Silencing of the Vasa gene by RNA Interference Affects Embryonic Development and Reproductive Output in the Sea Louse Caligus rogercresseyi.

The sea louse Caligus rogercresseyi is a major ectoparasitic copepod that causes significant economic losses in the salmon farming industry. Despite recent advancements, the mechanisms underlying germline and embryo development in this species remain poorly understood. The Vasa gene encodes a highly conserved DEAD box helicase that is required for germ cell formation and function in many species. In this study, the Vasa gene was characterized in C. rogercresseyi, and its expression and function were analyzed. Phylogenetic analysis showed that the Cr-Vasa gene product formed clusters in clades with Vasa proteins from closely related species of crustaceans. Cr-Vasa gene expression patterns were assessed by qPCR, and the results showed a significantly higher relative expression level in adult females compared to copepodid, chalimus, and adult male stages. Tissue-specific localization of Cr-Vasa mRNA in C. rogercresseyi was determined using chromogenic in situ hybridization, and strong positive signal was observed in male testes, but also in the intestine and cuticle, while in females, it was observed in the ovaries, oocytes, cuticle, intestine, and egg strings. RNAi-mediated gene silencing of Cr-Vasa impacted embryonic development and reproductive output in adult female lice. Females from the dsVasa-treated group displayed unusual phenotypes, including shorter egg strings with numerous extra-embryonic inclusions, irregularly shaped abnormal embryos, and aborted egg strings. This study provides insights into the role of the Vasa gene in C. rogercresseyi embryonic development and reproductive output, which may have implications for the control of this parasitic copepod in the salmon farming industry.

Restraint of Human Skin Fibroblast Motility, Migration, and Cell Surface Actin Dynamics, by Pannexin 1 and P2X7 Receptor Signaling.

Wound healing is a dynamic process required to maintain skin integrity and which relies on the precise migration of different cell types. A key molecule that regulates this process is ATP. However, the mechanisms involved in extracellular ATP management are poorly understood, particularly in the human dermis. Here, we explore the role, in human fibroblast migration during wound healing, of Pannexin 1 channels and their relationship with purinergic signals and in vivo cell surface filamentous actin dynamics. Using siRNA against Panx isoforms and different Panx1 channel inhibitors, we demonstrate in cultured human dermal fibroblasts that the absence or inhibition of Panx1 channels accelerates cell migration, increases single-cell motility, and promotes actin redistribution. These changes occur through a mechanism that involves the release of ATP to the extracellular space through a Panx1-dependent mechanism and the activation of the purinergic receptor P2X7. Together, these findings point to a pivotal role of Panx1 channels in skin fibroblast migration and suggest that these channels could be a useful pharmacological target to promote damaged skin healing.

Re-Epithelialization Appraisal of Skin Wound in a Porcine Model Using a Salmon-Gelatin Based Biomaterial as Wound Dressing.

The design of new functional materials for skin tissue engineering is an area of constant research. In this work, a novel wound-dressing biomaterial with a porous structure, previously formulated using salmon-gelatin as main component (called salmon-gelatin biomaterial (SGB)), was tested in vivo using pigs as skin wound models. Four weeks after cutaneous excision and implantation in the animals, the healing process did not show apparent symptoms of inflammation or infection. Interestingly, the temporal evolution of wound size from 100% to around 10% would indicate a faster recovery when SGB was compared against a commercial control. Histological analysis established that wounds treated with SGB presented similar healing and epithelialization profiles with respect to the commercial control. Moreover, vascularized granulation tissue and epithelialization stages were clearly identified, indicating a proliferation phase. These results showed that SGB formulation allows cell viability to be maintained. The latter foresees the development of therapeutic alternatives for skin repair based on SGB fabricated using low cost production protocols.

Design of a hybrid biomaterial for tissue engineering: Biopolymer-scaffold integrated with an autologous hydrogel carrying mesenchymal stem-cells.

Biologically active biomaterials as biopolymers and hydrogels have been used in medical applications providing favorable results in tissue engineering. In this research, a wound dressing device was designed by integration of an autologous clot hydrogel carrying mesenchymal stem-cells onto a biopolymeric scaffold. This hybrid biomaterial was tested in-vitro and in-vivo, and used in a human clinical case. The biopolymeric scaffold was made with gelatin, chitosan and hyaluronic acid, using a freeze-drying method. The scaffold was a porous material which was designed evaluating both physical properties (glass transition, melting temperature and pore size) and biological properties (cell viability and fibronectin expression). Two types of chitosan (120 and 300kDa) were used to manufacture the scaffold, being the high molecular weight the most biologically active and stable after sterilization with gamma irradiation (25kGy). A clot hydrogel was formulated with autologous plasma and calcium chloride, using an approach based on design of experiments. The optimum hydrogel was used to incorporate cells onto the porous scaffold, forming a wound dressing biomaterial. The wound dressing device was firstly tested in-vitro using human cells, and then, its biosecurity was evaluated in-vivo using a rabbit model. The in-vitro results showed high cell viability after one week (99.5%), high mitotic index (19.8%) and high fibronectin expression. The in-vivo application to rabbits showed adequate biodegradability capacity (between 1 and 2weeks), and the histological evaluation confirmed absence of rejection signs and reepithelization on the wound zone. Finally, the wound dressing biomaterial was used in a single human case to implant autologous cells on a skin surgery. The medical examination indicated high biocompatibility, partial biodegradation at one week, early regeneration capacity at 4weeks and absence of rejection signs.

Edible Scaffolds Based on Non-Mammalian Biopolymers for Myoblast Growth.

In vitro meat has recently emerged as a new concept in food biotechnology. Methods to produce in vitro meat generally involve the growth of muscle cells that are cultured on scaffolds using bioreactors. Suitable scaffold design and manufacture are critical to downstream culture and meat production. Most current scaffolds are based on mammalian-derived biomaterials, the use of which is counter to the desire to obviate mammal slaughter in artificial meat production. Consequently, most of the knowledge is related to the design and control of scaffold properties based on these mammalian-sourced materials. To address this, four different scaffold materials were formulated using non-mammalian sources, namely, salmon gelatin, alginate, and additives including gelling agents and plasticizers. The scaffolds were produced using a freeze-drying process, and the physical, mechanical, and biological properties of the scaffolds were evaluated. The most promising scaffolds were produced from salmon gelatin, alginate, agarose, and glycerol, which exhibited relatively large pore sizes (~200 µm diameter) and biocompatibility, permitting myoblast cell adhesion (~40%) and growth (~24 h duplication time). The biodegradation profiles of the scaffolds were followed, and were observed to be less than 25% after 4 weeks. The scaffolds enabled suitable myogenic response, with high cell proliferation, viability, and adequate cell distribution throughout. This system composed of non-mammalian edible scaffold material and muscle-cells is promising for the production of in vitro meat.

Designing a gelatin/chitosan/hyaluronic acid biopolymer using a thermophysical approach for use in tissue engineering.

Cell culture on biopolymeric scaffolds has provided treatments for tissue engineering. Biopolymeric mixtures based on gelatin (Ge), chitosan (Ch) and hyaluronic acid (Ha) have been used to make scaffolds for wound healing. Thermal and physical properties of scaffolds prepared with Ge, Ch and Ha were characterized. Thermal characterization was made by using differential scanning calorimetry (DSC), and physical characterization by gas pycnometry and scanning electron microscopy. The effects of Ge content and cross-linking on thermophysical properties were evaluated by means of a factorial experiment design (central composite face centered). Gelatin content was the main factor that affects the thermophysical properties (microstructure and thermal transitions) of the scaffold. The effect of Ge content of the scaffolds for tissue engineering was studied by seeding skin cells on the biopolymers. The cell attachment was not significantly modified at different Ge contents; however, the cell growth rate increased linearly with the decrease of the Ge content. This relationship together with the thermophysical characterization may be used to design scaffolds for tissue engineering.

One case of triphallia in the marine snail Echinolittorina peruviana (Caenogastropoda: Littorinidae) / Un caso de trifalia en el caracol marino Echinolittorina peruviana (Caenogastropoda: Littorinidae)

Gonochoric representatives of Littorinidae have a reproductive system organized with transit organs that connects the testicle with a penis specialized for internal fertilization. However, malformations at this level can produce, like triphallia (presence of triple penis). This description corresponds to one case of triphallia found in Echinolittorina peruviana (Lamarck, 1822) inhabitant of the South Pacific coasts. The macro and microscopic analysis reveals that every penis is an independent appendix whose individual morphology is similar to that in normal individuals, characterized by a non pigmented conic penis associated with a mammilliform gland located at the base of the right ocular tentacle. The cervical sperm groove lined by a simple ciliated and secretory columnar epithelium, runs along the neck of the animal, is subdivided into the base of each penis forming the penile seminal groove in each of them, and ascends dorsally from the base to the tip. The other organs that make up the reproductive system have a normal organization (no duplication or triplets), and the testicle organized in acini contains all the cell types of the male germ line. Although the cause of this condition is still unknown, might be associated with genetic causes and not of environmental kind...

Los representantes gonocóricos de Littorinidae presentan un sistema reproductor organizado con órganos de tránsito que conectan al testículo con un pene especializado para la fecundación interna. Sin embargo, pueden ocurrir malformaciones a este nivel, como la trifalia (presencia de pene triple). Esta descripción corresponde a un caso de trifalia encontrado en el representante gonocórico Echinolittorina peruviana (Lamarck, 1822), habitante de las costas del Pacífico Sur. El análisis macro y microscópico revela que cada pene es un apéndice independiente cuya morfología individual es semejante a la de individuos normales, caracterizada por un pene de forma cónica no pigmentado asociado a una glándula de tipo mamiliforme ubicada en la base del tentáculo ocular derecho. El surco espermático cervical revestido por un epitelio simple cilíndrico ciliado y secretor, recorre el cuello del animal, se subdivide en la base de cada pene conformando el surco espermático peneano en cada uno de ellos, y asciende dorsalmente desde la base hasta el ápice. Los otros órganos que conforman el sistema reproductor poseen una organización normal (sin duplicaciones o triplicaciones), y el testículo organizado en acinos contiene todos los tipos celulares de la línea germinal masculina. Aunque aún la causa de esta patología es desconocida podría estar asociada a causas genéticas y no de tipo ambiental...

Epipodium morphology of Prisogaster niger (Mollusca: Vetigastropoda): revealing potential autapomorphies of diagnostic value for the prisogasterinae / Morfología del epipodio de Prisogaster niger (mollusca: vetigastropoda): revelando autapomorfías potenciales de valor diagnóstico para prisogasterinae

In this work the epipodium morphology of the marine snail Prisogaster niger is described. In this gastropods the epipodium includes a complex of structures among which the neck lobes, epipodial ridge, epipodial tentacles and epipodial papillae constitute the most remarkable characters. Although these features are commonly found among trochoidean taxa, the observation of the epipodium, compared with the literature, revealed potential autapomorphic characters of diagnostic value for the genus and the subfamily Prisogasterinae, like epipodial lamellae and epipodial tuft.

En este trabajo se describe la morfología del epipodio del caracol marino Prisogaster niger. En esta especie el epipodio está conformado por un complejo de estructuras entre las cuales los lóbulos del cuello, rebordes epipodiales, tentáculos epipodiales y papilas epipodiales constituyen conspicuos caracteres. El estudio de la morfología del epipodio reveló potenciales autapomorfías de valor diagnóstico para la subfamilia Prisogasterinae.

Characterization of a gelatin/chitosan/hyaluronan scaffold-polymer

Gelatin, chitosan and hyaluronic acid are natural components used to prepare polymeric scaffold in tissue engineering. The physical properties of these materials confer an appropriate microenvironment for cells, which can be used as a regeneration system for skin and cartilage. In this work, we prepared and characterized a Gelatin/Chitosan/Hyaluronan lyophilized-polymer. Physical properties of lyophilized-polymer changed slightly with moisture, but when polymer was totally hydrated the elasticity changed significantly. Thermophysical characterisation indicated that temperatures higher than 30ºC could modify irreversibly the polymeric matrix probably due to protein denaturation. Besides, we used the polymer as scaffold to prepare a biosynthetic-skin, reporting biological behaviour and its mechanical properties.

The effect of an autologous cellular gel-matrix integrated implant system on wound healing.

BACKGROUND: This manuscript reports the production and preclinical studies to examine the tolerance and efficacy of an autologous cellular gel-matrix integrated implant system (IIS) aimed to treat full-thickness skin lesions. METHODS: The best concentration of fibrinogen and thrombin was experimentally determined by employing 28 formula ratios of thrombin and fibrinogen and checking clot formation and apparent stability. IIS was formed by integrating skin cells by means of the in situ gelification of fibrin into a porous crosslinked scaffold composed of chitosan, gelatin and hyaluronic acid. The in vitro cell proliferation within the IIS was examined by the MTT assay and PCNA expression. An experimental rabbit model consisting of six circular lesions was utilized to test each of the components of the IIS. Then, the IIS was utilized in an animal model to cover a 35% body surface full thickness lesion. RESULTS: The preclinical assays in rabbits demonstrated that the IIS was well tolerated and also that IIS-treated rabbit with lesions of 35% of their body surface, exhibited a better survival rate (p = 0,06). CONCLUSION: IIS should be further studied as a new wound dressing which shows promising properties, being the most remarkable its good biological tolerance and cell growth promotion properties.

Growth factor production from fibrin-encapsulated human keratinocytes.

Fibrin has been used extensively in cell encapsulation because it has important biological properties. Keratinocyte encapsulation in fibrin is a widely used technique in skin tissue engineering. The production of growth factors (EGF, TGF-beta1 and PDGF-BB) was evaluated when keratinocytes are encapsulated in fibrin. Secretions of TGF-beta1 and PDGF-BB increased more than five times compared to monolayer cultures. Encapsulated cells secreted about 80% active form of TGF-beta1 (monolayer cells only secreted inactive form). An enhanced secretion of TGF-beta1 and PDGF-BB was found in encapsulated cells, showing that fibrin capsules are favourable for the production of these growth factors.

Senescent cultures of human dermal fibroblasts modified phenotype when immobilized in fibrin polymer.

One of the limitations in tissue engineering is the restricted ability to expand the number of cells, because somatic cells can duplicate a limited number of times before they lose the ability to divide, leading to a senescent state. Here we report that the interaction of senescent fibroblasts with fibrin polymer can modify the senescent phenotype and partially restore the ability of growth-arrested cells to continue replicating. Primary human dermal fibroblasts were grown to >90% SA/beta-Gal (senescence associated beta-galactosidase). The senescent cells were immobilized in fibrin-polymers by mixing fibrinogen and thrombin solutions. Immobilized senescent cell cultures grew, however, their growth arrested after 24 h of immobilization. The percentage of cells with a positive reaction at SA/beta-Gal did not decrease significantly after immobilization, but the intensity of the stain decreased. The glycolytic activity in immobilized senescent fibroblast was re-established at pre-senescent levels. In conclusion, fibrin induces changes in the phenotype of senescent human fibroblasts. This simple procedure could complement available tissue-engineering techniques to increase the amount of biomass seeded on a fibrin scaffold, which could be beyond senescence.

A mathematical model for the design of fibrin microcapsules with skin cells.

The use of fibrin in tissue engineering has greatly increased over the last 10 years. The aim of this research was to develop a mathematical model to relate the microcapsule-size and cell-load to growth and oxygen depletion. Keratinocytes were isolated from rat skins and microencapsulated dropping fibrinogen and thrombin solutions. The cell growth was measured with MTT-assay and confirmed using histochemical technique. The oxygen was evaluated using a Clark sensor. It was found that Fick-Monod model explained the cell growth for the first 48 h, but overestimated the same thereafter. It was necessary to add a logistic equation to reach valid results. In relation to the preferred implant alternative, when considering large initial cell loads, the possibility to implant small loads of fast-growing cells arises from the simulations. In relation to the microcapsule size, it was found that a critical diameter could be established from which cell growth velocity is about the same.

Microscopic anatomy of the male reproductive system in Echinolittorina peruviana (Mollusca: Caenogastropoda) / Anatomía microscópica del sistema reproductor masculino de Echinolittorina peruviana (Mollusca: Caenogastropoda)

Echinolittorina peruviana (Lamarck, 1822), a gonochoric representative of the Littorinidae on the SE Pacific coast, has a male reproductive system adapted for internal fertilization. We describe this system at both macro- and microscopic levels, particularly the compartmentalized organization of the gonad, and the morphology of the penis. The male reproductive system has a variegated conical gonad-digestive gland complex. The gonad presents three compartments, 1) gametogenic acinar among the glandular acini, 2) periacinar with a layer of fusiform somatic cells and, 3) interacinar with glycogen storage cells shared with glandular acini. Spermatogenesis occurs within the acinar gametogenic compartment, with the germinal line organized in centripetal form towards the lumen. The seminal vesicle stores the products of spermatogenesis; in its cephalic region the euspermatozoa are united to the epithelium and the paraspermatozoa are distributed in the lumen. A short duct connects the seminal vesicle to the prostate gland that is open to the pallial cavity over its entire length. The anterior zone of the prostate gland is joined to the cervical spermatic groove that runs along the neck of the snail through the right pallial region; this continues as the penile spermatic groove, ascending from the base to the point of the penis. The penis is acutely conical and unpigmented; towards the anterior and adjacent to its base there is a glandular complex with a mamilliform process and a discoidal glandular region. The secretion from the discoidal region is transformed in a spicule of unknown function, whose histology is described here for the first time. The mamilliform process is formed by the spicular projection and a connective-muscle tissue band which surrounds it and separates it from the discoidal follicular glandular region; the follicular secretion crosses this band and is incorporated into the epithelium which lines the interior of the process.

Echinolittorina peruviana (Lamarck, 1822), representante gonocórico de Littorinidae en el Pacífico Sur, tiene un sistema reproductor masculino adaptado para la fecundación interna, que en este estudio se describe a niveles macrocoscópico y microscópico; enfatizando la organización compartimentalizada de la gónada y la morfología del pene. El sistema reproductor masculino se presenta como un complejo cónico gónada-glándula digestiva abigarrado. La gónada presenta tres compartimientos: 1) gametogénico acinar entre los acinos glandulares, 2) periacinar con una capa de células somáticas fusiformes y 3) interacinar con células almacenadoras de glicógeno, compartido con los acinos glandulares. En el compartimiento gametogénico acinar ocurre la espermatogénesis con la línea germinal organizada en forma centrípeta hacia el lumen. Hacia anterior, la vesícula seminal almacena los productos de la espermatogénesis; en su región cefálica los euespermatozoides se unen al epitelio y los paraespermatozoides se distribuyen en el lumen. Este órgano se conecta con un conducto corto a la glándula prostática, abierta hacia la cavidad paleal en toda su longitud. La zona anterior de la glándula prostática se une al surco espermático cervical, que recorre el cuello del animal por la región paleal derecha; éste continúa como surco espermático peniano ascendiendo desde su base hasta la punta. El pene es cónico aguzado no pigmentado; hacia anterior adyacente a su base, hay un complejo glandular con un proceso mamiliforme y una región glandular discoidal. Su producto de secreción sería una espícula de función desconocida, cuya histología es descrita por primera vez. En este complejo glandular, el proceso mamiliforme está constituido por la proyección espicular y una banda conjuntivo-muscular que lo rodea y separa de la región glandular folicular discoidal; su secreción atraviesa dicha banda, incorporándose al epitelio de revestimiento interno del proceso.

Anatomía microscópica del sistema reproductor en dos especies simpátricas del género fissurella bruguiére, 1789 (mollusca: vetigastropoda) / Microscopic anatomy of the reproductive system in two sympatric species of Fissurella Bruguiére, 1789 (Mollusca: Vetigastropoda

This study describes the microscopic anatomy of the reproductive systems of the keyhole limpets Fissurella latimarginata and Fissurella cumingi to test the existence of a possible isolating reproductive mechanism of morphophysiological type between species, and consequently to determine if the findings provide a valid taxonomic characteristic to distinguish these sympatric species. The results show that in both species the reproductive system is intimately associated with the excretory system. The gonad has a septa organization, and is connected with the distal portion of the right kidney by means of a renopericardial duct which functions as a genital duct. The gametes are evacuated to the mantle cavity through the right kidney opening. Besides transport of the gametes, the genital duct, which connects with the pericardium through the nephrostome, bears a gland attached to its inner wall both in males and females. No marked anatomical differences were noted between the reproductive systems of the two limpets which would allow their unequivocal taxonomic differentiation at the species level.

Este trabajo describe la anatomía microscópica del sistema reproductivo en las lapas Fissurella latimarginata y Fissurella cumingi, para examinar la posible existencia de un mecanismo de aislamiento reproductivo del tipo morfofisiológico y consecuentemente, determinar si el sistema reproductivo presenta algún carácter taxonómico válido que permita distinguir estas especies simpátricas. Los resultados muestran que en ambas especies y sexos el sistema reproductivo se relaciona con el sistema excretor. La gónada, de organización en septos, comunica con la porción distal del riñon derecho distal por medio del canal renopericárdico que funciona como conducto genital. Los gametos son evacuados a la cavidad del manto a través de la apertura renal derecha. Además de transportar los gametos, el conducto genital, que comunica con el pericardio a través del nefrostoma, presenta una glándula adosada a la pared interna en los machos y en las hembras. No se detectaron diferencias anatómicas marcadas del sistema reproductivo que permitan diferenciar las especies inequívocamente por lo que este sistema tiene bajo valor taxonómico a nivel específico.

Parasitic castration of Eurhomalea lenticularis (Bivalvia: Veneridae) by a digenetic trematode: quantitative histological analysis.

The clam Eurhomalea lenticularis may be parasitized by digenean trematodes of the family Plagiorchidae, specifically in the gonads (parasitic castration). A quantitative histological analysis of the parasitized gonads demonstrated a significant decrease in gonadal area, in the size of individual acini, and in the numbers of differentiated germ cells compared to unparasitized clams. Castration may be caused by mechanical compression due to trematode sporocyst growth. However, the uniform loss of germ cells in areas without sporocysts suggests that a more generalized mechanism is responsible. We suggest that parasitic castration has a primary effect on the host's neuroendocrine and gametogenic systems that regulate gamete production.

A homoeopathic proving of Candida parapsilosis

Candida parapsilosis is a common yeastlike organism that has been implicated in human infection. It has been recovered from respiratory secretions, urine, gastric washings, blood, vagina, oropharynx, skin, transtracheal aspiration, stool, pleural fluid, ear, and nails. It is implicated in the following human infections: Endopthalmitis, endocarditis, vaginitis, mycotic keratitis, external otomycosis, paronychia, and fungemia. In the vagina, it is found less frequently than Candida albicans and Torulopsis glabrata and is only rarely associated with vulvovaginal candidiasis as it is a less adherent organism