Extracellular matrix proteins: A positive feedback loop in lung fibrosis?

Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the development of bleomycin-induced lung fibrosis. We further report in vitro experiments clarifying both the effect of myofibroblast differentiation on this expression and the effect of extracellular elastin on myofibroblast differentiation.Lung fibrosis was induced in female C57Bl/6 mice by bleomycin instillation. Animals were sacrificed at zero to five weeks after fibrosis induction. Collagen synthesized during the week prior to sacrifice was labeled with deuterium. After sacrifice, lung tissue was collected for determination of new collagen formation, microarray analysis, and histology. Human lung fibroblasts were grown on tissue culture plastic or BioFlex culture plates coated with type I collagen or elastin, and stimulated to undergo myofibroblast differentiation by 0–10 ng/ml transforming growth factor (TGF)β₁. mRNA expression was analyzed by quantitative real-time PCR.New collagen formation during bleomycin-induced fibrosis was highly correlated to gene expression of elastin, type V collagen and tenascin C. At the protein level, elastin, type V collagen and tenascin C were highly expressed in fibrotic areas as seen in histological sections of the lung. Type V collagen and tenascin C were transiently increased. Human lung fibroblasts stimulated with TGFβ₁ strongly increased gene expression of elastin, type V collagen and tenascin C. The extracellular presence of elastin increased gene expression of the myofibroblastic markers α smooth muscle actin and type I collagen.The extracellular matrix composition changes dramatically during the development of lung fibrosis. The increased levels of elastin, type V collagen and tenascin C are probably the result of increased expression by fibroblastic cells; reversely, elastin influences myofibroblast differentiation. This suggests a reciprocal interaction between fibroblasts and the extracellular matrix composition that could enhance the development of lung fibrosis.     

Advanced glycation end-products diminish tendon collagen fiber sliding

Connective tissue aging and diabetes related comorbidity are associated with compromised tissue function, increased susceptibility to injury, and reduced healing capacity. This has been partly attributed to collagen cross-linking by advanced glycation end-products (AGEs) that accumulate with both age and disease. While such cross-links are believed to alter the physical properties of collagen structures and tissue behavior, existing data relating AGEs to tendon mechanics is contradictory. In this study, we utilized a rat tail tendon model to quantify the micro-mechanical repercussion of AGEs at the collagen fiber-level. Individual tendon fascicles were incubated with methylglyoxal (MGO), a naturally occurring metabolite known to form AGEs. After incubation in MGO solution or buffer only, tendons were stretched on the stage of a multiphoton confocal microscope and individual collagen fiber stretch and relative fiber sliding were quantified. Treatment by MGO yielded increased fluorescence and elevated denaturation temperatures as found in normally aged tissue, confirming formation of AGEs and related cross-links. No apparent ultrastructural changes were noted in transmission electron micrographs of cross-linked fibrils. MGO treatment strongly reduced tissue stress relaxation (p < 0.01), with concomitantly increased tissue yield stress (p < 0.01) and ultimate failure stress (p = 0.036). MGO did not affect tangential modulus in the linear part of the stress–strain curve (p = 0.46). Microscopic analysis of collagen fiber kinematics yielded striking results, with MGO treatment drastically reducing fiber-sliding (p < 0.01) with a compensatory increase in fiber-stretch (p < 0.01). We thus conclude that the main mechanical effect of AGEs is a loss of tissue viscoelasticity driven by matrix-level loss of fiber–fiber sliding. This has potentially important implications to tissue damage accumulation, mechanically regulated cell signaling, and matrix remodeling. It further highlights the importance of assessing viscoelasticity – not only elastic response – when considering age-related changes in the tendon matrix and connective tissue in general.     

Metabolic engineering of Saccharomyces cerevisiae for the biotechnological production of succinic acid

The production of bio-based succinic acid is receiving great attention, and several predominantly prokaryotic organisms have been evaluated for this purpose. In this study we report on the suitability of the highly acid- and osmotolerant yeast Saccharomyces cerevisiae as a succinic acid production host. We implemented a metabolic engineering strategy for the oxidative production of succinic acid in yeast by deletion of the genes SDH1, SDH2, IDH1 and IDP1. The engineered strains harbor a TCA cycle that is completely interrupted after the intermediates isocitrate and succinate. The strains show no serious growth constraints on glucose. In glucose-grown shake flask cultures, the quadruple deletion strain Δsdh1Δsdh2Δidh1Δidp1 produces succinic acid at a titer of 3.62 g L^?1 (factor 4.8 compared to wild-type) at a yield of 0.11 mol (mol glucose)^?1. Succinic acid is not accumulated intracellularly. This makes the yeast S. cerevisiae a suitable and promising candidate for the biotechnological production of succinic acid on an industrial scale.     

Isolation and partial characterization of collagen from outer skin of Sepia pharaonis (Ehrenberg, 1831) from Puducherry coast

Type I collagen from outer skin of Sepia pharaonis was extracted and partially characterized. Yield of Acid Soluble Collagen (ASC) and Pepsin Soluble Collagen (PSC) were calculated as 1.66% and 3.93% and the total protein content of ASC and PSC were found as 18.4% and 48.6%. FT-IR spectrum of ASC and PSC recorded 12 and 14 peaks, respectively. ¹H NMR spectrum of ASC showed singlets at 1.23 ppm, 3.1 ppm, 3.55 ppm and 3.7 ppm and PSC at 1.23 ppm and 2.08 ppm. The molecular weight for ASC was calculated as 102 kDa and for PSC as 110, 108 and 102 kDa through SDS-PAGE. Differential Scanning Calorimetry (DSC) results supported that PSC withstand high thermal stability (82.85 °C) than ASC (73.13 °C). Higher denaturation temperature with high molecular weight well support the property of type I collagen from skin of S. pharaonis and it could be used as another potent source for the extraction of collagen.     

Applying remote sensing techniques to monitoring seasonal and interannual changes of aquatic vegetation in Taihu Lake,China

Knowledge of the composition and areal distribution of aquatic vegetation types, as well as their seasonal and interannual variations, is crucial for managing and maintaining the balance of lake ecosystems. In this study, a series of remotely sensed images with a resolution of 30 m (HJ-CCD and Landsat TM) were collected and used to map the distribution of aquatic vegetation types in Taihu Lake, China. Seasonal and interannual dynamics of aquatic vegetation types were explored and analyzed. The distribution areas of Type I (emergent, floating-leaved and floating vegetation) and Type II (submerged vegetation) were used to model their growing season phenology by double logistic functions. The resulting double logistic models showed, the area of Type I reached its peak in mid-August, and the maximum area for Type II occurred in mid-September. From 1984 to 2013, Type I area increased continuously from 59.75 km² to 148.00 km² (R² = 0.84), whereas the area covered by Type II first increased and then decreased, with a trend conforming to a significant quadratic curve (R² = 0.83). The eutrophication and stable state of Taihu Lake was assessed using a simple indicator which was expressed as a ratio of Type II area to Type I area. The results showed that the eutrophication in the lake might have been increasing in the area studied since 2000. Additionally, the results showed that air temperature had likely a direct effect on the growth of Type I (R² = 0.66) and a significant, but delayed, effect on the growth of Type II.     

Isolation and characterization of collagen from the cartilage of Amur sturgeon (Acipenser schrenckii)

The collagen in Amur sturgeon cartilage was isolated using sodium chloride (salt-solubilized collagen, SSC, 2.18%), followed by acetic acid (acid-solubilized collagen, ASC, 27.04%) and then pepsin (pepsin-solubilized collagen, PSC, 55.92%). These collagens appeared to be dense sheet-like film linked by random-coiled filaments under SEM. The denaturation and melting temperatures of PSC (35.71 and 123.90 °C) were significantly higher than SSC (32.64 and 114.51 °C) and ASC (32.98 and 120.72 °C) assessed by circular dichroism and differential scanning calorimetry, which could be attributed to its high imino acid content (22.57%) and degree of hydroxylation (47.29%). Electrophoresis pattern showed that the SSC and ASC were type I collagen, while PSC was predominantly type II collagen along with other minor types. Infrared spectra confirmed their triple helical structure, and indicated more hydrogen bonding in ASC and more intermolecular crosslinks in PSC. These results provide some basis for their large-scale production and further application as alternatives to mammalian collagen.     

Unusual hepatic mitochondrial arginase in an Indian air-breathing teleost,Heteropneustes fossilis: Purification and characterization

A functional urea cycle with both cytosolic (ARG I) and mitochondrial (ARG II) arginase activity is present in the liver of an ureogenic air-breathing teleost, Heteropneustes fossilis. Antibodies against mammalian ARG II showed no cross-reactivity with the H. fossilis ARG II. ARG II was purified to homogeneity from H. fossilis liver. Purified ARG II showed a native molecular mass of 96 kDa. SDS–PAGE showed a major band at 48 kDa. The native enzyme, therefore, appears to be a homodimer. The pI value of the enzyme was 7.5. The purified enzyme showed maximum activity at pH 10.5 and 55 °C. The K_m of purified ARG II for l-arginine was 5.25 ± 1.12 mM. l-Ornithine and N^ω-hydroxy-l-arginine showed mixed inhibition with K_i values 2.16 ± 0.08 and 0.02 ± 0.004 mM respectively. Mn^+ 2 and Co^+ 2 were effective activators of arginase activity. Antibody raised against purified H. fossilis ARG II did not cross-react with fish ARG I, and mammalian ARG I and ARG II. Western blot with the antibodies against purified H. fossilis hepatic ARG II showed cross reactivity with a 96 kDa band on native PAGE and a 48 kDa band on SDS–PAGE. The molecular, immunological and kinetic properties suggest uniqueness of the hepatic mitochondrial ARG II in H. fossilis.     

Expression in SPARC-null mice of collagen type I lacking the globular domain of the α1(I) N-propeptide results in abdominal hernias and loss of dermal collagen

The sequence encoding the N-propeptide of collagen I is characterized by significant conservation of amino acids across species; however, the function of the N-propeptide remains poorly defined. Studies in vitro have suggested that one activity of this propeptide might be to act as a feedback inhibitor of collagen I synthesis. To determine whether the N-propeptide contributed to decreased collagen content in SPARC-null mice, mice carrying a deletion of exon 2, which encodes the globular domain of the N-propeptide of collagen I, were crossed to SPARC-null animals. Mice lacking SPARC and expressing collagen I without the globular domain of the N-propeptide were viable and fertile. However, a significant number of animals developed abdominal hernias within the first 2 months of life with an approximate 20% penetrance (~ 35% of males). The dermis of SPARC-null/exon 2-deleted mice was thinner and contained fewer large collagen fibers in comparison with wild-type or in either single transgenic animal. The average collagen fibril diameter of exon 2-deleted mice did not significantly differ from wild-type mice (WT: 87.9 nm versus exon 2-deleted: 88.2 nm), whereas SPARC-null/exon 2-deleted fibrils were smaller than that of SPARC-null dermis (SPARC-null: 60.2 nm, SPARC-null/exon 2-deleted: 40.8 nm). As measured by hydroxyproline analysis, double transgenic skin biopsies contained significantly less collagen than those of wild-type, those of exon 2-deleted, and those of SPARC-null biopsies. Acetic acid extraction of collagen from skin biopsies revealed an increase in the proportion of soluble collagen in the SPARC-null/exon 2-deleted mice. These results support a function of the N-propeptide of collagen I in facilitating incorporation and stabilization of collagen I into the insoluble ECM and argue against a primary function of the N-propeptide as a negative regulator of collagen synthesis.     

Stabilization of type I collagen using dialdehyde cellulose

Type I collagen from rat tail tendon (RTT) fibres was crosslinked with dialdehyde cellulose to bring about stabilization of the matrix. Dialdehyde cellulose (DAC) was prepared by periodate oxidation of hydrolyzed cellulose. Autoclaving of DAC resulted in hydrolysis and lower molecular weight oligomeric species. The formation of the crosslinked network between DAC and the collagen fibres has brought about significant thermal and enzymatic stability to collagen. DAC crosslinked collagen fibres exhibited an increase in hydrothermal stability by 20 °C with autoclaved DAC at pH 8. The collagen matrix resulted in an increase in denaturation peak temperature (T_D) and an increase in phase change of activation energy (E_a) and enthalpy change (ΔH) for the shinking process indicating intermolecular crosslinking arising from covalent interactions. Thermal stability and crosslinking efficiency was found to increase with pH and concentration of DAC. DAC treated collagen exhibited 93% resistance to collagenolytic hydrolysis.     

The vWFA2 domain of type VII collagen is responsible for collagen binding

Type VII collagen (Col7) is the major component of anchoring fibrils and very important for skin integrity. This is emphasized by the Col7 related skin blistering diseases dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita. Structural data that provides insights into the interaction network of Col7 and thus providing a basis for a better understanding of the pathogenesis of the diseases is missing.We proved that the von-Willebrand-factor A like domain 2 (vWFA2) of Col7 is responsible for type I collagen binding. The interaction has a K_D value of 90 μM as determined by SPR and is enthalpy driven as derived from the van’t Hoff equation. Furthermore, a hitherto unknown interaction of this domain with type IV collagen was identified. The interaction of vWFA2 with type I collagen is sensitive to the presence of magnesium ions, however, vWFA2 does not contain a magnesium binding site thus magnesium must bind to type I collagen.A lysine residue has been identified to be crucial for type I collagen binding. This allowed localization of the binding site. Mutational analysis suggests different interaction mechanisms in different species and that these interactions might be of covalent nature.     

Differential regulation of extracellular matrix constituents in myocardial remodeling with and without heart failure following pressure overload

Patients with aortic stenosis develop various degrees of myocardial hypertrophy and heart failure (HF) despite comparable transvalvular gradients. An important element in the transition from compensated hypertrophy to HF is dilatation of the left ventricle (LV). The molecular pathology associated with LV dilatation and development of HF is not known. Thus, we examined potential differences in the regulation of myocardial extracellular matrix (ECM) constituents in mice with hypertrophy only (ABnonHF) and with HF (ABHF) as response to comparable pressure overload. The ascending aorta was banded, or left loose in sham-operated mice. Increased lung weight and left atrial diameter indicating pulmonary congestion were used to identify ABHF mice. Cardiac function and geometry were evaluated by echocardiography. Despite comparable pressure gradients and cardiac output, ABHF had reduced fractional shortening (23%), reduced systolic (28%) and diastolic (32%) tissue velocity and increased LV internal dimension in diastole (10%) and systole (17%) (LVIDd/s) compared to ABnonHF (p ≤ 0.05). Microarray analyses identified 120 differently regulated genes related to ECM in ABHF compared to ABnonHF (p ≤ 0.05). Interestingly, in ABHF, we found a 24% (p ≤ 0.05) reduction of the LV collagen VIII protein levels despite increased levels of LV total collagen by 23% (p ≤ 0.05). LV collagen VIII correlated negatively with LVIDd (R = 0.55, p = 0.03) and LVIDs (R = 0.72, p = 0.002). As this protein may function as a “sealant” binding collagen fibrils together, reduction of collagen VIII could potentially contribute to LV dilatation and development of HF.     

Complete genome sequence,metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic,fast growing,xylose-utilizing bacterium

We have isolated a new extremely thermophilic fast-growing Geobacillus strain that can efficiently utilize xylose, glucose, mannose and galactose for cell growth. When grown aerobically at 72 °C, Geobacillus LC300 has a growth rate of 2.15 h⁻¹ on glucose and 1.52 h⁻¹ on xylose (doubling time less than 30 min). The corresponding specific glucose and xylose utilization rates are 5.55 g/g/h and 5.24 g/g/h, respectively. As such, Geobacillus LC300 grows 3-times faster than E. coli on glucose and xylose, and has a specific xylose utilization rate that is 3-times higher than the best metabolically engineered organism to date. To gain more insight into the metabolism of Geobacillus LC300 its genome was sequenced using PacBio׳s RS II single-molecule real-time (SMRT) sequencing platform and annotated using the RAST server. Based on the genome annotation and the measured biomass composition a core metabolic network model was constructed. To further demonstrate the biotechnological potential of this organism, Geobacillus LC300 was grown to high cell-densities in a fed-batch culture, where cells maintained a high xylose utilization rate under low dissolved oxygen concentrations. All of these characteristics make Geobacillus LC300 an attractive host for future metabolic engineering and biotechnology applications.     

Refolding,purification and characterization of an organic solvent-tolerant lipase from Serratia marcescens ECU1010

Expression of recombinant proteins as inclusion bodies in bacteria is one of the most efficient ways to produce cloned proteins, as long as the inclusion bodies can be successfully refolded. In this study, the different parameters were investigated and optimized on the refolding of denatured lipase. The maximum lipase activity of 5000 U/L was obtained after incubation of denatured enzyme in a refolding buffer containing 20 mM Tris–HCl (pH 7.0), 1 mM Ca²⁺ at 20 °C. Then, the refolded lipase was purified to homogeneity by anion exchange chromatography. The purified refolded lipase was stable in broad ranges of temperatures and pH values, as well as in a series of water-miscible organic solvents. In addition, some water-immiscible organic solvents, such as petroleum ether and isopropyl ether, could reduce the polarity and increase the nonpolarity of the refolding system. The results of Fourier transform infrared (FT-IR) microspectroscopy were the first to confirm that lipase refolding could be further improved in the presence of organic solvents. The purified refolded lipase could enantioselectively hydrolyze trans-3-(4-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM]. These features render the lipase attraction for biotechnological applications in the field of organic synthesis and pharmaceutical industry.     

Differences in the proportion of collagen and muscle in the canine lower urinary tract with regard to gonadal status and gender

Gonadectomy not only affects hormonal homeostasis but also alters the turnover of different components of the extracellular matrix in urogenital tissues. Collagen is an important component of the bladder and urethral walls and thus crucial for the mechanical properties of normal lower urinary tract (LUT) functions. This study aimed at investigating the possibility of differences in the proportion of collagen and muscle tissues in the LUT of intact and gonadectomised male and female dogs. Twenty clinically healthy dogs were used including 10 sexually intact dogs (5 males, 5 anoestrus females) and 10 gonadectomised dogs (4 males and 6 females). Four regions of the LUT, i.e. body and neck of the bladder as well as proximal and distal urethra were collected. The tissue sections were stained with Masson's Trichrome. Quantitative evaluation of the blue-stained area for collagen and red-counterstained area for muscle was performed using colour image analysis. The relative proportion of collagen and muscle significantly differed with the gonadal status, the gender and the region. Overall, gonadectomised dogs had a higher (P < 0.001) proportion of collagen and consequently a lower (P < 0.001) proportion of muscle than intact dogs. Regardless of gonadal statuses, females had a higher (P < 0.05) proportion of collagen and a lower (P < 0.05) proportion of muscle tissues than males. Gender differences were found in all four regions of the LUT in intact dogs but only in proximal urethra in gonadectomised dogs where spayed females had a higher (P < 0.05) proportion of collagen and less muscle (P < 0.05). Regional differences were observed in females; a higher proportion of collagen and therefore less muscle were found in the urethra compared with the bladder. Proportional differences in collagen and muscle between intact and gonadectomised animals suggest a relation of different hormonal statuses to structural changes in the canine LUT. Excessive collagen deposits and less muscular volume may impair structural and functional integrity of the LUT which may associate with the development of post-neutering urinary incontinence in the dog.     

Effect of ghrelin on chronic liver injury and fibrogenesis in male rats: Possible role of nitric oxide

Recent studies have revealed that ghrelin may be an antioxidant and anti-inflammatory agent in many organs, however its role in chronic liver injury (CLI) remains unclear. The role of nitric oxide (NO) in CLI is controversial as evidence suggests that NO is either a primary mediator of liver cell injury or exhibits a protective effect against injurious stimuli. Recent evidence demonstrated that the therapeutic potential for ghrelin was through eNOS activation and increase in NO production. However, its role on NO production in the liver has not been previously investigated. The aim of this study was to investigate the role of ghrelin in treatment of CLI, and whether this action is mediated through NO. Forty male rats were divided into four groups: Group I: Control; Group II: chronic liver injury (CLI); Group III: CLI + Ghrelin; and Group IV: CLI + Ghrelin + l-NAME. Liver enzymes and tumor necrosis factor alpha (TNF-α), were measured to assess hepatocellular injury. Liver tissue collagen content, malondialdehyde (MDA), gene expression of Bax, Bcl-2, and eNOS were assessed to determine the mechanism of ghrelin action. Results showed that ghrelin decreased serum liver enzymes and TNF-α levels. Ghrelin also reduced liver tissue collagen, MDA, and Bax gene expression, and increased Bcl-2 and eNOS gene expression. The effects on TNF-α, collagen, MDA, Bax, and eNOS were partially reversed in Group IV, suggesting that ghrelin's action could be through modulation of NO levels. Therefore, ghrelin's hepatoprotective effect is partially mediated by NO release.     

Purification,characterization and application of acidic lipase from Pseudomonas gessardii using beef tallow as a substrate for fats and oil hydrolysis

Beef tallow, a slaughter house waste was used as a substrate for lipase production, employing Pseudomonas gessardii. The strain, P. gessardii was isolated from the beef tallow acclimatized soil. The crude lipase activity at 139 U/ml by volume was obtained at optimized conditions of pH 5.0 and temperature of 37 °C. After purification, a 7.59-fold purity of lipase with specific activity of 1120 U/mg protein and molecular mass of 92 kDa was obtained. The purified lipase showed maximum activity and stability at pH 5.0 and 30 °C. Ca²⁺ had a stimulatory effect on the lipase activity compared to the other metal ions studied. The relative activity was enhanced with the addition of Triton X-100 with lower hydrophilic–lipophilic balance (HLB) value as 13.0 and DMSO with the lowest partition coefficient (log P) value, as 1.378. The amino acid composition and the functional groups of lipase were confirmed by HPLC and FT-IR spectroscopy. The purified lipase had the highest hydrolytic activity towards slaughterhouse wastes and vegetable oils. This work provides a potential biocatalyst for the wide applications in oleochemical and biotechnological industries.     

Purification and characterization of a highly selective glycyrrhizin-hydrolyzing β-glucuronidase from Penicillium purpurogenum Li-3

A novel β-glucuronidase from filamentous fungus Penicillium purpurogenum Li-3 was purified to electrophoretic homogeneity by ultrafiltration, ammonium sulfate precipitation, DEAE-cellulose ion exchange chromatography, and Sephadex G-100 gel filtration with an 80.7-fold increase in specific activity. The purified β-glucuronidase is a dimeric protein with an apparent molecular mass of 69.72 kDa (m/z = 69,717), determined by MALDI/TOF-MS. The optimal temperature and pH of the purified enzyme are 40 °C and 6.0, respectively. The enzyme is stable within pH 5.0–8.0, and the temperature up to 45 °C. Mg²⁺ ions enhanced the activity of the enzyme, Ca²⁺ and Al³⁺ showed no effect, while Mn²⁺, Zn²⁺, Hg²⁺ and Cu²⁺ substantially inhibited the enzymatic activity. The K_m and V_max values of the purified enzyme for glycyrrhizin (GL) were evaluated as 0.33 mM and 59.0 mmol mg^?1 min^?1, respectively. The purified enzyme displayed a highly selective glycyrrhizin-hydrolyzing property and converted GL directly to glycyrrhetic acid mono-glucuronide (GAMG), without producing byproduct glycyrrhetic acid (GA). The results suggest that the purified enzyme may have potential applications in bio-pharmaceutical and biotechnological industry.     

Isolation,purification and characterization of pepsin soluble collagen isolated from silvertip shark (Carcharhinus albimarginatus) skeletal and head bone

Type II pepsin soluble collagens (PSC) were isolated from skeletal and head bone of silvertip shark; and examined for their biochemical and structural properties. Among the raw materials, the protein content (8.99%) was high in skeletal bone and the ash content (28%) was high in head bone. After the collagen extraction, the raw materials contained higher amount of ash content ranging from 82 to 88%. The hydroxyproline content of skeletal and skeletal PSC (30 and 113 mg/g) was higher than those head and head PSC. Both collagens were composed of two different α-chains (α₁- and α₂-chains) and were characterized as type II collagen. Amino acid analysis of skeletal and head PSC indicated imino acid contents of 156 and 175 amino acid residues per 1000 residues, respectively. Similar, Fourier transform infrared spectra of SCII and HCII were observed, which suggested that the isolation process did not affect the secondary structure and molecular order of collagen, particularly the triple–helical structure. Denaturation temperature of skeletal PSC (31 °C) was higher than that of head PSC. SEM microstructure of the collagens depicted a porous, fibrillary and multi-layered structure. These results suggested that the PSC isolated from skeletal and head bone of silvertip shark were found to be suitable biomaterial in commercial applications as alternatives to mammalian collagen.     

Exploiting sequence and stability information for directing nanobody stability engineering

BackgroundVariable domains of camelid heavy-chain antibodies, commonly named nanobodies, have high biotechnological potential. In view of their broad range of applications in research, diagnostics and therapy, engineering their stability is of particular interest. One important aspect is the improvement of thermostability, because it can have immediate effects on conformational stability, protease resistance and aggregation propensity of the protein.MethodsWe analyzed the sequences and thermostabilities of 78 purified nanobody binders. From this data, potentially stabilizing amino acid variations were identified and studied experimentally.ResultsSome mutations improved the stability of nanobodies by up to 6.1 °C, with an average of 2.3 °C across eight modified nanobodies. The stabilizing mechanism involves an improvement of both conformational stability and aggregation behavior, explaining the variable degree of stabilization in individual molecules. In some instances, variations predicted to be stabilizing actually led to thermal destabilization of the proteins. The reasons for this contradiction between prediction and experiment were investigated.ConclusionsThe results reveal a mutational strategy to improve the biophysical behavior of nanobody binders and indicate a species-specificity of nanobody architecture.General significanceThis study illustrates the potential and limitations of engineering nanobody thermostability by merging sequence information with stability data, an aspect that is becoming increasingly important with the recent development of high-throughput biophysical methods.     

Horseradish peroxidase production from Spodoptera frugiperda larvae: A simple and inexpensive method

Horseradish peroxidase is used in many biotechnological fields including diagnostics, biocatalysts and biosensors. Horseradish peroxidase isozyme C (HRPC) was extracellularly expressed in Spodoptera frugiperda Sf9 cell culture and in intact larvae. At day 6 post-infection, the concentration of active HRPC in suspension cultures was 3.0 ± 0.1 μg per 1 × 10⁶ cells or 3.0 ± 0.1 mg l⁻¹ with a multiplicity of infection of 1 in the presence of 7.2 μM hemin. Similar yields were obtained in monolayer cultures. In larvae, the HRPC expression level was 137 ± 17 mg HRPC kg⁻¹ larvae at day 6 post-infection with a single larvae thus producing approximately 41 μg HRPC. The whole larval extract was separated by ion exchange chromatography and HRPC was purified in a single step with a yield of 75% and a purification factor of 117. The molecular weight of recombinant HRPC was 44,016 Da, and its glycosylation pattern agreed with that expected for invertebrates. The K_m and V_max were 12.1 ± 1.7 mM and 2673 ± 113 U mg⁻¹, respectively, similar to those of HRP purified from Armoracia rusticana roots. The method described in this study, based on overexpression of HRPC in S. frugiperda larvae, is a simple and inexpensive way to obtain high levels of active enzyme for research and other biotechnological applications.