Perspective on Agricultural Industrialization: Modification Strategies for Enhancing the Catalytic Capacity of Keratinase.

Keratinases is a special hydrolytic enzyme produced by microorganisms, which has the ability to catalyze the degradation of keratin. Currently, keratinases show great potential for application in many agricultural and industrial fields, such as biofermented feed, leather tanning, hair removal, and fertilizer production. However, these potentials have not yet been fully unleashed on an industrial scale. This paper reviews the sources, properties, and catalytic mechanisms of keratinases. Strategies for the molecular modification of keratinases are summarized and discussed in terms of improving the substrate specificity, thermostability, and pH tolerance of keratinases. The modification strategies are also enriched by the introduction of immobilized enzymes and directed evolution. In addition, the selection of modification strategies when facing specific industrial applications is discussed and prospects are provided. We believe that this review serves as a reference for the future quest to extend the application of keratinases from the laboratory to industry.

Bacillus subtilis: current and future modification strategies as a protein secreting factory.

Bacillus subtilis is regarded as a promising microbial expression system in bioengineering due to its high stress resistance, nontoxic, low codon preference and grow fast. The strain has a relatively efficient expression system, as it has at least three protein secretion pathways and abundant molecular chaperones, which guarantee its expression ability and compatibility. Currently, many proteins are expressed in Bacillus subtilis, and their application prospects are broad. Although Bacillus subtilis has great advantages compared with other prokaryotes related to protein expression and secretion, it still faces deficiencies, such as low wild-type expression, low product activity, and easy gene loss, which limit its large-scale application. Over the years, many researchers have achieved abundant results in the modification of Bacillus subtilis expression systems, especially the optimization of promoters, expression vectors, signal peptides, transport pathways and molecular chaperones. An optimal vector with a suitable promoter strength and other regulatory elements could increase protein synthesis and secretion, increasing industrial profits. This review highlights the research status of optimization strategies related to the expression system of Bacillus subtilis. Moreover, research progress on its application as a food-grade expression system is also presented, along with some future modification and application directions.

Comparative Probiogenomics Analysis of Limosilactobacillus fermentum 3872.

Limosilactobacillus fermentum is an important member of the lactic acid bacteria group and holds immense potential for probiotic properties in human health and relevant industries. In this study, a comparative probiogenomic approach was applied to analyze the genome sequence of L. fermentum 3872, which was extracted from a commercially available yogurt sample, along with 20 different publicly available strains. Results indicate that the genome size of the characterized L. fermentum 3892 strain is 2,057,839 bp, with a single- and circular-type chromosome possessing a G + C content of 51.69%. The genome of L. fermentum 3892 strain comprises a total of 2120 open reading frames (ORFs), two genes encoding rRNAs, and 53 genes encoding tRNAs. Upon comparative probiogenomic analysis, two plasmid sequences were detected among the study strains, including one for the L. fermentum 3872 genome, which was found between position 1,288,203 and 1,289,237 with an identity of 80.98. The whole-genome alignment revealed 2223 identical sites and a pairwise identity of 98.9%, indicating a significant difference of 1.1% among genome strains. Comparison of amino acid encoding genes among strains included in this study suggests that the strain 3872 exhibited the highest degree of amino acids present, including glutamine, glutamate, aspartate, asparagine, lysine, threonine, methionine, and cysteine. The comparative antibiotic resistome profiling revealed that strain 3872 exhibited a high resistant capacity only to ciprofloxacin antibiotics as compared to other strains. This study provides a genomic-based evaluation approach for comparative probiotic strain analysis in commercial foods and their significance to human health.

Molecular strategies to enhance the keratinase gene expression and its potential implications in poultry feed industry.

The tons of keratin waste are produced by the poultry and meat industry which is an insoluble and protein-rich material found in hair, feathers, wool, and some epidermal wastes. These waste products could be degraded and recycled to recover protein, which can save our environment. One of the potential strategy to achieve this target is use of microbial biotreatment which is more convenient, cost-effective, and environment-friendly by formulating hydrolysate complexes that could be administered as protein supplements, bioactive peptides, or animal feed ingredients. Keratin degradation shows great promise for long-term protein and amino acid recycling. According to the MEROPS database, known keratinolytic enzymes currently belong to at least 14 different protease families, including S1, S8, S9, S10, S16, M3, M4, M14, M16, M28, M32, M36, M38, and M55. In addition to exogenous attack (proteases from families S9, S10, M14, M28, M38, and M55), the various keratinolytic enzymes also function via endo-attack (proteases from families S1, S8, S16, M4, M16, and M36). Biotechnological methods have shown great promise for enhancing keratinase expression in different strains of microbes and different protein engineering techniques in genetically modified microbes such as bacteria and some fungi to enhance keratinase production and activity. Some microbes produce specific keratinolytic enzymes that can effectively degrade keratin substrates. Keratinases have been successfully used in the leather, textile, and pharmaceutical industries. However, the production and efficiency of existing enzymes need to be optimized before they can be used more widely in other processes, such as the cost-effective pretreatment of chicken waste. These can be improved more effectively by using various biotechnological applications which could serve as the best and novel approach for recycling and degrading biomass. This paper provides practical insights about molecular strategies to enhance keratinase expression to effectively utilize various poultry wastes like feathers and feed ingredients like soybean pulp. Furthermore, it describes the future implications of engineered keratinases for environment friendly utilization of wastes and crop byproducts for their better use in the poultry feed industry.

In silico design of a broad-spectrum multiepitope vaccine against influenza virus.

Influenza remains a global health concern due to its potential to cause pandemics as a result of rapidly mutating influenza virus strains. Existing vaccines often struggle to keep up with these rapidly mutating flu viruses. Therefore, the development of a broad-spectrum peptide vaccine that can stimulate an optimal antibody response has emerged as an innovative approach to addressing the influenza threat. In this study, an immunoinformatic approach was employed to rapidly predict immunodominant epitopes from different antigens, aiming to develop an effective multiepitope influenza vaccine (MEV). The immunodominant B-cell linear epitopes of seasonal influenza strains hemagglutinin (HA) and neuraminidase (NA) were predicted using an antibody-peptide microarray, involving a human cohort including vaccinees and infected patients. On the other hand, bioinformatics tools were used to predict immunodominant cytotoxic T-cell (CTL) and helper T-cell (HTL) epitopes. Subsequently, these epitopes were evaluated by various immunoinformatic tools. Epitopes with high antigenicity, high immunogenicity, non-allergenicity, non-toxicity, as well as exemplary conservation were then connected in series with appropriate linkers and adjuvants to construct a broad-spectrum MEV. Moreover, the structural analysis revealed that the MEV candidates exhibited good stability, and the docking results demonstrated their strong affinity to Toll-like receptors 4 (TLR4). In addition, molecular dynamics simulation confirmed the stable interaction between TLR4 and MEVs. Three injections with MEVs showed a high level of B-cell and T-cell immune responses according to the immunological simulations in silico. Furthermore, in-silico cloning was performed, and the results indicated that the MEVs could be produced in considerable quantities in Escherichia coli (E. coli). Based on these findings, it is reasonable to create a broad-spectrum MEV against different subtypes of influenza A and B viruses in silico.

The flexible linker and CotG were more effective for the spore surface display of keratinase KERQ7.

Feather, horn, hoof, and other keratin waste are protein-rich but limited by natural keratinase synthesis, activity, pH, and temperature stability. It is challenging to realize its large-scale application in industries. Bacillus subtilis spores are a safe, efficient, and highly resistant immobilized carrier, which can improve target proteins' resistance. In this research, KERQ7, the keratinase gene of Bacillus tequilensis strain Q7, was fused to the Bacillus subtilis genes coding for the coat proteins CotG and CotB, respectively, and displayed on the surface of B. subtilis spores. Compared with the free KERQ7, the immobilized KERQ7 showed a greater pH tolerance and heat resistance on the spore surface. The activity of CotG-KERQ7 is 1.25 times that of CotB-KERQ7, and CotG-KERQ7 is more stable. When the flexible linker peptide L3 was used to connect CotG and KERQ7, the activity was increased to 131.2 ± 3.4%, and the residual enzyme activity was still 62.5 ± 2.2% after being kept at 60 ℃ for 4 h. These findings indicate that the flexible linker and CotG were more effective for the spore surface display of keratinase to improve stress resistance and promote its wide application in feed, tanning, washing, and other industries.

An investigation of long-term outcome of rabbit anti-thymocyte globulin and cyclosporine therapy for pediatric severe aplastic anemia.

Children with severe aplastic anemia (SAA) face heterogeneous prognoses after immunosuppressive therapy (IST). There are few models that can predict the long-term outcomes of IST for these patients. The objective of this paper is to develop a more effective prediction model for SAA prognosis based on clinical electronic medical records from 203 children with newly diagnosed SAA. In the early stage, a novel model for long-term outcomes of SAA patients with IST was developed using machine-learning techniques. Among the indicators related to long-term efficacy, white blood cell count, lymphocyte count, absolute reticulocyte count, lymphocyte ratio in bone-marrow smears, C-reactive protein, and the level of IL-6, IL-8 and vitamin B12 in the early stage are strongly correlated with long-term efficacy (P < .05). Taken together, we analyzed the long-term outcomes of rabbit anti-thymocyte globulin and cyclosporine therapy for children with SAA through machine-learning techniques, which may shorten the observation period of therapeutic effects and reduce treatment costs and time.

Research progress on the degradation mechanism and modification of keratinase.

Keratin is regarded as the main component of feathers and is difficult to be degraded by conventional proteases, leading to substantial abandonment. Keratinase is the only enzyme with the most formidable potential for degrading feathers. Although there have been in-depth studies in recent years, the large-scale application of keratinase is still associated with many problems. It is relatively challenging to find keratinase not only with high activity but could also meet the industrial application environment, so it is urgent to exploit keratinase with high acid and temperature resistance, strong activity, and low price. Therefore, researchers have been keen to explore the degradation mechanism of keratinases and the modification of existing keratinases for decades. This review critically introduces the basic properties and mechanism of keratinase, and focuses on the current situation of keratinase modification and the direction and strategy of its future application and modification. KEY POINTS: •The research status and mechanism of keratinase were reviewed. •The new direction of keratinase application and modification is discussed. •The existing modification methods and future modification strategies of keratinases are reviewed.

Protective antigenic epitopes revealed by immunosignatures after three doses of inactivated SARS-CoV-2 vaccine.

Background: SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has infected millions of people around the world. Vaccination is a pillar in the strategy to control transmission of the SARS-CoV-2 spread. Immune responses to vaccination require elucidation. Methods: The immune responses to vaccination with three doses of inactivated SARS-CoV-2 vaccine were followed in a cohort of 37 healthy adults (18-59 years old). Blood samples were collected at multiple time points and submitted to peptide array, machine learning modeling, and sequence alignment analyses, the results of which were used to generate vaccine-induced antibody-binding region (VIABR) immunosignatures (Registration number: ChiCTR2200058571). Results: Antibody spectrum signals showed vaccination stimulated antibody production. Sequence alignment analyses revealed that a third vaccine dose generated a new highly represented VIABR near the A570D mutation, and the whole process of inoculation enhanced the VIABR near the N501Y mutation. In addition, the antigen conformational epitopes varied between short- and long-term samples. The amino acids with the highest scores in the short-term samples were distributed primarily in the receptor binding domain (RBD) and N-terminal domain regions of spike (S) protein, while in the long-term samples (12 weeks after the 2nd dose), some new conformational epitopes (CEs) were localized to crevices within the head of the S protein trimer. Conclusion: Protective antigenic epitopes were revealed by immunosignatures after three doses of inactivated SARS-CoV-2 vaccine inoculation. A third dose results in a new top-10 VIABR near the A570D mutation site of S protein, and the whole process of inoculation enhanced the VIABR near the N501Y mutation, thus potentially providing protection from strains that have gained invasion and immune escape abilities through these mutation.

The Biological Significance of Multi-copy Regions and Their Impact on Variant Discovery.

Identification of genetic variants via high-throughput sequencing (HTS) technologies has been essential for both fundamental and clinical studies. However, to what extent the genome sequence composition affects variant calling remains unclear. In this study, we identified 63,897 multi-copy sequences (MCSs) with a minimum length of 300 bp, each of which occurs at least twice in the human genome. The 151,749 genomic loci (multi-copy regions, or MCRs) harboring these MCSs account for 1.98% of the genome and are distributed unevenly across chromosomes. MCRs containing the same MCS tend to be located on the same chromosome. Gene Ontology (GO) analyses revealed that 3800 genes whose UTRs or exons overlap with MCRs are enriched for Golgi-related cellular component terms and various enzymatic activities in the GO biological function category. MCRs are also enriched for loci that are sensitive to neocarzinostatin-induced double-strand breaks. Moreover, genetic variants discovered by genome-wide association studies and recorded in dbSNP are significantly underrepresented in MCRs. Using simulated HTS datasets, we show that false variant discovery rates are significantly higher in MCRs than in other genomic regions. These results suggest that extra caution must be taken when identifying genetic variants in the MCRs via HTS technologies.