Protease Digestion Research Articles

Preparation of Healthy Single Neuron or Astrocyte Suspension from Adult Mouse Brain for RNA-seq.

Single-cell RNA sequencing (scRNA-seq) has been widely applied in neuroscience research, enabling the investigation of cellular heterogeneity at the transcriptional level, the characterization of rare cell types, and the detailed analysis of the stochastic nature of gene expression. Isolation of single nerve cells in good health, especially from the adult rodent brain, is the most difficult and critical process for scRNA-seq. Here, we describe methods to optimize protease digestion of brain slices, which enable yield of millions of cells in good health from the adult brain.

Mutations at proximal cysteine residues in PML impair ATO binding by destabilizing the RBCC domain.

Acute promyelocytic leukemia (APL) is characterized by the fusion gene promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) and is conventionally treated with arsenic trioxide (ATO). ATO binds directly to the RING finger, B-box, coiled-coil (RBCC) domain of PML and initiates degradation of the fusion oncoprotein PML-RARA. However, the mutational hotspot at C212-S220 disrupts ATO binding, leading to drug resistance in APL. Therefore, structural consequences of these point mutations in PML that remain uncertain require comprehensive analysis. In this study, we investigated the structure-based ensemble properties of the promyelocytic leukemia-RING-B-box-coiled-coil (PML-RBCC) domains and ATO-resistant mutations. Oligomeric studies reveal that PML-RBCC wild-type and mutants C212R, S214L, A216T, L217F, and S220G predominantly form tetramers, whereas mutants C213R, A216V, L218P, and D219H tend to form dimers. The stability of the dimeric mutants was lower, exhibiting a melting temperature (Tm) reduction of 30 °C compared with the tetrameric mutants and wild-type PML protein. Furthermore, the exposed surface of the C213R mutation rendered it more prone to protease digestion than that of the C212R mutation. The spectroscopic analysis highlighted ATO-induced structural alterations in S214L, A216V, and D219H mutants, in contrast to C213R, L217F, and L218P mutations. Moreover, the computational analysis revealed that the ATO-resistant mutations C213R, A216V, L217F, and L218P caused changes in the size, shape, and flexibility of the PML-RBCC wild-type protein. The mutations C213R, A216V, L217F, and L218P destabilize the wild-type protein structure due to the adaptation of distinct conformational changes. In addition, these mutations disrupt several hydrogen bonds, including interactions involving C212, C213, and C215, which are essential for ATO binding. The local and global structural features induced by these mutations provide mechanistic insight into ATO resistance and APL pathogenesis.

Sulfated polysaccharides accelerate gliadin digestion and reduce its toxicity

Celiac disease and other types of gluten intolerance significantly affect the life quality of patients making them restrict the diet removing all food produced from wheat, rye, oat, and barley flour, and some other products. These disorders arise from protease resistance of poorly soluble proteins prolamins, contained in gluten. Enhanced proteolytic digestion of gliadins might be considered as a prospective approach for the treatment of celiac disease and other types of gluten intolerance. Herein, we tested a range of sulfated polymers (kappa-carrageenan, dextran sulfate and different polysaccharides from brown seaweeds, and a synthetic polystyrene sulfonate) for the ability to activate gliadin digestion by human digestive proteases, pepsin and trypsin. Sulfated polysaccharide from Fucus evanescens enhanced proteolytic digestion of gliadins from wheat flour and reduced its cytotoxicity on intestinal epithelial Caco-2 cell culture. Regarding the non-toxic nature of fucoidans, the results provide a basis for polymer-based drugs or additives for the symptomatic treatment of gluten intolerance.

Promoting Growth and Health of African Catfish, Clarias gariepinus, Through Dietary Novel Supplement, Ginger, Zingiber officinale Rosc, Leaf Powder

African catfish is a popular freshwater aquaculture species as it can be farmed at high stocking density. However, high stocking density caused growth and health impairment. Hence, nutritional approach such as feed additive can be applied in fish farming to overcome this issue. This study evaluated the impacts of ginger (Zingiber officinale) leaf powder (GLP) on growth performance, digestive enzymes activities, antioxidative response, and disease resistance towards Edwardsiella tarda infection in African catfish. A feeding trial was performed using a control diet and four formulated diets with GLP at 1, 2, 3, and 4%. Ginger is an herb used in cuisine and traditional medicine. It was reported possesses medicinal values such as antimicrobial, anti-oxidant, and immune system modulator. After eight weeks, the growth performance of GLP-treated African catfish was significantly improved than control, and the best results were observed in the 2 and 3% groups (p<0.05). Meanwhile, fish supplemented with 1 and 4% GLP demonstrated comparable growth performance. There was a significant decreasing trend in FCR, HSI, and VI, where the lowest values were recorded by the 2 and 3% GLP diet groups, followed by 1% GLP, 4% GLP, and control groups (p<0.05). The digestive enzymes activity, including amylase, lipase, and protease, was significantly higher in dietary GLP groups than in control, where the highest activity was exhibited by groups 2 and 3% GLP (p<0.05), followed by 1% and 4% GLP groups. There was an increasing trend in the antioxidative response, where the GLP-treated groups had significantly higher catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activities than the control, and the highest was observed in fish fed with 2 and 3% GLP (p<0.05). Furthermore, the GLP-treated groups had a significantly higher cumulative survival rate than the control, where 2 and 3% GLP groups demonstrated the highest survival (p<0.05), followed by 1 and 4% GLP groups. In summary, this study revealed that dietary GLP potentially enhanced African catfish production at recommended doses of 2 and 3%.

Evaluation of Anti-Foodborne Bacterial Activity, Digestive Enzyme Secretion, and Antimicrobial Resistant Genes as Probiotic Strains Selection for Industrial Interest

Beneficial microbes, such as probiotic bacteria, are increasingly in demand in the food and feed industry. Lactic acid bacteria and bifidobacteria are commonly used as commercial probiotics, only a few species have been isolated from Southeast Asia areas. This study employed criteria including antimicrobial activity, the release of digestive enzymes, and the absence of antibiotic-resistant (AMR) genes to screen potential local isolates. The results revealed that 4 out of 16 isolates met these criteria, displaying anti-foodborne bacterial activities and a lack of fifty-one tested AMR genes. Furthermore, the four selected isolates demonstrated the production of extracellular digestive enzymes, including amylase, lipase, protease, β-glucanase, and cellulase, with enzyme indices ranging from 1.09–1.31. Among these isolates, two potential probiotics were identified as Bifidobacterium animalis subsp. lactis (strain H9-01) and Lactobacillus reuteri (strain P4-S03). Importantly, both species are approved for use as food and feed supplements in accordance with Thai regulations. This research outlines an approach for screening potential probiotics for industrial-scale applications.

Insights into the Mechanism of Installation of 5-Carboxymethylaminomethyl Uridine Hypermodification by tRNA-Modifying Enzymes MnmE and MnmG.

The evolutionarily conserved bacterial proteins MnmE and MnmG (and their homologues in Eukarya) install a 5-carboxymethylaminomethyl (cmnm5) or a 5-taurinomethyl (τm5) group onto wobble uridines of several tRNA species. The Escherichia coli MnmE binds guanosine-5'-triphosphate (GTP) and methylenetetrahydrofolate (CH2THF), while MnmG binds flavin adenine dinucleotide (FAD) and a reduced nicotinamide adenine dinucleotide (NADH). Together with glycine, MnmEG catalyzes the installation of cmnm5 in a reaction that also requires hydrolysis of GTP. In this letter, we investigated key steps of the MnmEG reaction using a combination of biochemical techniques. We show multiple lines of evidence supporting flavin-iminium FADH[N5═CH2]+ as a central intermediate in the MnmEG reaction. Using a synthetic FADH[N5═CD2]+ analogue, the intermediacy of the FAD in the transfer of the methylene group from CH2THF to the C5 position of U34 was unambiguously demonstrated. Further, MnmEG reactions containing the deuterated flavin-iminium intermediate and alternate nucleophiles such as taurine and ammonia also led to the formation of the anticipated U34-modified tRNAs, showing FAD[N5═CH2]+ as the universal intermediate for all MnmEG homologues. Additionally, an RNA-protein complex stable to urea-denaturing polyacrylamide gel electrophoresis was identified. Studies involving a series of nuclease (RNase T1) and protease (trypsin) digestions along with reverse transcription experiments suggest that the complex may be noncovalent. While the conserved MnmG cysteine C47 and C277 mutant variants were shown to reduce FAD, they were unable to promote the modified tRNA formation. Overall, this study provides critical insights into the biochemical mechanism underlying tRNA modification by the MnmEG.

GreenPhos, a universal method for in-depth measurement of plant phosphoproteomes with high quantitative reproducibility

Protein phosphorylation regulates a variety of important cellular and physiological processes in plants. In-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal phosphoproteomes. This is largely due to the need to improve protein extraction efficiency from plant cells, which have a dense cell wall, and to minimize sample loss resulting from the stringent sample clean-up steps required for the removal of a large amount of biomolecules interfering with phosphopeptide purification and mass spectrometry analysis. To this end, we developed a method with a streamlined workflow for highly efficient purification of phosphopeptides from tissues of various green organisms including Arabidopsis, rice, tomato, and Chlamydomonas reinhardtii, enabling in-depth identification with high quantitative reproducibility of about 11 000 phosphosites, the greatest depth achieved so far with single liquid chromatography-mass spectrometry (LC–MS) runs operated in a data-dependent acquisition (DDA) mode. The mainstay features of the method are the minimal sample loss achieved through elimination of sample clean-up before protease digestion and of desalting before phosphopeptide enrichment and hence the dramatic increases of time- and cost-effectiveness. The method, named GreenPhos, combined with single-shot LC–MS, enabled in-depth quantitative identification of Arabidopsis phosphoproteins, including differentially phosphorylated spliceosomal proteins, at multiple time points during salt stress and a number of kinase substrate motifs. GreenPhos is expected to serve as a universal method for purification of plant phosphopeptides, which, if samples are further fractionated and analyzed by multiple LC–MS runs, could enable measurement of plant phosphoproteomes with an unprecedented depth using a given mass spectrometry technology.

Validating blood microsampling for per- and polyfluoroalkyl substances quantification in whole blood

Microsampling allows the collection of blood samples using a method which is inexpensive, simple and minimally-invasive, without the need for specially-trained medical staff. Analysis of whole blood provides a more holistic understanding of per- and polyfluoroalkyl substances (PFAS) body burden. Capillary action microsamplers (Trajan hemaPEN®) allow the controlled collection of whole blood as dried blood spots (DBS) (four 2.74 µL ± 5 %). The quantification of 75 PFAS from DBS was evaluated by comparing five common extraction techniques. Spiked blood (5 ng/mL PFAS) was extracted by protein precipitation (centrifuged; filtered), acid-base liquid-liquid extraction, trypsin protease digestion, and weak anion exchange (WAX) solid-phase extraction with analysis by high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Filtered protein precipitation was the most effective extraction method, recovering 72 of the 75 PFAS within 70 to 130 % with method reporting limit (MRL) for PFOS of 0.17 ng/L and ranging between 0.05 ng/mL and 0.34 ng/mL for all other PFAS. The optimised method was applied to human blood samples to examine Inter- (n = 7) and intra-day (n = 5) PFAS blood levels in one individual. Sixteen PFAS were detected with an overall Σ16PFAS mean = 6.3 (range = 5.7–7.0) ng/mL and perfluorooctane sulfonate (branched and linear isomers, ΣPFOS) = 3.3 (2.8–3.7) ng/mL being the dominant PFAS present. To the authors knowledge, this minimally invasive self-sampling protocol is the most extensive method for PFAS in blood reported and could be a useful tool for large scale human biomonitoring studies.

Insights into the functional properties of a natural free amino acid mix: Effect on growth performance, nutrient metabolism, and immune response in a carnivorous fish, Asian seabass (Lates calcarifer)

Dietary supplements containing a functional feed additive have been shown to be beneficial to fish and shellfish aquaculture. However, the functional properties of aquafeed formulations have rarely been reported in fish. This study aimed to investigate the effects of natural free amino acid mix (FAAM) supplementation as a functional solution on the growth performance and nutrient utilization in a carnivorous fish, Asian seabass (Lates calcarifer). Five isonitrogenous and isolipidic diets were prepared with graded supplementation levels of FAAM at 0 % (control group), 0.25 %, 0.50 %, 0.75 %, and 1.0 %, denoted as FAAM0, FAAM0.25, FAAM0.5, FAAM0.75, and FAAM1.0, respectively. The experimental fish were fed different dietary FAAM supplementations to apparent satiation twice daily for eight weeks. Significant improvements were observed in the growth performance of fish among the five groups (P < 0.05). Fish fed with FAAM0.75 displayed significantly increased activities of lysozyme, myeloperoxidase, catalase, and glutathione peroxidase (P < 0.05). The activities of digestive enzymes, including amylase, protease, and lipase, were enhanced by the supplementation of FAAM in the feed (P < 0.05), especially for the groups that contained more than 0.5 % FAAM in the feed. Furthermore, the morphological profile of the intestinal tract, including the mucosal fold height, width, thickness, and goblet cell, increased in fish fed with FAAM at 1.0 % (P < 0.05). Moreover, FAAM supplementation in diets not only modulated the expression of immune-related genes (glutathione peroxidase (GPx), complement (C)3, C4, and C-reactive protein) in the liver but also positively impacted the growth-ralated genes, including growth hormone (GH), GH receptor (GHR), insulin-like growth factor I (IGF-I), and IGF-II. In addition, the amounts of monounsaturated fatty acids (mainly oleic acid (C18:1n9c)) and polyunsaturated fatty acids-especially γ-linolenic acid (C18:3 n6) and α-linolenic acid (C18:3n3)-increased in fish fed with diets containing FAAMs (P < 0.05). Interestingly, the diets supplemented with FAAMs also had a positive effect on the economic indices in terms of revenue-to-cost ratios. These findings provide a scientific basis for the application of FAAMs as a functional solution that can be used in feed formulations for Asian seabass.

Comprehensive analysis of the UDP-glucuronate decarboxylase (UXS) gene family in tobacco and functional characterization of NtUXS16 in Golgi apparatus in Arabidopsis

BackgroundUDP-glucuronate decarboxylase (also named UXS) converts UDP-glucuronic acid (UDP-GlcA) to UDP-xylose (UDP-Xyl) by decarboxylation of the C6-carboxylic acid of glucuronic acid. UDP-Xyl is an important sugar donor that is required for the synthesis of plant cell wall polysaccharides.ResultsIn this study, we first carried out the genome-wide identification of NtUXS genes in tobacco. A total of 17 NtUXS genes were identified, which could be divided into two groups (Group I and II), and the Group II UXSs can be further divided into two subgroups (Group IIa and IIb). Furthermore, the protein structures, intrachromosomal distributions and gene structures were thoroughly analyzed. To experimentally verify the subcellular localization of NtUXS16 protein, we transformed tobacco BY-2 cells with NtUXS16 fused to the monomeric red fluorescence protein (mRFP) at the C terminus under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The fluorescent signals of NtUXS16-mRFP were localized to the medial-Golgi apparatus. Contrary to previous predictions, protease digestion analysis revealed that NtUXS16 is not a type II membrane protein. Overexpression of NtUXS16 in Arabidopsis seedling in darkness led to a significant increase in hypocotyl length and a reduction in root length compared with the wild type. In summary, these results suggest Golgi apparatus localized-NtUXS16 plays an important role in hypocotyl and root growth in the dark.ConclusionOur findings facilitate our understanding of the novel functions of NtUXS16 and provide insights for further exploration of the biological roles of NtUXS genes in tobacco.

Secondary metabolites of Bacillus subtilis L2 show antiviral activity against pseudorabies virus

Bacillus subtilis (B. subtilis) is a commercially important probiotic known to produce secondary metabolites with antibacterial, antifungal and anti-inflammatory activities. However, the potential ability of B. subtilis to combat viruses, especially DNA viruses, has not been extensively investigated. In this study, we identified two distinct B. subtilis strains and examined the efficiency of their secondary metabolites against pseudorabies virus (PRV), a swine herpesvirus resulting in economic losses worldwide. We found that treatment with the secondary metabolites of B. subtilis L2, but not the metabolites of B. subtilis V11, significantly inhibited PRV replication in multiple cells. Notably, the antiviral activity of the metabolites of B. subtilis L2 was thermal stable, resistant to protease digestion. Moreover, these metabolites effectively impeded PRV binding, entry and replication. Importantly, oral administration of the metabolites of B. subtilis L2 protected mice from lethal PRV infection, rescuing weight loss and reducing the viral load in vivo. In summary, our results reveal that the metabolites of B. subtilis L2 exhibit anti-PRV activity both in vitro and in vivo, providing a potential candidate for novel antiviral drugs.

Screening and Production of Industrially Relevant Enzymes by Bacillus paranthracis Strain MHDS3, a Potential Probiotic

The digestive process and intestinal protein absorption are influenced by a variety of factors. Due to their numerous health advantages, including potential favorable effects on protein digestion and absorption, probiotics have gained increased attention in recent years. Probiotics can control the intestinal microflora, which in turn affects the intestinal bacteria responsible for proteolysis. Additionally, certain probiotics can release exoenzymes that aid in the digestion of proteins and others can stimulate the host’s digestive protease and peptidase activity. By boosting transport and enhancing the epithelium’s capacity for absorption, probiotics can also improve the absorption of tiny peptides and amino acids as well as lessen detrimental protein fermentation, which lowers the toxicity of metabolites. The present study explored the production of enzymes by Bacillus paranthracis strain MHDS3, a probiotic candidate isolated from Pellaea calomelanos. Bacillus paranthracis displayed enzyme activities of amylase (31,788.59 IU), cellulase (4487.486 IU), and pectinase (13.98986 IU) through submerged fermentation. The CAZyme analysis of B. paranthracis revealed 16 CAZyme gene clusters associated with cellulose, amylase, and pectinase activity. Thus, B. paranthracis is a promising probiotic strain that can produce enzymes with biotechnological applications.

Metformin inhibits digestive proteases and impairs protein digestion in mice

Metformin is among the most prescribed medications worldwide and the first-line therapy for type 2 diabetes. However, gastrointestinal side effects are common and can be dose limiting. The total daily metformin dose frequently reaches several grams, and poor absorption results in high intestinal drug concentrations. Here, we report that metformin inhibits the activity of enteropeptidase and other digestive enzymes at drug concentrations predicted to occur in the human duodenum. Treatment of mouse gastrointestinal tissue with metformin reduces enteropeptidase activity; further, metformin-treated mice exhibit reduced enteropeptidase activity, reduced trypsin activity, and impaired protein digestion within the intestinal lumen. These results indicate that metformin-induced protein maldigestion could contribute to the gastrointestinal side effects and other impacts of this widely used drug.

Emmer starch isolation by alkaline protease digestion of wet-milled seeds

In today's dynamic world, it is necessary to explore more alternative options for starch isolation and develop technologies to obtain better quality products. The presence of impurities in the composition of starch is an indicator that affects its properties. Starch was isolated from whole emmer seeds. The present study aims to compare the protein content of isolated emmer starch obtained by a standard alkaline method and a combined alkaline with an enzymatic method. The conducted method for the isolation of starch from emmer was with a duration of the alkaline treatment of 24 and 48 hours. The results were compared with the results obtained from the combination of alkaline (24 hours) and enzyme (with an amount of enzyme 0.05 - 2.2% for 1 - 8 h) treatment. The results showed that the long alkaline treatment could be replaced by a shorter one with 0.05% enzyme for 1 hThe lowest values of protein in the composition of starch were reported after treatment with 1.1% multienzyme preparation for 8 h.

Catalase-gold nanoaggregates manipulate the tumor microenvironment and enhance the effect of low-dose radiation therapy by reducing hypoxia

Radiotherapy as a standard method for cancer treatment faces tumor recurrence and antitumoral unresponsiveness. Suppressive tumor microenvironment (TME) and hypoxia are significant challenges affecting efficacy of radiotherapy. Herein, a versatile method is introduced for the preparation of pH-sensitive catalase-gold cross-linked nanoaggregate (Au@CAT) having acceptable stability and selective activity in tumor microenvironment. Combining Au@CAT with low-dose radiotherapy enhanced radiotherapy effects via polarizing protumoral immune cells to the antitumoral landscape. This therapeutic approach also attenuated hypoxia, confirmed by downregulating hypoxia hallmarks, such as hypoxia-inducible factor α-subunits (HIF-α), vascular endothelial growth factor (VEGF), and EGF. Catalase stability against protease digestion was improved significantly in Au@CAT compared to the free catalase. Moreover, minimal toxicity of Au@CAT on normal cells and increased reactive oxygen species (ROS) were confirmed in vitro compared with radiotherapy. Using the nanoaggregates combined with radiotherapy led to a significant reduction of immunosuppressive infiltrating cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (T-regs) compared to the other groups. While, this combined therapy could significantly increase the frequency of CD8+ cells as well as M1 to M2 macrophages (MQs) ratio. The combination therapy also reduced the tumor size and increased survival rate in mice models of colorectal cancer (CRC). Our results indicate that this innovative nanocomposite could be an excellent system for catalase delivery, manipulating the TME and providing a potential therapeutic strategy for treating CRC.

Physicochemical characterization, biodistribution, and bio‐effects of fluorescent carbon dots from roasted Spanish mackerel

AbstractNanoparticles with small sizes formed in the process of food thermal treatment have gained substantial attention due to their safety uncertainty and latent risks to human health. Herein, the physicochemical features of fluorescent carbon dots (CDs) from roasted Spanish mackerel were studied, it was found that the size distribution, fluorescence properties, surface groups, and formation process of CDs were highly dependent on the roasting time of Spanish mackerel. The interaction of CDs with digestive proteases resulted in the structural change and activity inhibition of proteases. When the concentration of CDs was 120 μg mL−1, the enzyme activity of pepsin and trypsin was reduced from 100% to 75.95% and 78.5%, respectively. The CDs caused static fluorescence quenching of pepsin and trypsin as confirmed by the fluorescence analysis. After oral administration, CDs can be transported to the main organs through the blood. The cytotoxicity assessment revealed that the cell viability decreased with the prolonging of roasting time. The CDs roasted at 230°C for 40 min showed a significant hemolytic effect even at a low concentration of 0.5 mg mL−1. Moreover, the cellular uptake level and pathway of CDs were significantly affected by blood protein adsorption. This study helps to evaluate the in vivo biodistribution, the interaction with proteases during gastrointestinal digestion, and the cellular uptake of CDs after transport through the blood, providing a theoretical basis for revealing the potential effects of foodborne nanoparticles.

A constitutive serine protease inhibitor suppresses herbivore performance in tea (Camellia sinensis).

Protease inhibitors promote herbivore resistance in diverse plant species. Although many inducible protease inhibitors have been identified, there are limited reports available on the biological relevance and molecular basis of constitutive protease inhibitors in herbivore resistance. Here, we identified a serine protease inhibitor, CsSERPIN1, from the tea plant (Camellia sinensis). Expression of CsSERPIN1 was not strongly affected by the assessed biotic and abiotic stresses. In vitro and in vivo experiments showed that CsSERPIN1 strongly inhibited the activities of digestive protease activities of trypsin and chymotrypsin. Transient or heterologous expression of CsSERPIN1 significantly reduced herbivory by two destructive herbivores, the tea geometrid and fall armyworm, in tea and Arabidopsis plants, respectively. The expression of CsSERPIN1 in Arabidopsis did not negatively influence the growth of the plants under the measured parameters. Our findings suggest that CsSERPIN1 can inactivate gut digestive proteases and suppress the growth and development of herbivores, making it a promising candidate for pest prevention in agriculture.

Partial characterization of digestive proteases in the river prawn Macrobrachium americanum () (Decapoda: Caridea: Palaemonidae) in Mexico

Abstract We characterized the digestive enzymes in adults of Macrobrachium americanum (Spence Bate, 1868) in southeastern Mexico. The digestive enzyme extracts were made from the specimens’ hepatopancreas. Alkaline proteases, trypsin, chymotrypsin, carboxypeptidase A, leucine aminopeptidase, lipases, α-amylase and alkaline phosphatase activities were determined, as well as the percentage of inhibition, pH, and temperature stabilities through biochemical and electrophoretic techniques. The maximum digestive activity of proteases was at 35 °C and pH 11. Alkaline digestive proteases were highly stable at pHs 4, 9 and 11 at 55 °C after 30 min pre-incubation. PMSF inhibited two bands with proteolytic activity (35.1 and 23.2 kDa), and SBT1 inhibited all bands, including the one with the highest molecular weight (64.2 kDa). We concluded that enzymes in M. americanum coincide with those of omnivorous decapods with a tendency to carnivory.

Effect of spirulina fortification on the digestive protease, carbohydrases and cocoons economic traits of mulberry silkworm Bombyx mori

Bombyx mori, the silkworm, is a voracious feeder that relies on digestive enzymes for mulberry digestion and its conversion in to silk. In the current study, fifth-instar larvae were given 2% spirulina supplementation with commercially graded spirulina samples, and a few digestive enzymes and cocoon traits were examined. The treatment groups of silkworms T1-control, T2-supplemented with spirulina powder graded for animal dietary consumption, T3- graded for human dietary consumption and T4-spirulina graded for medicinal applications. Spirulina supplementation was tested in silkworms for digestive protease, carbohydrases and cocoon traits. The results shows that the total protein content percentage difference (PD) in the gut wall was increased by 2.79% in T3, 1.96% in T4, and 0.78% in T2 compared to T1 (control). A similar trend was noticed in the gut content, with the PD of 15.81% in T3, 7.51% in T4, and 3.95% in T2. In total carbohydrate content, observed PD of 16.35% in T2, 11.27% in T3, and 9.38% in T4, while the gut content was 13% both in T2 and T3, and 1.22% in T4. The protease activity PD of gut wall tissue has 21.99% in T3, 5.13% in T2, and 4.30% in T4; similarly, the gut content has a PD of 112.38% in T3, 106.12% in T4, and 86.41% in T2. The amylase activity have shown highest PD in T4 (33.43%), T3 (23.37%), and moderate in T2 (19.23%) in the gut wall, and 93.10% in T3, 66.44% in T2, and 14.92% in T4 of gut content. Enzymes such as sucrase, cellulase, and pectinase have also improved with spirulina fortification. The sucrase activity in the gut wall and gut content were, respectively, 7.99% and 80.73% in T3 treatment. Noteworthy, the T2 showed 37.69% in the gut wall and 24.54% in the gut content for the cellulase activity. Unlike cellulase, pectinase activity has been observed with a PD of 93.15% in the gut wall and 64.63% in gut content. The study further confirms that the additional fortification of spirulina will have a beneficial effect on the digestive cells of the silkworm, Bombyx mori, resulting in the highest rate of enzyme activity and in turn, increasing survivability.

Conformational change in an engineered biliverdin-binding cyanobacteriochrome during the photoconversion process

Cyanobacteriochromes (CBCRs) derived from cyanobacteria are linear-tetrapyrrole-binding photoreceptors related to the canonical red/far-red reversible phytochrome photoreceptors. CBCRs contain chromophore-binding cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that are highly diverse in their primary sequences and are categorized into many subfamilies. Among this repertoire, the biliverdin (BV)-binding CBCR GAF domains receive considerable attention for their in vivo optogenetic and bioimaging applications because BV is a mammalian intrinsic chromophore and can absorb far-red light that penetrates deep into the mammalian body. The typical BV-binding CBCR GAF domain exhibits reversible photoconversion between far-red-absorbing dark-adapted and orange-absorbing photoproduct states. Herein, we applied various biochemical and spectral studies to identify the details of the conformational change during this photoconversion process. No oligomeric state change was observed, whereas the surface charge would change with a modification of the α-helix structures during the photoconversion process. Combinatorial analysis using partial protease digestion and mass spectrometry identified the region where the conformational change occurred. These results provide clues for the future development of optogenetic tools.