Bacillus subtilis extracellular protease production incurs a context-dependent cost.

Microbes encounter a wide range of polymeric nutrient sources in various environmental settings, which require processing to facilitate growth. Bacillus subtilis, a bacterium found in the rhizosphere and broader soil environment, is highly adaptable and resilient due to its ability to utilise diverse sources of carbon and nitrogen. Here, we explore the role of extracellular proteases in supporting growth and assess the cost associated with their production. We provide evidence of the essentiality of extracellular proteases when B. subtilis is provided with an abundant, but polymeric nutrient source and demonstrate the extracellular proteases as a shared public good that can operate over a distance. We show that B. subtilis is subjected to a public good dilemma, specifically in the context of growth sustained by the digestion of a polymeric food source. Furthermore, using mathematical simulations, we uncover that this selectively enforced dilemma is driven by the relative cost of producing the public good. Collectively, our findings reveal how bacteria can survive in environments that vary in terms of immediate nutrient accessibility and the consequent impact on the population composition. These findings enhance our fundamental understanding of how bacteria respond to diverse environments, which has importance to contexts ranging from survival in the soil to infection and pathogenesis scenarios.

Effects of exogenous protease on performance, economic evaluation, nutrient digestibility, fecal score, intestinal morphology, blood profile, carcass trait, and meat quality in broilers fed normal diets and diets considered with matrix value.

This study was conducted to estimate the effects of exogenous protease on performance, economic evaluation, nutrient digestibility, fecal score, intestinal morphology, blood profile, carcass traits, and meat quality in broilers fed normal diets and diets considered with matrix value. A total of 90, one-day-old Arbor Acres broiler chickens were randomly allocated to 3 dietary treatments with 6 replicates and each replicate of 5 broiler chickens. Treatments were as follows: 1) Basal diet (positive control, PC), 2) Basal diet formulated with full ProAct 360 matrix at 50 g/MT without addition of ProAct 360 (negative control, NC), 3) NC + 50 g/MT ProAct 360 (PA). Supplementation of exogenous protease to nutrient deficient NC diet by matrix values (PA) tended to increase growth performance and significantly improved intestinal morphology compared with the NC group. The PA group had significantly lower fecal score, and higher ATTD of crude protein and amino acids than those of the NC group. Furthermore, supplementation of exogenous protease to NC diet decreased feed cost, resulting in improved profit margin. However, there was no significant difference on carcass yield and relative organ weight. In conclusion, supplementation of exogenous protease using matrix value could be used as economic additive to improve growth, profit margin, digestibility, and gut health in broiler chickens.

From Capillary Electrophoresis to Deep Sequencing: An Improved HIV-1 Drug Resistance Assessment Solution Using In Vitro Diagnostic (IVD) Assays and Software.

BACKGROUND: Drug-resistance mutations were mostly detected using capillary electrophoresis sequencing, which does not detect minor variants with a frequency below 20%. Next-Generation Sequencing (NGS) can now detect additional mutations which can be useful for HIV-1 drug resistance interpretation. The objective of this study was to evaluate the performances of CE-IVD assays for HIV-1 drug-resistance assessment both for target-specific and whole-genome sequencing, using standardized end-to-end solution platforms. METHODS: A total of 301 clinical samples were prepared, extracted, and amplified for the three HIV-1 genomic targets, Protease (PR), Reverse Transcriptase (RT), and Integrase (INT), using the CE-IVD DeepChek® Assays; and then 19 clinical samples, using the CE-IVD DeepChek® HIV Whole Genome Assay, were sequenced on the NGS iSeq100 and MiSeq (Illumina, San Diego, CA, USA). Sequences were compared to those obtained by capillary electrophoresis. Quality control for Molecular Diagnostics (QCMD) samples was added to validate the clinical accuracy of these in vitro diagnostics (IVDs). Nineteen clinical samples were then tested with the same sample collection, handling, and measurement procedure for evaluating the use of NGS for whole-genome HIV-1. Sequencing analyzer outputs were submitted to a downstream CE-IVD standalone software tailored for HIV-1 analysis and interpretation. RESULTS: The limits of range detection were 1000 to 106 cp/mL for the HIV-1 target-specific sequencing. The median coverage per sample for the three amplicons (PR/RT and INT) was 13,237 reads. High analytical reproducibility and repeatability were evidenced by a positive percent agreement of 100%. Duplicated samples in two distinct NGS runs were 100% homologous. NGS detected all the mutations found by capillary electrophoresis and identified additional resistance variants. A perfect accuracy score with the QCMD panel detection of drug-resistance mutations was obtained. CONCLUSIONS: This study is the first evaluation of the DeepChek® Assays for targets specific (PR/RT and INT) and whole genome. A cutoff of 3% allowed for a better characterization of the viral population by identifying additional resistance mutations and improving the HIV-1 drug-resistance interpretation. The use of whole-genome sequencing is an additional and complementary tool to detect mutations in newly infected untreated patients and heavily experienced patients, both with higher HIV-1 viral-load profiles, to offer new insight and treatment strategies, especially using the new HIV-1 capsid/maturation inhibitors and to assess the potential clinical impact of mutations in the HIV-1 genome outside of the usual HIV-1 targets (RT/PR and INT).

Sustainable enzymatic treatment of organic waste in a framework of circular economy.

Enzymatic treatment of food and vegetable waste (FVW) is an eco-friendly approach for producing industrially relevant value-added products. This review describes the sources, activities and potential applications of crucial enzymes in FVW valorization. The specific roles of amylase, cellulase, xylanase, ligninase, protease, pectinase, tannase, lipase and zymase enzymes were explained. The exhaustive list of value-added products that could be produced from FVW is presented. FVW valorization through enzymatic and whole-cell enzymatic valorization was compared. The note on global firms specialized in enzyme production reiterates the economic importance of enzymatic treatment. This review provides information on choosing an efficient enzymatic FVW treatment strategy, such as nanoenzyme and cross-linked based enzyme immobilization, to make the process viable, sustainable and cheaper. Finally, the importance of life cycle assessment of enzymatic valorization of FVW was impressed to prove this approach is a better option to shift from a linear to a circular economy.

Assessment of probiotic and technological properties of Bacillus spp. isolated from Burkinabe Soumbala.

BACKGROUND: Soumbala is a highly loved alkaline traditional fermented food condiment in Burkina Faso. It harbors various microbiota dominated by fermentative Bacillus spp. as functional microorganism with little confirmed health-promoting properties. METHODS: The present study aimed to evaluate six Bacillus strains previously isolated and identified from soumbala. These strains were selected as presumptively safe bacteria for probiotic and technological characteristics. These strains were assessed for in vitro probiotic criteria (tolerance to acidic pH, gastric juice, 0.3% (m/v) bile salts, intestinal juice and 0.4% (w/v) phenol, cell surface hydrophobicity, auto-aggregation capacity, antimicrobial activity against foodborne pathogens, antibiotic susceptibility and biofilm production) and technological properties, including protease, amylase, lipase, and tannase activity, as well as poly-γ-glutamic acid (PGA) production and thermo-tolerance. RESULTS: All tested Bacillus strains (B54, F20, F24, F21, F26 and F44) presented variable relevant probiotic properties (good tolerance to pH 2 and pH 4, gastric juice, bile salts, intestinal juice and phenol), with marked differences in hydrophobicity and auto-aggregation capacity ranging from 73.62-94.71% and 49.35-92.30%, respectively. They exhibited a broad spectrum of activity against foodborne pathogens depending on target pathogen, with the highest activity exhibited by strain F20 (29.52 mm) against B. cereus 39 (p < 0.001). They also showed good biofilm production as well as variable hydrolytic enzyme activities, including protease (43.00-60.67 mm), amylase (22.59-49.55 mm), lipase (20.02-24.57 mm), and tannase (0-10.67 mm). All tested Bacillus strains tolerated temperature up to 50 °C, while only strains F26 and F44 showed the best PGA production. CONCLUSION: Overall, the tested cultures exhibiting potential probiotic and technological characteristics; particularly B. cereus F20, B. benzoevorans F21, B. cabrialessi F26, and B. tequilensis F44 could be a source of probiotic-starters of commercial interest in the production of high-quality soumbala.

Assessment of bacteriophage-encoded endolysin as a potent antimicrobial agent against antibiotic-resistant Salmonella Typhimurium.

This study was designed to evaluate the potential of using newly purified Salmonella phage-encoded endolysin LysPB32 as novel antibiotic alternative. The endolysin LysPB32 was characterized by analyzing pH and thermal stability, lytic spectrum, antimicrobial activity, and mutant frequency against Salmonella Typhimurium KCCM 40253 (STKCCM), S. Typhimurium ATCC 19585 (STATCC), S. Typhimurium CCARM 8009 (STCCARM), Klebsiella pneumoniae ATCC 23357 (KPATCC), K. pneumoniae CCARM 10237 (KPCCARM), Pseudomonas aeruginosa ATCC 27853 (PAATCC), Listeria monocytogenes ATCC 1911 (LMATCC), Staphylococcus aureus ATCC 25923 (SAATCC), and S. aureus CCARM 3080 (SACCARM). The molecular weight of LysPB32 is 17 kDa that was classified as N-acetyl-ß-d-muramidase. The optimum activity of LysPB32 against the outer membrane (OM) permeabilized STKCCM, STATCC, and STCCARM was observed at 37 °C and pH 6.5. LysPB32 had a broad spectrum of muralytic activity against antibiotic-sensitive STKCCM (41 mOD/min), STATCC (32 mOD/min), and SBKACC (25 mOD/min) and antibiotic-resistant STCCARM (35 mOD/min) and KPCCARM (31 mOD/min). The minimum inhibitory concentrations (MICs) of polymyxin B against STKCCM, STCCARM, and STATCC were decreased by 4-, 4-, and 8-folds, respectively, when treated with LysPB32. The combination of LysPB32 and polymyxin B effectively inhibited the growth of STKCCM, STCCARM, and STATCC after 24 h of incubation at 37 °C, showing 4.9-, 4.4-, and 3.3-log reductions, respectively. The mutant frequency was low in STKCCM, STCCARM, and STATCC treated with combination of LysPB32-polymyxin B system. The results suggest the LysPB32-polymyxin system can be a potential candidate for alternative therapeutic agent to control antibiotic-resistant pathogens.

Efficacy assessment of a novel endolysin PlyAZ3aT for the treatment of ceftriaxone-resistant pneumococcal meningitis in an infant rat model.

BACKGROUND: Treatment failure in pneumococcal meningitis due to antibiotic resistance is an increasing clinical challenge and alternatives to antibiotics warrant investigation. Phage-derived endolysins efficiently kill gram-positive bacteria including multi-drug resistant strains, making them attractive therapeutic candidates. The current study assessed the therapeutic potential of the novel endolysin PlyAZ3aT in an infant rat model of ceftriaxone-resistant pneumococcal meningitis. METHODS: Efficacy of PlyAZ3aT was assessed in a randomized, blinded and controlled experimental study in infant Wistar rats. Meningitis was induced by intracisternal infection with 5 x 107 CFU/ml of a ceftriaxone-resistant clinical strain of S. pneumoniae, serotype 19A. Seventeen hours post infection (hpi), animals were randomized into 3 treatment groups and received either (i) placebo (phosphate buffered saline [PBS], n = 8), (ii) 50 mg/kg vancomycin (n = 10) or (iii) 400 mg/kg PlyAZ3aT (n = 8) via intraperitoneal injection. Treatments were repeated after 12 h. Survival at 42 hpi was the primary outcome; bacterial loads in cerebrospinal fluid (CSF) and blood were secondary outcomes. Additionally, pharmacokinetics of PlyAZ3aT in serum and CSF was assessed. RESULTS: PlyAZ3aT did not improve survival compared to PBS, while survival for vancomycin treated animals was 70% which is a significant improvement when compared to PBS or PlyAZ3aT (p<0.05 each). PlyAZ3aT was not able to control the infection, reflected by the inability to reduce bacterial loads in the CSF, whereas Vancomycin sterilized the CSF and within 25 h. Pharmacokinetic studies indicated that PlyAZ3aT did not cross the blood brain barrier (BBB). In support, PlyAZ3aT showed a peak concentration of 785 µg/ml in serum 2 h after intraperitoneal injection but could not be detected in CSF. CONCLUSION: In experimental pneumococcal meningitis, PlyAZ3aT failed to cure the infection due to an inability to reach the CSF. Optimization of the galenic formulation e.g. using liposomes might enable crossing of the BBB and improve treatment efficacy.

Porcine Epidemic Diarrhea: Causative Agent, Epidemiology, Clinical Characteristics, and Treatment Strategy Targeting Main Protease.

AIMS: This aimed to study the causative agent, epidemiology, clinical characteristics, and treatment strategy targeting the main protease in porcine epidemic diarrhea. BACKGROUND: Porcine epidemic diarrhea (PED) is a contagious intestinal viral infection causing severe diarrhea, vomiting, and dehydration in pigs. High rates of mortalities and severe morbidities, approaching 100%, are reported in piglets infected with PEDV. In recent years, PED has been observed to influence the swine-farming nations in Europe, Asia, the USA, South Korea, and Canada. The PED virus (PEDV) transmission takes place through a faecal-oral route. OBJECTIVE: The objective is to review the characteristics of PEDV and its role in the disease. In addition, we aim to outline some possible methods to combat PED infection, including targeting the main protease of coronavirus and their future perspectives. METHODS: This study is a review of literature on the PED virus. RESULTS: Apart from symptomatic treatment and supportive care, there is no available specific treatment for PEDV. Appropriate disinfectants and cleaning are pivotal for the control of PEDV. To date, apart from anti-PEDV inhibitors, there are no specific drugs available commercially to treat the disease. Therefore, 3C-like protease (3CLpro) in PEDV that has highly conserved structure and catalytic mechanism serves as an alluring drug as it plays a vital role during viral polyprotein processing at the time of infection. CONCLUSION: A well synchronized and collective effort of scientists, swine veterinarians, pork industry experts, and associated authorities is essential for the accomplishment of proper execution of these required measures.

Design, Synthesis and Preclinical Assessment of 99mTc-iFAP for In Vivo Fibroblast Activation Protein (FAP) Imaging.

Fibroblast activation protein (FAP) is expressed in the microenvironment of most human epithelial tumors. 68Ga-labeled FAP inhibitors based on the cyanopyrrolidine structure (FAPI) are currently used for the detection of the tumor microenvironment by PET imaging. This research aimed to design, synthesize and preclinically evaluate a new FAP inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-iFAP) structure for SPECT imaging. Molecular docking for affinity calculations was performed using the AutoDock software. The chemical synthesis was based on a series of coupling reactions of 6-hidrazinylnicotinic acid (HYNIC) and D-alanine to a boronic acid derivative. The iFAP was prepared as a lyophilized formulation based on EDDA/SnCl2 for labeling with 99mTc. The radiochemical purity (R.P.) was verified via ITLC-SG and reversed-phase radio-HPLC. The stability in human serum was evaluated by size-exclusion HPLC. In vitro cell uptake was assessed using N30 stromal endometrial cells (FAP positive) and human fibroblasts (FAP negative). Biodistribution and tumor uptake were determined in Hep-G2 tumor-bearing nude mice, from which images were acquired using a micro-SPECT/CT. The iFAP ligand (Ki = 0.536 nm, AutoDock affinity), characterized by UV-Vis, FT-IR, 1H-NMR and UPLC-mass spectroscopies, was synthesized with a chemical purity of 92%. The 99mTc-iFAP was obtained with a R.P. >98%. In vitro and in vivo studies indicated high radiotracer stability in human serum (>95% at 24 h), specific recognition for FAP, high tumor uptake (7.05 ± 1.13% ID/g at 30 min) and fast kidney elimination. The results found in this research justify additional dosimetric and clinical studies to establish the sensitivity and specificity of the 99mTc-iFAP.

Assessment of gelatinolytic proteinases in chilled grass carp (Ctenopharyngodon idellus) fillets: characterization and contribution to texture softening.

BACKGROUND: Texture softening is always a problem during chilling of grass carp fillets. To solve this problem and provide for better quality of flesh, understanding the mechanism of softening is necessary. Gelatinolytic proteinases are suspected to play an essential role in the disintegration of collagen in softening of fish flesh. In the present study, the types and contribution of gelatinolytic proteinases in chilled fillets were investigated. RESULTS: Four active bands (G1, 250 kDa; G2, 68 kDa; G3, 66 kDa; G4, 29 kDa) of gelatinolytic proteinases were identified in grass carp fillets by gelatin zymography. The effect of inhibitors and metal ions revealed that G1 was possibly a serine proteinase, G2 and G3 were calcium-dependent metalloproteinases and G4 was a cysteine proteinase. The effect of the inhibitors phenylmethanesulfonyl fluoride (PMSF), l-3-carboxy-trans-2,3-epoxy-propionyl-l-leucine-4-guanidinobutylamide (E-64) and 1,10-phenanthroline (Phen) on chilled fillets revealed that gelatinolytic proteinase activities were significantly suppressed. Collagen solubility indicated that metalloproteinase and serine proteinase played critical roles in collagen breakdown during the first 3 days, and cysteine proteinase revealed its effect after 3 days. Meanwhile, during chilled storage for 11 days, the final values of shear force increased 19.68% and 24.33% in PMSF and E-64 treatments when compared to control fillets respectively, whereas the increase after Phen treatment was 49.89%. CONCLUSION: Our study concluded that the disintegration of collagen in post-mortem softening of grass carp fillets was mainly mediated by metalloproteinase and to a lesser extent by serine proteinase and cysteine proteinase. © 2021 Society of Chemical Industry.

Public health research needs for molecular epidemiology and to emphasize homeostasis - could the omnipotent endopeptidase inhibitor α-2-macroglobulin be a meaningful biomarker?

Public health authorities in low- and middle-income countries face dramatic challenges in handling rapidly increasing non-communicable diseases (NCDs), due to the epidemiological- and particularly nutritional transition. Among major reasons for the development of NCDs are smoking and alcohol, but overnutrition and obesity are also major threats to population health. Obesity is related to diabetes and cancer, but also has a genetic background. It is difficult to recommend a healthy nutrition. This is because of conflicting nutritional conceptions, and given the complexity of human metabolism understanding this topic can be difficult for the laymen.  Public health measures advocating physical activity and refraining from high intake of energy, sugar and soft drinks need to be enhanced by supporting the 'intrinsic motivation' to preserve a good health. The mission of public health should be to increase awareness about the complexity of human metabolism, and the involvement of genetic and epigenetics in health and diseases. To maintain homeostasis, means to keep an optimal relationship between catabolism and synthesis, seems to be of particular interest. Preconditions for this is, that public health institutions within the administration- and academic sector follow up developments in life science and molecular biology and conduct population-based research making use of molecular epidemiology, especially those related to key metabolic steps and maintenance of 'homeostasis', in balancing catabolism and anabolism. A prospective biomarker for this situation might be α-2-macroglobulin.

Assessment of platelet-rich fibrin in the maintenance and recovery of cell viability of the periodontal ligament.

This study analyzed the efficacy of autologous platelet-rich fibrin (PRF) in maintaining and recovering cell viability of the periodontal ligament (PDL). The PDL cells were isolated from 45 extracted teeth randomly distributed among 6 groups: 5 min, 1 h, 2 h, PRF 30 min, PRF 1 h and PRF 2 h. In the groups 5 min, 1 h and 2 h (n = 5), the teeth were kept dry in extra-alveolar times of 5 min, 1 h and 2 h respectively. The teeth of the groups PRF 30 min, PRF 1 h and PRF 2 h (n = 10) were kept dry at extra-alveolar times of 30 min, 1 and 2 h followed by immersion in PRF for 45 min. PDL cells were isolated by enzymatic digestion with type II collagenase and dispase, counted and analyzed for viability with Trypan blue vital dye in Neubauer chamber. The variables total number of cells and cell viability demonstrated that in the 5 min, 1 h and 2 h groups there was a decrease after the extra-alveolar dry times of 1 and 2 h. In comparison with the total number of cells, group 1 h, considered immediate reimplantation, did not present statistical difference when compared to the groups PRF 30 min, PRF 1 h and 2 h, a result that demonstrates that PRF assists in cell maintenance and recovery. PRF provided increased cell viability in relation to the different dry extra-alveolar times analyzed (p < 0.001). Autologous PRF presented effectiveness in maintaining and recovering PDL cells from extracted teeth and kept dry for up to 2 h.

First behavioural assessment of a novel Immp2l knockdown mouse model with relevance for Gilles de la Tourette syndrome and Autism spectrum disorder.

Gilles de la Tourette syndrome (GTS) is a neurodevelopmental disorder, which shares some clinical features with Autism spectrum disorder (ASD). The genetic factors relevant to the development of both disorders are yet to be fully understood, however, some genetic association studies have identified inner mitochondrial membrane peptidase subunit 2 (IMMP2L) as a potential risk gene for both GTS and ASD. The impact of Immp2l deficiency on behavioural domains is currently unknown. A new genetic mouse model for Immp2l was developed. Adult heterozygous (HET) and homozygous (HOMO) Immp2l knockdown (Immp2l KD) mice of both sexes were compared to wild type-like (WT) littermates in the open field (OF), social interaction, novel object recognition, marble burying, and prepulse inhibition (PPI). The effect of acute dexamphetamine (2 mg/kg) on OF behaviour was also determined. OF locomotion was significantly higher in HET compared to HOMO male littermates. Male and female HOMO mice were much more sensitive to the locomotor-stimulating effects of dexamphetamine (DEX), whereas only HOMO males exhibited significant increased DEX-induced OF exploration compared to control groups. HOMO females failed to habituate to an acoustic startle stimulus. Furthermore, compared to HOMO females, HET females showed reduced social interaction, and a similar trend was seen in HET males. The Immp2l KD mouse model possesses moderate face validity for preclinical research into GTS and ASD, in particular as dysfunctional dopaminergic neurotransmission appears to be one mechanism leading to disease presentation. The sex-dependent differences observed in most findings reinforce the strong influence of sex in the pathophysiology of GTS and ASD.

CRDSAT Generated by pCARGHO: A New Efficient Lectin-Based Affinity Tag Method for Safe, Simple, and Low-Cost Protein Purification.

Purification of recombinant proteins remains a bottleneck for downstream processing. The authors engineered a new galectin 3 truncated form (CRDSAT ), functionally and structurally characterized, with preserved solubility and lectinic activity. Taking advantage of these properties, the authors designed an expression vector (pCARGHO), suitable for CRDSAT -tagged protein expression in prokaryotes. CRDSAT binds to lactose-Sepharose with a high specificity and facilitates solubilization of fusion proteins. This tag is structurally stable and can be easily removed from fusion proteins using TEV protease. Furthermore, due to their basic isoelectric point (pI), CRDSAT , and TEV are efficiently eliminated using cationic exchange chromatography. When pI of the protein of interest (POI) and CRDSAT are close, other chromatographic methods are successfully tested. Using CRDSAT tag, the authors purified several proteins from prokaryote and eukaryote origin and demonstrated as examples, the preservation of both Escherichia coli Thioredoxin 1 and human CDC25Bcd activities. Overall, yields of proteins obtained after tag removal are about 5-50 mg per litre of bacterial culture. Our purification method displays various advantages described herein that may greatly interest academic laboratories, biotechnology, and pharmaceutical companies.

Assessment of the Fusion Tags on Increasing Soluble Production of the Active TEV Protease Variant and Other Target Proteins in E. coli.

In this study, five fusion tags affecting soluble production and cleavage activity of the tobacco etch virus (TEV) protease (TEVp) variant in Escherichia coli strains BL21 (DE3) and Rosetta™ (DE3) are investigated. Combination of the augmenting rare transfer RNAs (tRNAs) and the fused expressivity tag (N-terminal seven amino acid residues of E. coli translation initiation factor II) promotes the soluble TEVp partner expressed at relatively high level. Attachment of the maltose-binding protein (MBP) tag increases soluble expression of the protease released from the fusion protein in E. coli cells, but the incorporated TEVp recognition sequence slightly decreases expressivity of the fusion construct. Except for the green fluorescent protein, the attached expressivity tag shows less efficiency than the MBP tag in enhancing expression levels of the selected five target proteins in the Rosetta™ (DE3) cells under different induction conditions. Our results identified that high-level production of the functional target protein as the fusion partner in E. coli is combined with the intrinsic property of fusion tag, fusion protein stability, inherent folding of target protein, rare tRNA abundance, and the incorporated linker. Purified TEVp fusion constructs with the N-terminal expressivity tag, as well as the MBP partner, are the ideal alternatives for removing fusion tag.

Wide bandwidth nanomechanical assessment of murine cartilage reveals protection of aggrecan knock-in mice from joint-overuse.

Aggrecan loss in human and animal cartilage precedes clinical symptoms of osteoarthritis, suggesting that aggrecan loss is an initiating step in cartilage pathology. Characterizing early stages of cartilage degeneration caused by aging and overuse is important in the search for therapeutics. In this study, atomic force microscopy (AFM)-based force-displacement micromechanics, AFM-based wide bandwidth nanomechanics (nanodynamic), and histologic assessments were used to study changes in distal femur cartilage of wildtype mice and mice in which the aggrecan interglobular domain was mutated to make the cartilage aggrecanase-resistant. Half the animals were subjected to voluntary running-wheel exercise of varying durations. Wildtype mice at three selected age groups were compared. While histological assessment was not sensitive enough to capture any statistically significant changes in these relatively young populations of mice, micromechanical assessment captured changes in the quasi-equilibrium structural-elastic behavior of the cartilage matrix. Additionally, nanodynamic assessment captured changes in the fluid-solid poroelastic behavior and the high frequency stiffness of the tissue, which proved to be the most sensitive assessment of changes in cartilage associated with aging and joint-overuse. In wildtype mice, aging caused softening of the cartilage tissue at the microscale and at the nanoscale. Softening with increased animal age was found at high loading rates (frequencies), suggesting an increase in hydraulic permeability, with implications for loss of function pertinent to running and impact-injury. Running caused substantial changes in fluid-solid interactions in aggrecanase-resistant mice, suggestive of tissue degradation. However, higher nanodynamic stiffness magnitude and lower hydraulic permeability was observed in running aggrecanase-resistant mice compared to running wildtype controls at the same age, thereby suggesting protection from joint-overuse.

Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins.

Improvement of glycemic control in streptozotocin-induced diabetic rats by Atlantic salmon skin gelatin hydrolysate as the dipeptidyl-peptidase IV inhibitor.

In our previous study, Atlantic salmon skin gelatin hydrolysed with flavourzyme possessed 42.5% dipeptidyl-peptidase (DPP)-IV inhibitory activity at a concentration of 5 mg mL(-1). The oral administration of the hydrolysate (FSGH) at a single dose of 300 mg per day in streptozotocin (STZ)-induced diabetic rats for 5 weeks was evaluated for its antidiabetic effect. During the 5-week experiment, body weight increased, and the food and water intake was reduced by FSGH in diabetic rats. The daily administration of FSGH for 5 weeks was effective for lowering the blood glucose levels of diabetic rats during an oral glucose tolerance test (OGTT). After the 5-week treatment, plasma DPP-IV activity was inhibited; the plasma activity of glucagon-like peptide-1 (GLP-1), insulin, and the insulin-to-glucagon ratio were increased by FSGH in diabetic rats. The results indicate that FSGH has the function of inhibiting GLP-1 degradation by DPP-IV, resulting in the enhancement of insulin secretion and improvement of glycemic control in STZ-induced diabetic rats.

Two novel antioxidant nonapeptides from protein hydrolysate of skate (Raja porosa) muscle.

In the current study, the preparation conditions of neutrase hydrolysate (SMH) from skate (Raja porosa) muscle protein were optimized using orthogonal L9(3)4 tests, and R values indicated that pH was the most important factor affecting HO· scavenging activity of SMH. Under the optimum conditions of pH 7.0, enzymolysis temperature 60 °C, enzyme/substrate ratio (E/S) 2%, and enzymolysis time 5 h, EC50 of SMH on HO· was 2.14 ± 0.17 mg/mL. Using ultrafiltration, gel filtration chromatography, and RP-HPLC, two novel antioxidant nonapeptides (SP-A and SP-B) were isolated from SMH and their amino acid sequences were found to be APPTAYAQS (SP-A) and NWDMEKIWD (SP-B) with calculated molecular masses of 904.98 Da and 1236.38 Da, respectively. Both showed strong antioxidant activities. SP-A and SP-B exhibited good scavenging activities on HO· (EC50 0.390 and 0.176 mg/mL), DPPH· (EC50 0.614 and 0.289 mg/mL), and O2-· (EC50 0.215 and 0.132 mg/mL) in a dose-dependent manner. SP-B was also effective against lipid peroxidation in the model system. The aromatic (2Trp), acidic (2Asp and Glu), and basic (Lys) amino acid residues within the sequences of SP-B might account for its pronounced antioxidant activity. The results of this study suggested that protein hydrolysate and peptides from skate muscle might be effective as food additives for retarding lipid peroxidation occurring in foodstuffs.

Protease increases fermentation rate and ethanol yield in dry-grind ethanol production.

The effects of acid protease and urea addition during the fermentation step were evaluated. The fermentations were also tested with and without the addition of urea to determine if protease altered the nitrogen requirements of the yeast. Results show that the addition of the protease had a statistically significant effect on the fermentation rate and yield. Fermentation rates and yields were improved with the addition of the protease over the corresponding controls without protease. Protease addition either with or with added urea resulted in a higher final ethanol yield than without the protease addition. Urea addition levels >1200 ppm of supplemental nitrogen inhibited ethanol production. The economic effects of the protease addition were evaluated by using process engineering and economic models developed at the Eastern Regional Research Center. The decrease in overall processing costs from protease addition was as high as $0.01/L (4 ¢/gal) of denatured ethanol produced.