Search Results (22)

The arid mountainous region of Hail in Saudi Arabia has a variety of desert vegetation, some of which are conventionally used in Bedouin traditional medicine. These plants need scientific examination. This research seeks to examine Blepharis ciliaris using a thorough multi-analytical methodology that includes antibacterial and antioxidant assessments as well as computational modeling. GC–MS analysis of the methanolic extract revealed 17 organic compounds, including pentadecanoic acid, ethyl methyl ester (2.63%); hexadecanoic acid, methyl ester (1.00%); 9,12-octadecadienoic acid (Z,Z)-, methyl ester (2.74%); 9-octadecenoic acid, methyl ester (E) (2.78%); octadecanoic acid (5.88%); 9-tetradecenoic acid (Z) (3.22%); and undec-10-enoic acid, undec-2-n-1-yl ester (5.67%). The DPPH test evaluated antioxidant activity, revealing a notable increase with higher concentrations of the methanolic extract, achieving maximum inhibition of 81.54% at 1000 µg/mL. The methanolic extract exhibited moderate antibacterial activity, with average inhibition zones of 10.33 ± 1.53 mm, 13.33 ± 1.53 mm, 10.67 ± 1.53 mm, and 10.00 ± 2.00 mm against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Serratia marcescens, respectively, as determined by the disk diffusion method. The minimum inhibitory concentration (MIC) values were 500 µg/mL for S. aureus and B. subtilis, whereas E. coli and S. marcescens showed susceptibility at 1000 µg/mL. Computational simulations were employed to assess the toxicity, drug-likeness, and ADMET profiles of compounds derived from Blepharis ciliaris. Thirteen bioactive compounds were assessed in silico against Staphylococcus aureus sortase A (PDB: 1T2O), Bacillus subtilis BsFabHb (PDB: 8VDB), Escherichia coli LPS assembly protein (LptD) (PDB: 4RHB), and a modeled Serratia marcescens outer-membrane protein TolC, focusing on cell wall and membrane structures. Compound 3, (+)-Ascorbic acid 2,6-dihexadecanoate, shown significant binding affinities to B. subtilis BsFabHb, E. coli LPS assembly protein, and S. marcescens TolC. Full article

Background: Endogenous antimicrobial peptides (AMPs) are evolutionarily ancient molecular factors of innate immunity that play a key role in host defense. The study of the diversity of animal defense peptides has important applications in the context of the growing global antimicrobial resistance. Methods: In this study using a transcriptome mining approach, we found three novel thanatin-like β-hairpin AMPs in the bean bug Riptortus pedestris, named Rip-2, Rip-3, and Rip-4. The peptides were expressed in the bacterial system, and their antimicrobial activities were evaluated both in vitro and in vivo. Results: Homologs of the discovered AMPs are widely distributed among different members of the infraorder Pentatomomorpha. Rip-2 was shown to have the most similar structure and LptA-targeting mechanism of action to those of thanatin, but the former peptides demonstrated a higher activity against key Gram-negative ESKAPE pathogens and also displayed a significant efficacy in a lethal model of septicemia caused by E. coli in mice at daily doses greater than 5 mg/kg. In contrast, Rip-3 and Rip-4 peptides caused bacterial membrane damage, did not induce bacterial resistance, and exhibited a strong selectivity against Bacillus and Mycobacterium spp. Conclusions: This study extends the knowledge of the structure and functions of insect host defense AMPs. Each of the novel β-hairpin peptides has a potential to be a template for the development of selective antibiotic drugs. Full article

Herein, we investigated the toxicity and membrane-permeabilizing capabilities of Lpt and Lpt-like peptides, belonging to type I toxin–antitoxin systems carried by plasmid DNA of Lacticaseibacillus strains. These 29 amino acid peptides are predicted to form α-helical structures with a conserved central hydrophobic sequence and differently charged hydrophilic termini. Like Lpt, the expression of Lpt-like in E. coli induced growth arrest, nucleoid condensation, and cell membrane damage, suggesting membrane interaction as the mode of action. The membrane permeabilization activity of both peptides was evaluated by using liposome leakage assays, dynamic light scattering, and CD spectroscopy. Lpt and Lpt-like showed liposome leakage activity, which did not lead to liposome disruption but depended on peptide concentration. Lpt was generally more effective than Lpt-like, probably due to different physical chemical properties. Leakage was significantly reduced in larger liposomes and increased with negatively charged PCPS liposomes, indicating that electrostatic interactions and membrane curvature influence peptide activity. Contrary to most membrane-active peptides, Lpt an Lpt-like progressively lost their α-helical structure upon interaction with liposomes. Our data are inconsistent with the formation of membrane-spanning peptide pores but support a mechanism relying on the transient failure of the membrane permeability barrier possibly through the formation of “lipid pores”. Full article

Currently, viable antibiotics available to mitigate infections caused by drug-resistant Gram-negative bacteria are highly limited. Thanatin, a 21-residue-long insect-derived antimicrobial peptide (AMP), is a promising lead molecule for the potential development of novel antibiotics. Thanatin is extremely potent, particularly against the Enterobacter group of Gram-negative pathogens, e.g., E. coli and K. pneumoniae. As a mode of action, cationic thanatin efficiently permeabilizes the LPS-outer membrane and binds to the periplasmic protein LptA_m to inhibit outer membrane biogenesis. Here, we have utilized N-terminal truncated 16- and 14-residue peptide fragments of thanatin and investigated structure, activity, and selectivity with correlating modes of action. A designed 16-residue peptide containing D-Lys (dk) named VF16 (V1PIIYCNRRT-dk-KCQRF16) demonstrated killing activity in Gram-negative bacteria. The VF16 peptide did not show any detectable toxicity to the HEK 293T cell line and kidney cell line Hep G2. As a mode of action, VF16 interacted with LPS, permeabilizing the outer membrane and binding to LptA_m with high affinity. Atomic-resolution structures of VF16 in complex with LPS revealed cationic and aromatic surfaces involved in outer membrane interactions and permeabilization. Further, analyses of an inactive 14-residue native thanatin peptide (IM14: IIYCNRRTGKCQRM) delineated the requirement of the β-sheet structure in activity and target interactions. Taken together, this work would pave the way for the designing of short analogs of thanatin-based antimicrobials. Full article

Stochastic variations of the operation conditions and the resultant variations of the aerodynamic performance in Low-Pressure Turbine (LPT) can often be found. This paper studies the aerodynamic performance impact of the uncertain variations of flow parameters, including inlet total pressure, inlet flow angle, and turbulence intensity for an LPT cascade. Flow simulations by solving the Reynolds-averaged Navier–Stokes equations, the SST turbulence model, and $\gamma -{\widetilde{Re}}_{\theta t}$ transition model equations are first carried out. Then, a Gaussian process (GP) based on an adaptive sampling technique is introduced. The accuracy of adaptive GP (ADGP) is proven to be high through a function experiment. Using ADGP, the uncertainty propagation models between the performance parameters, including total pressure-loss coefficient, outlet flow angle, Zweifel number, and the uncertain inlet flow parameters, are established. Finally, using the propagation models, uncertainty quantifications of the performance changes are conducted. The results demonstrate that the total pressure-loss coefficient and Zweifel number are sensitive to uncertainties, while the outlet flow angle is almost insensitive. Statistical analysis of the flow field by direct Monte Carlo simulation (MCS) shows that flow transition on the suction side and viscous shear stress are rather sensitive to uncertainties. Moreover, Sobol sensitivity analysis is carried out to specify the influence of each uncertainty on the performance changes in the turbine cascade. Full article

The aerodynamics of a high-speed low-pressure turbine (LPT) cascade were investigated under steady and unsteady inlet flows. The tests were performed at outlet Mach (M) and Reynolds numbers ($Re$) of $0.90$ and 70k, respectively. Unsteady wakes were simulated by means of a wake generator equipped with bars. A bar reduced frequency ($f^{+}$) of ∼0.95 was used for the unsteady case. The inlet flow field was characterized in terms of the total pressure profile and incidence. The blade aerodynamics at midspan and the secondary flow region were investigated by means of pneumatic taps and hot-film sensors. The latter provided a novel view into the impact of the secondary flows on the heat transfer topology on the blade suction side (SS). The cascade performance was quantified in terms of the outlet flow angle and losses by means of a directional multi-hole probe. The results report the phase-averaged impact of unsteady wakes on the secondary flow structures in an open test case high-speed LPT geometry. Full article

It is widely expected that electric vehicles will be able to satisfy most road transport needs. The aim of this paper is to provide an answer to how far the electrification of buses used for local public transport can be pursuable through insight into the engineering problems and range, i.e., the autonomy on battery, as well as the efficiency of recharging systems. At first, a wide survey of the main solutions that are present on the market, or foreseen for the near future, concerning the electrification of fleets for LPT is provided. Thereafter, such solutions are compared through numerical applications and by using a practical case study, pertaining to the city of Turin (IT), where static inductive charging has been extensively experienced. Particular attention is paid to engineering problems and to the autonomy on battery of the vehicles as a function of their mass, electric energy storage system, charging opportunities and infrastructure, while comparing the time and efficiency of recharging systems. The authors conclude by recommending the most promising alternatives for battery electric buses while outlining their limits, striving to provide for the literature a research instrument, which is lacking, for delimitating the applicability of electric buses for LPT while outlining the viable solutions. Full article

The large differential-thermal extrusion (LDTE) process, a novel approach for efficiently fabricating a high-strength Mg-10.3Gd-4.4Y-0.9Zn-0.7Mn (wt.%) alloy, is introduced in this work. Unlike typical isothermal extrusion processes, where the ingot and die temperatures are kept the same, LDTE involves significantly higher ingot temperatures (~120 °C) compared to the die temperature. For high-strength Mg-RE alloys, the maximum isothermal extrusion ram speed is normally limited to 1 mm/s. This research uses the LDTE process to significantly increase the ram speed to 2.0 mm/s. The LPTE-processed alloy possesses a phase composition that is similar to that of isothermal extruded alloys, including α-Mg, 14H-type long-period stacking ordered (LPSO) and β-Mg₅(Gd, Y) phases. The weakly preferentially oriented α-Mg grains in the LDTE-processed alloy have $<10\overline{1}0>$_Mg//ED fibrous and <0001>_Mg//ED anomalous textures as their two main constituents. After isothermal aging, high quantitative densities of prismatic β′ and basal γ′ precipitates are produced, which have the beneficial effect of precipitation hardening. With a yield tensile strength of 344 MPa, an ultimate tensile strength of 488 MPa, and an elongation of 9.7%, the alloy produced by the LDTE process exhibits an exceptional strength–ductility balance, further demonstrating the potential of this method for efficiently producing high-strength Mg alloys. Full article

Herein, we developed a bio-functionalized solution-immersed silicon (SIS) sensor at the single-cell level to identify Erwinia amylovora (E. amylovora), a highly infectious bacterial pathogen responsible for fire blight, which is notorious for its rapid spread and destructive impact on apple and pear orchards. This method allows for ultra-sensitive measurements without pre-amplification or labeling compared to conventional methods. To detect a single cell of E. amylovora, we used Lipopolysaccharide Transporter E (LptE), which is involved in the assembly of lipopolysaccharide (LPS) at the surface of the outer membrane of E. amylovora, as a capture agent. We confirmed that LptE interacts with E. amylovora via LPS through in-house ELISA analysis, then used it to construct the sensor chip by immobilizing the capture molecule on the sensor surface modified with 3′-Aminopropyl triethoxysilane (APTES) and glutaraldehyde (GA). The LptE-based SIS sensor exhibited the sensitive and specific detection of the target bacterial cell in real time. The dose–response curve shows a linearity (R² > 0.992) with wide dynamic ranges from 1 to 10⁷ cells/mL for the target bacterial pathogen. The sensor showed the value change (dΨ) of approximately 0.008° for growing overlayer thickness induced from a single-cell E. amylovora, while no change in the control bacterial cell (Bacillus subtilis) was observed, or negligible change, if any. Furthermore, the bacterial sensor demonstrated a potential for the continuous detection of E. amylovora through simple surface regeneration, enabling its reusability. Taken together, our system has the potential to be applied in fields where early symptoms are not observed and where single-cell or ultra-sensitive detection is required, such as plant bacterial pathogen detection, foodborne pathogen monitoring and analysis, and pathogenic microbial diagnosis. Full article

The cationic antimicrobial ß-hairpin, thanatin, was recently developed into drug-like analogues active against carbapenem-resistant Enterobacteriaceae (CRE). The analogues represent new antibiotics with a novel mode of action targeting LptA in the periplasm and disrupting LPS transport. The compounds lose antimicrobial efficacy when the sequence identity to E. coli LptA falls below 70%. We wanted to test the thanatin analogues against LptA of a phylogenetic distant organism and investigate the molecular determinants of inactivity. Acinetobacter baumannii (A. baumannii) is a critical Gram-negative pathogen that has gained increasing attention for its multi-drug resistance and hospital burden. A. baumannii LptA shares 28% sequence identity with E. coli LptA and displays an intrinsic resistance to thanatin and thanatin analogues (MIC values > 32 µg/mL) through a mechanism not yet described. We investigated the inactivity further and discovered that these CRE-optimized derivatives can bind to LptA of A. baumannii in vitro, despite the high MIC values. Herein, we present a high-resolution structure of A. baumannii LptAm in complex with a thanatin derivative 7 and binding affinities of selected thanatin derivatives. Together, these data offer structural insights into why thanatin derivatives are inactive against A. baumannii LptA, despite binding events in vitro. Full article

The ability to monitor lymphocyte responses is critical for developing our understanding of the immune response in humans. In the current clinical setting, relying on the metabolic incorporation of [³H] thymidine into cellular DNA via a lymphocyte proliferation test (LPT) is the only method that is routinely performed to determine cell proliferation. However, techniques that measure DNA synthesis with a radioactive material such as [³H] thymidine are intrinsically more sensitive to the different stages of the cell cycle, which could lead to over-analyses and the subsequent inaccurate interpretation of the information provided. With cell proliferation assays, the output should preferably provide a direct and accurate measurement of the number of actively dividing cells, regardless of the stimuli properties or length of exposure. In fact, an ideal technique should have the capacity to measure lymphocyte responses on both a quantitative level, i.e., cumulative magnitude of lymphoproliferative response, and a qualitative level, i.e., phenotypical and functional characterization of stimulated immune cells. There are many LPT alternatives currently available to measure various aspects of cell proliferation. Of the nine techniques discussed, we noted that the majority of these LPT alternatives measure lymphocyte proliferation using flow cytometry. Across some of these alternatives, the covalent labelling of cells with a high fluorescence intensity and low variance with minimal cell toxicity while maximizing the number of detectable cell divisions or magnitude of proliferation was achieved. Herein, we review the performance of these different LPT alternatives and address their compatibility with the [³H] thymidine LPT so as to identify the “best” alternative to the [³H] thymidine LPT. Full article

The need for novel antibiotics has become imperative with the increasing prevalence of antibiotic resistance in Gram-negative bacteria in clinics. Acting as a permeability barrier, lipopolysaccharide (LPS) protects Gram-negative bacteria against drugs. LPS is synthesized in cells and transported to the outer membrane (OM) via seven lipopolysaccharide transport (Lpt) proteins (LptA–LptG). Of these seven Lpt proteins, LptC interacts with LptA to transfer LPS from the inner membrane (IM) to the OM, and assembly is aided by LptD/LptE. This interaction among the Lpt proteins is important for the biosynthesis of LPS; therefore, the Lpt proteins, which are significant in the assembly process of LPS, can be a potential target for new antibiotics. In this study, a yeast two-hybrid (Y2H) system was used to screen compounds that could block LPS transport by inhibiting LptA/LptC interaction, which finally disrupts the biosynthesis of the OM. We selected the compound IMB-0042 for this study. Our results suggest that IMB-0042 disrupts LptA/LptC interaction by binding to both LptA and LptC. Escherichia coli cells, when treated with IMB-0042, showed filament morphology, impaired OM integrity, and an accumulation of LPS in the periplasm. IMB-0042 inhibited the growth of Gram-negative bacteria and showed synergistic sensitization to other antibiotics, with low cytotoxicity. Thus, we successfully identified a potential antibacterial agent by using a Y2H system, which blocks the transport of LPS by targeting LptA/LptC interaction in Escherichia coli. Full article

Background: In this experimental study, we aimed to assess the acaricidal effects of Elettaria cardamomum L. essential oil (ECEO) against Hyalomma anatolicum tick in cattle from Saudi Arabia. Methods: Gas chromatography-mass spectrometry (GC-MS) was performed to identify the chemical composition of ECEO. The acaricidal, larvicidal, and repellent activity of ECEO against H. anatolicum was studied through the adult immersion test (AIT), the larval packet test (LPT), the vertical movement behavior of tick’s larvae technique, anti-acetylcholinesterase (AChE) activity, and oxidative enzyme activity. Results: By GC/MS, the most compounds were 1,8-cineole (34.3%), α-terpinyl acetate (23.3%), and α-pinene (17.7%), respectively. ECEO significantly (p < 0.001) increased the mortality rate as a dose-dependent response. After ECEO Treatment, number of eggs, egg weight, and hatchability significantly declined as a dose-dependent response. ECEO at concentrations of 5 µL/mL and above completely killed the larva. The LC₅₀ and LC₉₀ values for ECEO were 1.46 and 2.68 µL/mL, respectively. ECEO at concentrations of 10, 20, and 40 µL/mL showed 100% repellency activity up to 60, 120, and 360 min incubation, respectively. ECEO, especially at ½ LC₅₀ and LC_50, significantly inhibited GST and AChE activities of H. anatolicum larvae compared to the control group. Conclusions: We found promising adulticidal, larvicidal, and repellent effects of ECEO against H. anatolicum as a vector of theileriosis in Saudi Arabia. We also found that ECEO displayed these activities through inhibiting AChE and GST. Nevertheless, additional investigations are required to confirm the accurate mechanisms and the relevance of ECEO in practical application. Full article

Zoonotic and antimicrobial-resistant Escherichia coli (hereafter, E. coli) is a global public health threat which can lead to detrimental effects on human health. Here, we aim to investigate the antimicrobial resistance and the presence of mcr-1 gene in E. coli isolated from chicken feces. Ninety-four E. coli isolates were obtained from samples collected from different locations in Bangladesh, and the isolates were identified using conventional microbiological tests. Phenotypic disk diffusion tests using 20 antimicrobial agents were performed according to CLSI-EUCAST guidelines, and minimum inhibitory concentrations (MICs) were determined for a subset of samples. E. coli isolates showed high resistance to colistin (88.30%), ciprofloxacin (77.66%), trimethoprim/sulfamethoxazole (76.60%), tigecycline (75.53%), and enrofloxacin (71.28%). Additionally, the pathotype eaeA gene was confirmed in ten randomly selected E. coli isolates using primer-specific polymerase chain reaction (PCR). The presence of mcr-1 gene was confirmed using PCR and sequencing analysis in six out of ten E. coli isolates. Furthermore, sequencing and phylogenetic analyses revealed a similarity between the catalytic domain of Neisseria meningitidis lipooligosaccharide phosphoethanolamine transferase A (LptA) and MCR proteins, indicating that the six tested isolates were colistin resistant. Finally, the findings of the present study showed that E. coli isolated from chicken harbored mcr-1 gene, and multidrug and colistin resistance. These findings accentuate the need to implement strict measures to limit the imprudent use of antibiotics, particularly colistin, in agriculture and poultry farms. Full article

A large number of bacterial toxin–antitoxin (TA) systems have been identified so far and different experimental approaches have been explored to investigate their activity and regulation both in vivo and in vitro. Nonetheless, a common feature of these methods is represented by the difficulty in cell transformation, culturing, and stability of the transformants, due to the expression of highly toxic proteins. Recently, in dealing with the type I Lpt/RNAII and the type II YafQ/DinJ TA systems, we encountered several of these problems that urged us to optimize methodological strategies to study the phenotype of recombinant Escherichia coli host cells. In particular, we have found conditions to tightly repress toxin expression by combining the pET expression system with the E. coli C41(DE3) pLysS strain. To monitor the RNase activity of the YafQ toxin, we developed a fluorescence approach based on Thioflavin-T which fluoresces brightly when complexed with bacterial RNA. Fluorescence microscopy was also applied to reveal loss of membrane integrity associated with the activity of the type I toxin Lpt, by using DAPI and ethidium bromide to selectively stain cells with impaired membrane permeability. We further found that atomic force microscopy can readily be employed to characterize toxin-induced membrane damages. Full article