila genetics, DeadEasy expands the tools that enable the use of Drosophila to analyse gene function, model disease and test drugs in the intact nervous system and whole animal.

DeadEasy Mito-Glia: Automatic Counting of Mitotic Cells and Glial Cells in Drosophila

Cell number changes during normal development, and in disease (e.g., neurodegeneration, cancer). Many genes affect cell number, thus functional genetic analysis frequently requires analysis of cell number alterations upon loss of function mutations or in gain of function experiments. Drosophila is a most powerful model organism to investigate the function of genes involved in development or disease in vivo. Image processing and pattern recognition techniques can be used to extract information from microscopy images to quantify automatically distinct cellular features, but these methods are still not very extended in this model organism. Thus cellular quantification is often carried out manually, which is laborious, tedious, error prone or humanly unfeasible. Here, we present DeadEasy Mito-Glia, an image processing method to count automatically the number of mitotic cells labelled with anti-phospho-histone H3 and of glial cells labelled with anti-Repo in Drosophila embryos. This programme belongs to the DeadEasy suite of which we have previously developed versions to count apoptotic cells and neuronal nuclei. Having separate programmes is paramount for accuracy. DeadEasy Mito-Glia is very easy to use, fast, objective and very accurate when counting dividing cells and glial cells labelled with a nuclear marker. Although this method has been validated for Drosophila embryos, we provide an interactive window for biologists to easily extend its application to other nuclear markers and other sample types. DeadEasy MitoGlia is freely available as an ImageJ plug-in, it increases the repertoire of tools for in vivo genetic analysis, and it will be of interest to a broad community of developmental, cancer and neuro-biologists.

DeadEasy neurons: automatic counting of HB9 neuronal nuclei in Drosophila.

Research into the genetic basis of nervous system development and neurodegenerative diseases requires counting neurons to find out the extent of neurogenesis or neuronal loss. Drosophila is a widely used model organism for in vivo studies. However, counting neurons throughout the nervous system of the intact animal is humanly unfeasible. Automatic methods for cell counting in intact Drosophila are desirable. Here, we show a method called DeadEasy Neurons to count the number of neurons stained with anti‐HB9 antibodies in Drosophila embryos. DeadEasy Neurons employs image filtering and mathematical morphology techniques in 2D and 3D, followed by identification of nuclei in 3D based on minimum volume, to count automatically the number of HB9 neurons in vivo. The resultant method has been validated for Drosophila embryos and we show here how it can be used to address biological questions. Counting neurons with DeadEasy is very fast, extremely accurate, and objective, and it enables analyses otherwise humanly unmanageable. DeadEasy Neurons can be modified by the user for other applications, and it will be freely available as an ImageJ plug‐in. DeadEasy Neurons will be of interest to the microscopy, image processing, Drosophila, neurobiology, and biomedical communities.