Exploring the historical roots, advantages and efficacy of phage therapy in plant diseases management.

In the drug-resistance era, phage therapy has received considerable attention from worldwide researchers. Phage therapy has been given much attention in public health but is rarely applied to control plant diseases. Herein, we discuss phage therapy as a biocontrol approach against several plant diseases. The emergence of antibiotic resistance in agriculturally important pathogenic bacteria and the toxic nature of different synthetic compounds used to control microbes has driven researchers to rethink the century-old strategy of phage therapy''. Compared to other treatment strategies, phage therapy offers remarkable advantages such as high specificity, less chances of drug resistance, non-harmful nature, and benefit to soil microbial flora. The optimizations and protective formulations of phages are significant accomplishments; however, steps towards a better understanding of the physiologic characteristics of phages need to be preceded to commercialize their use. The future of phage therapy in the context of plant disease management is promising and could play a significant role in sustainable agriculture. Ongoing research will likely affirm the safety of phage therapy, ensuring that it does not harm non-target organisms, including beneficial soil microbes. Phage therapy could become vital in addressing global food security challenges, particularly in regions heavily impacted by plant bacterial diseases. Efforts to create formulations that enhance the stability and shelf-life of phages will be crucial, especially for their use in varied environmental conditions.

Development of a Universal One-Step Purification and Activation Method to Engineer Protein-Glutaminase through Rational Design.

Cytotoxic enzymes often exist as zymogens containing prodomains to keep them in an inactive state. Protein-glutaminase (PG), which can enhance various functional characteristics of food proteins, is an enzyme containing pro-PG and mature-PG (mPG). However, poor activity and stability limit its application while tedious purification and activation steps limit its high-throughput engineering. Here, based on structural analysis, we replaced the linker sequence between pro-PG and mPG with the HRV3C protease recognition sequence and then coexpressed it with HRV3C protease in Escherichia coli to develop an efficient one-step purification and activation method for PG. We then used this method to obtain several mutants designed by a combination of computer-aided approach and beneficial point mutations. The specific activity (131.6 U/mg) of the best variant D1 was 4.14-fold that of the wild type, and t1/2 and T5010 increased by 13 min and 7 °C, respectively. D1 could effectively improve the solubility and emulsification of wheat proteins, more than twice the effect of the wild type. We also discussed the mechanism underlying the improved properties of D1. In summary, we not only provide a universal one-step purification and activation method to facilitate zymogen engineering but also obtain an excellent PG mutant.

Revolutionizing Treatment Strategies for Autoimmune and Inflammatory Disorders: The Impact of Dipeptidyl-Peptidase 4 Inhibitors.

DPP4 (Dipeptidyl-peptidase 4) a versatile protease, emerges as a prominent player in soluble and membrane-bound forms. Its heightened expression has been intimately linked to the initiation and severity of diverse autoimmune diseases, spanning rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis (SSc), inflammatory bowel disease, autoimmune diabetes, and even SARS-CoV-2 infection. Operating as a co-stimulator of T cell activity, DPP4 propels T cell proliferation by binding adenosine deaminase (ADA), thereby augmenting the breakdown of adenosine-an influential inhibitor of T cell proliferation. However, the discovery of a wide range of DPP4 inhibitors has shown promise in alleviating these diseases' signs, symptoms, and severity. The available DPP4 inhibitors have demonstrated significant effectiveness in blocking DPP4 activity. Based on the characterization of their binding mechanisms, three distinct groups of DPP4 inhibitors have been identified: saxagliptin, alogliptin, and sitagliptin, each representing a different class. Elevated levels of angiotensin-converting enzyme 2 (ACE2) expression are associated with producing various coronavirus peptidases. With its anti-inflammatory properties, Sitagliptin may assist COVID-19 patients in preventing and managing cytokine storms. This comprehensive review delves into the burgeoning realm of DPP4 inhibitors as therapeutic interventions for diverse autoimmune diseases. With a discerning focus on their efficacy, the investigation sheds light on their remarkable capacity to alleviate the burdensome signs and symptoms intricately linked to these conditions.

Identification of Natural Lead Compounds against Hemagglutinin-Esterase Surface Glycoprotein in Human Coronaviruses Investigated via MD Simulation, Principal Component Analysis, Cross-Correlation, H-Bond Plot and MMGBSA.

The pandemic outbreak of human coronavirus is a global health concern that affects people of all ages and genders, but there is currently still no effective, approved and potential drug against human coronavirus, as many other coronavirus vaccines have serious side effects while the development of small antiviral inhibitors has gained tremendous attention. For this research, HE was used as a therapeutic target, as the spike protein displays a high binding affinity for both host ACE2 and viral HE glycoprotein. Molecular docking, pharmacophore modelling and virtual screening of 38,000 natural compounds were employed to find out the best natural inhibitor against human coronaviruses with more efficiency and fewer side effects and further evaluated via MD simulation, PCA, DCCR and MMGBSA. The lead compound 'Calceolarioside B' was identified on the basis of pharmacophoric features which depict favorable binding (ΔGbind -37.6799 kcal/mol) with the HE(5N11) receptor that describes positive correlation movements in active site residues with better stability, a robust H-bond network, compactness and reliable ADMET properties. The Fraxinus sieboldiana Blume plant containing the Calceolarioside B compound could be used as a potential inhibitor that shows a higher efficacy and potency with fewer side effects. This research work will aid investigators in the testing and identification of chemicals that are effective and useful against human coronavirus.

Whole-genome sequence of a putative pathogenic Bacillus sp. strain SD-4 isolated from cattle feed.

OBJECTIVES: The present study describes the draft genome sequence of a novel Bacillus sp. strain SD-4 isolated from animal feed. The study aims to get a deeper insight into antimicrobial resistance and secondary metabolite biosynthetic gene clusters (BGCs) and the association between them. METHODS: The strain SD-4 was preliminarily evaluated for antibacterial activities, motility, biofilm formation, and enterotoxin production using in vitro assays. The genome of strain SD-4 was sequenced using the Illumina HiSeq 2500 platform with paired-end reads. The reads were assembled and annotated using SPAdes and PGAP, respectively. The genome was further analysed using several bioinformatics tools, including TYGS, AntiSMASH, RAST, PlasmidFinder, VFDB, VirulenceFinder, CARD, PathogenFinder, MobileElement finder, IslandViewer, and CRISPRFinder. RESULTS: In vitro assays showed that the strain is motile, synthesises biofilm, and produces an enterotoxin and antibacterial metabolites. The genome analysis revealed that the strain SD-4 carries antimicrobial resistance genes (ARGs), virulence factors, and beneficial secondary metabolite BGCs. Further genome analysis showed interesting genome architectures containing several mobile genetic elements, including two plasmid replicons (repUS22 and rep20), five prophages, and at least four genomic islands (GIs), including one Listeria pathogenicity island LIPI-1. Moreover, the strain SD-4 is identified as a putative human pathogen. CONCLUSION: The genome of strain SD-4 harbours several BGCs coding for biologically active metabolites. It also contains antimicrobial resistance genes and is identified as a potential human pathogen. These results can be used to better comprehend antibiotic resistance in environmental bacteria that are not influenced by human intervention.

Improved Production of Xylanase in Pichia pastoris and Its Application in Xylose Production From Xylan.

Xylanases with high specific activity has been focused with great interest as a useful enzyme in biomass utilization. The production of recombinant GH11 xylanase (MYCTH_56237) from Myceliophthora thermophila has been improved through N-terminal signal peptide engineering in P. pastoris. The production of newly recombinant xylanase (termed Mtxyn11C) was improved from 442.53 to 490.7 U/mL, through a replacement of α-factor signal peptide with the native xylanase signal peptide segment (MVSVKAVLLLGAAGTTLA) in P. pastoris. Scaling up of Mtxyn11C production in a 7.5 L fermentor was improved to the maximal production rate of 2503 U/mL. In this study, the degradation efficiency of Mtxyn11C was further examined. Analysis of the hydrolytic mode of action towards the birchwood xylan (BWX) revealed that Mtxyn11C was clearly more effective than commercial xylanase and degrades xylan into xylooligosaccharides (xylobiose, xylotriose, xylotetraose). More importantly, Mtxyn11C in combination with a single multifunctional xylanolytic enzyme, improved the hydrolysis of BWX into single xylose by 40%. Altogether, this study provided strategies for improved production of xylanase together with rapid conversion of xylose from BWX, which provides sustainable, cost-effective and environmental friendly approaches to produce xylose/XOSs for biomass energy or biofuels production.

Role of the fungus-specific flavin carrier Flc1 in antifungal resistance in the fungal pathogen Cryptococcus neoformans.

FLC family, a conserved fungus-specific family of integral membrane proteins, has been demonstrated to play important roles in flavin transport, growth, and virulence in several fungi but not yet in Cryptococcus neoformans. In this study, we have identified the single homologue of flavin adenine dinucleotide transporter in the opportunistic pathogen C. neoformans. The computational and phylogenetic analysis confirmed the fungal specificity of cryptococcal Flc1 protein, thus providing a promising drug target for clinical treatment of cryptococcosis. Disruption of FLC1 conferred sensitivity to 1% Congo red and 0.02% SDS, as well as leading to impaired chitin distribution in cell wall as observed with Calcofluor White staining, which collectively indicated the roles of FLC1 in maintenance of cell wall integrity. Further investigations revealed the defects of flc1Δ mutant in resistance to poor nutrition and elevated temperatures, and the ability to undergo invasive growth under nutrient-depleted conditions was reduced as well in flc1Δ mutant, suggesting the roles of Flc1 in response to environmental stresses. More importantly, our results showed that flc1Δ mutant exhibited severe susceptibility to antifungal aminoglycosides (hygromycin B and geneticin) and amphotericin B, but developed multidrug resistance to flucytosine and rapamycin, which provided great hints for therapeutic failure of cryptococcosis in clinic with the standard combination therapy. Finally, typical virulence factors including melanin biosynthesis and capsule formation in flc1Δ mutant were reduced as well, indicating the possible involvement of Flc1 in virulence.

Characterization of Two Endo-ß-1, 4-Xylanases from Myceliophthora thermophila and Their Saccharification Efficiencies, Synergistic with Commercial Cellulase.

The xylanases with high specific activity and resistance to harsh conditions are of high practical value for biomass utilization. In the present study, two new GH11 xylanase genes, MYCTH_56237 and MYCTH_49824, have been cloned from thermophilic fungus Myceliophthora thermophila and expressed in Pichia pastoris. The specific activities of purified xylanases reach approximately 1,533.7 and 1,412.5 U/mg, respectively. Based on multiple template-based homology modeling, the structures of their catalytic domains are predicted. Enzyme activity was more effective in 7.5 L fermentor, yielding 2,010.4 and 2,004.2 U/mL, respectively. Both enzymes exhibit optimal activity at 60°C with pH of 6.0 and 7.0, respectively. Their activities are not affected by EDTA and an array of metal ions. The kinetic constants have been determined for MYCTH_56237 (Km = 8.80 mg/mL, Vmax = 2,380 U/mg) and MYCTH_49824 (Km = 5.67 mg/mL, Vmax = 1,750 U/mg). More importantly, both xylanases significantly cooperate with the commercial cellulase Celluclast 1.5 L in terms of the saccharification efficiency. All these biochemical properties of the xylanases offer practical potential for future applications.

Genome-Wide Identification of circRNAs in Pathogenic Basidiomycetous Yeast Cryptococcus neoformans Suggests Conserved circRNA Host Genes over Kingdoms.

Circular RNAs (circRNAs), a novel class of ubiquitous and intriguing noncoding RNA, have been found in a number of eukaryotes but not yet basidiomycetes. In this study, we identified 73 circRNAs from 39.28 million filtered RNA reads from the basidiomycete Cryptococcus neoformans JEC21 using next-generation sequencing (NGS) and the bioinformatics tool circular RNA identification (CIRI). Furthermore, mapping of newly found circRNAs to the genome showed that 73.97% of the circRNAs originated from exonic regions, whereas 20.55% were from intergenic regions and 5.48% were from intronic regions. Enrichment analysis of circRNA host genes was conducted based on the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway databases. The results reveal that host genes are mainly responsible for primary metabolism and, interestingly, ribosomal protein production. Furthermore, we uncovered a high-level circRNA that was a transcript from the guanosine triphosphate (GTP)ase gene CNM01190 (gene ID: 3255052) in our yeast. Coincidentally, YPT5, CNM01190's ortholog of the GTPase in Schizosaccharomyces pombe, protists, and humans, has already been proven to generate circRNAs. Additionally, overexpression of RNA debranching enzyme DBR1 had varied influence on the expression of circRNAs, indicating that multiple circRNA biosynthesis pathways exist in C. neoformans. Our study provides evidence for the existence of stable circRNAs in the opportunistic human pathogen C. neoformans and raises a question regarding their role related to pathogenesis in this yeast.

Thermophilic xylanases: from bench to bottle.

Lignocellulosic biomass is a valuable raw material. As technology has evolved, industrial interest in new ways to take advantage of this raw material has grown. Biomass is treated with different microbial cells or enzymes under ideal industrial conditions to produce the desired products. Xylanases are the key enzymes that degrade the xylosidic linkages in the xylan backbone of the biomass, and commercial enzymes are categorized into different glycoside hydrolase families. Thermophilic microorganisms are excellent sources of industrially relevant thermostable enzymes that can withstand the harsh conditions of industrial processing. Thermostable xylanases display high-specific activity at elevated temperatures and distinguish themselves in biochemical properties, structures, and modes of action from their mesophilic counterparts. Natural xylanases can be further improved through genetic engineering. Rapid progress with genome editing, writing, and synthetic biological techniques have provided unlimited potential to produce thermophilic xylanases in their natural hosts or cell factories including bacteria, yeasts, and filamentous fungi. This review will discuss the biotechnological potential of xylanases from thermophilic microorganisms and the ways they are being optimized and produced for various industrial applications.

Identification of a basidiomycete-specific Vilse-like GTPase activating proteins (GAPs) and its roles in the production of virulence factors in Cryptococcus neoformans.

Cryptococcus neoformans is a basidiomycetous pathogenic yeast that causes fatal infections in both immunocompetent and immunocompromised patients. Regulation on the production of its virulence factors is not fully understood. Here we reported the characterization of a gene, named CVH1(CNA06260), encoding a Drosophila Vilse-like RhoGAP homolog, which is hallmarked by three conserved functional domains: WW, MyTH4 and RhoGAP. Phylogenetic analysis suggests that CVH1 is highly conserved from protists to mammals and interestingly in basidiomycetes, but absent in plants or Ascomycota and other lower fungi. This phylogenetic distribution indicates an evolutionary link among these groups of organisms. Functional analyses demonstrated that CVH1 was involved in stress tolerance and virulence factor production. By disrupting CVH1, we created a second mutant cvh1Δ with the CRISPR-Cas9 editing tool. The mutant strain exhibited hypersensitivity to osmotic stress by 2 M sorbitol and NaCl, suggesting defects in the HOG signaling pathway and an interaction of Cvh1 with the HOG pathway. Hypersensitivity of cvh1Δ to 1% Congo red and 0.01% SDS suggests that the cell wall integrity was impaired in the mutant. And cvh1Δ hardly produced the pigment melanin and capsule. Our study for the first time demonstrates that the fungal Vilse-like RhoGAP CVH1 is an important regulator of multiple biological processes in C. neoformans, and provides novel insights into the regulatory circuit of stress resistance/cell wall integrity, and laccase and capsule synthesis in C. neoformans.

First report on molecular characterization of Leishmania species from cutaneous leishmaniasis patients in southern Khyber Pakhtunkhwa province of Pakistan.

OBJECTIVE: To report presence of Leishmania major in Khyber Pakhtunkhwa of Pakistan, where cutaneous leishmaniasis (CL) is endemic and was thought to be caused by Leishmania tropica only. METHODS: Biopsy samples from 432 CL suspected patients were collected from 3 southern districts of Khyber Pakhtunkhwa during years 2011-2016. Microscopy on Giemsa stained slides were done followed by amplification of the ribosomal internal transcribed spacer 1 gene. RESULTS: Leishmania amastigotes were detected by microscopy in 308 of 432 samples (71.3%) while 374 out of 432 samples (86.6%) were positive by ribosomal internal transcribed spacer 1 PCR. Subsequent restriction fragment length polymorphism confirmed L. tropica in 351 and L. major in 6 biopsy samples. CONCLUSIONS: This study is the first molecular characterization of Leishmania species in southern Khyber Pakhtunkhwa. It confirmed the previous assumptions that anthroponotic CL is the major CL form present in Khyber Pakhtunkhwa province. Furthermore, this is the first report of L. major from a classical anthroponotic CL endemic focus identified in rural areas of Kohat district in southern Khyber Pakhtunkhwa.

Bacterial Contribution in Chronicity of Wounds.

A wound is damage of a tissue usually caused by laceration of a membrane, generally the skin. Wound healing is accomplished in three stages in healthy individuals, including inflammatory, proliferative, and remodeling stages. Healing of wounds normally starts from the inflammatory phase and ends up in the remodeling phase, but chronic wounds remain in an inflammatory stage and do not show progression due to some specific reasons. Chronic wounds are classified in different categories, such as diabetic foot ulcer (DFU), venous leg ulcers (VLU) and pressure ulcer (PU), surgical site infection (SSI), abscess, or trauma ulcers. Globally, the incidence rate of DFU is 1-4 % and prevalence rate is 5.3-10.5 %. However, colonization of pathogenic bacteria at the wound site is associated with wound chronicity. Most chronic wounds contain more than one bacterial species and produce a synergetic effect that results in previously non-virulent bacterial species becoming virulent and causing damage to the host. While investigating bacterial diversity in chronic wounds, Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia were found most frequently in chronic wounds. Recently, it has been observed that bacteria in chronic wounds develop biofilms that contribute to a delay in healing. In a mature biofilm, bacteria grow slowly due to deficiency of nutrients that results in the resistance of bacteria to antibiotics. The present review reflects the reasons why acute wounds become chronic. Interesting findings include the bacterial load, which forms biofilms and shows high-level resistance toward antibiotics, which is a threat to human health in general and particularly to some patients who have acute wounds.

Geographic distribution of hepatitis C virus genotypes in pakistan.

BACKGROUND: Distribution of Hepatitis C Virus (HCV) genotypes may be changed over time. Epidemiological Studies on distribution patterns of HCV genotypes in Pakistani population might assist for better treatment options and preventive strategies. OBJECTIVES: This study was conducted to determine distribution patterns of HCV genotypes in different geographical regions of Pakistan. PATIENTS AND METHODS: In this cross-sectional study, 1818 randomly selected patients from different geographical regions of Pakistan, diagnosed with HCV infection by the third generation Enzyme Linked Immunosorbent Assay (ELISA), were included between April 2011 and December 2013. HCV RNA was detected in serum samples of patients by Reverse Transcription Polymerase Chain Reaction (RT- PCR) of the core region. Qualitative PCR was performed to determine viral load. HCV genotyping was performed by RT-nested PCR using type-specific primers of the core region. Frequency of different genotypes among patients was assessed according to gender, age and geographical region at the time of sampling. RESULTS: Of 1818 HCV RNA positive samples, HCV genotypes PCR fragments were detected in 1552 (85.5%) samples. HCV genotype 3a was the predominant genotype (39.4%) followed by genotype 2a (24.93%). HCV genotype 3 was the predominant genotype in Punjab and Sindh regions, while genotype 2 was the most predominant genotype in Khyber Pakhtunkhwa region and the second predominant genotype after genotype 3 in Sindh region. The incidence of genotype 2a is increasing in our country with decrease in the incidence of genotype 3a. A higher incidence of HCV various genotypes were observed among male patients and those younger than 45 years. CONCLUSIONS: This study may facilitate treatment options and preventive strategies in Pakistan.