Mammalian Sources Research Articles

Highly Efficient Methods to Culture Mouse Cholangiocytes and Small Intestine Organoids.

BackgroundOrganoids, which enable disease modeling and drug screening closer to an in vivo environment, can be isolated and grown from organs such as the brain, small intestine, kidney, lungs, and liver. To facilitate the establishment of liver and small intestinal organoids, we developed efficient protocols for cholangiocytes and intestine crypts collecting and organoid culturing.MethodsCholangiocytes were collected from intrahepatic bile ducts, the gallbladder, and small intestine crypts by gravity settling and multistep centrifugation methods. The cells isolated were embedded with Matrigel and grew in three-dimensional spheroids in a suitable culture medium. The stability of organoid cells was assessed by subculture, cryopreservation, and thawing. RNA and DNA extraction of organoids, as well as immunostaining procedure, were also optimized. Hand-picking procedures were developed and performed to ensure similar growth characteristics of organoids.ResultsA large number of cholangiocytes and small intestine crypts were collected under these protocols. Cholangiocytes developed into cyst-like structures after 3–4 days in Matrigel. After 1–2 weeks of cultivation, small intestinal organoids (in-orgs) developed buds and formed a mature structure. Compared to organoids derived from the gallbladder, cholangiocyte organoids (Cho-orgs) from intrahepatic the bile ducts grew more slowly but had a longer culture term, expressed the cholangiocytes markers Krt19 and Krt7, and recapitulated in vivo tissue organization.ConclusionsOur protocols simplified the cell collection procedure and avoided the possibility of exposing tissue-derived stem cells to mechanical damage or chemical injury by gravity settling and multistep centrifugation. In addition, our approach allowed similar growth characteristics of organoids from different mammalian tissue sources. The protocol requires 2–4 weeks to establish a stable organoid growth system. Organoids could be stably passaged, cryopreserved, and recovered under protocol guidance. Besides, the organoids of cholangiocytes and small intestines retained their original tissue characteristics, such as tissue-specific marker expression, which prepares them for further experiments such as preclinical in vitro trials and mechanism research studies.

The Production of Gelatin from Snapper Scales (Lutjanus camphecanus Sp.) through Enzymatic Pretreatment

Fish wastes such as fish bones and scales have a potential to be used as a gelatin source. It can be used as an alternative material to replace pork or bovine which ordinarily conflicted with religion issue. Gelatin from fish has different characteristics compared with mammalian source. It has several advantageous properties such as high amount of hydrophobic amino acids, less proline and hydroxy proline, lower gelling ability, and melting point. In this study, gelatin was produced from fish waste especially snapper scales using the bromelain enzyme at the pre-treatment stage to improve its characteristic. The snapper scales soaking in hot water at 80⁰C for about 30 minutes to remove the fat which attached the scales. A pre-treatment has been conducted using bromelain enzyme solution at various concentrations of 1%, 2%, 3%, 4% and 5% for 6 hours to remove non-collagenous proteins and loosen the bonds of the fish scales. The next step is demineralization using a 15% concentration of citric acid solution for 3 days. The ossein formed in the demineralization stage was then hydrolyzed at 50⁰C and 70⁰C for 9 hours with stirring at 300 rpm. The best gelatin yield was obtained from the pretreatment of bromelain enzyme solution with a concentration of 3% at a hydrolysis temperature of 70⁰C with a yield of 9.13%, pH value of 3.86, water content of 8%, ash content of 0.80%. FTIR spectra shows the presence of functional groups correspond to gelatin formation, such as carbon, hydrogen, hydroxyl group (O-H), carbonyl group (C=O), amine group (N-H) and alkene group (C=C). The heavy metal content test showed that the gelatin contained no heavy metal content of lead (Pb), mercury (Hg), cadmium (Cd) and arsenic (As).

Fish Gelatin: Current Nutritional, Medicinal, Tissue Repair Applications, and as a Carrier of Drug Delivery.

Gelatin is obtained via partial denaturation of collagen and is extensively used in various industries. The majority of gelatin utilized globally is derived from a mammalian source. Several health and religious concerns associated with porcine/bovine gelatin have been reported. Therefore, gelatin from a marine source is widely being investigated for its efficiency and utilization in a variety of applications as a potential substitute for porcine/bovine gelatin. Although fish gelatin is less durable and possesses lower melting and gelling temperatures compared to mammal-derived gelatin, various modifications have been reported to promote its rheological and functional properties to be efficiently employed. The present review describes in detail the current innovative applications of fish gelatin involving the food industry, drug delivery, and possible therapeutic applications. Gelatin bioactive molecules may be utilized as carriers for drug delivery. Due to its versatility, gelatin can be used in different carrier systems, such as microparticles, nanoparticles, fibers, and hydrogels. The present review also provides a perspective on the other potential pharmaceutical applications of fish gelatin, such as tissue regeneration, antioxidant supplementation, and antihypertensive and anticancer treatments.

Alternatives to Cow's Milk-Based Infant Formulas in the Prevention and Management of Cow's Milk Allergy.

Cow’s milk-based infant formulas are the most common substitute to mother’s milk in infancy when breastfeeding is impossible or insufficient, as cow’s milk is a globally available source of mammalian proteins with high nutritional value. However, cow’s milk allergy (CMA) is the most prevalent type of food allergy among infants, affecting up to 3.8% of small children. Hypoallergenic infant formulas based on hydrolysed cow’s milk proteins are commercially available for the management of CMA. Yet, there is a growing demand for more options for infant feeding, both in general but especially for the prevention and management of CMA. Milk from other mammalian sources than the cow, such as goat, sheep, camel, donkey, and horse, has received some attention in the last decade due to the different protein composition profile and protein amino acid sequences, resulting in a potentially low cross-reactivity with cow’s milk proteins. Recently, proteins from plant sources, such as potato, lentil, chickpeas, quinoa, in addition to soy and rice, have gained increased interest due to their climate friendly and vegan status as well as potential lower allergenicity. In this review, we provide an overview of current and potential future infant formulas and their relevance in CMA prevention and management.

Taxon-specific zeta-crystallin of camel eye lens: A comparative in silico studies

The eye lens is a specialized organ of the visual system, which is transparent due to its complex geometry and unique protein composition. Nature has designed it to focus light on the retina throughout the life span. Several factors (post-translational modifications, thermal and solar radiations) causes aggregation of the lens proteins and result in early-onset of cataract. A cataract is one of the major causes of blindness worldwide. It's interesting how camel eyes maintain lens transparency in a harsh desert climate? The camel eye lenses contain a novel protein zeta (ζ)-crystallin in a bulk amount that is also present in two other animals (guinea pig and Japanese frog) but is adopted for milder habitats. The camel lens ζ-crystallin is a poorly characterized protein. This study has investigated the physicochemical and structural properties of camel, guinea pig, and Japanese frog’s ζ-crystallin. The docking results showed a strong affinity between NADPH and ζ-crystallin. RMSD, RMSF, radius of gyration, and SASA analysis in the ligand-free and bound states showed distinct prope6rties in these ζ-crystallins. Moreover, the hydrophobicity of camel and Japanese frog ζ-crystallin is lower than ζ-crystallin of other mammalian sources. The unique physicochemical and structural properties of taxon-specific ζ-crystallin are likely to maintain lens transparency.

Extracellular vesicles in human milk.

Milk-derived extracellular vesicles (MDEVs) are nanovesicles that carry microRNA (miRNA) DNA, RNA, proteins and lipids. MDEVs have a potential of therapeutic targets, based on their properties and cargo profile. The present review summarizes recent studies on MDEVs, their cargo and potential role in mammalian development. The detailed characterization of their miRNA cargo leads to the conclusion of their potential importance in the regulation of gene expression, immune function, development and infant growth.While their miRNAs are important regulatory elements and their profile expression was characterized in various mammalian milk sources, little is known about their effect on infant health and development. MiRNA activity in breast milk is likely influenced by the overall ecosystem of the early environment, including maternal characteristics, behaviors, and health. MDEVs may have an important role in early child development and infant future health. Understanding benefits of MDEVs characteristics have potential role on gut maturation, immune system development and the prevention of metabolic disorders.

Repurposing fish waste into gelatin as a potential alternative for mammalian sources: A review.

Mammalian gelatin is extensively utilized in the food industry because of its physicochemical properties. However, its usage is restricted and essentially prohibited for religious people. Fish gelatin is a promising alternative with no religious and social restrictions. The desirable properties of fish gelatin can be significantly improved by various methods, such as the addition of active compounds, enzymes, and natural crosslinking agents (e.g., plant phenolics and genipin), and nonthermal physical treatments (e.g., ionizing radiation and high pressure). The aim of this study was to explore whether the properties of fish gelatin (gel strength, melting or gelling temperature, odor, viscosity, sensory properties, film-forming ability, etc.) could be improved to make it comparable to mammalian gelatin. The structure and properties of gelatins obtained from mammalian and fish sources are summarized. Moreover, the modification methods used to ameliorate the properties of fish gelatin, including rheological (gelling temperature from 13-19°C to 23-25°C), physicochemical (gel strengths from ∼200 to 250g), and thermal properties (melting points from ∼25 to 30°C), are comprehensively discussed. The relevant literature reviewed and the technological advancements in the industry can propel the development of fish gelatin as a potential alternative to mammalian gelatin, thereby expanding its competitive market share with increasing utility.

Effect of Mammalian Tissue Source on the Molecular and Macroscopic Characteristics of UV-Cured Type I Collagen Hydrogel Networks

The tissue source of type I collagen is critical to ensure scalability and regulation-friendly clinical translation of new medical device prototypes. However, the selection of a commercial source of collagen that fulfils both aforementioned requirements and is compliant with new manufacturing routes is challenging. This study investigates the effect that type I collagen extracted from three different mammalian tissues has on the molecular and macroscopic characteristics of a new UV-cured collagen hydrogel. Pepsin-solubilised bovine atelocollagen (BA) and pepsin-solubilised porcine atelocollagen (PA) were selected as commercially available raw materials associated with varying safety risks and compared with in-house acid-extracted type I collagen from rat tails (CRT). All raw materials displayed the typical dichroic and electrophoretic characteristics of type I collagen, while significantly decreased lysine content was measured on samples of PA. Following covalent functionalisation with 4-vinylbenzyl chloride (4VBC), BA and CRT products generated comparable UV-cured hydrogels with significantly increased averaged gel content (G ≥ 97 wt.%), while the porcine variants revealed the highest swelling ratio (SR = 2224 ± 242 wt.%) and an order of magnitude reduction in compression modulus (Ec = 6 ± 2 kPa). Collectively, these results support the use of bovine tissues as a chemically viable source of type I collagen for the realisation of UV-cured hydrogels with competitive mechanical properties and covalent network architectures.

Artificial Cornea: Past, Current, and Future Directions.

Corneal diseases are a leading cause of blindness with an estimated 10 million patients diagnosed with bilateral corneal blindness worldwide. Corneal transplantation is highly successful in low-risk patients with corneal blindness but often fails those with high-risk indications such as recurrent or chronic inflammatory disorders, history of glaucoma and herpetic infections, and those with neovascularisation of the host bed. Moreover, the need for donor corneas greatly exceeds the supply, especially in disadvantaged countries. Therefore, artificial and bio-mimetic corneas have been investigated for patients with indications that result in keratoplasty failure. Two long-lasting keratoprostheses with different indications, the Boston type-1 keratoprostheses and osteo-odonto-keratoprostheses have been adapted to minimise complications that have arisen over time. However, both utilise either autologous tissue or an allograft cornea to increase biointegration. To step away from the need for donor material, synthetic keratoprostheses with soft skirts have been introduced to increase biointegration between the device and native tissue. The AlphaCor™, a synthetic polymer (PHEMA) hydrogel, addressed certain complications of the previous versions of keratoprostheses but resulted in stromal melting and optic deposition. Efforts are being made towards creating synthetic keratoprostheses that emulate native corneas by the inclusion of biomolecules that support enhanced biointegration of the implant while reducing stromal melting and optic deposition. The field continues to shift towards more advanced bioengineering approaches to form replacement corneas. Certain biomolecules such as collagen are being investigated to create corneal substitutes, which can be used as the basis for bio-inks in 3D corneal bioprinting. Alternatively, decellularised corneas from mammalian sources have shown potential in replicating both the corneal composition and fibril architecture. This review will discuss the limitations of keratoplasty, milestones in the history of artificial corneal development, advancements in current artificial corneas, and future possibilities in this field.

Scalable Nucleic Acid Storage and Retrieval Using Barcoded Microcapsules.

Rapid advances in nucleic acid sequencing and synthesis technologies have spurred a major need to collect, store, and sequence the DNA and RNA from viral, bacterial, and mammalian sources and organisms. However, current approaches to storing nucleic acids rely on a low-temperature environment and require robotics for access, posing challenges for scalable and low-cost nucleic acid storage. Here, we present an alternative method for storing nucleic acids, termed Preservation and Access of Nucleic aciDs using barcOded micRocApsules (PANDORA). Nucleic acids spanning kilobases to gigabases and from different sources, including animals, bacteria, and viruses, are encapsulated into silica microcapsules to protect them from environmental denaturants at room temperature. Molecular barcodes attached to each microcapsule enable sample pooling and subsequent identification and retrieval using fluorescence-activated sorting. We demonstrate quantitative storage and rapid access to targeted nucleic acids from a pool emulating standard retrieval operations implemented in conventional storage systems, including recovery of 100,000-200,000 samples and Boolean logic selection using four unique barcodes. Quantitative polymerase chain reaction and short-read sequencing of the retrieved samples validated the sorting experiments and the integrity of the released nucleic acids. Our proposed approach offers a scalable long-term, room-temperature storage and retrieval of nucleic acids with high sample fidelity.

Non culturable bacteria associated with COVID-19: More details are demanded

Coronavirus disease 2019 (COVID-19) is a zoonotic infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with the extreme acute respiratory syndrome. Coronavirus is a zoonotic infection with a positive polarity RNA envelope that belongs to the Coronaviridae family. There are four recognized genera of coronavirus, however on January 10, 2020, a new coronavirus arose in Wuhan, China, causing a serious pulmonary outbreak. Severe acute respiratory syndrome coronavirus 2 appears to be the third highly deadly human coronavirus to develop in the recent two decades, following SARS coronavirus and Middle East respiratory syndrome (MERS) coronavirus. Severe acute respiratory syndrome coronavirus 2 infection associated with a bacterial pathogen (combined viral and bacterial pneumonia) was described in COVID-19. Secondary bacterial pneumonia can develop after a viral respiratory illness has cleared up or during the healing period. In fatal instances, bacterial infections are more prevalent than in recovered patients. If bacterial infections are the cause of death in COVID-19, this has significant implications for patient treatment. The death rate for patients who acquired secondary infections was 56.7 %, compared to 10.6 % for all COVID-19 patients hospitalized. In 78 % of the cases, Gram-negative bacteria were found. The most common pathogen was Klebsiella pneumoniae (29 %), followed by Acinetobacter baumannii (21%). Pseudomonas aeruginosa (9.6%) and Escherichia coli (8.2%). methicillin-resistant Staphylococcus aureus was isolated only in 1.29%. High levels of carbapenem resistance were seen in Acinetobacter baumannii (92.6%) followed by Klebsiella pneumoniae (72.8%). According to recent research, bacterial co-infection occurred in 3.1–3.5% of COVID-19 patients at admission, with subsequent bacterial infections occurring in up to 15% of patients following hospitalization. Atypical organisms linked to COVID-19 are organisms that are difficult to grow and do not show up on a gram stain. Because antibiotics must be able to enter intracellularly to reach their intended target, they are difficult to separate and treat due to their intracellular nature. Because atypical organisms lack cell walls, such as Mycoplasma pneumoniae, the most prevalent, beta-lactam antibiotics are not indicated. Antibiotic resistance, poor compliance, and an inability to take oral medicines are all factors that contribute to treatment failure. Furthermore, some individuals may have obstructive lung lesions or a misdiagnosis. Atypical pneumonia can be contracted from a variety of causes. Mycoplasma pneumoniae, which is associated with close living conditions such as at school and military barracks, legionella from stagnant water sources, Chlamydophila pneumoniae, Coxiella brunette, and Francisella tularensis from various mammalian sources are among the most commonly identified atypical pathogens. Conclusion: Secondary infections in COVID-19 patients have been linked to poor health outcomes. Non-culturable bacteria have been associated with death in COVID-19 patients, and they may require a specific antibiotic to preserve the patient's life. More research is needed to validate this issue, as there is currently little evidence available.

Comparative study on the rheological properties of myofibrillar proteins from different kinds of meat

In this study, the gel properties of myofibrillar proteins (MPs) from four meat sources (fish, beef, sheep, and pork) were compared. Oscillatory rheology measurements including temperature sweep, frequency sweep, and strain sweep were conducted to characterise the small and large deformation rheological properties of the MPs. In addition, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscopy (SEM) were used to evaluate differences in the molecular weight distribution as well as the microstructures in gel among different MPs. Frequency sweep measurements showed that all MP gels were weak gels. MPs extracted from pork exhibited the highest gel strength and most compact gel structure, whereas those from fish exhibited the lowest gel strength and loosest gel structure. In addition, the MP extracted from pork (PSM) had the highest content of myosin heavy chain (MHC) and actin. In conclusion, the MPs extracted from fish source and mammalian sources varied significantly in terms of rheological properties and microstructural characteristics. These results provided useful information for developing mixed gel products with different gel strengths.

Synthesis and Application of Fish Gelatin for Hydrogels/ Composite Hydrogels: A Review

Hydrogels are one of the biopolymers that have been applied and have excellent potential to be developed as a raw material in future food technology, biomedicine, and three-dimensional (3D) bioprinting. Even stigmatized that hydrogels are the only source of bioink for 3D bioprinting. Among natural sources, protein-based hydrogels have advantages in the aspects of biocompatibility, biodegradability, tunability, molecular binding ability, and bioactive properties. Gelatin is a natural protein-based biopolymer that offers potential. Besides its advantages as a natural protein-based hydrogel, gelatin is also inexpensive, usually extracted from processing by-products such as skins and bones. Studies also mentioned that gelatin has the tripeptide motif that promotes cell attachment for subsequent cellular processes, like migration, differentiation, and proliferation. However, most gelatin is derived from mammalian sources, while these sources are limited considering socio-religion, cultural, health aspects. Fish gelatin is the most potential source for alternative gelatin. They have uniqueness and viscosity for bio-fabrication and injectable hydrogels. Therefore, this paper will review the hydrogels based on fish gelatin studied in recent years and the last decade. Here also described the stages in the fabrication of fish gelatin hydrogels/hydrogel composites with different co-polymers, composite materials, polymerization methods, and future intended use of obtained fish gelatin hydrogels/composites.

The two faces of cyanide: an environmental toxin and a potential novel mammalian gasotransmitter.

Cyanide is traditionally viewed as a cytotoxic agent, with its primary mode of action being the inhibition of mitochondrial Complex IV (cytochrome c oxidase). However, recent studies demonstrate that the effect of cyanide on Complex IV in various mammalian cells is biphasic: in lower concentrations (nanomolar to low micromolar) cyanide stimulates Complex IV activity, increases ATP production and accelerates cell proliferation, while at higher concentrations (high micromolar to low millimolar) it produces the previously known (‘classic’) toxic effects. The first part of the article describes the cytotoxic actions of cyanide in the context of environmental toxicology, and highlights pathophysiological conditions (e.g., cystic fibrosis with Pseudomonas colonization) where bacterially produced cyanide exerts deleterious effects to the host. The second part of the article summarizes the mammalian sources of cyanide production and overviews the emerging concept that mammalian cells may produce cyanide, in low concentrations, to serve biological regulatory roles. Cyanide fulfills many of the general criteria as a ‘classical’ mammalian gasotransmitter and shares some common features with the current members of this class: nitric oxide, carbon monoxide, and hydrogen sulfide.

Isolation and Culture of Neonatal Murine Primary Cardiomyocytes.

The cardiomyocyte is the main cell type in the heart responsible for its contractile function. Culturing primary cardiomyocytes from mammalian sources to study their function remains challenging as they are terminally differentiated and cease to multiply soon after birth. The major technical hurdles associated with primary cardiomyocyte culture include attaining high yields, obtaining healthy/viable cells that show spontaneous contractions upon culture, and avoiding contamination by non-myocyte cardiac cell types such as fibroblasts and endothelial cells. The yield and the quality of the cardiomyocytes obtained are impacted by a variety of factors, such as the purity of the reagents, composition of the digestion mixture, the digestion conditions, and the temperature of the tissue during different steps of isolation. Here, we provide a simplified workflow to isolate, culture, and maintain neonatal primary cardiomyocytes from rats/mice in culture dishes, which can then be used to study, for instance, cardiac hypertrophy and drug-induced cardiotoxicity. © 2021 Wiley Periodicals LLC. Basic Protocol: Isolation and culture of primary cardiomyocytes from rat/mouse pups Support Protocol: Coating of tissue culture plates with extracellular matrix substrates for efficient cardiomyocyte attachment.

Model-based control for column-based continuous viral inactivation of biopharmaceuticals.

Batch low-pH hold is a common processing step to inactivate enveloped viruses for biologics derived from mammalian sources. Increased interest in the transition of biopharmaceutical manufacturing from batch to continuous operation resulted in numerous attempts to adapt batch low-pH hold to continuous processing. However, control challenges with operating this system have not been directly addressed. This article describes a low-cost, column-based continuous viral inactivation system constructed with off-the-shelf components. Model-based, reaction-invariant pH controller is implemented to account for the nonlinearities with Bayesian estimation addressing variations in the operation. The residence time distribution is modeled as a plug flow reactor with axial dispersion in series with a continuously stirred tank reactor, and is periodically estimated during operation through inverse tracer experiments. The estimated residence time distribution quantifies the minimum residence time, which is used to adjust feed flow rates. Controller validation experiments demonstrate that pH and minimum residence time setpoint tracking and disturbance rejection are achieved with fast and accurate response and no instability. Viral inactivation testing demonstrates tight control of logarithmic reduction values over extended operation. This study provides tools for the design and operation of continuous viral inactivation systems in service of increasing productivity, improving product quality, and enhancing patient safety.

Synthesis and characterization of C2C12-laden gelatin methacryloyl (GelMA) from marine and mammalian sources

Gelatin methacryloyl (GelMA) is widely used for tissue engineering applications as an extracellular matrix (ECM) mimicking scaffold due to its cost-effectiveness, ease of synthesis, and high biocompatibility. GelMA is widely synthesized from porcine skin gelatin, which labors under clinical, religious, and economical restrictions. In order to overcome these limitations, GelMA can be produced from fish skin gelatin, which is eco-friendly as well. Here, we present a comparative study of the physicochemical (structural, thermal, water uptake, swelling, rheological, and mechanical) and biological (cell viability, proliferation, and spreading) properties of porcine and fish skin GelMA with low and high methacrylation degrees, before and after crosslinking, to check whether fish skin can replace porcine skin as the source of GelMA. Porcine and fish skin GelMA presented similar structural, thermal, and water uptake properties prior to crosslinking. However, subsequent to crosslinking, fish skin GelMA hydrogels exhibited a higher mass swelling ratio and a lower elastic and compressive Young's moduli than porcine skin GelMA hydrogels of similar methacrylation level. Both types of GelMA hydrogels showed great biocompatibility toward encapsulated mouse myoblast cells (C2C12), however, improved cell spreading was observed in fish skin GelMA hydrogels, and cell proliferation was only induced in low methacrylated GelMA. These results suggest that fish skin GelMA is a promising substitute for porcine skin GelMA for biomedical applications and that low methacrylated fish skin GelMA can be used as a potential scaffold for skeletal muscle tissue engineering.

Gelatin Extraction from the Bangladeshi Pangas Catfish (Pangasius pangasius) Waste and Comparative Study of Their Physicochemical Properties with a Commercial Gelatin

Abstract- Production of gelatin from the aquatic source is gradually replacing the mammalian sources because of some socio-cultural and religious issues. The Pangasius pangasius catfish is a native species and very popular in Bangladesh due to their availability and cheap price. The perspective of this study was to extract the gelatin from the skin and bones of this catfish and compare them with commercial gelatin. Gelatin was extracted by applying the acid-base extraction process and the resultant gelatins were evaluated based on some physical properties. The gelatin yield was found significantly higher from the skin sample (10.85±0.93%) than the bone (5.23±0.39%) of Pangasius. The extracted skin gelatin had higher moisture and fat content than the bone and Commercial gelatin, while the ash content was significantly higher in bone gelatin. Protein content was noted in skin gelatin (81.34±3.45%), bone gelatin (73.44±2.58%), Where commercial gelatin (92.38±3.89%). Skin gelatin exerted significantly higher (p<0.05) viscosity (4.62±0.3 mPa.s) than the extracted bone gelatin (3.11±.24 mPa.s) and lower than the commercial gelatin (5.76±0.34 mPa.s). The melting and setting temperature of this catfish skin and bone gelatin were very near to each other and significantly lower than the commercial gelatin. Skin gelatin had exerted higher water holding capacity (2.36±0.11 ml/g), fat binding capacity (3.23±0.05 ml/g), foaming capacity ratio (1.88±0.07), and foam stability (1.51±0.04). Both the skin and bone gelatins were acidic. In this comparative study, it was noticed that the skin gelatin had better physical properties than the bone gelatin of native Pangasius catfish. Pangasius skin may be recognized as a potential aquatic source of edible gelatin with good yield and desirable physical properties comparing with commercial gelatin.

Novel Aryl-Substituted Pyrimidones as Inhibitors of 3-Mercaptopyruvate Sulfurtransferase with Antiproliferative Efficacy in Colon Cancer

The enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is one of the more recently identified mammalian sources of H2S. A recent study identified several novel 3-MST inhibitors with micromolar potency. Among those, (2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one) or HMPSNE was found to be the most potent and selective. We now took the central core of this compound and modified the pyrimidone and the arylketone sides independently. A 63-compound library was synthesized; compounds were tested for H2S generation from recombinant 3-MST in vitro. Active compounds were subsequently tested to elucidate their potency and selectivity. Computer modeling studies have delineated some of the key structural features necessary for binding to the 3-MST's active site. Six novel 3-MST inhibitors were tested in cell-based assays: they exerted inhibitory effects in murine MC38 and CT26 colon cancer cell proliferation; the antiproliferative effect of the compound with the highest potency and best cell-based activity (1b) was also confirmed on the growth of MC38 tumors in mice.

The crystal structure of yeast regulatory subunit reveals key evolutionary insights into Protein Kinase A oligomerization

Protein Kinase A (PKA) is a widespread enzyme that plays a key role in many signaling pathways from lower eukaryotes to metazoans. In mammals, the regulatory (R) subunits sequester and target the catalytic (C) subunits to proper subcellular locations. This targeting is accomplished by the dimerization and docking (D/D) domain of the R subunits. The activation of the holoenzyme depends on the binding of the second messenger cAMP. The only available structures of the D/D domain proceed from mammalian sources. Unlike dimeric mammalian counterparts, the R subunit from Saccharomyces cerevisiae (Bcy1) forms tetramers in solution. Here we describe the first high-resolution structure of a non-mammalian D/D domain. The tetramer in the crystals of the Bcy1 D/D domain is a dimer of dimers that retain the classical D/D domain fold. By using phylogenetic and structural analyses combined with site-directed mutagenesis, we found that fungal R subunits present an insertion of a single amino acid at the D/D domain that shifts the position of a downstream, conserved arginine. This residue participates in intra-dimer interactions in mammalian D/D domains, while due to this insertion it is involved in inter-dimer contacts in Bcy1, which are crucial for the stability of the tetramer. This surprising finding challenges well-established concepts regarding the oligomeric state within the PKAR protein family and provides important insights into the yet unexplored structural diversity of the D/D domains and the molecular determinants of R subunit oligomerization.