Drawing blood from rats through the saphenous vein and by cardiac puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available (1-3). Here, we present two techniques for drawing blood from rats, each with its specific applications. Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies (4,5), determining plasma chemistry, or blood counts (6). Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro (4-9).     

Salt-induced changes in the plasma membrane proteome of the halotolerant alga Dunaliella salina as revealed by blue native gel electrophoresis and nano-LC-MS/MS analysis

The halotolerant alga Dunaliella salina is a recognized model photosynthetic organism for studying plant adaptation to high salinity. The adaptation mechanisms involve major changes in the proteome composition associated with energy metabolism and carbon and iron acquisition. To clarify the molecular basis for the remarkable resistance to high salt, we performed a comprehensive proteomics analysis of the plasma membrane. Plasma membrane proteins were recognized by tagging intact cells with a membrane-impermeable biotin derivative. Proteins were resolved by two-dimensional blue native/SDS-PAGE and identified by nano-LC-MS/MS. Of 55 identified proteins, about 60% were integral membrane or membrane-associated proteins. We identified novel surface coat proteins, lipid-metabolizing enzymes, a new family of membrane proteins of unknown function, ion transporters, small GTP-binding proteins, and heat shock proteins. The abundance of 20 protein spots increased and that of two protein spots decreased under high salt. The major salt-regulated proteins were implicated in protein and membrane structure stabilization and within signal transduction pathways. The migration profiles of native protein complexes on blue native gels revealed oligomerization or co-migration of major surface-exposed proteins, which may indicate mechanisms of stabilization at high salinity.     

Exposure of neural crest cells to elevated glucose leads to congenital heart defects, an effect that can be prevented by N-acetylcysteine

BACKGROUND: Diabetes mellitus during pregnancy increases the risk for congenital heart disease in the offspring. The majority of the cardiovascular malformations occur in the outflow tract and pharyngeal arch arteries, where neural crest cells are essential for normal development. We studied the effects of specific exposure of neural crest cells to elevated glucose on heart development. Antioxidants reduce the damaging effect of glucose on neural crest cells in vitro; therefore, we investigated the effect of supplementing N-acetylcysteine in vivo. METHODS: Cardiac neural crest of HH 8-12 chicken embryos was directly exposed by a single injection in the neural tube with 30 mM D-glucose (or 30 mM L-glucose as a control). To examine the effect of a reduction in oxidative stress, we added 2 mM N-acetylcysteine to the injected D-glucose. RESULTS: Exposure of neural crest cells to elevated D-glucose-induced congenital heart malformations in 82% of the embryos. In the embryos injected with L-glucose, only 9% developed a heart malformation. As expected, all malformations were located in the outflow tract and pharyngeal arch arteries. The frequency of heart malformations decreased from 82% to 27% when 2 mM N-acetylcysteine was added to the injected D-glucose. CONCLUSIONS: These data are the first to confirm that the vulnerability of neural crest cells to elevated glucose induces congenital heart malformations. The fact that N-acetylcysteine limits the teratogenicity of glucose implies that its damaging effect is mediated by an increase of oxidative stress in the neural crest cells.     

Nod factor-independent ‘crack-entry’ symbiosis in dalbergoid legume Arachis hypogaea

Dalbergoids are typified by crack-entry symbiosis which is evidenced to be Nod Factor (NF)-independent in several Aeschynomene legumes. Natural symbionts of the dalbergoid legume Arachis hypogaea are always NF-producing, prompting us to check whether symbiosis in this legume could also be NF-independent. For this, we followed the symbiosis with two NF-containing bradyrhizobial strains – SEMIA6144, a natural symbiont of Arachis and ORS285, a versatile nodulator of Aeschynomene legumes, along with their corresponding nodulation (nod) mutants. Additionally, we investigated NF-deficient bradyrhizobia like BTAi1, a natural symbiont of Aeschynomene indica and the WBOS strains that were natural endophytes of Oryza sativa, collected from an Arachis-Oryza intercropped field. While SEMIA6144ΔnodC was non-nodulating, both ORS285 and ORS285ΔnodB could induce functional nodulation, although with lower efficiency than SEMIA6144. On the other hand, all the NF-deficient strains – BTAi1, WBOS2 and WBOS4 showed comparable nodulation with ORS285 indicating Arachis to harbour an NF-independent mechanism of symbiosis. Intriguingly, symbiosis in Arachis, irrespective of whether it was NF-dependent or independent, was always associated with the curling or branching of the rosette root hairs at the lateral root bases. Thus, despite being predominantly described as an NF-dependent legume, Arachis does retain a vestigial, less-efficient form of NF-independent symbiosis.     

Phylogeography of the Endangered Franciscana Dolphin: Timing and Geological Setting of the Evolution of Populations

Pontoporia blainvillei (Gervais & d’Orbigny, 1844), the franciscana dolphin, is the most endangered small cetacean in the Western South Atlantic. It is an endemic species with a coastal and estuarine distribution that has been divided into four Franciscana Management Areas (FMAs). We used the mitochondrial DNA control region to conduct a phylogeographic analysis to evaluate the population structure of the franciscana and the influence of paleoceanographic events on its biogeographic history. We found nine populations along the entire distribution (Φ_ST?=?0.41, Φ_CT?=?0.38, p?<?10^–5), with estimated migration rates resulting in less than one female per generation. Populations from FMAIII and FMAIV in the south (including the Río de La Plata Estuary) showed higher long-term migration rates and effective population sizes than northern populations. The phylogeographic analysis supports the franciscana origin in the Río de La Plata Estuary, with further dispersal south and northwards. The first lineage split happened around 2.5 Ma, with lineage radiation throughout the Pleistocene until recent fragmentation events shaped current-day populations. We suggest that Pleistocene glaciations influenced the dispersion and population structure of the franciscana. Specifically, that the shift of the Brazil-Malvinas Confluence drove the dispersion northwards. Then, low sea-level periods caused either the isolation in estuarine refugia or local extinctions, followed by re-colonizations.

     

Species diversity in Gymnogeophagus (Teleostei: Cichlidae) and comparative biogeography of cichlids in the Middle Paraná basin,an emerging hotspot of fish endemism

Hydrobiologia - We address the diversity of two species groups of the cichlid genus Gymnogeophagus in the Middle Paraná basin using molecular phylogeography and traditional morphological...     

Variations in carotenoid content and acyl chain composition in exponential,stationary and biofilm states of Staphylococcus aureus,and their influence on membrane biophysical properties

Bacteria are often found in close association with surfaces, resulting in the formation of biofilms. In Staphylococcus aureus (S. aureus), biofilms are implicated in the resilience of chronic infections, presenting a serious clinical problem world-wide. Here, S. aureus biofilms are grown under flow within clinical catheters at 37 °C. The lipid composition and biophysical properties of lipid extracts from these biofilms are compared with those from exponential growth and stationary phase cells. Biofilms show a reduction in iso and anteiso branching compensated by an increase in saturated fatty acids compared to stationary phase. A drastic reduction in carotenoid levels is also observed during biofilm formation. Thermotropic measurements of Laurdan GP and DPH polarization, show a reduction of lipid packing at 37 °C for biofilms compared to stationary phase. We studied the effects of carotenoid content on DMPG and DPPG model membranes showing trends in thermotropic behavior consistent with those observed in bacterial isolates, indicating that carotenoids participate in modulating lipid packing. Additionally, bending elastic constant (k_c) measurements using vesicle fluctuation analysis (VFA) show that the presence of carotenoids can increase membrane bending rigidity. The antimicrobial peptide Magainin H2 was less activity on liposomes composed of stationary phase compared to biofilms or exponential growth isolates. This study contributes to an understanding of how Staphylococcus aureus modulates the composition of its membrane lipids, and how those changes affect the biophysical properties of membranes, which in turn may play a role in its virulence and its resistance to different membrane-active antimicrobial agents.     

Mycosis Due to Tropicoporus tropicalis (= Inonotus tropicalis) in a Domestic Dog

Mycopathologia - Mycelial basidiomycetes rarely produce mycoses in animals including humans. We report a case of a 9-year-old female mongrel dog with lesions in the prescapular lymph nodes. The...     

GABAA Modulation of S100B Secretion in Acute Hippocampal Slices and Astrocyte Cultures

Astrocytes are the major glial cells in brain tissue and are involved, among many functions, ionic and metabolic homeostasis maintenance of synapses. These cells express receptors and transporters for neurotransmitters, including GABA. GABA signaling is reportedly able to affect astroglial response to injury, as evaluated by specific astrocyte markers such as glial fibrillary acid protein and the calcium-binding protein, S100B. Herein, we investigated the modulatory effects of the GABA_A receptor on astrocyte S100B secretion in acute hippocampal slices and astrocyte cultures, using the agonist, muscimol, and the antagonists pentylenetetrazol (PTZ) and bicuculline. These effects were analyzed in the presence of tetrodotoxin (TTX), fluorocitrate (FLC), cobalt and barium. PTZ positively modify S100B secretion in hippocampal slices and astrocyte cultures; in contrast, bicuculline inhibited S100B secretion only in hippocampal slices. Muscimol, per se, did not change S100B secretion, but prevented the effects of PTZ and bicuculline. Moreover, PTZ-induced S100B secretion was prevented by TTX, FLC, cobalt and barium indicating a complex GABA_A communication between astrocytes and neurons. The effects of two putative agonists of GABA_A, β-hydroxybutyrate and methylglyoxal, on S100B secretion were also evaluated. In view of the neurotrophic role of extracellular S100B under conditions of injury, our data reinforce the idea that GABA_A receptors act directly on astrocytes, and indirectly on neurons, to modulate astroglial response.

     

Shared KEGG pathways of icariin-targeted genes and osteoarthritis

The beneficial effects of icariin in the management of many diseases, such as chronic renal failure and heart failure, are well known. Icariin has also been shown to ameliorate osteoarthritis (OA) symptoms; however, the underlying mechanisms remain unclear. In this study, a bioinformatics analysis was performed to investigate the KEGG pathways of icariin-targeted genes involved in OA. Our study suggests that icariin plays a role in OA by regulating inflammatory cytokine production, insulin resistance, and cell survival through modulation of the NF-κB, MAPK, and Akt signaling pathways. Importantly, IKBKB, NFKBIA, MAPK8, MAPK9, and MAPK10 may be the hub genes affected by icariin when providing its beneficial effects on OA. In addition, we found that icariin decreases proinflammatory factors and inhibits chondrocyte apoptosis through suppression of the NF-κB pathway. Our study highlights a set of KEGG pathways that could explain the molecular mechanism of icariin's action on OA, suggesting that icariin could be considered as a promising therapeutic option for OA.