Visualizing the 3D architecture of multiple erythrocytes infected with Plasmodium at nanoscale by focused ion beam-scanning electron microscopy

Different methods for three-dimensional visualization of biological structures have been developed and extensively applied by different research groups. In the field of electron microscopy, a new technique that has emerged is the use of a focused ion beam and scanning electron microscopy for 3D reconstruction at nanoscale resolution. The higher extent of volume that can be reconstructed with this instrument represent one of the main benefits of this technique, which can provide statistically relevant 3D morphometrical data. As the life cycle of Plasmodium species is a process that involves several structurally complex developmental stages that are responsible for a series of modifications in the erythrocyte surface and cytoplasm, a high number of features within the parasites and the host cells has to be sampled for the correct interpretation of their 3D organization. Here, we used FIB-SEM to visualize the 3D architecture of multiple erythrocytes infected with Plasmodium chabaudi and analyzed their morphometrical parameters in a 3D space. We analyzed and quantified alterations on the host cells, such as the variety of shapes and sizes of their membrane profiles and parasite internal structures such as a polymorphic organization of hemoglobin-filled tubules. The results show the complex 3D organization of Plasmodium and infected erythrocyte, and demonstrate the contribution of FIB-SEM for the obtainment of statistical data for an accurate interpretation of complex biological structures.     

Genotype-specific differences between mouse CNS stem cell lines expressing frontotemporal dementia mutant or wild type human tau

Stem cell (SC) lines that capture the genetics of disease susceptibility provide new research tools. To assess the utility of mouse central nervous system (CNS) SC-containing neurosphere cultures for studying heritable neurodegenerative disease, we compared neurosphere cultures from transgenic mice that express human tau with the P301L familial frontotemporal dementia (FTD) mutation, rTg(tau(P301L))4510, with those expressing comparable levels of wild type human tau, rTg(tau(wt))21221. rTg(tau(P301L))4510 mice express the human tau(P301L) variant in their forebrains and display cellular, histological, biochemical and behavioral abnormalities similar to those in human FTD, including age-dependent differences in tau phosphorylation that distinguish them from rTg(tau(wt))21221 mice. We compared FTD-hallmark tau phosphorylation in neurospheres from rTg(tau(P301L))4510 mice and from rTg(tau(wt))21221 mice. The tau genotype-specific phosphorylation patterns in neurospheres mimicked those seen in mice, validating use of neurosphere cultures as models for studying tau phosphorylation. Genotype-specific tau phosphorylation was observed in 35 independent cell lines from individual fetuses; tau in rTg(tau(P301L))4510 cultures was hypophosphorylated in comparison with rTg(tau(wt))21221 as was seen in young adult mice. In addition, there were fewer human tau-expressing cells in rTg(tau(P301L))4510 than in rTg(tau(wt))21221 cultures. Following differentiation, neuronal filopodia-spine density was slightly greater in rTg(tau(P301L))4510 than rTg(tau(wt))21221 and control cultures. Together with the recapitulation of genotype-specific phosphorylation patterns, the observation that neurosphere lines maintained their cell line-specific-differences and retained SC characteristics over several passages supports the utility of SC cultures as surrogates for analysis of cellular disease mechanisms.     

Comprehensive biothreat cluster identification by PCR/electrospray-ionization mass spectrometry

Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.     

Biological properties of solid free form designed ceramic scaffolds with BMP-2: in vitro and in vivo evaluation

Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures.     

Negative chromatography on agarose-TREN as a technique for purification of protein spiked in soybean seeds extract

Alkyl amines and polyamines have been used as ligands for protein purification by mixed-mode chromatography. The adsorption of proteins onto these ligands seems to be governed by multiple effects such as electrostatic, hydrophobic, and affinity interactions. In this work we investigated the adsorption of proteins extracted from soybean onto the adsorbent agarose-Tris(2-aminoethyl)amine (TREN). The effects of flow rate, buffer system, and extract concentration on the capture of proteins extracted from soybean were evaluated. Experiments using Mes at pH 6.5 as adsorption buffer allowed the adsorption of almost the totality of native soybean protein with a dynamic adsorption capacity of 13.50 mg mL^?1 adsorbent. Experiments with human IgG (pI in the range of 5.8–9.0) and human serum albumin (HSA, pI of 4.9) spiked into these extracts lead to the conclusion that electrostatic forces play a major role in the interaction between protein and agarose-TREN. Based on this work, negative chromatography with agarose-TREN should be considered as a method for purification of basic recombinant protein produced in transgenic soybean seeds.     

A Paracoccidioides brasiliensis glycan shares serologic and functional properties with cryptococcal glucuronoxylomannan

The cell wall of the yeast form of the dimorphic fungus Paracoccidioides brasiliensis is enriched with α1,3-glucans. In Cryptococcus neoformans, α1,3-glucans interact with glucuronoxylomannan (GXM), a heteropolysaccharide that is essential for fungal virulence. In this study, we investigated the occurrence of P. brasiliensis glycans sharing properties with cryptococcal GXM. Protein database searches in P. brasiliensis revealed the presence of sequences homologous to those coding for enzymes involved in the synthesis of GXM and capsular architecture in C. neoformans. In addition, monoclonal antibodies (mAbs) raised to cryptococcal GXM bound to P. brasiliensis cells. Using protocols that were previously established for extraction and analysis of C. neoformans GXM, we recovered a P. brasiliensis glycan fraction composed of mannose and galactose, in addition to small amounts of glucose, xylose and rhamnose. In comparison with the C. neoformans GXM, the P. brasiliensis glycan fraction components had smaller molecular dimensions. The P. brasiliensis components, nevertheless, reacted with different GXM-binding mAbs. Extracellular vesicle fractions of P. brasiliensis also reacted with a GXM-binding mAb, suggesting that the polysaccharide-like molecule is exported to the extracellular space in secretory vesicles. An acapsular mutant of C. neoformans incorporated molecules from the P. brasiliensis extract onto the cell wall, resulting in the formation of surface networks that resembled the cryptococcal capsule. Coating the C. neoformans acapsular mutant with the P. brasiliensis glycan fraction resulted in protection against phagocytosis by murine macrophages. These results suggest that P. brasiliensis and C. neoformans share metabolic pathways required for the synthesis of similar polysaccharides and that P. brasiliensis yeast cell walls have molecules that mimic certain aspects of C. neoformans GXM. These findings are important because they provide additional evidence for the sharing of antigenically similar components across phylogenetically distant fungal species. Since GXM has been shown to be important for the pathogenesis of C. neoformans and to elicit protective antibodies, the finding of similar molecules in P. brasiliensis raises the possibility that these glycans play similar functions in paracoccidiomycosis.     

Protection by DHA of Early Hippocampal Changes in Diabetes: Possible Role of CREB and NF-κB

The mechanisms underlying diabetic encephalopathy, are only partially understood. In this study, we try to address the mechanisms of diabetes induced damage and whether docosahexaenoic acid (DHA) could attenuate the degenerative changes in diabetic hippocampus in a rodent model of diabetes. Diabetes was induced in rats by an intraperitoneal injection of streptozotocin. Animals were divided into the following experimental groups: control rats; control animals treated with DHA; untreated diabetic rats; diabetic rats treated with insulin; diabetic rats treated with DHA; diabetic rats treated with insulin and DHA. At the end of week 12, rats were killed and one of the hemispheres was cryosectioned and the other was dissected and hippocampi homogenized. The number of bromodeoxyuridine positive cells in the hippocampus of diabetic rats was decreased, and the latency time to find the platform in the Morris Water maze was significantly increased in the diabetic rats when compared to controls. No changes where observed in the expression of p21 in the hippocampus of control and diabetic rats. Biochemical markers of oxidative stress were altered in hippocampus of diabetic rats, and NFκB-positive cells were increased in the hippocampus of diabetic rats when compared to controls. Treatment with DHA, or the combination of DHA with insulin, significantly restored to control levels all the values mentioned above. Our findings confirm a pivotal role for oxidative stress as well as NF-κB, but not p21, in diabetes-induced hippocampal impairments. Administration of DHA as well as insulin prevented the changes induced by diabetes in hippocampus.     

Nitrosative stress mediated misfolded protein aggregation mitigated by Na-d-β-hydroxybutyrate intervention

Mitochondrial dysfunction, leading to elevated levels of reactive oxygen species, is associated with the pathogenesis of neurodegenerative disorders. Rotenone, a mitochondrial stressor induces caspase-9 and caspase-3 activation leading proteolytic cleavage of substrate nuclear poly(ADP-ribose) polymerase (PARP). PARP cleavage is directly related to apoptotic cell death. In this study, we have monitored the aggregation of green-fluorescent protein (GFP)-tagged synphilin-1, as a rotenone-induced Parkinsonia-onset biomarker. We report that the innate ketone body, Na-d-β-hydroxybutyrate (NaβHB) reduces markedly the incidence of synphilin-1 aggregation. Furthermore, our data reveal that the metabolic byproduct also prevents rotenone-induced caspase-activated apoptotic cell death in dopaminergic SH-SY5Y cells. Together, these results suggest that NaβHB is neuroprotective; it attenuates effects originating from mitochondrial insult and can serve as a scaffold for the design and development of sporadic neuropathies.     

A zinc-dependent mechanism regulates meiotic progression in mammalian oocytes

Precise coordination of meiotic progression is a critical determinant of an egg's capacity to be fertilized successfully, and zinc has emerged as a key regulatory element in this process. An early manifestation of a regulatory role for this transition metal is the significant increase in total intracellular zinc. This accumulation is essential for meiotic progression beyond telophase I and the establishment of meiotic arrest at metaphase II. The subsequent developmental event, fertilization, induces a rapid expulsion of labile zinc that is a hallmark event in meiotic resumption. In the present study, we show that the zinc fluxes work, in part, by altering the activity of the cytostatic factor (CSF), the cellular activity required for the establishment and maintenance of metaphase II arrest in the mature, unfertilized egg. We propose a model in which zinc exerts concentration-dependent regulation of meiosis through the CSF component EMI2, a zinc-binding protein. Together, the data support the conclusion that zinc itself, through its interaction with EMI2, is a central component of the CSF.