K1 Strain Research Articles

Enhancement of Repeat-Mediated Deletion Rearrangement Induced by Particle Irradiation in a RecA-Dependent Manner in Escherichia coli.

Repeat-mediated deletion (RMD) rearrangement is a major source of genome instability and can be deleterious to the organism, whereby the intervening sequence between two repeats is deleted along with one of the repeats. RMD rearrangement is likely induced by DNA double-strand breaks (DSBs); however, it is unclear how the complexity of DSBs influences RMD rearrangement. Here, a transgenic Escherichia coli strain K12 MG1655 with a lacI repeat-controlled amp activation was used while taking advantage of particle irradiation, such as proton and carbon irradiation, to generate different complexities of DSBs. Our research confirmed the enhancement of RMD under proton and carbon irradiation and revealed a positive correlation between RMD enhancement and LET. In addition, RMD enhancement could be suppressed by an intermolecular homologous sequence, which was regulated by its composition and length. Meanwhile, RMD enhancement was significantly stimulated by exogenous λ-Red recombinase. Further results investigating its mechanisms showed that the enhancement of RMD, induced by particle irradiation, occurred in a RecA-dependent manner. Our finding has a significant impact on the understanding of RMD rearrangement and provides some clues for elucidating the repair process and possible outcomes of complex DNA damage.

Role of Ibea Gene in Hbmecs Invasion and Biofilm Formation Induced By Neonatal Meningitis Escherichia coli

Background: Bacterial meningitis is a severe infectious disease that affects the central nervous system and can result in high mortality rates and neurological complications. In neonates, meningitis is mainly caused by neonatal meningitis Escherichia coli (NMEC), which invades the intestine, leading to hematogenous spreadand potential bacterial meningitis through blood-brain barrier. Previous studies indicated that the ability of NMEC to adhere and invade is associated with biofilm formation and a deficiency in ibeA may result in decreased virulence and biofilm-forming ability of avian pathogenic Escherichia coli (APEC). Furthermore, the ibeA gene is positioned downstream of ibeR. Our previous studies have shown that ibeR and ibeA genes can both regulate the virulence and relevant phenotypes in E. coli K1. However, the effects of ibeR and ibeA on the biofilm formation and invasion of K1 strain remain to be further studied. Recent studies suggest that indole may act as a signaling molecule to regulate the expression of adhesion and biofilm-promoting factors in E. coli. However, the mechanism by which indole affects biofilms is still unclear. This study aimed to investigate the mechanism of ibeA inhibition of indole-induced NMEC invasion and biofilm formation. Methods: The viability and growth of the wild strain E44 and ibeA-deletion mutant strain ZD1 were assessed by MTT cell proliferation assay kit (Coolaber). The absorbance values were measured at 490 nm and 540 nm by ELISA. The invasion activities and the biofilm formation ability of E44 and ZD1 were detected in vitro studies. We conducted a cell invasion test using the Human Brain Microvascular Endothelial Cells (HBMEC) model. The ability to form a biofilm was measured at 600 nm using a specific biofilm formation index. Biofilm phenotypic was observed by an confocal laser-scanning microscope(Olympus FV-1000, Japan). Then, we used indole as a regulatory agency to compare its biofilm inhibitory efficacy between E44 and ibeR-deletion mutant strain BR2. The experimental data were analyzed using the SPSS 19.0 statistical package. A statistically significant difference was indicated by P<0.05. Results: No difference was found in bacterial growth between the E. coli strains E44 and ZD1. The biofilm formation and HBMECs invasion of ZD1 was significantly lower than that of E44. After treatment with 0.5mM indole, the biofilm formation ability and the ibeA protein expressions of E44 were significantly inhibited. However, indole at different concentrations had no obvious inhibition on BR2 biofilm formation and the ibeA protein expressions. Conclusion: In summary, the study found that ibeA had a significant regulatory effect on HBMECs invasion and biofilm formation of NMEC, which may inhibit by indole via regulating ibeA through ibeR. ( Acknowledgements: Grant from School of Public Health of Southern Medical University, Guangzhou, China, No. GW202329; J.Z., Q.L. and L.W. contributed equally to this work; Corresponding author: Hong Cao, gzhcao@smu.edu.cn)

Structural informatics approach for designing an epitope-based vaccine against the brain-eating Naegleria fowleri.

Primary Amoebic Meningoencephalitis (PAM), a severe lethal brain disease, is caused by a parasite, Naegleria fowleri, also known as the "brain-eating amoeba". The chances of a patient's recovery after being affected by this parasite are very low. Only 5% of people are known to survive this life-threatening infection. Despite the fact that N. fowleri causes a severe, fatal infection, there is no proper treatment available to prevent or cure it. In this context, it is necessary to formulate a potential vaccine that could be able to combat N. fowleri infection. The current study aimed at developing a multi-epitope subunit vaccine against N. fowleri by utilizing immunoinformatics techniques and reverse vaccinology approaches. The T- and B-cell epitopes were predicted by various tools. In order to choose epitopes with the ability to trigger both T- and B-cell-mediated immune responses, the epitopes were put through a screening pipeline including toxicity, antigenicity, cytokine-inductivity, and allergenicity analysis. Three vaccine constructs were designed from the generated epitopes linked with linkers and adjuvants. The modeled vaccines were docked with the immune receptors, where vaccine-1 showed the highest binding affinity. Binding affinity and stability of the docked complex were confirmed through normal mode analysis and molecular dynamic simulations. Immune simulations developed the immune profile, and in silico cloning affirmed the expression probability of the vaccine construct in Escherichia coli (E. coli) strain K12. This study demonstrates an innovative preventative strategy for the brain-eating amoeba by developing a potential vaccine through immunoinformatics and reverse vaccinology approaches. This study has great preventive potential for Primary Amoebic Meningoencephalitis, and further research is required to assess the efficacy of the designed vaccine.

Rational design of multi-epitope-based vaccine by exploring all dengue virus serotypes proteome: an immunoinformatic approach.

Millions of people's lives are being devastated by dengue virus (DENV), a severe tropical and subtropical illness spread by mosquitoes and other vectors. Dengue fever may be self-limiting like a common cold or can rapidly progress to catastrophic dengue hemorrhagic fever or dengue shock syndrome. With four distinct dengue serotypes (DENV1-4), each with the potential to contain antibody-boosting complicated mechanisms, developing a dengue vaccine has been an ambitious challenge. Here, we used a computational pan-vaccinomics-based vaccine design strategy (reverse vaccinology) for all 4 DENV serotypes acquired from different regions of the world to develop a new and safe vaccine against DENV. Consequently, only five mapped epitopes from all the 4 serotypes were shown to be extremely effective for the construction of multi-epitope vaccine constructs. The suggested vaccine construct V5 from eight vaccine models was thus classified as an antigenic, non-allergenic, and stable vaccine model. Moreover, molecular docking and molecular dynamics simulation was performed for the V5 vaccine candidate against the HLAs and TRL2 and 4 immunological receptors. Later, the vaccine sequence was transcribed into the cDNA to generate an expression vector for the Escherichia coli K12 strain. Our research suggests that this vaccine design (V5) has promising potential as a dengue vaccine. However, further experimental analysis into the vaccine's efficacy might be required for the V5 proper validation to combat all DENV serotypes.

The IbeA protein from adherent invasive Escherichia coli isa flavoprotein sharing structural homology with FAD-dependent oxidoreductases.

Invasion of brain endothelium protein A (IbeA) is a virulence factor specific to pathogenic Escherichia coli. Originally identified in the K1 strain causing neonatal meningitis, it was more recently found in avian pathogenic Escherichia coli (APEC) and adherent invasive Escherichia coli (AIEC). In these bacteria, IbeA facilitates host cell invasion and intracellular survival, in particular, under harsh conditions like oxidative stress. Furthermore, IbeA from AIEC contributes to intramacrophage survival and replication, thus enhancing the inflammatory response within the intestine. Therefore, this factor is a promising drug target for anti-AIEC strategies in the context of Crohn's disease. Despite such an important role, the biological function of IbeA remains largely unknown. In particular, its exact nature and cellular localization, i.e., membrane-bound invasin versus cytosolic factor, are still of debate. Here, we developed an efficient protocol for recombinant expression of IbeA under native conditions and demonstrated that IbeA from AIEC is a soluble, homodimeric flavoprotein. Using mass spectrometry and tryptophan fluorescence measurements, we further showed that IbeA preferentially binds flavin adenine dinucleotide (FAD), with an affinity in the one-hundred nanomolar range and optimal binding under reducing conditions. 3D-modeling with AlphaFold revealed that IbeA shares strong structural homology with FAD-dependent oxidoreductases. Finally, we used ligand docking, mutational analyses, and molecular dynamics simulations to identify the FAD binding pocket within IbeA and characterize possible conformational changes occurring upon ligand binding. Overall, we suggest that the role of IbeA in the survival of AIEC within host cells, notably macrophages, is linked to modulation of redox processes.

Coexistence of c-rmpA with p-rmpA and p-rmpA2 rather than excessive siderophores confers higher virulence in K1 Klebsiella pneumoniae

Pyogenic liver abscess (PLA), which is particularly endemic in East Asia, is a relatively common and fatal infectious disease. Over the last 30-40 years, Klebsiella pneumoniae has replaced Escherichia coli as the dominant and overwhelming pathogen. To investigate the survival advantage of serotype K1 K.pneumoniae, we determined sequence types (STs), serotypes, and 11 virulence genes (allS, entB, irp2, iroN, iucA, fimH, mrkD, p-rmpA2, c-rmpA, p-rmpA, and peg-344). Virulence genes c-rmpA, p-rmpA, and p-rmpA2 in K.pneumoniae NTUH-K2044, which all confer hypercapsule and consequent hypervirulence, were deleted individually, and the consequent effects were evaluated. The lethality of various K1 K.pneumoniae strains was compared by using the Galleria mellonella model. In total, 31 K1 K.pneumoniae strains causing PLA and 30 causing non-PLA were identified. A significantly higher rate of c-rmpA was presented in PLA-derived K.pneumoniae strains than in non-PLA-derived strains. Similar ST23 (which dominates K1 strains) and string test-positive rates were observed in the two groups. Deletion of c-rmpA, p-rmpA, and p-rmpA2 individually did not confer significant effects on morphologies, such as positive string test, hypercapsule, and growth speed. Δc-rmpA presented weaker expressions of p-rmpA/p-rmpA2 than NTUH-K2044 and showed a higher expression of manC than Δp-rmpA and Δp-rmpA2. Three rmpAs conferred more virulence than one or two rmpAs, which presented an equally lethal effect in K1 K.pneumoniae. Klebsiella pneumoniae strains (H19 and H34) with the same genetic backgrounds except for siderophores showed equal virulence, but were less virulent than strain NTUH-K2044. Thus, the coexistence of c-rmpA with p-rmpA and p-rmpA2 enhances the lethality of K1 K.pneumoniae strains and the development of PLA. Excessive siderophores are not vital for the hypervirulence of K1 K.pneumoniae strains, although K1 strains usually harbour them on a molecular basis.

Evaluation of 4-Aminoquinoline Hydrazone Analogues as Potential Leads for Drug-Resistant Malaria.

The emergence of resistance to first-line antimalarial drugs calls for the development of new therapies for drug-resistant malaria. The efficacy of quinoline-based antimalarial drugs has prompted the development of novel quinolines. A panel of 4-aminoquinoline hydrazone analogues were tested on the multidrug-resistant K1 strain of Plasmodium falciparum: IC50 values after a 48 h cycle ranged from 0.60 to 49 µM, while the 72 h cycle ranged from 0.026 to 0.219 μM. Time-course assays were carried out to define the activity of the lead compounds, which inhibited over 50% growth in 24 h and 90% growth in 72 h. Cytotoxicity assays with HepG2 cells showed IC50 values of 0.87-11.1 μM, whereas in MDBK cells, IC50 values ranged from 1.66 to 11.7 μM. High selectivity indices were observed for the lead compounds screened at 72 h on P. falciparum. Analyses of stage specificity revealed that the ring stages of the parasite life cycle were most affected. Based on antimalarial efficacy and in vitro safety profiles, lead compound 4-(2-benzylidenehydrazinyl)-6-methoxy-2-methylquinoline 2 was progressed to drug combination studies for the detection of synergism, with a combinatory index of 0.599 at IC90 for the combination with artemether, indicating a synergistic antimalarial activity. Compound 2 was screened on different strains of P. falciparum (3D7, Dd2), which maintained similar activity to K1, suggesting no cross-resistance between multidrug resistance and sensitive parasite strains. In vivo analysis with 2 showed the suppression of parasitaemia with P. yoelii NL (non-lethal)-treated mice (20 mg/kg and 5 mg/kg).

Molecular Mechanisms of Pseudomonas-Assisted Plant Nitrogen Uptake: Opportunities for Modern Agriculture.

Pseudomonas spp. make up 1.6% of the bacteria in the soil and are found throughout the world. More than 140 species of this genus have been identified, some beneficial to the plant. Several species in the family Pseudomonadaceae, including Azotobacter vinelandii AvOP, Pseudomonas stutzeri A1501, Pseudomonas stutzeri DSM4166, Pseudomonas szotifigens 6HT33bT, and Pseudomonas sp. strain K1 can fix nitrogen from the air. The genes required for these reactions are organized in a nitrogen fixation island, obtained via horizontal gene transfer from Klebsiella pneumoniae, Pseudomonas stutzeri, and Azotobacter vinelandii. Today, this island is conserved in Pseudomonas spp. from different geographical locations, which, in turn, have evolved to deal with different geo-climatic conditions. Here, we summarize the molecular mechanisms behind Pseudomonas-driven plant growth promotion, with particular focus on improving plant performance at limiting nitrogen (N) and improving plant N content. We describe Pseudomonas-plant interaction strategies in the soil, noting that the mechanisms of denitrification, ammonification, and secondary metabolite signaling are only marginally explored. Plant growth promotion is dependent on the abiotic conditions and differs at sufficient and deficient N. The molecular controls behind different plant responses are not fully elucidated. We suggest that superposition of transcriptome, proteome, and metabolome data and their integration with plant phenotype development through time will help fill these gaps. The aim of this review is to summarize the knowledge behind Pseudomonas-driven nitrogen fixation and to point to possible agricultural solutions. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Agrobacterium rhizogenes-Mediated Genetic Transformation and Establishment of CRISPR/Cas9 Genome-Editing Technology in Limonium bicolor

Limonium bicolor is a perennial herbaceous plant belonging to the Plumbaginaceae family. It can be used as a dried flower or in cut flower arrangements and serves as a model recretohalophyte. Its genome sequencing has been recently completed. However, the research on L. bicolor is limited by the absence of a highly efficient genetic transformation system. In this study, we established a highly efficient Agrobacterium rhizogenes-mediated L. bicolor genetic transformation method. The transgenic hairy roots were induced from the hypocotyl of L. bicolor using A. rhizogenes strain K599 harboring pRdGa4Cas9 plasmid (which carries an expression cassette of 35S::DsRed2). The transgenic shoots were regenerated from hairy root segments (~0.1 cm diameter), and induction efficiency was achieved at 100%. The transgenic shoots with 4–5 rosette leaves were directly planted into the soil to induce the transgenic roots. Therefore, transgenic plantlets were produced. The DsRed2 can be used as a reliable reporter gene in screening transgenic plantlets. Furthermore, we also established a CRISPR/Cas9 system in L. bicolor employing the A. rhizogenes-mediated genetic transformation approach. The highly efficient transformation method and CRIPSP/Cas9 system established will provide a valuable tool for functional genomics investigation and trait improvement in L. bicolor.

Delineating multi-epitopes vaccine designing from membrane protein CL5 against all monkeypox strains: a pangenome reverse vaccinology approach

The recently identified monkeypox virus (MPXV or mpox) is a zoonotic orthopox virus that infects humans and causes diseases with traits like smallpox. The world health organization (WHO) estimates that 3-6% of MPXV cases result in death. As it might impact everyone globally, like COVID, and become the next pandemic, the cure for this disease is important for global public health. The high incidence and disease ratio of MPXV necessitates immediate efforts to design a unique vaccine candidate capable of addressing MPXV diseases. Here, we used a computational pan-genome-based vaccine design strategy for all currently reported 19 MPXV strains acquired from different regions of the world. Thus, this study's objective was to develop a new and safe vaccine candidate against MPXV by targeting the membrane CL5 protein; identified after the pangenome analysis. Proteomics and reverse vaccinology have covered up all of the MPXV epitopes that would usually stimulate robust host immune responses. Following this, only two mapped (MHC-I, MHC-II, and B-cell) epitopes were observed to be extremely effective that can be used in the construction of CL5 protein vaccine candidates. The suggested vaccine (V5) candidate from eight vaccine models was shown to be antigenic, non-allergenic, and stable (with 213 amino acids). The vaccine's candidate efficacy was evaluated by using many in silico methods to predict, improve, and validate its 3D structure. Molecular docking and molecular dynamics simulations further reveal that the proposed vaccine candidate ensemble has a high interaction energy with the HLAs and TRL2/4 immunological receptors under study. Later, the vaccine sequence was used to generate an expression vector for the E. coli K12 strain. Further study uncovers that V5 was highly immunogenic because it produced robust primary, secondary, and tertiary immune responses. Eventually, the use of computer-aided vaccine designing may significantly reduce costs and speed up the process of developing vaccines. Although, the results of this research are promising, however, more research (experimental; in vivo, and in vitro studies) is needed to verify the biological efficacy of the proposed vaccine against MPXV. Communicated by Ramaswamy H. Sarma

A therapeutic epitopes-based vaccine engineering against Salmonella enterica XDR strains for typhoid fever: a Pan-vaccinomics approach

A prevalent food-borne pathogen, Salmonella enterica serotypes Typhi, is responsible for gastrointestinal and systemic infections globally. Salmonella vaccines are the most effective, however, producing a broad-spectrum vaccine remains challenging due to Salmonella’s many serotypes. Efforts are urgently required to develop a novel vaccine candidate that can tackle all S. Typhi strains because of their high resistance to multiple kinds of antibiotics (particularly the XDR H58 strain). In this work, we used a computational pangenome-based vaccine design technique on all available (n = 119) S. Typhi reference genomes and identified one TonB-dependent siderophore receptor (WP_001034967.1) as highly conserved and prospective vaccine candidates from the predicted core genome (n = 3,351). The applied pan-proteomics and Immunoinformatic approaches help in the identification of four epitopes that may trigger adequate host body immune responses. Furthermore, the proposed vaccine ensemble demonstrates a stable binding conformation with the examined immunological receptor (HLAs and TRL2/4) and has large interaction energy determined via molecular docking and molecular dynamics simulation techniques. Eventually, an expression vector for the Escherichia. coli K12 strain was constructed from the vaccine sequence. Additional analysis revealed that the vaccine may help to elicit strong immune responses for typhoid infections, however, experimental analysis is required to verify the vaccine’s effectiveness based on these results. Moreover, the applied computer-assisted vaccine design may considerably decrease vaccine development costs and speed up the process. The study’s findings are intriguing, but they must be evaluated in the experimental labs to confirm the developed vaccine’s biological efficiency against XDR S. Typhi. Communicated by Ramaswamy H. Sarma

Palladium-catalyzed facile synthesis of imidazo[1,2-a]pyridine-flavone hybrids and evaluation of their antiplasmodial activity

An efficient cross-coupling reaction between imidazo[1,2-a]pyridine derivatives and 6-bromoflavones has been well established. This reaction proceeds through a Palladium-catalyzed cross-coupling reaction to provide imidazo[1,2-a]pyridine-flavone hybrids in good to excellent yield. Short reaction time, high yield, and wide substrate scope are the major advantages of this method. Using SYBR Green I assay, these hybrid molecules were examined for anti-plasmodial activity against Chloroquine-sensitive 3D7 and Chloroquine-resistant K1 strains of Plasmodium falciparum. The compounds 9o {IC50s (µM) 1.983D7, 1.98K1} and 9p {IC50s (µM) 1.923D7, 5.45K1} were found to be the most potent anti-plasmodial compounds in the series. Also, all these compounds were found to be non-cytotoxic towards Vero cells with their CC50s > 100 µM. We have conducted microscopic examination experiments on compounds 9o and 9p, which resulted in a drastic impact on parasite growth of the malaria parasite. The interaction of these two potent hybrids was also examined in the binding site of wild-type Pf-DHFR-TS (Plasmodium falciparum dihydrofolate reductase-thymidylate synthase) using molecular docking studies. The computational ADME studies of the hybrid compounds confirmed their significant physicochemical, pharmacokinetic, and drug-likeness properties confirming these hybrids as a new oral antimalarial agent. As a result, these new hybrid compounds could be valuable in the creation of future antimalarial drugs.

The Arabinose 5-Phosphate Isomerase KdsD Is Required for Virulence in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, which is endemic primarily in Southeast Asia and northern Australia but is increasingly being seen in other tropical and subtropical regions of the world. Melioidosis is associated with high morbidity and mortality rates, which is mediated by the wide range of virulence factors encoded by B. pseudomallei. These virulence determinants include surface polysaccharides such as lipopolysaccharide (LPS) and capsular polysaccharides (CPS). Here, we investigated a predicted arabinose-5-phosphate isomerase (API) similar to KdsD in B. pseudomallei strain K96243. KdsD is required for the production of the highly conserved 3-deoxy-d-manno-octulosonic acid (Kdo), a key sugar in the core region of LPS. Recombinant KdsD was expressed and purified, and API activity was determined. Although a putative API paralogue (KpsF) is also predicted to be encoded, the deletion of kdsD resulted in growth defects, loss of motility, reduced survival in RAW 264.7 murine macrophages, and attenuation in a BALB/c mouse model of melioidosis. Suppressor mutations were observed during a phenotypic screen for motility, revealing single nucleotide polymorphisms or indels located in the poorly understood CPS type IV cluster. Crucially, suppressor mutations did not result in reversion of attenuation in vivo. This study demonstrates the importance of KdsD for B. pseudomallei virulence and highlights further the complex nature of the polysaccharides it produces. IMPORTANCE The intrinsic resistance of B. pseudomallei to many antibiotics complicates treatment. This opportunistic pathogen possesses a wide range of virulence factors, resulting in severe and potentially fatal disease. Virulence factors as targets for drug development offer an alternative approach to combat pathogenic bacteria. Prior to initiating early drug discovery approaches, it is important to demonstrate that disruption of the target gene will prevent the development of disease. This study highlights the fact that KdsD is crucial for virulence of B. pseudomallei in an animal model of infection and provides supportive phenotypic characterization that builds a foundation for future therapeutic development.

Oral Probiotics, Streptococcus salivarius K12 and M18, Suppress the Release of Volatile Sulfur Compounds and a Virulent Protease from Oral Bacteria: An In-Vitro Study.

To evaluate the inhibitory effects of Streptococcus salivarius K12 and M18 strains on the growth of six oral pathogens as well as their release of volatile sulfur compounds (VSCs), and whether these probiotics can inhibit the expression of arginine-specific gingipain A (RgpA), a protease secreted by Porphyromonas gingivalis. After six halitogenic oral pathogens (P. gingivalis, Treponema denticola, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, and Eikenella corrodens) were cultured with or without S. salivarius K12 and M18, the concentration of two VSCs was measured. Moreover, the antimicrobial activity of S. salivarius K12 and M18 against these pathogens and the suppressive effect on RgpA release by P. gingivalis were assessed. In the co-culture of S. salivarius K12 or M18 with oral pathogenic bacteria, the growth of all six oral pathogens was significantly inhibited (p < 0.01). Additionally, S. salivarius K12 and M18 had an inhibitory effect on the production of the halitogenic substances H2S and CH3SH (p < 0.01) as well as the expression of P. gingivalis RgpA. Finally, we demonstrated that the addition of only culture supernatants of the two strains K12/M18 to oral pathogen cultures was sufficient to mimic the effects of K12/M18 co-cultures upon VSCs production and protease expression. S. salivarius K12 and M18 inhibited VSC release by all six of the major oral pathogens that were assayed and reduced the expression of RgpA.

Bioassay-Guided Isolation and Identification of Antiplasmodial Compounds from the Stem Bark of Clausena excavata.

Clausena excavata is a medicinal plant widely distributed in Southeast Asia. It is used for a variety of indications, including to treat malaria. In our present study, a phytochemical study of the methanol extract from the stem bark of C. excavata led to the isolation of five pyranocoumarins, nordentatin (1: ), dentatin (2: ), kinocoumarin (3: ), clausarin (4: ), and clausenidin (5: ), and a coumarin, 8-hydroxy-3″,4″-dihydrocapnolactone-2',3'-diol (6: ). The isolation of compound 6: from C. excavata and the antiplasmodial activities against a multidrug-resistant K1 strain of Plasmodium falciparum of 1, 3: , and 5: were reported for the first time. Compounds 3: and 4: exhibited potent antiplasmodial activities with EC50 values of 1.10 and 0.58 µM, respectively, while 1: and 5: had EC50 values of 5.62 and 7.15 µM, respectively. A prenyl group attached to the C-3 or C-12 position on the pyranocoumarin ring probably plays an important role on the activity. A hydroxyl group at the C-10 position is also likely to enhance the activity.

Photoprotection Against UV-Induced Skin Damage Using Hyaluronic Acid Produced by Lactiplantibacillus plantarum and Enterococcus durans

Exposure to ultraviolet (UV) radiation is one of the major factors that causes skin aging, erythema, sunburns, and skin cancer. This study aimed to select probiotic bacterial isolates able to produce high yield of hyaluronic acid (HA) to be employed for skin photoprotection and other possible biological applications. The selected isolates K11 and St3 were able to produce the highest yields of HA 4.8 and 4.4 mg/ml, respectively. Both isolates were identified as Enterococcus durans strain K11 and Lactiplantibacillus plantarum strain St3 using 16S rRNA gene sequencing. The antioxidant activity of HA produced by E. durans strain K11 and L. plantarum strain St3 was (65.4 0.2%) and (66.6 0.1%), respectively. The viability of UVB-irradiated keratinocytes pre-treated with HA produced by E. durans strain K11 and L. plantarum strain St3 was 91.3 and 91.4%, respectively, compared with the control. While the viability of UVB-irradiated keratinocytes post-treated with HA produced by E. durans strain K11 and L. plantarum strain St3 was 86 and 88.5%, respectively. To the best of our knowledge, this is the first recordation of HA production by Enterococcus durans and Lactiplantibacillus plantarum which revealed a significant radioprotection of the human keratinocytes against UVB radiation.

Evaluation of Antimicrobial Activities against Various E. coli Strains of a Novel Hybrid Peptide-LENART01.

Finding the ideal antimicrobial drug with improved efficacy and a safety profile that eliminates antibiotic resistance caused by pathogens remains a difficult task. Indeed, there is an urgent need for innovation in the design and development of a microbial inhibitor. Given that many promising antimicrobial peptides with excellent broad-spectrum antibacterial properties are secreted by some frog species (e.g., bombesins, opioids, temporins, etc.), our goal was to identify the antimicrobial properties of amphibian-derived dermorphin and ranatensin peptides, which were combined to produce a hybrid compound. This new chimera (named LENART01) was tested for its antimicrobial activity against E. coli strains K12 and R1-R4, which are characterized by differences in lipopolysaccharide (LPS) core oligosaccharide structure. The results showed that LENART01 had superior activity against the R2 and R4 strains compared with the effects of the clinically available antibiotics ciprofloxacin or bleomycin (MIC values). Importantly, the inhibitory effect was not concentration dependent; however, LENART01 showed a time- and dose-dependent hemolytic effect in hemolytic assays.

Antiplasmodial Activity Evaluation of a Bestatin-Related Aminopeptidase Inhibitor, Phebestin.

The increasing burden and spread of resistant malaria parasites remains an immense burden to public health. These factors have driven the demand to search for a new therapeutic agent. From our screening, phebestin stood out with nanomolar efficacy against Plasmodium falciparum 3D7. Phebestin was initially identified as an aminopeptidase N inhibitor. Phebestin inhibited the in vitro multiplication of the P. falciparum 3D7 (chloroquine-sensitive) and K1 (chloroquine-resistant) strains at IC50 values of 157.90 ± 6.26 nM and 268.17 ± 67.59 nM, respectively. Furthermore, phebestin exhibited no cytotoxic against human foreskin fibroblast cells at 2.5 mM. In the stage-specific assay, phebestin inhibited all parasite stages at 100 and 10-fold its IC50 concentration. Using 72-h in vitro exposure of phebestin at concentrations of 1 μM on P. falciparum 3D7 distorted the parasite morphology, showed dying signs, shrank, and prevented reinvasion of RBCs, even after the compound was washed from the culture. An in silico study found that phebestin binds to P. falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP), as observed for bestatin. In vivo evaluation using P. yoelii 17XNL-infected mice with administrations of 20 mg/kg phebestin, once daily for 7 days, resulted in significantly lower parasitemia peaks in the phebestin-treated group (19.53%) than in the untreated group (29.55%). At the same dose and treatment, P. berghei ANKA-infected mice showed reduced parasitemia levels and improved survival compared to untreated mice. These results indicate that phebestin is a promising candidate for development as a potential therapeutic agent against malaria.

Characterisation of the E. coli HMS174 and BLR host cell proteome to guide purification process development.

Mass-spectrometry-based proteomics is increasingly employed to monitor purification processes or to detect critical host cell proteins in the final drug substance. This approach is inherently unbiased and can be used to identify individual host cell proteins without prior knowledge. In process development for the purification of new biopharmaceuticals, such as protein subunit vaccines, a broader knowledge of the host cell proteome could promote a more rational process design. Proteomics can establish qualitative and quantitative information on the complete host cell proteome before purification (i.e., protein abundances and physicochemical properties). Such information allows for a more rational design of the purification strategy and accelerates purification process development. In this study, we present an extensive proteomic characterisation of two E.coli host cell strains widely employed in academia and industry to produce therapeutic proteins, BLR and HMS174. The established database contains the observed abundance of each identified protein, information relating to their hydrophobicity, the isoelectric point, molecular weight, and toxicity. These physicochemical properties were plotted on proteome property maps to showcase the selection of suitable purification strategies. Furthermore, sequence alignment allowed integration of subunit information and occurrences of post-translational modifications from the well-studied E.coli K12 strain.

Purification and Identification of EPS Produced by Five Lactic Acid Bacteria and Evaluation of Their Effects on the Texture of Fermented Goat Milk

Extracellular polysaccharide (EPS) produced by five lactic acid bacteria (Limosilactobacillus fermentum B55, Limosilactobacillus fermentum B62, Lactiplantibacillus plantarum 7830, Pediococcus acidilactici B30, and Lactobacillus helveticus K2) were purified and identified, and their effects on the texture of fermented goat milk were evaluated. The purified EPS fractions EPS 1a, EPS 2b, EPS 3c, EPS 4d, and EPS 5e of strains B62, 7830, K2, B55, and B30 were obtained with ion exchange chromatography, and their molecular weights were 2.41 × 104, 1.62 × 104, 6.42 × 103, 6.45 × 103, and 1.26 × 104 Da, found using gel permeation chromatography. The infrared spectrum results showed that these substances all contained polysaccharide characteristic absorption peaks, most of which contained O-H bonds, C-H bonds, hydroxyl and carboxyl bonds, and groups. The analysis of monosaccharide composition presented that EPS1a was composed of guluronic acid, rhamnose, and galactose, with a molar ratio of 2.7:1:2.4; EPS2b and EPS3c were composed of guluronic acid; EPS4d was composed of guluronic acid, glucose, and galactose, with a molar ratio of 1:1.1:1.2; and EPS5e was composed of glucose and galactose, with a molar ratio of 1.6:1, indicating the differences in the composition and structure of EPS produced by various strains. Compared with the control group that only had the starter added, adding EPS-producing strains could promote acid production and improve the texture of fermented goat milk, and its acidity, hardness, consistency, viscosity, and viscosity index were higher.