K1 Strain Research Articles

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.

In silico design and immunoinformatics analysis of a universal multi-epitope vaccine against monkeypox virus.

Monkeypox virus (MPXV) outbreaks have been reported in various countries worldwide; however, there is no specific vaccine against MPXV. In this study, therefore, we employed computational approaches to design a multi-epitope vaccine against MPXV. Initially, cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), linear B lymphocytes (LBL) epitopes were predicted from the cell surface-binding protein and envelope protein A28 homolog, both of which play essential roles in MPXV pathogenesis. All of the predicted epitopes were evaluated using key parameters. A total of 7 CTL, 4 HTL, and 5 LBL epitopes were chosen and combined with appropriate linkers and adjuvant to construct a multi-epitope vaccine. The CTL and HTL epitopes of the vaccine construct cover 95.57% of the worldwide population. The designed vaccine construct was found to be highly antigenic, non-allergenic, soluble, and to have acceptable physicochemical properties. The 3D structure of the vaccine and its potential interaction with Toll-Like receptor-4 (TLR4) were predicted. Molecular dynamics (MD) simulation confirmed the vaccine's high stability in complex with TLR4. Finally, codon adaptation and in silico cloning confirmed the high expression rate of the vaccine constructs in strain K12 of Escherichia coli (E. coli). These findings are very encouraging; however, in vitro and animal studies are needed to ensure the potency and safety of this vaccine candidate.

Identification of three capsule depolymerases in a bacteriophage infecting Klebsiella pneumoniae capsular types K7, K20, and K27 and therapeutic application

BackgroundKlebsiella pneumoniae capsular types K1, K2, K5, K20, K54, and K57 are prevalent hypervirulent types associated with community infections, and worrisomely, hypervirulent strains that acquired drug resistance have been found. In the search for alternative therapeutics, studies have been conducted on phages that infect K. pneumoniae K1, K2, K5, and K57-type strains and their phage-encoded depolymerases. However, phages targeting K. pneumoniae K20-type strains and capsule depolymerases capable of digesting K20-type capsules have rarely been reported. In this study, we characterized a phage that can infect K. pneumoniae K20-type strains, phage vB_KpnM‐20.MethodsA phage was isolated from sewage water in Taipei, Taiwan, its genome was analyzed, and its predicted capsule depolymerases were expressed and purified. The host specificity and capsule-digesting activity of the capsule depolymerases were determined. The therapeutic effect of the depolymerase targeting K. pneumoniae K20-type strains was analyzed in a mouse infection model.ResultsThe isolated Klebsiella phage, vB_KpnM‐20, infects K. pneumoniae K7, K20, and K27-type strains. Three capsule depolymerases, K7dep, K20dep, and K27dep, encoded by the phage were specific to K7, K20, and K27-type capsules, respectively. K20dep also recognized Escherichia coli K30-type capsule, which is highly similar to K. pneumoniae K20-type. The survival of K. pneumoniae K20-type-infected mice was increased following administration of K20dep.ConclusionsThe potential of capsule depolymerase K20dep for the treatment of K. pneumoniae infections was revealed using an in vivo infection model. In addition, K7dep, K20dep, and K27dep capsule depolymerases could be used for K. pneumoniae capsular typing.

In Silico and In Vitro Investigation of the Distribution and Expression of Key Genes in the Fucose Operon of Escherichia coli

Many gut bacteria degrade polysaccharides, providing nutritional advantages to their hosts. Fucose, a mucin degradation product, was suggested as a communication molecule between the resident microbiota and external pathogens. However, the precise role and variants of the fucose utilization pathway remain to be elucidated. Here, we computationally and experimentally investigated the fucose utilization operon of E. coli. While the operon is conserved among E. coli genomes, a variant pathway, in which an ABC transporter system replaces the fucose permease gene (fucP), was computationally identified in 50 out of 1058 genomes. Comparative genomics and subsystems analysis results were confirmed by polymerase chain reaction-based screening of 40 human E. coli isolates, which indicated the conservation of fucP in 92.5% of the isolates (vs. 7.5% of its suggested alternative, yjfF). The in silico predictions were confirmed by in vitro experiments comparing the growth of E. coli strains K12, BL21, and isogenic fucose-utilization K12 mutants. Additionally, fucP and fucI transcripts were quantified in E. coli K12 and BL21, after in silico analysis of their expression in 483 public transcriptomes. In conclusion, E. coli utilizes fucose by two pathway variants, with measurable transcriptional differences. Future studies will explore this variation’s impact on signaling and virulence.

Construction of a multi-epitope in silico vaccine against Anaplasma Marginale using immunoinformatics approach

Anaplasma Marginale, a rickettsial gram-negative bacterium, is responsible for causing bovine anaplasmosis which has widely spread among the different parts of the world causing death among cattle. It is mainly spread through the transmission of infected ticks. The manifestation of infected ticks could be controlled to a limited extent. However, subsequently, it is very much necessary for the cattle to develop strong immunity against A. marginale. It could be possible if a subunit vaccine is created against the rickettsial bacteria and introduced into the blood of the cattle for trials. In this paper, we have conducted a study to develop a multi-epitope subunit vaccine against A.marginale by using reverse vaccinology process. Five highly immunogenic outer membrane proteins of A. marginale were selected such as MSP1a, MSP2, Vir B10, OMP1 and OMP11. These proteins bind to the MHC class I molecules of the epithelial cells of mammals and MHC class II molecules of the antigen-presenting cells such as B-cells. The MHC class I molecules present antigens to the cytotoxic t-lymphocytes whereas the MHC class II molecules present antigens to the helper t-lymphocytes thus, initiating a strong immune response among the bovine species. We constructed three vaccine sequences from which two vaccine constructs V2 and V3, were found to be stable with high solubility and negative allergenicity. We also performed molecular docking of the two refined vaccine constructs with Rp-105 (30% resemblance in sequence to TLR4) and TLR-9. The vaccines interacted with the receptors with high binding energy. The best-designed vaccine construct was then reverse transcribed and adapted for the host E.coli K12 strain. The reverse transcribed sequence was later inserted into the pET28a (+) vector for the cloning and expression of the vaccine. The study could be a better initiative for in vitro and in vivo practice in developing an effective vaccine against A. marginale.

Flax black spot caused by Alternaria alternata, a new disease afflicting leaves and the exocarps of flax in China

AbstractFlax black spot was found in Ordos City in 2022, mainly infecting leaves and the exocarps, causing leaves turn yellow and brownish with black leaf tips before drying and damage, and exocarps became brown or black with a layer of black mould which significantly affected the yield and quality. The Y3 and K1 strains from infected leaves and the exocarps were the same pathogen of flax black spot which was identified as Alternaria alternata by morphological characteristics and molecular identification. In the pathogenicity test, A. alternata caused severe black spot on leaves and brown or black exocarps with mould of flax consistent with those observed in the field. The study for pathogenic identification ascertained a new disease of flax in China which lays the foundation for subsequent studies on disease control.

Structural vaccinology-based design of multi-epitopes vaccine against Streptococcus gordonii and validation using molecular modeling and immune simulation approaches

Streptococcus gordonii is an oral bacterium colonizing the dental cavity and leading to plaque formation. This pervasive colonizer is also the etiologic agent of bacterial endocarditis and has a major role in infective endocarditis. The bacteria reach the heart through oral bleeding, leading to inflammation of cardiovascular valves. Over the past 50 years, it has shown a significant pathogenic role in immunocompromised and neutropenic patients. Since antibiotic resistance has created prophylaxis failure towards infective endocarditis, a potent therapeutic candidate is needed. Therefore, multi-epitopes vaccine offers advantages over the other approaches. Thus, herein, numerous molecular-omics tools were exploited to mine immunogenic peptides, i.e., T-cell and B-cell epitopes, and construct a vaccine sequence. Our findings revealed a total of 24 epitopes, including CTL, HTL, and B-cell are responsible for imparting immune responses, which were combined with the help of different linkers, and MEVC was constructed. Multifactorial validation of the candidate vaccine was performed to minimize the risk factors. The final sequence was docked with TLR2 to validate its conformation compatibility with receptor and long-term interactions stability. Our analysis revealed that the vaccine construct is immunogenic and non-allergenic. The construct also established various contacts with the immune receptor. Finally, the vaccine sequence was reverse-translated, optimized for codon usage, and analyzed for expression in the Escherichia coli K12 strain. Maximum expression was noted with a CAI score of 0.95. In silico immune simulation revealed that the antigen was neutralized on the 3rd day after injection. In conclusion, the current study warrants validation of the vaccine construct both in in vitro and in vivo models for accurate therapeutic intervention.

Functional Characterization of Plasmid-Borne rmpADC Homologues in Klebsiella pneumoniae.

Expression of the hypermucoviscosity (HMV) phenotype and capsular polysaccharide (CPS) biosynthesis in Klebsiella pneumoniae were reported to be encoded by genes located in the chromosomal rmp locus. However, the functions of the rmp locus in the virulence plasmid remained unclear, and most of the rmp loci in clinical K. pneumoniae are plasmid carried. In this study, we investigated the functional characteristics of plasmid-borne rmp homologues in clinical hypervirulent K. pneumoniae (hvKP) strains by cloning and introducing such gene homologues into K. pneumoniae strains of different capsule types, followed by the evaluation of phenotypic changes in these strains. Acquisition of the plasmid-borne prmpADC and prmpA2D2 loci were found to result in an increase in mucoviscosity and CPS production in K1 and K2 K. pneumoniae, while only the prmpA2D2 locus contributed to phenotypic changes in the ST11/KL64 strain. Consistently, both rmpD and rmpD2 increased HMV in K1 and K2 K. pneumoniae, while only rmpD2 contributed to HMV in the ST11/KL64 strain; rmpC contributed to CPS overproduction in K1 and K2 strains but not in the ST11/KL64 strain. Furthermore, we proposed a logistic molecular basis of the HMV phenotype of K. pneumoniae on which prmpD2-mediated HMV is attributed to the increase of cell-free CPS production. Our data confirm that the rmp homologues carried by the virulence plasmid play a key role in virulence expression in K. pneumoniae, but the phenotype is highly dependent on the genetic background of the host strain and explained why most of the clinical ST11 strains carry only the prmpA2D2 locus. IMPORTANCE Klebsiella pneumoniae has become the most frequently isolated bacterial pathogen in hospital settings, with a very high mortality rate worldwide. Factors contributing to the virulence of K. pneumoniae are the overproduction of capsular polysaccharide (CPS) as well as the hypermucoviscosity (HMV) phenotype. These two phenotypes were reported to be regulated by rmpA/A2 homologues, which are often carried by virulence plasmids. Here, we determined the functional role of two plasmid-borne rmpA in mediating expression of the HMV phenotype and CPS production in K. pneumoniae. Different capsule types exhibited differences in the expression of HMV and CPS production although they harbored an identical plasmid-borne rmpA or rmpA2 locus, indicating that these virulence-related phenotypes are strongly related to the genetic background of the host strains. Our study provides a novel understanding of the regulation of virulence-related phenotypes and clinical management of K. pneumoniae infections.

A combined evaluation of the characteristics and antibiotic resistance induction potential of antibiotic wastewater during the treatment process

Antibiotic wastewater contains a variety of pollutant stressors that can induce and promote antibiotic resistance (AR) when released into the environment. Although these substances are mostly in concentrations lower than those known to induce AR individually, it is possible that antibiotic wastewater discharge might still promote the AR transmission risk via additive or synergistic effects. However, the comprehensive effect of antibiotic wastewater on AR development has rarely been evaluated, and its treatment efficiency remains unknown. Here, samples were collected from different stages of a cephalosporin production wastewater treatment plant, and the potential AR induction effect of their chemical mixtures was explored through the exposure of the antibiotic-sensitive Escherichia coli K12 strain. Incubation with raw cephalosporin production wastewater significantly promoted mutation rates (3.6 × 103–9.3 × 103-fold) and minimum inhibition concentrations (6.0–6.7-fold) of E. coli against ampicillin and chloramphenicol. This may be attributed to the inhibition effect and oxidative stress of cephalosporin wastewater on E. coli. The AR induction effect of cephalosporin wastewater decreased after the coagulation sedimentation treatment and was completely removed after the full treatment process. A Pearson correlation analysis revealed that the reduction in the AR induction effect had a strong positive correlation with the removal of organics and biological toxicity. This indicates that the antibiotic wastewater treatment had a collaborative processing effect of conventional pollutants, toxicity, and the AR induction effect. This study illustrates the potential AR transmission risk of antibiotic wastewater and highlights the need for its adequate treatment.

Proteomics-based vaccine targets annotation and design of subunit and mRNA-based vaccines for Monkeypox virus (MPXV) against the recent outbreak

Monkeypox Virus (MPXV) is a growing public health threat with increasing cases and fatalities globally. To date, no specific vaccine or small molecule therapeutic choices are available for the treatment of MPXV disease. In this work, we employed proteomics and structural vaccinology approaches to design mRNA and multi-epitopes-based vaccines (MVC) against MPXV. We first identified ten proteins from the whole proteome of MPXV as potential vaccine targets. We then employed structural vaccinology approaches to map potential epitopes of these proteins for B cell, cytotoxic T lymphocytes (CTL), and Helper T lymphocytes (HTL). Finally, 9 CTL, 6 B cell, and 5 HTL epitopes were joined together through suitable linkers to construct MVC (multi-epitope vaccine) and mRNA-based vaccines. Molecular docking, binding free energy calculation, and in silico cloning revealed robust interaction of the designed MVC with toll-like receptor 2 (TLR2) and efficient expression in E. Coli K12 strain. The immune simulation results revealed that the antigen titer after the injection reached to the maximum level on the 5th day and an abrupt decline in the antigen titer was observed upon the production of IgM, IgG and IgM + IgG, dendritic cells, IFN-gamma, and IL (interleukins), which suggested the potential of our designed vaccine candidate for inducing an immune response against MPXV.

Production of recombinant expansin and detection by SDS page analysis in Escherichia coli

The study aims to produce Expansin protein isolated from a young tomato plant by using Escherichia coli which is used in recombinant protein production. Continuous culture is the most common method used to grow cells for recombinant protein production. In the study, the K12 strain of E. coli was used as a culture for the production of Expansin protein. The used LeExp1 gene was isolated from a young tomato plant. Since the related gene is found in very small amounts in plants, it has been reproduced using the PCR method and has been made workable with this method. T17 vector (T7 RNA polymerase system), which is frequently used in the production of recombinant protein, was used as the bacterial expression vector. The T7 RNA polymerase system is a commonly used vector in E. coli. With the transfer, the E. coli bacterium was given the ability to produce recombinant protein. Whether the obtained recombinant protein expressed the appropriate protein was determined by SDS Page analysis.

Antimalarial Imidazopyridines Incorporating an Intramolecular Hydrogen Bonding Motif: Medicinal Chemistry and Mechanistic Studies

We previously identified a novel class of antimalarial benzimidazoles incorporating an intramolecular hydrogen bonding motif. The frontrunner of the series, analogue A, showed nanomolar activity against the chloroquine-sensitive NF54 and multi-drug-resistant K1 strains of Plasmodium falciparum (PfNF54 IC50 = 0.079 μM; PfK1 IC50 = 0.335 μM). Here, we describe a cell-based medicinal chemistry structure-activity relationship study using compound A as a basis. This effort led to the identification of novel antimalarial imidazopyridines with activities of <1 μM, favorable cytotoxicity profiles, and good physicochemical properties. Analogue 14 ( PfNF54 IC50 = 0.08 μM; PfK1 IC50 = 0.10 μM) was identified as the frontrunner of the series. Preliminary mode of action studies employing molecular docking, live-cell confocal microscopy, and a cellular heme fractionation assay revealed that 14 does not directly inhibit the conversion of heme to hemozoin, although it could be involved in other processes in the parasite's digestive vacuole.

MERS virus spike protein HTL-epitopes selection and multi-epitope vaccine design using computational biology

MERS-CoV, a zoonotic virus, poses a serious threat to public health globally. Thus, it is imperative to develop an effective vaccination strategy for protection against MERS-CoV. Immunoinformatics and computational biology tools provide a faster and more cost-effective strategy to design potential vaccine candidates. In this work, the spike proteins from different strains of MERS-CoV were selected to predict HTL-epitopes that show affinity for T-helper MHC-class II HTL allelic determinant (HLA-DRB1:0101). The antigenicity and conservation of these epitopes among the selected spike protein variants in different MERS-CoV strains were analyzed. The analysis identified five epitopes with high antigenicity: QSIFYRLNGVGITQQ, DTIKYYSIIPHSIRS, PEPITSLNTKYVAPQ, INGRLTTLNAFVAQQ and GDMYVYSAGHATGTT. Then, a multi-epitope vaccine candidate was designed using linkers and adjuvant molecules. Finally, the vaccine construct was subjected to molecular docking with TLR5 (Toll-like receptor-5). The proposed vaccine construct had strong binding energy of −32.3 kcal/mol when interacting with TLR5.Molecular dynamics simulation analysis showed that the complex of the vaccine construct and TLR5 is stable. Analysis using in silico immune simulation also showed that the prospective multi-epitope vaccine design had the potential to elicit a response within 70 days, with the immune system producing cytokines and immunoglobulins. Finally, codon adaptation and in silico cloning analysis showed that the candidate vaccine could be expressed in the Escherichia coli K12 strain. Here we also designed support vaccine construct MEV-2 by using B-cell and CD8+ CTL epitopes to generate the complete immunogenic effect. This study opens new avenues for the extension of research on MERS vaccine development. Communicated by Ramaswamy H. Sarma

Probiotic Lactobacillus paragasseri K7 Nanofiber Encapsulation Using Nozzle-Free Electrospinning.

Probiotics are live microorganisms that can have beneficial effects on humans. Encapsulation offers them a better chance of survival. Therefore, nozzle-free electrospinning was introduced for their embedding in nanofibrous material. Probiotic Lactobacillus paragasseri K7 in lyophilized and fresh form, with and without inulin as prebiotic, was added to a polymer solution of sodium alginate (NaAlg) and polyethylene oxide (PEO). Conductivity, viscosity, pH, and surface tension were determined to define the optimal concentration and volume ratio for smooth electrospinning. The success of the formed nanoscale materials was examined by scanning electron microscope (SEM), while the entrapment of probiotics in the nanofibrous mats was detected by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). Spontaneous diffusion of bacteria from electrospun samples in PBS buffer pH 7.4 was studied by plate counting on MRS agar. By exposing polymer solutions containing L. paragasseri K7 and inulin to a high electric field, the nanofilm was formed on a polypropylene substrate, used as collecting material. When polymer solutions without inulin were used, the bead-like nanofibers may have become visible. The SEM results suggest that inulin, in addition to K7 strain, additionally lowers the conductivity of spinning macromolecular solution and hinders the nanofiber formation. The results of ATR-FTIR confirmed the presence of L. paragasseri K7 embedded in nanocomposites by the appearance of characteristic peaks. The samples containing the probiotic regardless of its form with inulin had similar surface composition, except that the sodium content was higher in the samples with fresh probiotic, probably due to greater and thus less easy embedding of the bacteria in NaAlg. Within 2 h, the largest amount of probiotic strain K7 was spontaneously released from the electrospun sample containing the inulin and probiotic in freeze-dried form (44%), while the amount released from the nanofibrous sample, which also contained the inulin and probiotic in fresh form, was significantly lower (21%). These preliminary results demonstrate the potential of nozzle-free electrospinning technology for the development of probiotic delivery systems for short-term use, such as feminine hygiene materials (tampons, pads, napkins).

Effect of liquiritigenin on chloroquine accumulation in digestive vacuole leading to apoptosis-like death of chloroquine-resistant P. falciparum

BackgroundMalaria remains one of the major health concerns, especially in tropical countries. Although drugs such as artemisinin-based combinations are efficient for treating Plasmodium falciparum, the growing threat from multi-drug resistance has become a major challenge. Thus, there is a constant need to identify and validate new combinations to sustain current disease control strategies to overcome the challenge of drug resistance in the malaria parasites. To meet this demand, liquiritigenin (LTG) has been found to positively interact in combination with the existing clinically used drug chloroquine (CQ), which has become unfunctional due to acquired drug resistance. PurposeTo evaluate the best interaction between LTG and CQ against CQ- resistant strain of P. falciparum. Furthermore, the in vivo antimalarial efficacy and possible mechanism of action of the best combination was also assessed. MethodsThe in vitro anti-plasmodial potential of LTG against CQ- resistant strain K1 of P. falciparum was tested using Giemsa staining method. The behaviour of the combinations was evaluated using the fix ratio method and evaluated the interaction of LTG and CQ by calculating the fractional inhibitory concentration index (FICI). Oral toxicity study was carried out in a mice model. In vivo antimalarial efficacy of LTG alone and in combination with CQ was evaluated using a four-day suppression test in a mouse model. The effect of LTG on CQ accumulation was measured using HPLC and the rate of alkalinization of the digestive vacuole. Cytosolic Ca2+ level, mitochondrial membrane potential, caspase-like activity, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and Annexin V Apoptosis assay to assess anti-plasmodial potential. Proteomics analysis was evaluated by LC-MS/MS analysis. ResultsLTG possesses anti-plasmodial activity on its own and it showed to be an adjuvant of CQ. In in vitro studies, LTG showed synergy with CQ only in the ratio (CQ: LTG-1:4) against CQ-resistant strain (K1) of P. falciparum. Interestingly, in vivo studies, LTG in combination with CQ showed higher chemo-suppression and enhanced mean survival time at much lower concentrations compared to individual doses of LTG and CQ against CQ- resistant strain (N67) of Plasmodium yoelli nigeriensis. LTG was found to increase the CQ accumulation into digestive vacuole, reducing the rate of alkalinization, in turn increasing cytosolic Ca2+ level, loss of mitochondrial potential, caspase-3 activity, DNA damage and externalization of phosphatidylserine of the membrane (in vitro). These observations indicate the involvement of apoptosis-like death of P. falciparum that might be due to the accumulation of CQ. ConclusionLTG showed synergy with CQ in the ratio LTG: CQ, 4:1) in vitro and was able to curtail the IC50 of CQ and LTG. Interestingly, in vivo in combination with CQ, LTG showed higher chemo-suppression as well as enhanced mean survival time at a much lower concentrations of both the partners as compared to an individual dose of CQ and LTG. Thus, synergistic drug combination offers the possibility to enhance CQ efficacy in chemotherapy.

Peat-based hairy root transformation using Rhizobium rhizogenes as a rapid and efficient tool for easily exploring potential genes related to root-knot nematode parasitism and host response

BackgroundRoot-knot nematodes (RKNs) pose a worldwide threat to agriculture of many crops including cucumber. Genetic transformation (GT) has emerged as a powerful tool for exploration of plant-RKN interactions and genetic improvement of RKN resistance. However, it is usually difficult to achieve a highly efficient and stable GT protocol for most crops due to the complexity of this process.ResultsHere we firstly applied the hairy root transformation system in exploring root-RKN interactions in cucumber plants and developed a rapid and efficient tool transformation using Rhizobium rhizogenes strain K599. A solid-medium-based hypocotyl-cutting infection (SHI) method, a rockwool-based hypocotyl-cutting infection (RHI) method, and a peat-based cotyledon-node injection (PCI) method was evaluated for their ability to induce transgenic roots in cucumber plants. The PCI method generally outperformed the SHI and RHI methods for stimulating more transgenic roots and evaluating the phenotype of roots during nematode parasitism. Using the PCI method, we generated the CRISPR/Cas9-mediated malate synthase (MS) gene (involved in biotic stress responses) knockout plant and the LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16, a potential host susceptibility gene for RKN) promoter-driven GUS expressing plant. Knockout of MS in hairy roots resulted in effective resistance against RKNs, while nematode infection induced a strong expression of LBD16-driven GUS in root galls. This is the first report of a direct link between these genes and RKN performance in cucumber.ConclusionTaken together, the present study demonstrates that the PCI method allows fast, easy and efficient in vivo studies of potential genes related to root-knot nematode parasitism and host response.

Design, synthesis, and in silico-in vitro antimalarial evaluation of 1,2,3-triazole-linked dihydropyrimidinone quinoline hybrids

In response to the malaria parasite’s resistance towards quinoline-based antimalarial drugs, we have employed quinoline-containing compounds in combination with dihydropyrimidinone (DHPM) analogues as resistance reversal agents (RAs) and investigated their antimalarial activities based on DHPM’s resistance reversal abilities. The present study employed click chemistry to link DHPM and quinoline compounds which offered several synthetic advantages over the previously used amide coupling for the same hybrids. Among the synthesised compounds, 4 hybrids with the 7-chloroquinoline moiety showed antimalarial activity below 1 µM while compounds with the mefloquine moiety showed lower antimalarial activity than chloroquine (CQ) and the 7-chloroquinoline hybrids. Among the tested hybrids for the IC50 determination, four compounds displayed good antimalarial activity with increased sensitivity against the CQ-resistant K1 strain between 421 and 567 nM and showed higher activity between 138 and 245 nM against the NF54 CQ-sensitive strain, while three compounds have IC50 values greater than 5 µM. Additionally, in silico molecular docking and molecular dynamics studies were conducted to investigate the binding affinities of all the synthesised compounds as glutathione reductase protein competitive inhibitors. Further optimisation of the compound with the highest binding affinity generated 16 compounds with higher binding affinities than the flavine adenine dinucleotide (FAD) cofactor.

First Report of Enterobacter roggenkampii and E. cloacae complex sp. Caused Tomato Pith Necrosis in Guangdong Province, China.

Tomato (Solanum lycopersicum L.) is an important greenhouse and field-grown vegetable. During 2019 to 2021, a new bacterial pith necrosis broke out in tomato producing areas in China. The disease incidence rate in the field was approximately 10% to 30% in a few tomato planting areas of Guangdong province, and even 100% in Dianbai distinct, Maoming city. Diseased plants showed yellowing of the lower leaves, brown vascular tissues, and wilting along with brown necrotic spots and a large number of adventitious roots on the stem. Diseased plants were collected, and short fragments of the diseased stems were sterilized with 75% alcohol for 2 minutes, washed with sterile water twice, and stripped the cortex (Fang 1998). Dilutions of xylem specimen soaking solution were plated onto the TTC medium (peptone 10.0 g, acid hydrolyzed casein 1.0 g, glucose 5.0 g, agar 15.0 g, distilled water 1000 mL, 0.5% 2, 3, 5-triphenyltetrazolium chloride, pH7.0), and cultured at 28℃ for 24 h. Three pink single colonies (A2 from Guangzhou (113°21' E, 23°9' N), Guangdong, and K6, and K7 from Maoming (110°55' E, 21°25' N), Guangdong) were selected and purified. Strains A2, K6, and K7 were Gram-negative, motile, and showed white fluidal colonies with pink center on TTC medium, and white, round, and smooth-surface colonies on NA medium (peptone 10 g, beef extract 3 g, sodium chloride 5 g, agar 15 g, distilled water 1000 mL, pH7.0) at 28℃ for 24 h. Three strains could utilize citrate, sorbitol, lactose and arginine, and were negative for methylred reaction test, determination of phenylalanine amino acid deaminase, lysine decarboxylase, urease, soluble starch decomposition and gelatin liquefaction, whereas were positive for Voges-Proskauer test, which conformed to the characteristics of genus Enterobacter (Davin-Regli et al. 2019). To determine the species of the Enterobacter isolates, partial sequences 16S rDNA, gyrB, and rpoB of strain A2, K6, and K7 were amplified. The PCR products were purified, sequenced, and deposited to GenBank. The BLASTN analysis of 16S rDNA, rpoB and gyrB sequences showed strain A2 (MW785888, OL364948, OL364943) was 99.20%, 99.17% and 98.57% identity with E. roggenkampii DSM16690, respectively, strain K6 (MW785890, OL364950, OL364945) was 99.73%, 99.63%, 99.63% identity with E. cloacae complex sp. N13-01531, and strain K7 (MW785893, OL364951, OL364946) was 99.8%, 98.81%, 98.99% identity with the E. roggenkampii Ed-982 and Ek140. Nucleotide sequences of 3 strains were aligned using ClustalW program, and neighbor-joining method (NJ) was used in the construction of a phylogenetic tree using MEGA7 program. Phylogenetic trees based on gyrB sequence, rpoB sequence, and the concatenated sequence of 16S rDNA-rpoB-gyrB and rpoB-gyrB showed strain A2 and K7 were clustered to E. roggenkampii, strain K6 was clustered to E. cloacae complex sp. The roots of tomato material 'Moneymaker' at stage of 4-5 true leaves were cut and irrigated 10 mL bacterial suspension (OD600=0.6) of strains A2, K6, and K7, respectively. As a control, the tomato roots were treated with 10 mL sterile water. All plants were incubated at 30°C. The experiments were conducted with 20 tomato seedlings for each tested strain and control, and repeated twice. All plants inoculated showed yellowing in the lower leaves 6-7 days after inoculation (DAI), subsequently the stems of some plants were rotten, along with bacterial pus in the internodes. The plants wilted, and stems were hollow 20 DAI, which is similar to the field symptoms. No symptoms were observed in control plants. Strains were successfully reisolated from wilting plants, and identified as A2, K6, and K7, respectively, based on gyrB sequence analysis, fulfilling Koch's postulates. Zhou et al. (2021) reported that E. roggenkampii caused bacterial wilt of mulberry in Guangxi, China. Chen et al. (2021) reported E. asburiae caused tomatoes pith necrosis in Fujian and Zhejiang, China. To our knowledge, this is the first report of E. roggenkampii and E. cloacae complex sp. causing bacterial pith necrosis of tomato. Further research would focus on exploring the pathogenic mechanism of the pathogen, and providing reference of controlling the disease.

Forces Governing the Transport of Pathogenic and Nonpathogenic Escherichia coli in Nitrogen and Magnesium Doped Biochar Amended Sand Columns

Background: Access to safe drinking water remains a global issue with fecal indicator bacteria being major pollutants. Biochars offer low-cost adsorbents for bacterial pathogens. A fundamental understanding of how biochars interact with bacterial pathogens is essential to designing effective biofilters. Methods: Water-saturated sand columns amended with Magnesium and Nitrogen-doped biochars produced by pyrolysis at 400, 500, 600, and 700 °C were used to Quantify the transport of pathogenic Escherichia coli O157:H7 and nonpathogenic E. coli k12 strains in porous media. Measured data were modeled using DLVO theory of colloidal stability. were explored. Results: (1) Biochar is hydrophobic while sand and bacteria are hydrophilic; (2) all Gibbs free energy values quantified between E. coli O157:H7 and biochar were negative except for biochar produced at 700 °C; (3) all types of forces investigated (van der Waals, electrostatic, and acid-base interactions) played a role in governing the interactions between bacteria and biochar. Conclusions: (1) Adding doped biochar to sand at a 2% weight ratio enhanced the retention of bacterial cells in the sand/biochar columns; (2) bacterial transport is strain-dependent and mediated by various types of forces resulting from interactions between the various functional groups displayed on bacteria and biochar/sand. Our findings emphasize the importance of monitoring biochar’s functionality to eliminate bacterial pollutants from contaminated water.