In Vivo Isolation and Characterization of Stem Cells with Diverse Phenotypes Using Growth Factor Impregnated Biomatrices

BACKGROUND: The stimulation to differentiate into specific cell types for somatic stem cells is largely due to a series of internal and external signals coming from the microenvironment that surrounds the stem cell. Even though intensive research has been made, the basic mechanisms of plasticity and/or the molecules regulating stem cells proliferation and differentiation are not completely determined. Potential answers concerning the problems could be derived from the studies of stem cells in culture. METHODOLOGY/PRINCIPLE FINDINGS: We combine a new procedure (using the matrigel biopolymer supplemented with a selected cytokine/growth factor) with classic techniques such as light, confocal and electron microscopy, immunohistochemistry and cell culture, to perform an analysis on stem cells involved in the leech (Hirudo medicinalis) repair tissues. The leech has a relative anatomical simplicity and is a reliable model for studying a variety of basic events, such as tissue repair, which has a striking similarity with vertebrate responses. Our data demonstrate that the injection of an appropriate combination of the matrigel biopolymer supplemented with a selected cytokine/growth factor in the leech Hirudo medicinalis is a remarkably effective tool for isolating a specific cell population in vivo. A comparative analysis of biopolymer in vivo sorted stem cells indicates that VEGF recruited cells of a hematopoietic/endothelial phenotype whereas MCP-1/CCL2 isolated cells that were of an early myeloid lineage. CONCLUSION: Our paper describes, for the first time, a method allowing not only the isolation of a specific cell population in relation to the cytokine utilized but also the possibility to culture a precise cell type whose isolation is otherwise quite difficult. This approach could be broadly applied to isolate stem cells of diverse origins based on the recruitment stimuli employed

Functional amyloid formation in LPS activated cells from invertebrates to vertebrates

LPS stimulation provokes serious cellular stress with an increase of cytoplasmic reactive oxygen species (ROS). We have investigated, among the different cellular defenses, amyloidogenesis as common physiological response to attenuate oxidative stress. Optical and electron microscopic observations of the following LPS activated cell lines [insect (larval hemocytes, IPLB-LdFB and Drosophila Schneider\u2019s S2 cells); mouse (NIH3T3 embryonic fibroblasts); Human (Human Umbilical Vein Endothelial Cells (HUVEC), neutrophils, and mesenchymal stem cells] reveal that, all are characterized by irregular profiles, cytoplasmic empty vacuoles or by cisternae containing fibrillar material. The compartmentalized fibrillar material shows staining properties typical of amyloid fibrils. LPS activation leads to ROS generation, resulting in pH acidification. Stimulated cells show pink cytoplasm in May-Gr\ufcnwald Giemsa differential staining, giving a gross indication of a lower intracellular pH. Moreover the activation of amyloidogenesis is also linked with an extensive production of ACTH and \u3b1-MSH in all cultured cell types. We suggest that amyloidogenesis is a common, physiological cellular response to weak ROS, starting when other anti-stress cellular systems failed to restore homeostasis. The morphological evidence and/or functional characterization of synthesized amyloid fibrils could be an early indicator of oxidative stress that may lead to a general inflammatory process

New value from food and industrial wastes - bioaccumulation of omega-3 fatty acids from an oleaginous microbial biomass paired with a brewery by-product using black soldier fly (Hermetia illucens) larvae.

Research on bioconversion based on insects is intensifying as it addresses the problem of reducing and reusing food and industrial waste. To reach this goal, we need to find more means of pairing waste to insects. With this goal, brewers\u2019 spent grains (BSG) - a food waste of the brewing industry - paired with the oleaginous biomass of the thraustochytrid Schizochytrium limacinum cultivated on crude glycerol - a major waste of biodiesel production - were successfully used to grow Hermetia illucens larvae. Combining BSG and S. limacinum in the diet in an attempt to design the lipid profile of H. illucens larvae to contain a higher percentage of omega-3 fatty acids is novel. Insect larvae were grown on three different substrates: i) standard diet for Diptera (SD), ii) BSG, and iii) BSG + 10% S. limacinum biomass. The larvae and substrates were analyzed for fatty acid composition and larval growth was measured until 25% of insects reached the prepupal stage. Our data showed that including omega-3-rich S. limacinum biomass in the BSG substrate promoted an increase in larval weight compared to larvae fed on SD or BSG substrates. Furthermore, it was possible, albeit in a limited way, to incorporate omega-3 fatty acids, principally docosahexaenoic acid (DHA) from BSG + S. limacinum substrate containing 20% of DHA into the larval fat (7% DHA). However, H. illucens with this level of DHA may not be suitable if the aim is to get larvae with high omega-3 lipids to feed carnivorous fish

Amyloid/Melanin distinctive mark in invertebrate immunity

Protostomes and Deuterostomes show the same nexus between melanin production, and amyloid fibril production, i.e., the presence of melanin is indissolubly linked to amyloid scaffold that, in turn, is conditioned by the redox status/cytoplasmic pH modification, pro-protein cleavage presence, adrenocorticotropin hormone (ACTH), melanocyte-stimulating hormone (\u3b1-MSH), and neutral endopeptidase (NEP) overexpressions. These events represent the crucial component of immune response in invertebrates, while in vertebrates these series of occurrences could be interpreted as a modest and very restricted innate immune response. On the whole, it emerges that the mechanisms involving amyloid fibrils/pigment synthesis in phylogenetically distant metazoan (viz, cnidaria, molluscs, annelids, insects, ascidians and vertebrates) are evolutionary conserved. Furthermore, our data show the relationship between immune and neuroendocrine systems in amyloid/melanin synthesis. Indeed the process is closely associated to ACTH-\u3b1-MSH production, and their role in stress responses leading to pigment production reflects and confirms again their ancient phylogeny

Modelling long-distance route choice using mobile phone call detail record data: A case study of Senegal

The growing mobile phone penetration rates have led to the emergence of large-scale call detail records (CDRs) that could serve as a low-cost data source for travel behaviour modelling. However, to the best of our knowledge, there is no previous study evaluating the potential of CDR data in the context of route choice behaviour modelling. Being event-driven, the data are discontinuous and only able to yield partial trajectories, thus presenting serious challenges for route identification. This paper proposes techniques for inferring the users' chosen routes or subsets of their likely routes from partial CDR trajectories, as well as data fusion with external sources of information such as route costs, and then adapts the broad choice framework to the current modelling scenario. The model results show that CDR data can capture the expected travel behaviour and the derived values of travel time are found to be realistic for the study area

Molecular and functional characterization of Hermetia illucens larval midgut

The increase in global demand for meat and the management of organic waste are huge global issues. In addition to policies to contain the excessive meat consumption and the production of food waste, a possible perspective is to consider insects as agents for organic waste reduction and as source of protein for monogastric feed production. The larvae of Hermetia illucens (Diptera: Stratiomyidae) are good candidates for bioconversion of vegetal waste and feed production because they grow on different organic substrates and the dry-matter of the prepupa contains a very high percentage of protein with high nutritional value. The study of H. illucens midgut physiology is essential to understand the extraordinary feeding plasticity of this insect and to best exploit this ability. We characterized the digestive enzymes involved in the initial phase of digestion in the three regions of the midgut (anterior, middle and posterior). The proteolytic activity was assayed using chromogenic substrates and specific inhibitors. The total proteolytic activity is highest in the posterior midgut and the major activity is due to serine proteases. We measured the activity and the transcript levels of the two main serine proteases involved in insect digestion: trypsin-like and chymotrypsin-like proteases. Moreover, we determined the total amylolytic activity and the highest value was recorded in the anterior midgut. Thanks to our data, we propose the first model of the functional activity of H. illucens midgut. This work was supported by Fondazione Cariplo (Insect bioconversion: from vegetable waste to protein production for fish feed, ID 2014-0550)

The larval midgut of Hermetia illucens is characterized by a highly complex structural organization

The demand for food of animal origin is expected to increase by 70-80% within 2050, with a consequent rise in feed requirement. Another serious concern is represented by food waste disposal: in fact, it is estimated that 1.3 billion tons/year of food are globally wasted. The dipteran Hermetia illucens, also known as black soldier fly (BSF), is a promising insect species to tackle these critical challenges because of the ability of the larvae to grow on different organic substrates and their efficiency in the bioconversion process. Moreover, the high nutritional value of the larvae and pupae makes them an alternative protein source for the production of fish feed. A deep understanding of the biology of the larval midgut, which is implicated in food digestion and nutrient absorption, is essential to better comprehend the extraordinary dietary plasticity of the larva. In the present study, we performed a morphological characterization of the midgut of last instar larvae. Our results demonstrate that the larval midgut is composed of three distinct anatomical regions with different luminal pH. These regions are characterized by different cell types that accomplish digestion and absorption activities (columnar cells), acidification of the midgut lumen (cuprophilic cells), regulation processes (endocrine cells), and growth of the epithelium (stem cells). Moreover, we are investigating the expression of different enzymes along BSF midgut epithelium, which are involved in transport mechanisms. This work was supported by Fondazione Cariplo (Insect bioconversion: from vegetable waste to protein production for fish feed, ID 2014-0550)

Leiomiosarcoma cutáneo primario: a próposito de un caso

El leiomiosarcoma es un tumor maligno mesenquimatoso infrecuente. Su incidencia es menor al 10% de los casos de los sarcomas de tejidos blandos. Afecta a ambos sexos y su presenta entre la quinta y sexta década de la vida, los pacientes inmunocomprometidos son más propensos a desarrollar este tumor. Se lo clasifica en leiomiosarcoma cutáneo (derivado del músculo piloerector del pelo y ubicado histológicamente a nivel dérmico) y el leiomiosarcoma subcutáneo (derivado del músculo liso de las paredes vasculares). Su diagnóstico es clínico e histológico siendo esencial la inmunohistoquímica. Los diagnósticos diferenciales son diversos tumores cutáneos. El tratamiento de elección de este tumor es con la cirugía dónde debe tomarse un margen de seguridad de 3 cm o la cirugía micrográfica de Mohs. El fracaso terapéutico está dadopor la recurrencia que es muy frecuente en este tumor.Facultad de Ciencias Médica