Surface-enhanced Raman spectroscopy and ultrastructural analysis of penicillin-producing Penicillium rubens strains.

Raman spectroscopy, transmission electron microscopy (TEM) and atomic force microscopy (AFM) techniques can perform chemical analyses and acquire high-resolution images of cell samples. For this reason, in this study, semi-thin sections of a single Penicillium rubens cell were analysed by Raman enhanced surface spectroscopy. The spectra showed peaks corresponding to the macromolecules that make up the cellular components. In addition, the various organelles were analysed by TEM and AFM to observe the cellular nanostructures. With the use of these techniques, it is possible to identify molecules in semi-thin sections, which provides a wide potential for biomedical applications and for the analysis of cell dynamics. The observation of the most detailed possible structure of cells is used as a starting point in numerous studies to identify and localise some biochemical processes. Given that the function of eukaryotic cells depends on the location, shape, structure and function of the subcellular organelles (and on the interaction between them), the sum of the data obtained allows a complete analysis of what happens in the cell. This article addresses, from a multidisciplinary point of view, what happens in a single cell of a filamentous fungus (Penicillium rubens) while it is in a physiological moment (secondary metabolism) that allows the biosynthesis of an antibiotic (penicillin). For this purpose, different types of microscopies were used (TEM: transmission electron microscopy, and AFM: atomic force microscopy, which allow visualising small details in the cell) and a spectroscopy method (Raman, which allows detecting certain characteristics of the macromolecules and some stretching bonds). Regarding the results, during the synthesis of penicillin, the antibiotic-producing Penicillium rubens cells showed significant changes compared to the non-producing cells: the cell wall is observed to be significantly thickened in the production phase, organelles such as peroxisomes grow in number and size since it is known that the final route of metabolite synthesis takes place in them. When penicillin is released from peroxisomes, they must be degraded to release the load from the cell; this is done by vacuoles, which are active and engulf peroxisomes. The newly synthesised penicillin is found within secretory vesicles that travel towards the cell membrane and both membranes fuse creating ripples. On the other hand, and given that a single cell is being studied, it is essential to increase the signal to detect biomolecules employing the Raman-SERS technique, using a silver substrate to obtain the increased signal.

Latexin expression correlated with mineralization of articular cartilage during progression of post-traumatic osteoarthritis in a rat model.

As latexin has been linked with chondrocyte hypertrophic differentiation it is possible that this protein may also be involved in the mineralization of cartilage in OA. Therefore, we correlated latexin expression with the mineralization marker, alkaline phosphatase and determined the mineral deposition in the articular cartilage by analyzing the Ca/P ratio and the collagen fibrils pattern, during the progression of post-traumatic OA in a rat model. OA was induced by medial meniscectomy and post-surgery exercise for 5, 10, 20 and 45 days. Protein expression in articular cartilage was evaluated by immunofluorescence, histochemistry and Western blot. Minerals and structure of collagen fibrils in the superficial zone of cartilage were analyzed by energy dispersive X-ray spectroscopy (EDX) and atomic force microscopy (AFM) respectively. Protein expression analysis showed time-dependent up-regulation of latexin during OA progression. In the cartilage, latexin expression correlated with the expression and activity of alkaline phosphatase. EDX of the superficial zone of cartilage showed a Ca/P ratio closer to theoretical values for basic calcium phosphate minerals. The presence of minerals was also analyzed indirectly with AFM, as the collagen fibril pattern was less evident in the mineralized tissue. Latexin is expressed in articular cartilage from the early stages of post-traumatic OA; however, minerals were detected after latexin expression was up-regulated, indicating that its activity precedes and remains during the pathological mineralization of cartilage. Thus, our results contribute to the identification of molecules involved in the mineralization of articular chondrocytes.

Isolation and characterization of exosomes released from mosquito cells infected with dengue virus.

Exosomes are endocytic origin small-membrane vesicles secreted to the extracellular space by most cell types. Exosomes released from virus infected-cells can mediate the cell-to-cell communication to promote or modulate viral transmission. Dengue virus (DENV) is an arbovirus transmitted by Aedes mosquitoes bite to humans. Interestingly, the role of exosomes during the DENV infection in mammalian cells has already been described. However, little is known about exosomes derived from infected mosquito cells. Thus, the exosomes released from DENV-infected C6/36 cells were isolated, purified and analyzed using an antibody against the tetraspanin CD9 from human that showed cross-reactivity with the homologs to human CD9 found in Aedes albopictus (AalCD9). The exosomes from DENV infected cells were larger than the exosomes secreted from uninfected cells, contained virus-like particles, and they were able to infect naïve C6/36 cells, suggesting that exosomes are playing a role in virus dissemination.

The dengue virus non-structural protein 1 (NS1) is secreted efficiently from infected mosquito cells.

Dengue virus NS1 is a glycoprotein of 46-50kDa which associates as a dimer to internal and cytoplasmic membranes and is also secreted, as a hexamer, to the extracellular milieu. However, the notion exist that NS1 is secreted only from infected vertebrate and not mosquito cells. In this work, evidence is presented showing that NS1 is secreted efficiently by infected mosquito cells. NS1 was detected in cell supernatants starting at 6hpi with a continuous concentration increase up to 24hpi. Nevertheless, cell viability showed an average cell survival of 97%. At variance with observations with vertebrate cells, NS1 does not seems to associate with the cytoplasmic membrane of insect cells. Finally, evidence is presented indicating that NS1 is secreted from insect cells as a barrel-shaped hexamer. These findings provide new insights into the biology of NS1 and open questions about the role of secreted NS1 in the vector mosquito.

Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy.

Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves a dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica.

Chondrocytes interconnecting tracks and cytoplasmic projections observed within the superficial zone of normal human articular cartilage--a transmission electron microscopy, atomic force microscopy, and two-photon excitation microscopy studies.

The morphology of the normal human and rat articular cartilage was assessed using transmission electron microscopy (TEM), atomic force microscopy (AFM), and two-photon excitation (2PE) microscopy. Spurr-embedded sections from fixed human cartilage were simultaneously evaluated using TEM and AFM. The presences of tracks among the chondrocytes from the superficial zone of the cartilage were observed. In order to ratify the presence of interconnecting tracks among superficial zone chondrocytes, whole fixed human and rat cartilage, as well as fresh whole rat cartilage, were examined under the 2PE. In all cases, these tracks were observed. In addition, porous matrix, well-defined lacunae, and cytoplasmic projections anchored to the extracellular matrix (ECM) were also detected. We conclude that normal human and rat flattened superficial chondrocytes might be interconnected by tracks running through the ECM. In addition, cytoplasmic projections were observed anchored to the ECM. All these structures may possibly be related to cell/cell and ECM/chondrocytes signaling. Our findings provide new information that possibly will be of relevant importance for a more profound study of normal cartilage physiology and eventually, the pathogenesis of osteoarthritis.

Purkinje cell density in cerebella of alcoholized and non-alcoholized male rat offspring

The effect of alcohol intake by male rats was evaluated on Purkinje cell morphology and number in their offspring. Forty five male Wistar rats, 45 days old, were used and divided into three groups of 15 rats each: control group (CG), fed with conventional Purina rodent feed (CPRF) and water ad libitum; experimental group (EG), fed with CPRF ad libitum and a mixture of water/ethanol, which represented 36 per cent of kiloclories in food; and an equinergetic intake control group (ECG), which was given CPRF (in grams) and sugar in their drinking water, in order to substitute the energetic value provided by alcohol. Five subgroups (n=3) were created to be used for different treatment periods: 60, 90,120, 150 and 180 days; all groups started treatment period when they were 70 days old. At the end of each treatment period, male rats were mated with nulliparous females not having undergone treatment. Offspring were obtained and studied at 14 and 21 days of age. The Purkinje cells of the cerebella of 14-and 21-day-old offspring belonging to the CG and ECG showed no morphological changes. On the other hand, in 14-day-old offspring belonging to the experimental group of parents alcoholized during 90, 120, and 180 days, a large number of hyperchromatic Purkinje cells were seen, forming zones of cells undergoing a degenerative process. No significant differences in cellular density were determined between the CG and the ECG. When comparing the CG vs. EG and the ECG vs. Eg, significant differences were found in the 14-day-old offspring as well as in the 21-day-old ones with a p<0.05 of rats belonging to parents alcoholized for 90, 120, and 180 days. The results may indicate that there are changes in the germinal plasma of males fue to alcohol of males due to alcohol sunsumption; therefore, reflecting this effect on a drecrease of Purkinje cells and probably on ther cell populations