Outer-Membrane Permeabilization, LPS Transport Inhibition: Activity, Interactions, and Structures of Thanatin Derived Antimicrobial Peptides.

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 LptAm 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 LptAm 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 ß-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.

Suppressor mutants demonstrate the metabolic plasticity of unsaturated fatty acid synthesis in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa PAO1 has two aerobic pathways for synthesis of unsaturated fatty acids (UFAs), DesA and DesB plus the oxygen independent FabAB pathway. The DesA desaturase acts on saturated acyl chains of membrane phospholipid bilayers whereas the substrates of the DesB desaturase are thought to be long chain saturated acyl-CoA thioesters derived from exogeneous saturated fatty acids that are required to support DesB-dependent growth. Under suitable aerobic conditions either of these membrane-bound desaturates can support growth of P. aeruginosa ∆fabA strains lacking the oxygen independent FabAB pathway. We previously studied function of the desA desaturase of P. putida in a P. aeruginosa ∆fabA ∆desA strain that required supplementation with a UFA for growth and noted bypass suppression of the P. aeruginosa ∆fabA ∆desA strain that restored UFA synthesis. We report three genes encoding lipid metabolism proteins that give rise to suppressor strains that bypass loss of the DesA and oxygen independent FabAB pathways.

Early Molecular Insights into Thanatin Analogues Binding to A. baumannii LptA.

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.

Synthesis and Biological Evaluation of Quinoline Derivatives as a Novel Class of Broad-Spectrum Antibacterial Agents.

Nineteen new quinoline derivatives were prepared via the Mannich reaction and evaluated for their antibacterial activities against both Gram-positive (G⁺) and Gram-negative (G-) bacteria, taking compound 1 as the lead. Among the target compounds, quinolone coupled hybrid 5d exerted the potential effect against most of the tested G⁺ and G- strains with MIC values of 0.125⁻8 µg/mL, much better than those of 1. Molecular-docking assay showed that compound 5d might target both bacterial LptA and Top IV proteins, thereby displaying a broad-spectrum antibacterial effect. This hybridization strategy was an efficient way to promote the antibacterial activity of this kind, and compound 5d was selected for the further investigation, with an advantage of a dual-target mechanism of action.

Dimerization of isolated Pseudomonas aeruginosa lipopolysaccharide transporter component LptA.

LptA is a soluble periplasmic component of the lipopolysaccharide (LPS) transport system of Gram-negative bacteria that transports newly synthesized LPS from the inner membrane to the outer leaflet of the outer membrane. LptA links the inner membrane components (LptBFGC) to the outer membrane components (LptDE), but it is uncertain whether LptA is a freely moving LPS shuttle or part of a stable trans-periplasm structure. Escherichiacoli LptA forms highly polymerized head-to-tail oligomers in solution, but dimers in vivo. We studied the oligomerization of purified Pseudomonasaeruginosa LptA. Size-exclusion chromatography showed that P. aeruginosa LptA, unlike E. coli LptA, is a dimer over a wide range of concentrations. Chemical crosslinking with bis(sulfosuccinimidyl) suberate confirmed that dimers were the predominant species even at sub-micromolar LptA concentrations, which was unaffected by LPS binding. Mass spectrometry of crosslinked dimers showed that crosslinks occurred between the N-terminal α-amino group and either Lys-172 or Lys-173 near the C-terminus. These results support a hypothetical structure for the dimer of isolated P. aeruginosa LptA in which the N-terminus of one monomer is in close proximity to the C-terminus of the other, and the same surface of each monomer forms the interface between them, preventing further oligomerization.

Correlating quadriceps patellar tendon angle and lateral patellar tilt angle in patients with irregular alignment: a cross-sectional study with retrospective data.

Background: The newly defined angle, quadriceps-patella angle (QPA), reflects the combined force transmitted to the patella by the quadriceps muscles and patellar tendon. An increase in QPA may correlate with an increased force on the patella, which is significant in diagnosing patellofemoral instability and pain syndrome. In our study, we examined how various angles and pathologies vary depending on lateral patellar tilt angle (LPTA). QPA and patellar malalignment was investigated. Thus, the importance of understanding patellar malalignment and the research gap. Methods: Three hundred and fifty patients who underwent knee magnetic resonance imaging (MRI) examinations were included. The cross-sectional study conducted retrospectively between the years of 2018-2020 in a tertiary care outpatient clinic. Shapiro-Wilk normality, Chi-square, Mann-Whitney-U, Spearman correlation and receiver operating characteristic (ROC) curve analysis, statistical tests used for analysis. The patellar tendon length, patellar height, tibial tubercle-trochlear groove distance (TT-TG), patella angle, trochlear sulcus angle, trochlear groove depth (TGD), medial trochlea length (MT), lateral trochlea length (LT), medial trochlear/lateral trochlear length ratio (MT/LT), LPTA, patella-patellar tendon angle (PPTA), QPA, Insall-Salvati index (ISI), medial trochlear inclination (MTI), lateral trochlear inclination (LTI) were among these measurements. In addition, we aim to reveal whether there is a significant relationship between two important angles LPTA and QPA. Whether there is a significant increase in the development of chondromalacia for the patient group with LPTA >5°. We examined how the frequency of chondromalacia changes in the patient group with LPTA >5°. Results: Two hundred and seventy seven patients included in the study and many measurements were performed on MRI. Fad-pad edema was found to be significantly higher in the group with LPTA <5° (P=0.046). TT-TG distance was significantly higher, TGD and MT were significantly lower in patients with higher LPTA (P=0.001, P=0.002 and P=0.017, respectively). A low level of significant positive correlation was found between QPA and patellar tendon length. There is no significant difference between QPA and PPTA angles between the groups with LPTA <5° and >5° (P=0.503, P=0.188). In the ROC analysis performed to determine the cut-off value, the LPTA value ≤14.2° which significantly predicted the presence of fad-pad edema, had the highest sensitivity and specificity [sensitivity: 76.71%, specificity: 39.90%, area under the curve (AUC): 0.588, P=0.024]. Conclusions: QPA is independent from many angles of the knee and does not change significantly. As the patellar tendon length increases, QPA angle also increases. In patients with abnormal LPTA, the frequency of TT-TG distance and chondromalacia increased, while TGD and MT decreased. Patients with a low LPTA can be more carefully examined for chondromalacia and fad-pad edema in clinical and MRI examination.

Discovery, characterization, and redesign of potent antimicrobial thanatin orthologs from Chinavia ubica and Murgantia histrionica targeting E. coli LptA.

Podisus maculiventris thanatin has been reported as a potent antimicrobial peptide with antibacterial and antifungal activity. Its antibiotic activity has been most thoroughly characterized against E. coli and shown to interfere with multiple pathways, such as the lipopolysaccharide transport (LPT) pathway comprised of seven different Lpt proteins. Thanatin binds to E. coli LptA and LptD, thus disrupting the LPT complex formation and inhibiting cell wall synthesis and microbial growth. Here, we performed a genomic database search to uncover novel thanatin orthologs, characterized their binding to E. coli LptA using bio-layer interferometry, and assessed their antimicrobial activity against E. coli. We found that thanatins from Chinavia ubica and Murgantia histrionica bound tighter (by 3.6- and 2.2-fold respectively) to LptA and exhibited more potent antibiotic activity (by 2.1- and 2.8-fold respectively) than the canonical thanatin from P. maculiventris. We crystallized and determined the LptA-bound complex structures of thanatins from C. ubica (1.90 Å resolution), M. histrionica (1.80 Å resolution), and P. maculiventris (2.43 Å resolution) to better understand their mechanism of action. Our structural analysis revealed that residues A10 and I21 in C. ubica and M. histrionica thanatin are important for improving the binding interface with LptA, thus overall improving the potency of thanatin against E. coli. We also designed a stapled variant of thanatin that removes the need for a disulfide bond but retains the ability to bind LptA and antibiotic activity. Our discovery presents a library of novel thanatin sequences to serve as starting scaffolds for designing more potent antimicrobial therapeutics.

Binding and transport of LPS occurs through the coordinated combination of an array of sites across the entire Escherichia coli LPS transport protein LptA.

The outer leaflet of the outer membrane (OM) of bacteria such as Escherichia coli, Pseudomonas aeruginosa, and other important pathogens is largely composed of lipopolysaccharide (LPS), which is essential to nearly all Gram-negative bacteria. LPS is transported to the outer leaflet of the OM through a yet unknown mechanism by seven proteins that comprise the LPS transport system. LptA, the only entirely periplasmic Lpt protein, bridges the periplasmic space between the IM LptB2 FGC and the OM LptDE complexes. LptA is postulated to protect the hydrophobic acyl chains of LPS as it crosses the hydrophilic periplasm, is essential to cell viability, and contains many conserved residues distributed across the protein. To identify which side chains are required for function of E. coli LptA in vivo, we performed a systematic, unbiased, high-throughput screen of the effect of 172 single alanine substitutions on cell viability utilizing an engineered BL21 derivative with a chromosomal knockout of the lptA gene. Remarkably, LptA is highly tolerant to amino acid substitution with alanine. Only four alanine mutants could not complement the chromosomal knockout; CD spectroscopy showed that these substitutions resulted in proteins with significantly altered secondary structure. In addition, 29 partial loss-of-function mutants were identified that led to OM permeability defects; interestingly, these sites were solely located within ß-strands of the central core of the protein and each resulted in misfolding of the protein. Therefore, no single residue within LptA is responsible for LPS binding, supporting previous EPR spectroscopy data indicating that sites across the entire protein work in concert to bind and transport LPS.

A latent profile transition analysis and influencing factors of internet addiction for adolescents: A short-term longitudinal study.

Internet addiction for adolescent, which is widely concerned by the whole society, has become a public health problem. Internet addiction not only had a negative impact on physical and mental development of adolescents, but also was harmful to their study, life, interpersonal communication and personality formation, and so on. In recent years, the data analysis methods of longitudinal research have developed rapidly. It not only focused on the overall average growth trend, but also considered the differences in the individual trends. Latent profile transition analysis (LPTA) is an extension of latent profile analysis (LPA) and latent transition analysis (LTA), and is a longitudinal data analysis method. LPTA can simultaneously estimate group membership in multiple time points and their latent transition tendency among these subgroups between each two time points. This study used LPTA to explore the development trend of adolescent internet addiction over time and its influencing factors. 1033 adolescents participated in a short-term 6-month longitudinal study with a total of three tests. Participants completed internet addiction test, self-rating anxiety scale and self-rating depression scale. The results showed that: (1) There are three categories of adolescent internet addiction, namely non-internet addiction group, low-internet addiction group and high-internet addiction group. (2) Non-internet addiction group has a strong stability. Low-internet addiction group has a high probability to become non-internet addiction group or high-internet addiction group. (3) Boys are more likely than girls to develop into high-internet addiction group. Anxiety and depression both affect the development of adolescent internet addiction.

Linking dual mode of action of host defense antimicrobial peptide thanatin: Structures, lipopolysaccharide and LptAm binding of designed analogs.

At present, antibiotics options to cure infections caused by drug resistant Gram-negative pathogens are highly inadequate. LPS outer membrane, proteins involved in LPS transport and biosynthesis pathways are vital targets. Thanatin, an insect derived 21-residue long antimicrobial peptide may be exploited for the development of effective antibiotics against Gram-negative bacteria. As a mode of bacterial cell killing, thanatin disrupts LPS outer membrane and inhibits LPS transport by binding to the periplasmic protein LptAm. Here, we report structure-activity correlation of thanatin and analogs for the purpose of rational design. These analogs of thanatin are investigated, by NMR, ITC and fluorescence, to correlate structure, antibacterial activity and binding with LPS and LptAm, a truncated monomeric variant. Our results demonstrate that an analog thanatin M21F exhibits superior antibacterial activity. In LPS interaction analyses, thanatin M21F demonstrate high affinity binding to outer membrane LPS. The atomic resolution structure of thanatin M21F in LPS micelle reveals four stranded ß-sheet structure in a dimeric topology whereby the sidechain of aromatic residues Y10, F21 sustained mutual packing at the interface. Strikingly, LptAm binding affinity of thanatin M21F has been significantly increased with an estimated Kd ~ 0.73 nM vs 13 nM for thanatin. Further, atomic resolution structures and interactions of Ala based thanatin analogs define plausible correlations with antibacterial activity and LPS, LptAm interactions. Taken together, the current work provides a frame-work for the designing of thanatin based potent antimicrobial peptides for the treatment of drug resistance Gram-negative bacteria.

Identification of a Small Molecule That Inhibits the Interaction of LPS Transporters LptA and LptC.

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.

Use of Site-Directed Spin Labeling EPR Spectroscopy to Study Protein-LPS Interactions.

Site-directed spin labeling EPR (electron paramagnetic resonance) spectroscopy is a technique used to identify the local conformational changes at a specific residue of interest within a purified protein in response to a ligand. Here, we describe the site-directed spin labeling EPR spectroscopy methodology to monitor changes in the side-chain motion in soluble lipopolysaccharide transport proteins upon the addition of lipopolysaccharide (LPS). A comparison of the spectral overlays of the spin-labeled protein in the absence and presence of LPS provides a qualitative visualization of how LPS binding affects the motion of each spin-labeled site tested within the protein. No change in the spectral lineshapes of a spin-labeled protein in the absence and presence of LPS indicates that the site is not affected by LPS binding, while differences in the spectral lineshapes indicate that LPS does affect the mobility of the spin label side chain within the protein structure. This is a powerful readout of conformational changes at specific residues of interest that can be used to identify a specific site as a reporter of changes induced by ligand binding and to map out the effects of ligand binding through an array of reporter sites within a protein. With the use of AquaStar tubing, protein concentrations as low as 2 µM allow for up to a 100-fold excess of LPS. This methodology may also be applied to other protein-ligand or protein-protein interactions with minor adaptations.

[Development of a BLI assay-based method for detecting LptA/LptC interaction].

In Gram-negative bacteria, lipopolysaccharide transport (Lpt) protein LptA and LptC form a complex to transport LPS from the inner membrane (IM) to the outer membrane (OM). Blocking the interaction between LptA and LptC will lead to the defect of OM and cell death. Therefore, Lpt protein interaction could be used as a target to screen new drugs for killing Gram-negative bacteria. Here we used biolayer interferometry (BLI) assay to detect the interaction between LptA and LptC, with the aim to develop a method for screening the LptA/LptC interaction blockers in vitro. Firstly, LptC and LptA with or without signal peptide (LptAfull or LptAno signal) were expressed in E. coli BL21(DE3). The purified proteins were then labeled with biotin and the super streptavidin (SSA) biosensor was blocked with diluent. The biotin labeled protein sample was mixed with the sensor, and then the binding of the protein with a series of diluted non biotinylated protein was detected. At the same time, non-biotinylated protein was used as a control. The binding of biotinylated protein to a small molecule IMB-881 and the blocking of interaction were also detected by the same method. In the blank control, the biosensor without biotinylated protein was used to detect the serially diluted samples. The signal response constant was calculated by using steady analysis. The results showed that biotinylated LptC had a good binding activity with LptAfull and LptAno signal with KD value 2.9e⁻7±7.9e⁻8 and 6.0e⁻7±2.8e⁻8, respectively; biotinylated LptAno signal had a good binding activity with LptC, with a KD value of 9.6e⁻7±7.2e⁻8. All binding curves showed obvious fast binding and fast dissociation morphology. The small molecule compound IMB-881 can bind to LptA to block the interaction between LptA and LptC, but has no binding activity with LptC. In summary, we developed a method for detecting the LptA/LptC interaction based on the BLI technology, and confirmed that this method can be used to evaluate the blocking activity of small molecule blockers, providing a new approach for the screening of LptA/LptC interaction blockers.

Multidrug Antimicrobial Resistance and Molecular Detection of mcr-1 Gene in Salmonella Species Isolated from Chicken.

Colistin (polymyxin E) is widely used in animal and human medicine and is increasingly used as one of the last-resort antibiotics against Gram-negative bacilli. Due to the increased use of colistin in treating infections caused by multidrug-resistant Gram-negative bacteria, resistance to this antibiotic ought to be monitored. The study was undertaken to elucidate the molecular mechanisms, genetic relationships and phenotype correlations of colistin-resistant isolates. Here, we report the detection of the mcr-1 gene in chicken-associated Salmonella isolates in Bangladesh and its in-silico functional analysis. Out of 100 samples, 82 Salmonella spp. were isolated from chicken specimens (liver, intestine). Phenotypic disc diffusion and minimum inhibitory concentration (MIC) assay using different antimicrobial agents were performed. Salmonella isolates were characterized using PCR methods targeting genus-specific invA and mcr-1 genes with validation for the functional analysis. The majority of the tested Salmonella isolates were found resistant to colistin (92.68%), ciprofloxacin (73.17%), tigecycline (62.20%) and trimethoprim/sulfamethoxazole (60.98%). When screened using PCR, five out of ten Salmonella isolates were found to carry the mcr-1 gene. One isolate was confirmed for Salmonella enterica subsp. enterica serovar Enteritidis, and other four isolates were confirmed for Salmonella enterica subsp. enterica serovar Typhimurium. Sequencing and phylogenetic analysis revealed a divergent evolutionary relationship between the catalytic domain of Neisseria meningitidis lipooligosaccharide phosphoethanolamine transferase A (LptA) and MCR proteins, rendering them resistant to colistin. Three-dimensional homology structural analysis of MCR-1 proteins and molecular docking interactions suggested that MCR-1 and LptA share a similar substrate binding cavity, which could be validated for the functional analysis. The comprehensive molecular and in-silico analyses of the colistin resistance mcr-1 gene of Salmonella spp. of chicken origin in the present study highlight the importance of continued monitoring and surveillance for antimicrobial resistance among pathogens in food chain animals.

Emerging peptide antibiotics with therapeutic potential.

This review covers some of the recent progress in the field of peptide antibiotics with a focus on compounds with novel or established mode of action and with demonstrated efficacy in animal infection models. Novel drug discovery approaches, linear and macrocyclic peptide antibiotics, lipopeptides like the polymyxins as well as peptides addressing targets located in the plasma membrane or in the outer membrane of bacterial cells are discussed.

Hormonal Function Responses to Moderate Aerobic Exercise in Older Adults with Depression.

BACKGROUND: Poor daily life physical activities among older people were related to depressive mood especially memory loss. In addition to that, the change in physical ability is significantly associated with the score of depression among older age. OBJECTIVE: The present study aimed to evaluate the effects of a supervised aerobic training program with moderate intensity for 12 weeks on mood profiles and hormonal levels of the hypothalamus-pituitary-adrenal axis (HPA axis) of older adults. METHODS: A total of 80 individuals of both gender (90 males, 110 females) of ages ranged between 65 and 95 years were recruited for this study. Based upon the profile of mood states (POMS) analysis, the participants were classified into two groups: control group (n=30) and depressive group (n=50). Leisure-time physical activity (LTPA), adrenal hormones such as ACTH, corticosterone (CORT), cortisol, DHEA/S, and cortisol:DHEA/S ratio were measured at baseline and post-intervention of moderate aerobic exercise for 12 weeks. RESULTS: Older adults with higher depressive scores showed a remarkable change in the level of adrenal hormones compared to control. There was a significant increase in the level of ACTH, CORT, cortisol, and cortisol:DHEA/S ratio, and decrease in DHEA/S. Compared to females, males showed an improvement in depressive mood score along with an increase in LPTA, DHEA/S and decrease in ACTH, CORT, cortisol, cortisol:DHEA/S ratio following 12 weeks of supervised aerobic training, respectively. CONCLUSION: The findings of this study showed that 12 weeks of supervised exercise interventions are promising non-drug therapeutic strategies in improving depression among older adults. The potential performance in a psychological state occurs physiologically via optimizing the levels of the hormones of the HPA axis.

Identification of an anti-Gram-negative bacteria agent disrupting the interaction between lipopolysaccharide transporters LptA and LptC.

INTRODUCTION: The emergence of drug-resistant Gram-negative bacteria is a serious clinical problem that causes increased morbidity and mortality. However, the slow discovery of new antibiotics is unable to meet the need for treating bacterial infections caused by drug-resistant strains. Lipopolysaccharide (LPS) is synthesized in the cytoplasm and transported to the cell envelope by the LPS transport (Lpt) system. LptA and LptC form a complex that transports LPS from the inner membrane to the outer membrane. METHODS: This study performed a screen for agents that disrupt the transport of LPS in Gram-negative bacteria Escherichia coli. It established a yeast two-hybrid system to detect LptA-LptC interaction and used this system to identify a compound, IMB-881, that blocks this interaction and shows antibacterial activity. RESULTS: This study demonstrated that the IMB-881 compound specifically binds to LptA to disrupt LptA-LptC interaction using surface plasmon resonance assay. Overproduction of LptA protein but not that of LptC lowered the antibacterial activity of IMB-881. Strikingly, Escherichia coli cells accumulated 'extra' membrane material in the periplasm and exhibited filament morphology after treatment with IMB-881. CONCLUSION: This study successfully identified, by using a yeast two-hybrid system, an antibacterial agent that likely blocks LPS transport in Gram-negative bacteria.

Expression of aggrecan components in perineuronal nets in the mouse cerebral cortex.

Specific regions of the cerebral cortex are highly plastic in an organism's lifetime. It is thought that perineuronal nets (PNNs) regulate plasticity, but labeling for Wisteria floribunda agglutinin (WFA), which is widely used to detect PNNs, is observed throughout the cortex. The aggrecan molecule-a PNN component-may regulate plasticity, and may also be involved in determining region-specific vulnerability to stress. To clarify cortical region-specific plasticity and vulnerability, we qualitatively analyzed aggrecan-positive and glycosylated aggrecan-positive PNNs in the mature mouse cerebral cortex. Our findings revealed the selective expression of both aggrecan-positive and glycosylated aggrecan-positive PNNs in the cortex. WFA-positive PNNs expressed aggrecan in a region-specific manner in the cortex. Furthermore, we observed variable distributions of PNNs containing WFA- and aggrecan-positive molecules. Together, our findings suggest that PNN components and their function differ depending on the cortical region, and that aggrecan molecules may be involved in determining region-specific plasticity and vulnerability in the cortex.

Lipopolysaccharide binding to the periplasmic protein LptA.

Lipopolysaccharide (LPS) and the periplasmic protein, LptA, are two essential components of Gram-negative bacteria. LPS, also known as endotoxin, is found asymmetrically distributed in the outer leaflet of the outer membrane of Gram-negative bacteria such as Escherichia coli and plays a role in the organism's natural defense in adverse environmental conditions. LptA is a member of the lipopolysaccharide transport protein (Lpt) family, which also includes LptC, LptDE, and LptBFG2 , that functions to transport LPS through the periplasm to the outer leaflet of the outer membrane after MsbA flips LPS across the inner membrane. It is hypothesized that LPS binds to LptA to cross the periplasm and that the acyl chains of LPS bind to the central pocket of LptA. The studies described here are the first to comprehensively characterize and quantitate the binding of LPS by LptA. Using site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy, data were collected for 15 spin-labeled residues in and around the proposed LPS binding pocket on LptA to observe the mobility changes caused by the presence of exogenous LPS and identify the binding location of LPS to LptA. The EPR data obtained suggest a 1:1 ratio for the LPS:LptA complex and allow the first calculation of dissociation constants for the LptA-LPS interaction. The results indicate that the entire protein is affected by LPS binding, the N-terminus unfolds in the presence of LPS, and a mutant LptA protein unable to form oligomers has an altered affinity for LPS.

Improving diffraction resolution using a new dehydration method.

The production of high-quality crystals is one of the major obstacles in determining the three-dimensional structure of macromolecules by X-ray crystallography. It is fairly common that a visually well formed crystal diffracts poorly to a resolution that is too low to be suitable for structure determination. Dehydration has proven to be an effective post-crystallization treatment for improving crystal diffraction quality. Several dehydration methods have been developed, but no single one of them is suitable for all crystals. Here, a new convenient and effective dehydration method is reported that makes use of a dehydrating solution that will not dry out in air for several hours. Using this dehydration method, the resolution of Archaeoglobus fulgidus Cas5a crystals has been increased from 3.2 to 1.95 Šand the resolution of Escherichia coli LptA crystals has been increased from <5 to 3.4 Å.