Synthesis of Human Bone Morphogenetic Protein-2 (hBMP-2) in E. coli Periplasmic Space: Its Characterization and Preclinical Testing

Human BMP-2, a homodimeric protein that belongs to the TGF- β family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

Thunderstorm allergy and asthma: state of the art

Thunderstorm-triggered asthma (TA) can be defined as the occurrence of acute asthma attacks immediately following a thunderstorm during pollen seasons. Outbreaks have occurred across the world during pollen season with the capacity to rapidly inundate a health care service, resulting in potentially catastrophic outcomes for allergicpatients. TA occurs when specific meteorological and aerobiological factors combine to affect predisposed atopic patients with IgE-mediated sentitization to pollen allergens. Thunderstorm outflows can concentrate aeroallergens, most commonly grass pollen but also other pollens such as Parietaria and moulds in TA, at ground level to release respirable allergenic particles after rupture by osmotic shock related to humidity and rainfall. Inhalation of high concentrations of these aeroallergens by sensitized individuals can induce early asthmatic responses which can be followed by a late inflammatory phase. There is evidence that, during pollen season, thunderstorms can induce allergic asthma outbreaks, sometimes also severe asthma crisis and sometimes deaths in patients suffering from pollen allergy. It has been observed that changes in the weather such as rain or humidity may induce hydratation of pollen grains during pollen seasons and sometimes also their fragmentation which generates atmospheric biological aerosols carrying allergens. Asthma attacks are induced for the high concentration at ground level of pollen grains which may release allergenic particles of respirable size after rupture by osmotic shock. In other words, it is a global health problem observed in several cities and areas of the world that can strike without sufficient warning, inducing sometimes severe clinical consequences also with deaths of asthma patients. Due to constant climate change, future TA events are likely to become more common, more disastrous and more unpredictable, as a consequence it is important to have deep knowledge on this topic to prevent asthma attacks. Other environmental factors, such as rapid changes in temperature and agricultural practices, also contribute to causing TA.

The Dysferlin Transcript Containing the Alternative Exon 40a is Essential for Myocyte Functions

Dysferlinopathies are a group of muscular dystrophies caused by recessive mutations in the DYSF gene encoding the dysferlin protein. Dysferlin is a transmembrane protein involved in several muscle functions like T-tubule maintenance and membrane repair. In 2009, a study showed the existence of fourteen dysferlin transcripts generated from alternative splicing. We were interested in dysferlin transcripts containing the exon 40a, and among them the transcript 11 which contains all the canonical exons and exon 40a. This alternative exon encodes a protein region that is cleaved by calpains during the muscle membrane repair mechanism. Firstly, we tested the impact of mutations in exon 40a on its cleavability by calpains. We showed that the peptide encoded by the exon 40a domain is resistant to mutations and that calpains cleaved dysferlin in the first part of DYSF exon 40a. To further explore the implication of this transcript in cell functions, we performed membrane repair, osmotic shock, and transferrin assay. Our results indicated that dysferlin transcript 11 is a key factor in the membrane repair process. Moreover, dysferlin transcript 11 participates in other cell functions such as membrane protection and vesicle trafficking. These results support the need to restore the dysferlin transcript containing the alternative exon 40a in patients affected with dysferlinopathy.

A new approach to the determination of tubular membrane capacitance: passive membrane electrical properties under reduced electrical conductivity of the extracellular solution

The tubular system of cardiomyocytes plays a key role in excitation-contraction coupling. To determine the area of the tubular membrane in relation to the area of the surface membrane, indirect measurements through the determination of membrane capacitances by electrophysiological measurements are currently used in addition to microscopic methods. Unlike existing electrophysiological methods based on an irreversible procedure (osmotic shock), the proposed approach uses a reversible short-term intermittent increase in the electrical resistance of the extracellular medium. The resulting increase in the lumen resistance of the tubular system makes it possible to determine separately capacitances of the tubular and surface membranes from altered capacitive current responses to subthreshold voltage-clamped rectangular pulses. Based on the analysis of the time course of capacitive current, computational relations were derived which allow to quantify elements of the electrical equivalent circuit of the measured cardiomyocyte including both capacitances. The exposition to isotonic low-conductivity sucrose solution is reversible which is the main advantage of the proposed approach allowing repetitive measurements on the same cell under control and sucrose solutions. In addition, it might be possible to identify changes in both surface and tubular membrane capacitances caused by various interventions. Preliminary experiments in rat ventricular cardiomyocytes (n = 10) resulted in values of the surface and tubular capacitances 72.3 ± 16.4 and 42.1 ± 14.7 pF, respectively, implying the fraction of tubular capacitance/area of 0.36 ± 0.08. We conclude that the newly proposed method provides results comparable to those reported in literature and, in contrast to the currently used methods, enables repetitive evaluation of parameters describing the surface and tubular membranes. It may be used to study alterations of the tubular system resulting from various interventions including associated cardiac pathologies.

Conflicting effects of haemolysis on plasma sodium and chloride are due to different haemolysis study protocols: A case for standardisation

Background Haemolysis has been reported as having a positive, negative or no effect on plasma sodium (PNa) and chloride (PCl). We investigated the haemoltytic effect of different haemolysis protocols on PNa and PCl using modelling and laboratory experiments. Methods In a modelling experiment, percentage change and recovery due to dilution in routinely ( in vitro) haemolysed samples were compared against shear stress haemolysis and samples spiked with haemolysate from whole blood freeze–thaw, packed cells freeze–thaw and osmotic shock protocols. The results were compared against a control base pool. Additionally, for the osmotic shock method, results were compared against saline- and deionised water (DIW)-spiked controls. In a laboratory experiment, percentage change and recovery were similarly compared using haemolysate from whole blood freeze–thaw and osmotic shock protocols. PNa, PCl and H-index were measured on the Abbott Architect and haemoglobin on the Sysmex XN-9000. Results In the modelling experiment, the percentage decrease in PNa and PCl was similar in in vitro haemolysis, shear stress haemolysis, whole blood freeze–thaw haemolysis and packed cells freeze–thaw haemolysis and this was lower compared to the osmotic shock method. In the laboratory experiment, the change in PNa compared to the base pool was less ( p < 0.001) per unit increase in H-index in the freeze–thaw method (−0.33 mmol, 95% CI −0.35 to −0.31) compared to the osmotic shock method (−0.65 mmol, 95% CI −0.66 to −0.64). PCl did not change with haemolysis in the freeze–thaw method and changed by −0.21 ± 0.01 mmol per unit increase in the H-index in the osmotic shock method. Recovery of PNa and PCl increased with increasing H-index in both methods. Conclusion The osmotic shock protocol is inappropriate for haemolysis studies because of dilution with DIW used for cell lysis. Recovery calculations may incorrectly compensate for genuine dilution caused by haemolysis.

Analyzing and optimizing the carbon utilization and lipid yield at different light intensities for Scenedesmus arcuatus

Microalgae, the photosynthetic microorganism growing abundantly in marine and aquatic ecosystems, are potential source for biological sequestration of CO2. The carbon uptake differs in the presence of other nutrients, light intensity etc. The biomass yield of Scenedesmus arcuatus var capitatus was studied based on the Face Centred Central Composite design (FCCD) of Response Surface Methodology (RSM) for nitrate, phosphate and carbonate under different conditions (laboratory, room and sunlight conditions). Various pre-treatments (osmotic shock, autoclaving, microwave and ultrasonication) were employed to find the best method for maximum lipid yield. The biomass yield reached a maximum of 1 g/L under sunlight conditions of nitrate concentration 500 ppm and carbonate 2000 ppm. The laboratory conditions resulted in a biomass yield of 0.59 g/L at 500 ppm nitrate, 1000 ppm carbonate and 250 ppm phosphate. Under room conditions, the yield was very low (0.11 g/L). Osmotic shock resulted in higher lipid yield than the other pre-treatment methods. The ability of Scenedesmus arcuatus to uptake high carbon under sunlight conditions and to adapt to high light intensity and fluctuations in light intensity concludes that this species is suitable for large-scale open pond cultivation for CO2 sequestration and production of metabolites.

Novel elasticity measurement techniques reveal that the C. elegans cuticle is required for physical integrity with age

Changes in biomechanical properties have profound impacts on human health. C. elegans might serve as a model for studying the molecular genetics of mammalian tissue decline. Previously, we found that collagens are required for insulin signaling mutants’ long lifespan and that overexpression of specific collagens extends wild-type lifespan. However, whether these effects on lifespan are due to mechanical changes during aging has not yet been established. Here, we have developed two novel methods to study the cuticle: we measure mechanical properties of live animals using osmotic shock (OS), and we directly perform the tensile test (TT) on isolated cuticles using microfluidic technology. Using these tools, we find that cuticle, not the muscle, is responsible for changes in ‘stretchiness’ of C. elegans, and that cuticle stiffness is highly non-linear and anisotropic. We also found that collagen mutations alter integrity of the cuticle by significantly altering elasticity. Additionally, aging stiffens the cuticle under mechanical loads beyond the cuticle’s healthy stretched state. Measurements of elasticity showed that long-lived daf-2 mutants were considerably better at preventing progressive mechanical changes with age. These tests of C. elegans biophysical properties suggest that the cuticle is responsible for their resilience.

The virulome of Streptomyces scabiei in response to cello-oligosaccharides elicitors

The development of spots or lesions symptomatic of the common scab disease on root and tuber crops is caused by few pathogenic Streptomyces with Streptomyces scabiei 87-22 as the model species. Thaxtomin phytotoxins are the primary virulence determinants, mainly acting by impairing cellulose synthesis, and their production in S. scabiei is in turn boosted by the cello-oligosaccharides released from host plants. In this work we aimed to determine which molecules and which biosynthetic gene clusters (BGCs) of the specialized metabolism of S. scabiei 87-22 show a production and/or transcriptional response to cello-oligosaccharides. Comparative metabolomic and transcriptomic analyses revealed that molecules of the virulome of S. scabiei induced by cellobiose and cellotriose include i) thaxtomins and concanamycins phytotoxins (and to a lesser extent N-coronafacoyl-L-isoleucine), ii) desferrioxamines, scabichelin and turgichelin siderophores in order to acquire iron essential for housekeeping functions, iii) ectoine for protection against osmotic shock once inside the host, and iv) bottromycins and concanamycins antimicrobials possibly to prevent other microorganisms from colonizing the same niche. Importantly, both cell-oligosaccharides reduced the production of the spore germination inhibitors germicidins and the plant growth regulators rotihibins. The metabolomic study also revealed that cellotriose is in general a more potent elicitor of the virulome compared to cellobiose. This result supports an earlier hypothesis that suggested that the trisaccharide would be the real virulence-triggering factor released from the plant cell wall through the action of thaxtomins. Interestingly, except for thaxtomins, none of these BGCs expression seems to be under direct control of the cellulose utilization repressor CebR suggesting the existence of another master regulator sensing the internalization of cello-oligosaccharides. Finally, we found nine additional cryptic and orphan BGCs that have their expression awakened by cello-oligosaccharides, demonstrating that other and yet to be discovered metabolites are part of the virulome of S. scabiei.

Discovery of a Diverse Set of Bacteria That Build Their Cell Walls without the Canonical Peptidoglycan Polymerase aPBP

Peptidoglycan (PG) is a large, cross-linked polymer that forms the cell wall of most bacterial species and confers shape, rigidity, and protection from osmotic shock. It is also a potent stimulator of the immune response in animals.