Ideonella alba sp. nov. and Ideonella aquatica sp. nov. isolated from an aquaculture farm.

The three novel bacterial strains designated as 3Y2T, 4Y16 and 4Y11T were isolated from an aquaculture farm and characterized using a polyphasic taxonomic approach. These strains were determined to be catalase- and oxidase-positive and to hydrolyze gelatin and aesculin. The results of 16S rRNA gene-based phylogenetic analysis indicated that the three strains were related to members of the genus Ideonella. The phylogenomic results further indicated that the three strains formed two independent branches distinct from reference type strains within this genus. The digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) and average amino acid identity (AAI) values between the three strains and their relatives were far below the thresholds of 70 % dDDH, 95-96 % ANI and 95 % AAI for species definition, respectively, indicating that the three strains represent two novel genospecies. The results of chemotaxonomic characterization indicated that the major cellular fatty acids of the three strains were summed feature 3 (C16 : 1ω6c and/or C16 : 1 ω7c) and C16 : 0; the common main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol; the respiratory quinone was ubiquinone-8. The genomic DNA G+C contents of the three strains were 70.2, 70.1 and 69.7%, respectively. On the basis of the different genotypes and distinctive phenotypes such as the phosphatidylcholine and glycolipid only in 3Y2T and the utilization of malic acid and trisodium citrate only in 4Y11T, strains 3Y2T and 4Y11T are concluded to represent two novel species of the genus Ideonella, for which the names Ideonella alba sp. nov. (type strain 3Y2T = GDMCC 1.2584T = KCTC 82813T) and Ideonella aquatica sp. nov. (type strain 4Y11T = GDMCC 1.1935T = JCM 34285T) are proposed.

Recent developments in sustainably sourced protein-based biomaterials.

Research into the development of sustainable biomaterials is increasing in both interest and global importance due to the increasing demand for materials with decreased environmental impact. This research field utilises natural, renewable resources to develop innovative biomaterials. The development of sustainable biomaterials encompasses the entire material life cycle, from desirable traits, and environmental impact from production through to recycling or disposal. The main objective of this review is to provide a comprehensive definition of sustainable biomaterials and to give an overview of the use of natural proteins in biomaterial development. Proteins such as collagen, gelatin, keratin, and silk, are biocompatible, biodegradable, and may form materials with varying properties. Proteins, therefore, provide an intriguing source of biomaterials for numerous applications, including additive manufacturing, nanotechnology, and tissue engineering. We give an insight into current research and future directions in each of these areas, to expand knowledge on the capabilities of sustainably sourced proteins as advanced biomaterials.

Gelatin expression from an engineered Saccharomyces cerevisiae CUP1 promoter in Pichia pastoris.

The methylotrophic yeast Pichia pastoris (reclassified as Komagataella phaffii) is a versatile protein expression system, yet many commonly used promoters have attributes undesirable for fermentation or its optimization. Hence, the copper-inducible CUP1 gene promoter from the related yeast Saccharomyces cerevisiae was used to express human gelatin. Multimerization of a potential copper response element in the CUP1 promoter, a S. cerevisiae Ace1p binding site, significantly increased gelatin expression. Expression was induced by copper in a dose-dependent fashion and was not dependent on cell density. Gelatin was additionally induced in standard copper-containing fermentation basal salts media. Removal of a S. cerevisiae heat shock factor (Hsf1p) binding site reduced copper-dependent gelatin induction suggesting that a similar protein may regulate this promoter in P. pastoris. This engineered copper inducible promoter expands the yeast recombinant protein production tool kit.

Corneal Opacity Induced by Light in a Mouse Model of Gelatinous Drop-Like Corneal Dystrophy.

Gelatinous drop-like corneal dystrophy (GDLD) is a severe inherited corneal dystrophy characterized by subepithelial corneal amyloid deposition. We had previously succeeded in identifying the responsible gene, TACSTD2, and subsequently found that the epithelial barrier function is significantly decreased. As with GDLD patients, the knockout mice showed severe loss of tight junction, progressive opacity, and neovascularization in the cornea. We devised an easy method to confirm the loss of the corneal barrier function even before corneal opacity is observed. Furthermore, by using knockout mice, we were able to verify clinical findings, such as the wound healing delay and light-induced acceleration of the disease. This mouse model should prove to be a highly useful tool for investigating the pathology of GDLD and for developing new therapies.

Morphology, structure, properties and applications of starch ghost: A review.

Starch ghost, an insoluble structure of gelatinized starch, plays an important role in the applications of starch. In this review, we summarized the preparation, morphology, structure, properties and applications of starch ghost. The preparation steps of starch ghost include gelatinization, purification and preservation, and many factors influence the yield of starch ghost. The morphology and content of starch ghost can be influenced by many factors like starch resource and amylose content. Ghosts from non-waxy starches are composed of amylopectin with long branch-chains and amylose. These molecules cross-link to each other to reinforce the structure, and tend to form B-type double helix in ghosts from high-amylose starches. Some surface proteins that bind tightly to starch granules are also present in starch ghost. Protein and lipid are thought to have limited effects on the structural stability, but they make a big difference in the morphology of starch ghost. Starch ghost shows a different resistance to amylase among various starches, but it can be further digested under the high shear force. The mechanical, enzymatic hydrolysis and electrochemical properties of starch ghost make it widely used as emulsifier, stabilizer, thickener and starch-based films or gels in food and non-food processing industries.

Development and verification of a quantitative real-time PCR method to identify and quantify gelatin derived from animal hide.

The species origin of hide gelatin is a crucial issue with respect to health concerns and religious restrictions. Analysis of the animal-derived ingredients of gelatin by reliable methods is necessary to ensure its authenticity. However, due to the highly processed nature of gelatin, it remains a challenge to identify gelatin end products accurately and robustly. Our study established and verified a quantitative real-time PCR (qPCR) method based on careful selection of target genes and a DNA extraction method. The middle products of the gelatin production streamline were investigated to explore the influence of each critical processing step on the method. Gelatin reference samples were used to quantify the levels of target species. Commercial gelatin commodities were surveyed to highlight the mislabeling situation. In summary, the qPCR method was demonstrated to be highly specific and sensitive, with limits of detection (LOD) of 0.1 to 1 pg/µL and gelatin LODs of 0.1% to 5% (w/w). The transition from decoction to concentrated gel was found to have the most severe effect on the qPCR. Intensification of pressure or temperature or employment of enzyme hydrolysis aggravated the DNA damage, resulting in elevated Cq values. Quantitation of gelatin products was feasible; gelatin products produced from 5% target hide and 95% matrix hide mixtures showed 2.9% to 5.2% target species. The 26% relative error for low gelatin content is acceptable for semiquantitation purposes. A market survey showed that 52.6% of the gelatin products were mislabeled as being of animal origin.

Heterologous Expression of Recombinant Transglutaminase in Bacillus subtilis SCK6 with Optimized Signal Peptide and Codon, and Its Impact on Gelatin Properties.

Microbial transglutaminases (MTGs) are widely used in the food industry. In this study, the MTG gene of Streptomyces sp. TYQ1024 was cloned and expressed in a food-grade bacterial strain, Bacillus subtilis SCK6. Extracellular activity of the MTG after codon and signal peptide (SP Ync M) optimization was 20 times that of the pre-optimized enzyme. After purification, the molecular weight of the MTG was 38 kDa and the specific activity was 63.75 U/mg. The optimal temperature and pH for the recombinant MTG activity were 50°C and 8.0, respectively. MTG activity increased 1.42- fold in the presence of ß-ME and 1.6-fold in the presence of DTT. Moreover, 18% sodium chloride still resulted in 83% enzyme activity, which showed good salt tolerance. Cross-linking gelatin with the MTG increased the strength of gelatin 1.67 times and increased the thermal denaturation temperature from 61.8 to 75.8°C. The MTG also significantly increased the strength and thermal stability of gelatin. These characteristics demonstrated the huge commercial potential of MTG, such as for applications in salted protein foods.

Tks5 SH3 domains exhibit differential effects on invadopodia development.

The Src substrate Tks5 helps scaffold matrix-remodeling invadopodia in invasive cancer cells. Focus was directed here on how the five SH3 domains of Tks5 impact that activity. Mutations designed to inhibit protein-protein interactions were created in the individual SH3 domains of Tks5, and the constructs were introduced into the LNCaP prostate carcinoma cell line, a model system with intrinsically low Tks5 expression and which our lab had previously showed the dependence of Src-dependent Tks5 phosphorylation on invadopodia development. In LNCaP cells, acute increases in wild-type Tks5 led to increased gelatin matrix degradation. A similar result was observed when Tks5 was mutated in its 4th or 5th SH3 domains. This was in contrast to the 1st, 2nd, and 3rd SH3 domain mutations of Tks5 where each had a remarkable accentuating effect on gelatin degradation. Conversely, in the invadopodia-competent Src-3T3 model system, mutations in any one of the first three SH3 domains had a dominant negative effect that largely eliminated the presence of invadopodia, inhibited gelatin degradation activity, and redistributed both Src, cortactin, and Tks5 to what are likely endosomal compartments. A hypothesis involving Tks5 conformational states and the regulation of endosomal trafficking is presented as an explanation for these seemingly disparate results.

Hybridization between two high Arctic cetaceans confirmed by genomic analysis.

In 1990, a skull from a morphologically unusual Monodontid was found in West Greenland and collected for the Natural History Museum of Denmark, University of Copenhagen. From its intermediate morphology, the skull was hypothesized to be a beluga/narwhal hybrid. If confirmed, the specimen would, to our knowledge, represent the sole evidence of hybridization between the only two toothed whale species endemic to the Arctic. Here we present genome-wide DNA sequence data from the specimen and investigate its origin using a genomic reference panel of eight belugas and eight narwhals. Our analyses reveal that the specimen is a male, first-generation hybrid between a female narwhal and a male beluga. We use stable carbon and nitrogen isotope analysis to investigate the dietary niche of the hybrid and find a higher δ13C value than in both belugas and narwhals, suggesting a foraging strategy unlike either parental species. These results further our understanding of the interaction between belugas and narwhals, and underscore the importance of natural history collections in monitoring changes in biodiversity. In addition, our study exemplifies how recent major advances in population genomic analyses using genotype likelihoods can provide key biological and ecological insights from low-coverage data (down to 0.05x).

An optimized TaqMan real-time PCR method for authentication of ASINI CORII COLLA (donkey-hide gelatin).

In this study, probe/primers of high specificity and sensitivity were selected to analyze donkey-hide gelatin for donkey DNA and to look for horse, ox, and pig DNA as possible adulterants. The mitochondrial CO I genes in donkey, horse, and ox were selected as target sequences for design and synthesis of three pairs of specific probes and primers. In addition, eight pairs of probe/primers were obtained via literature search. Out of these eleven groups of probe/primers, those with the highest specificity and sensitivity were selected, which was fulfilled by the screening firstly with animal hide samples then the hide-glue samples. Other parameters that might affect detection specificity and efficiency-such as the amount of sampling and final concentration of primers-were also optimized. Replication tests were also conducted. The results showed that the selected probe/primers could accurately detect donkey DNA and horse, ox, and pig DNA in gelatin samples with good reproducibility. Analysis of four samples of on-market gelatin using this assay showed that two of the four samples indeed contained only donkey DNA, whereas the other two samples contained both donkey and horse DNA, indicating adulteration of these samples with horse hide. These results indicate that the TaqMan probe real-time PCR method can be used for identifying the purity of donkey DNA in gelatin samples, and can provide technical support for identifying adulterations in the gelatin market.

Novel multiplex PCR-RFLP assay discriminates bovine, porcine and fish gelatin substitution in Asian pharmaceuticals capsule shells.

Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus' consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.

Rapid detection of collagens using a closed-tube LAMP method.

Identification methods of collagens and gelatins have been studied many years due to religious and food safety issues. Some researchers detected the collagen while others took up their study based on DNA at the first time. In this work, we used a closed-tube loop-mediated isothermal amplification (LAMP) technique to differentiate collagen and gelatin samples. DNA was extracted by DNeasy mericon Food Kit and was dissolved in 30 µl elution buffer, optimum concentration of Mg2+, deoxyribonucleoside triphosphates(dNTPs), betaine in LAMP reaction is 6.0 mmol/L, 2.0 mmol/L, and 0.8 mmol/L, respectively. After LAMP reaction, samples being detected changed their initial color to green, others' were colorless or brown slightly. The research offered a simple, fast detection technique to differentiate collagen and gelatin samples derived from porcine, bovine and channel catfish (Ictalurus punctatus) , the collagens' species can be determined by color variation in reaction tubes within two hour.

Molecular beacon-based real-time PCR method for detection of porcine DNA in gelatin and gelatin capsules.

BACKGROUND: The pharmaceutical industry has boosted gelatin consumption worldwide. This is supported by the availability of cost-effective gelatin production from porcine by-products. However, cross-contamination of gelatin materials, where porcine gelatin was unintentionally included in the other animal sources of gelatin, has caused significant concerns about halal authenticity. The real-time polymerase chain reaction (PCR) has enabled a highly specific and sensitive animal species detection method in various food products. Hence, such a technique was employed in the present study to detect and quantify porcine DNA in gelatin using a molecular beacon probe, with differences in performance between mitochondrial (cytochrome b gene) and chromosomal DNA-(MPRE42 repetitive element) based porcine-specific PCR assays being compared. RESULTS: A higher sensitivity was observed in chromosomal DNA (MPRE-PCR assay), where this assay allows the detection of gelatin DNA at amounts as as low as 1 pg, whereas mitochondrial DNA (CBH-PCR assay) can only detect at levels down to 10 pg of gelatin DNA. When an analysis with commercial gelatin and gelatin capsule samples was conducted, the same result was observed, with a significantly more sensitive detection being provided by the repetitive element of chromosomal DNA. CONCLUSION: The present study has established highly sensitive DNA-based porcine detection systems derived from chromosomal DNA that are feasible for highly processed products such as gelatin and gelatin capsules containing a minute amount of DNA. This sensitive detection method can also be implemented to assist the halal authentication process of various food products available on the market. © 2018 Society of Chemical Industry.

Gelatin/carboxymethyl cellulose mucoadhesive films with lysozyme: Development and characterization.

The goal of our study is to develop and characterize mucoadhesive films with entrapped lysozyme based on gelatin/sodium carboxymethyl cellulose as perspective antimicrobial preparation. Lysozyme in mucoadhesive films retains more than 95% of its initial activity for 3 years of storage. Different physical-chemical and biochemical characteristics of entrapped enzyme were evaluated, such as film thickness, weight, time of dissolution in water, bioadhesive force, in vitro lysozyme release, pH- and thermoprofiles of hydrolytic activity, effect of γ-sterilization, etc. We have shown that gelatin/sodium carboxymethyl cellulose films have adhesive force on the level of 4380Pa. Scanning electron microscopy images shows the relative uniformity of the gelatin surface with entrapped lysozyme. Mucoadhesive films with lysozyme have 100% bactericidal effect on the test strain, Staphylococcus aureus ATCC 25923 F-49 and thus could be considered as a perspective antimicrobial preparation.

Effects of substrate stiffness on adipogenic and osteogenic differentiation of human mesenchymal stem cells.

Substrate mechanical properties, in addition to biochemical signals, have been shown to modulate cell phenotype. In this study, we inspected the effects of substrate stiffness on human mesenchymal stem cells (hMSCs) derived from adult human bone marrow differentiation into adipogenic and osteogenic cells. A chemically modified extracellular matrix derived and highly biocompatible hydrogel, based on thiol functionalized hyaluronic acid (HA-SH) and thiol functionalized recombinant human gelatin (Gtn-SH), which can be crosslinked by poly (ethylene glycol) tetra-acrylate (PEGTA), was used as a model system. The stiffness of the hydrogel was controlled by adjusting the crosslinking density. Human bone marrow MSCs were cultured on the hydrogels with different stiffness under adipogenic and osteogenic conditions. Oil Red O staining and F-actin staining were applied to assess the change of cell morphologies under adipogenic and osteogenic differentiation, respectively. Gene expression of cells was determined with reverse transcription polymerase chain reaction (RT-PCR) as a function of hydrogel stiffness. Results support the hypothesis that adipogenic and osteogenic differentiation of hMSCs are inclined to occur on substrate with stiffness similar to their in vivo microenvironments.

Hyaluronic acid-recombinant gelatin gels as a scaffold for soft tissue regeneration.

An array of different types of hyaluronic acid (HA)- and collagen-based products is available for filling soft-tissue defects. A major drawback of the current soft-tissue fillers is their inability to induce cell infiltration and new tissue formation. Our aim is to develop novel biodegradable injectable gels which induce soft tissue regeneration, initially resulting in integration and finally replacement of the gel with new autologous tissue. Two reference gels of pure HA, monophasic HA-1 and micronised HA-2, were used. Furthermore, both gels were mixed with recombinant gelatin (RG) resulting in HA-1+RG and HA-2+RG. All gels were subcutaneously injected on the back of rats and explanted after 4 weeks. Addition of RG to HA-1 resulted in stroma formation (neovascularisation and ECM deposition) which was restricted to the outer rim of the HA-1+RG gel. In contrast, addition of RG to HA-2 induced stroma formation throughout the gel. The RG component of the gel was degraded by macrophages and giant cells and subsequently replaced by new vascularised tissue. Immunohistochemical staining showed that the extracellular matrix components collagen I and III were deposited throughout the gel. In conclusion, this study shows the proof of principle that addition of RG to HA-2 results in a novel injectable gel capable of inducing soft tissue regeneration. In this gel HA has a scaffold function whereas the RG component induces new tissue formation, resulting in proper vascularisation and integration of the HA-2+RG gel with the autologous tissue.

Detection of porcine DNA in gelatine and gelatine-containing processed food products-Halal/Kosher authentication.

A commercially available real-time PCR, based on a multi-copy target cytochrome b (cyt b) using porcine specific primers, has been validated for the Halal/Kosher authentication of gelatine. Extraction and purification of DNA from gelatine were successfully achieved using the SureFood® PREP Animal system, and real-time PCR was carried out using SureFood® Animal ID Pork Sens kit. The minimum level of adulteration that could be detected was 1.0% w/w for marshmallows and gum drops. A small survey was undertaken of processed food products such as gum drops, marshmallows and Turkish delight, believed to contain gelatine. Of fourteen food products from Germany, two samples were found to contain porcine gelatine, whereas of twenty-nine samples from Turkey twenty-eight were negative. However, one product from Turkey contained porcine DNA and thus was not Halal, and neither was the use of porcine gelatine indicated on the product label.

New strategy for expression of recombinant hydroxylated human-derived gelatin in Pichia pastoris KM71.

Gelatin is a well-known biopolymer, and it has a long history of use mainly as a gelling agent in the food industry. This paper reports a new method for producing recombinant hydroxylated human-derived gelatin in Pichia pastoris KM71. Three independent expression cassettes encoding for specific length of gelatin, prolyl 4-hydroxylase (P4H, EC 1.14.11.2), α-subunit (αP4H), and protein-disulfide isomerase (PDI) were individually cloned in one expression vector, pPIC9K. The modified gelatin gene and two subunit genes of P4H were under the control of two different inducible promoters, namely, alcohol oxidase 1 promoter (PAOX1) and formaldehyde dehydrogenase 1 promoter (PFLD1), respectively. The results of sodium dodecylsulfate-polyacrylamide gel electrophoresis show that a recombinant gelatin was successfully expressed in P. pastoris KM71 by methanol induction. Liquid chromatography coupled with tandem mass spectrometry analysis indicates that the expressed gelatin was hydroxylated with approximately 66.7% of proline residues in the Y positions of Gly-X-Y triplets. The results of nuclear magnetic resonance spectroscopy of recombinant gelatin test show that the (1)H and (13)C spectra have many corresponding characteristic displacement peaks, and amino acids composition analysis shows that it contains hydroxyproline and its UV absorption is consistent with the characteristics of gelatin.

Clinical and basic aspects of gelatinous drop-like corneal dystrophy.

Gelatinous drop-like corneal dystrophy (GDLD) was first reported in 1914 as a peculiar corneal dystrophy with an autosomal recessive inheritance mode. GDLD is rare in many countries, but relatively prevalent in Japan. The typical finding of GDLD is grayish, mulberry-like, protruding subepithelial depositions with a prominent hyperfluorescence of the cornea. Histologically, GDLD corneas are characterized by subepithelial amyloid depositions that were identified as lactoferrin by amino acid sequencing analysis. In 1998, the TACSTD2 gene was identified as a causative gene for this disease through a linkage analysis and a candidate gene approach. To date, 14 reports have demonstrated 21 mutations comprised of 9 missense, 6 nonsense, and 6 frameshift mutations from 9 ethnic back grounds. Currently, it is hypothesized that the loss of TACSTD2 gene function causes decreased epithelial barrier function, thereby facilitating tear fluid permeation into corneal tissue, the permeated lactoferrin then transforming into amyloid depositions via an unknown mechanism. For the visual rehabilitation of patients with GDLD, ophthalmologists currently employ various types of keratoplasties; however, almost all patients will experience a recurrence of the disease within a few years after such interventions. Wearing of a soft contact lens is sometimes considered as an alternative treatment for GDLD.

Nano self-assembly of recombinant human gelatin conjugated with α-tocopheryl succinate for Hsp90 inhibitor, 17-AAG, delivery.

A wide variety of drug delivery systems have been developed for the delivery of anticancer agents. One of the most frequently used natural biomaterials in drug delivery systems is polysaccharides; however, they are difficult to digest and to eliminate from the body after systemic administration due to their high molecular weight natures and the absence of degrading enzymes. Therefore, the development of degradable and eliminable natural biomaterials is critical for successful in vivo applications. In the present study, we report the development of self-assembled biodegradable nanoparticles based on recombinant human gelatin (rHG) modified with alpha-tocopheryl succinate (TOS). The rHG-TOS nanoparticles efficiently encapsulated 17-AAG (17-allylamino-17-demethoxygeldanamycin), a small molecular anticancer drug targeting heat shock protein 90. The formation of 17-AAG-loaded nanoparticles was confirmed using TEM and dynamic light scattering analysis and found to be within the size of 90-220 nm. The loading efficiency, sustained release pattern, and stability of 17-AAG from the rHG-TOS nanoparticles were determined using HPLC. Furthermore, the passive targeting of rHG-TOS nanoparticles to the tumor area via enhanced permeability and retention effect was examined by noninvasive live animal imaging in a tumor mouse model. Finally, the 17-AAG-loaded nanoparticles were nonimmunogenic and more efficient than free 17-AAG in manifesting an anticancer effect in the tumor model. Overall, our data demonstrate rHG-TOS as a promising tool for the delivery of 17-AAG featuring therapeutic efficacy and biocompatibility.