Potentiation of Vancomycin: Creating Cooperative Membrane Lysis through a "Derivatization-for-Sensitization" Approach.

Gram-negative bacteria, especially the ones with multidrug resistance, post dire challenges to antibiotic treatments due to the presence of the outer membrane (OM), which blocks the entry of many antibiotics. Current solutions for such permeability issues, namely lipophilic-cationic derivatization of antibiotics and sensitization with membrane-active agents, cannot effectively potentiate the large, globular, and hydrophilic antibiotics such as vancomycin, due to ineffective disruption of the OM. Here, we present our solution for high-degree OM binding of vancomycin via a hybrid "derivatization-for-sensitization" approach, which features a combination of LPS-targeting lipo-cationic modifications on vancomycin and OM disruption activity from a sensitizing adjuvant. 106- to 107-fold potentiation of vancomycin and 20-fold increase of the sensitizer's effectiveness were achieved with a combination of a vancomycin derivative and its sensitizer. Such potentiation is the result of direct membrane lysis through cooperative membrane binding for the sensitizer-antibiotic complex, which strongly promotes the uptake of vancomycin and adds to the extensive antiresistance effectiveness. The potential of such derivatization-for-sensitization approach was also supported by the combination's potent in vivo antimicrobial efficacy in mouse model studies, and the expanded application of such strategy on other antibiotics and sensitizer structures.

Benzo[c][1,2,5]thiadiazole Donor-Acceptor Dyes: A Synthetic, Spectroscopic, and Computational Study.

The synthesis, optical characterization and computational modeling of seven benzo[c][1,2,5]thiadiazole (BTD) donor-acceptor dyes are reported. These dyes have been studied using electrochemical analysis, electronic absorption, emission, and Raman and resonance Raman spectroscopies coupled with various density functional theoretical approaches. Crystal structure geometries on a number of these compounds are also reported. The optical spectra are dominated by low energy charge-transfer states; this may be modulated by the coupling between donor and acceptor through variation in donor energy, variation of the donor-acceptor torsion angle, and incorporation of an insulating bridge. These modifications result in a perturbation of the excitation energy for this charge-transfer transition of up to ∼2000 cm(-1). Emission spectra exhibit significant solvatochromisim, with Lippert-Mataga analysis yielding Δµ between 8 and 33 D. Predicted λmax, ε, and Raman cross sections calculated by M06L, B3LYP, PBE0, M06, CAM-B3LYP, and ωB97XD DFT functionals were compared to experimental results and analyzed using multivariate analysis, which shows that hybrid functionals with 20-27% HF best predict ground state absorption, while long-range corrected functionals best predict molecular polarizabilities.

Bromazolam Blood Concentrations in Postmortem Cases-A British Columbia Perspective.

Bromazolam is a designer benzodiazepine that was first detected in British Columbia in January 2021. Postmortem cases were analyzed using a comprehensive blood drug screening procedure by liquid chromatography-high-resolution mass spectrometry before being retrospectively analyzed using an in-house novel psychoactive substances data processing method. Bromazolam was detected in 41 postmortem cases in 2021 and quantitatively confirmed by standard addition, using liquid chromatography-tandem mass spectrometry. The mean bromazolam concentration observed was 11.4 ± 53.7 ng/mL (median concentration: 1.6 ng/mL), with a range from 0.5 to 319.3 ng/mL and the majority of cases co-occurring with fentanyl. These low concentrations may be indicative of a presumed enhancement of opioid effects, rather than being used as a stand-alone drug. Bromazolam was always detected with opioids (fentanyl and carfentanil), stimulants (methamphetamine) and/or other benzodiazepines (etizolam and flualprazolam). To our knowledge, this is the first report to provide concentrations of bromazolam in postmortem blood samples in Canada.

Unique territorial and sub-chromosomal organization revealed in the holocentric moth Bombyx mori.

The hallmarks of chromosome organization in multicellular eukaryotes are chromosome territories (CT), chromatin compartments, and different types of domains, including topologically associated domains (TADs). Yet, most of these concepts derive from analyses of organisms with monocentric chromosomes. Here we describe the 3D genome architecture of an organism with holocentric chromosomes, the silkworm Bombyx mori . At the genome-wide scale, B. mori chromosomes form highly separated territories and lack substantial trans contacts. As described in other eukaryotes, B. mori chromosomes segregate into an active A and an inactive B compartment. Remarkably, we also identify a third compartment, Secluded "S", with a unique contact pattern. Compartment S shows strong enrichment of short-range contacts and depletion of long-range contacts. It hosts a unique combination of genetic and epigenetic features, localizes at the periphery of CTs and shows developmental plasticity. Biophysical modeling shows that formation of such secluded domains requires a new mechanism - a high density of extruded loops within them along with low level of extrusion and compartmentalization of A and B. Together with other evidence of loop extrusion in interphase, this suggests SMC-mediated loop extrusion in this insect. Overall, our analyses highlight the evolutionary plasticity of 3D genome organization driven by a new combination of known processes.

Amphiphilic Nano-Swords for Direct Penetration and Eradication of Pathogenic Bacterial Biofilms.

Bacterial biofilms are major causes of persistent and recurrent infections and implant failures. Biofilms are formable by most clinically important pathogens worldwide, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, causing recalcitrance to standard antibiotic therapy or anti-biofilm strategies due to amphiphilic impermeable extracellular polymeric substances (EPS) and the presence of resistant and persistent bacteria within the biofilm matrix. Herein, we report our design of an oligoamidine-based amphiphilic "nano-sword" with high structural compacity and rigidity. Its rigid, amphiphilic structure ensures effective penetration into EPS, and the membrane-DNA dual-targeting mechanism exerts strong bactericidal effect on the dormant bacterial persisters within biofilms. The potency of this oligoamidine is shown in two distinct modes of application: it may be used as a coating agent for polycaprolactone to fully inhibit surface biofilm growth in an implant-site mimicking micro-environment; meanwhile, it cures model mice of biofilm infections in various ex vivo and in vivo studies.

Improving the Hemocompatibility of Antimicrobial Peptidomimetics through Amphiphilicity Masking Using a Secondary Amphiphilic Polymer.

Antimicrobial peptidomimetics (AMPMs) have received widespread attention as potentially powerful weapons against antibiotic resistance. However, AMPMs' membrane disruption mechanism not only brings resistance-resistant nature, but also nonspecific binding and disruption toward eukaryotic cell membranes, and consequently, their hemolytic activity is the primary concern on clinical applications. Here, the preparation and screening of an AMPM library is reported, through which a best-performing hit, PT-b1, can be obtained. To further improve PT-b1's hemocompatibility, a strategy is devised to mask the amphiphilicity of the AMPM using a charge-free, FDA-approved amphiphilic polymer, Pluronic F-127 (PF127). A PF127 solution containing PT-b1 can form a temperature-sensitive, absorbable hydrogel at higher concentration, but dissolve and complex with PT-b1 through hydrophobic interactions at lower concentration or lower temperature. The complexation from PF127 can mask the amphiphilicity of PT-b1 and render it extremely hemocompatible, yet the reversibility in such nanocomplexation and the existence of a secondary mechanism of action ensure that the AMPM's potency remains unchanged. The in vivo effectiveness of this antimicrobial hydrogel system is demonstrated using a mice wound infection model established with Methicillin-resistant Staphylococcus aureus, and observations indicate the hydrogel can promote wound healing and suppress bacteria-caused inflammation even when resistant pathogens are involved.

Cooperative Membrane Damage as a Mechanism for Pentamidine-Antibiotic Mutual Sensitization.

Most Gram-positive-selective antibiotics have low activity against Gram-negative bacteria due to the presence of an outer membrane barrier. There is, therefore, interest in developing combination therapies that can penetrate the outer membrane (OM) with known antibiotics coupled with membrane-active sensitizing adjuvants. However, two unanswered questions hinder the development of such combination therapies: the sensitization spectrum of the sensitizer and the mechanism of antibiotic-sensitizer mutual potentiation. Here, with pentamidine as an example, we screened a library of 170 FDA-approved antibiotics in combination with pentamidine, a compound known to disturb the OM of Gram-negative bacteria. We found that four antibiotics, minocycline, linezolid, valnemulin, and nadifloxacin, displaced enhanced activity in combination with pentamidine against several multidrug-resistant Gram-negative bacteria. Through a descriptor-based structural-activity analysis and multiple cell-based biochemical assays, we found that hydrophobicity, partial charge, rigidity, and surface rugosity were key factors that affected sensitization via a cooperative membrane damage mechanism in which lipopolysaccharides and phospholipids were identified as sites of synergy. Finally, in vitro experiments showed that the linezolid-pentamidine combination slowed the generation of drug resistance, and there was also potent activity in in vivo experiments. Overall, our results highlight the importance of the physicochemical properties of antibiotics and cooperative membrane damage for synergistic pentamidine-antibiotic drug combinations.

Local activation of α2 adrenergic receptors is required for vagus nerve stimulation induced motor cortical plasticity.

Vagus nerve stimulation (VNS) paired with rehabilitation training is emerging as a potential treatment for improving recovery of motor function following stroke. In rats, VNS paired with skilled forelimb training results in significant reorganization of the somatotopic cortical motor map; however, the mechanisms underlying this form of VNS-dependent plasticity remain unclear. Recent studies have shown that VNS-driven cortical plasticity is dependent on noradrenergic innervation of the neocortex. In the central nervous system, noradrenergic α2 receptors (α2-ARs) are widely expressed in the motor cortex and have been critically implicated in synaptic communication and plasticity. In current study, we examined whether activation of cortical α2-ARs is necessary for VNS-driven motor cortical reorganization to occur. Consistent with previous studies, we found that VNS paired with motor training enlarges the map representation of task-relevant musculature in the motor cortex. Infusion of α2-AR antagonists into M1 blocked VNS-driven motor map reorganization from occurring. Our results suggest that local α2-AR activation is required for VNS-induced cortical reorganization to occur, providing insight into the mechanisms that may underlie the neuroplastic effects of VNS therapy.

Choice of a spin singlet or triplet: electronic properties of Bis-Co(II), Bis-Ni(II), Bis-Cu(II) and Bis-Zn(II) oxygen doubly N-confused hexaphyrin (1.1.1.1.1.1).

Fused hexaphyrins have many physical and chemical properties and can coordinate transition metal ions. In this study, we investigated the geometric structure, charge decomposition analysis (CDA), spin density, frontier molecular orbital (FMO) compositions and absorption spectra of four oxygen doubly N-confused hexaphyrin (1.1.1.1.1.1) (ONCP) complexes with the metal ions Co(II), Ni(II), Cu(II) and Zn(II) (designated ONCP-d-Co, ONCP-d-Ni, ONCP-d-Cu and ONCP-d-Zn). Based on their energies, geometric structures, FMO characteristics and comparison to experiments, ONCP-d-Co and ONCP-d-Cu have the mix-states of the triplet state and broken-symmetry state (antiferromagnetic state) rather than the spin singlet of a closed shell as previously reported. Moreover, based on analyses of the spin density and spin population of the spin triplet ONCP-d-Co and ONCP-d-Cu complexes, the charge transfer in ONCP-d-Cu is greater than that in ONCP-d-Co because the spin density in ONCP-d-Cu is concentrated not only on the Cu ion but also on the ONCP ligand. Thus, the CDA value for ONCP-d-Cu is larger. Finally, through comparative analysis of the FMO compositions and absorption spectra, the complexes and ligand are shown to have very similar absorption spectra with characteristics that arise mainly from π → π* transitions both in the B-band and the Q-band, which is due to the FMO compositions being dominated by the highly delocalized conjugated system, rather than by the metal ions. The absorption maxima of the Q-band are ONCP-d-Co (1020 nm) > ONCP-d-Zn (1012 nm) > ONCP-d-Ni (997 nm) > ONCP-d-Cu (988 nm), which is inversely proportional to the energy gap in their FMOs. Graphical Abstract The present work investigates the geometric structure, charge decomposition analysis (CDA), spin density, frontier molecular orbital (FMO) compositions and absorption spectra of four oxygen doubly N-confused hexaphyrin (1.1.1.1.1.1) (ONCP) complexes with the metal ions Co(II), Ni(II), Cu(II) and Zn(II) (designated ONCP-d-Co, ONCP-d-Ni, ONCP-d-Cu and ONCP-d-Zn). Based on their energies, geometric structures, FMO characteristics and comparison to experiments, ONCP-d-Co and ONCP-d-Cu have the mix-state of the triplet state and broken-symmetry state (antiferromagnetic state) rather than the spin singlet of a close shell as were previously reported. Meanwhile, ONCP-d-Ni and ONCP-d-Zn show spin singlet structure. The calculated CDA shows the following order: ONCP-d-Cu (1.487) > ONCP-d-Ni (1.255) > ONCP-d-Co (1.211) > ONCP-d-Zn (1.201). Through comparisons of spin density and spin populations of ONCP-d-Co and ONCP-d-Cu, charge transfer between Cu and ligand ONCP is greater than that of Co and ONCP, which makes the CDA value of ONCP-d-Cu obviously larger than that of the other complexes.

Doping control analysis of 121 prohibited substances in equine hair by liquid chromatography-tandem mass spectrometry.

Equine hair is becoming an increasingly popular biological matrix for doping control of horse sports; one of the reasons for this is the significantly longer detection window hair can offer. Hair analysis opens up the opportunity for longitudinal monitoring of drug exposure which would otherwise not be possible with the more traditional and common biological matrices, such as urine and blood. As such, there is a need for more multi-target screening methods covering a broad range of prohibited substances in equine hair at the required sensitivities for equine doping control. This paper describes a sensitive ultra-high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) method for the detection of 121 drugs and/or their metabolites in equine hair covering ten classes of prohibited substances with estimated limits of detection between 0.1 and 10 pg/mg. To our knowledge, this is the first report of a screening method in equine hair which can cover such a broad range and well over one hundred prohibited substances in a single analytical run. This method has been validated for its specificity, precision and extraction recovery. Applicability of this method has been demonstrated by: (i) the successful identification of clenbuterol, 2-(1-hydroxyethyl) promazine sulfoxide, acepromazine and tetrahydrozoline in genuine equine mane samples; as well as (ii) the detection of drugs from artificially incurred mane hair samples which have been prepared by soaking blank hair samples in solutions of drug targets.

The effects of bupropion on hybrid striped bass brain chemistry and predatory behavior.

Increased use of antidepressants has led to an increase in their detection in final treated wastewater effluents and receiving streams. Antidepressants are intended to modify human behavior by altering brain chemistry, and because of the high functional conservation of antidepressant target receptors in vertebrates, aquatic organisms may be at risk. The antidepressant bupropion is designed to alter brain norepinephrine and dopamine concentrations in humans. The objective of the present study was to understand if alteration of dopaminergic neurotransmitter concentrations in the hybrid striped bass (Morone saxatilis × Morone chrysops) brain by bupropion would alter this predator's ability to capture prey. The authors exposed hybrid striped bass to bupropion in a static system for 6 d, followed by a 6-d recovery period. During the present study's 12-d experiment, each hybrid striped bass was fed 4 unexposed fathead minnows every 3 d, and the time it took the hybrid striped bass to consume each of those 4 fathead minnows was quantified. After each feeding event, hybrid striped bass brains were harvested and analyzed for changes in several brain neurotransmitter concentrations, including serotonin, norepinephrine, dopamine, and many of their metabolites. Although bupropion altered the concentration of dopamine and many of the dopaminergic neurotransmitter metabolite concentrations in the brains on day 3 of the exposure, it did not alter the time to capture prey. This suggests that alteration of dopaminergic neurotransmitter concentrations in the hybrid striped bass brain does not alter a predator's ability to capture prey. Environ Toxicol Chem 2016;35:2058-2065. © 2016 SETAC.

Carbon nanotubes dispersed in aqueous solution by ruthenium(ii) polypyridyl complexes.

Cationic ruthenium(ii) polypyridyl complexes with appended pyrene groups have been synthesized and used to disperse single-walled carbon nanotubes (SWCNT) in aqueous solutions. To this end, planar pyrene groups enable association by means of π-stacking onto carbon nanotubes and, in turn, the attachment of the cationic ruthenium complexes. Importantly, the ionic nature of the ruthenium complexes allows the formation of stable dispersions featuring individualized SWCNTs in water as confirmed in a number of spectroscopic and microscopic assays. In addition, steady-state photoluminescence spectroscopy was used to probe the excited state interactions between the ruthenium complexes and SWCNTs. These studies show that the photoluminescence of both, that is, of the ruthenium complexes and of SWCNTs, are quenched when they interact with each other. Pump-probe transient absorption experiments were performed to shed light onto the nature of the photoluminescence quenching, showing carbon nanotube-based bands with picosecond lifetimes, but no new bands which could be unambigously assigned to photoinduced charge transfer process. Thus, from the spectroscopic data, we conclude that quenching of the photoluminescence of the ruthenium complexes is due to energy transfer to proximal SWCNTs.

Elevated Levels of SOX10 in Serum from Vitiligo and Melanoma Patients, Analyzed by Proximity Ligation Assay.

BACKGROUND: The diagnosis of malignant melanoma currently relies on clinical inspection of the skin surface and on the histopathological status of the excised tumor. The serum marker S100B is used for prognostic estimates at later stages of the disease, but analyses are marred by false positives and inadequate sensitivity in predicting relapsing disorder. OBJECTIVES: To investigate SOX10 as a potential biomarker for melanoma and vitiligo. METHODS: In this study we have applied proximity ligation assay (PLA) to detect the transcription factor SOX10 as a possible serum marker for melanoma. We studied a cohort of 110 melanoma patients. We further investigated a second cohort of 85 patients with vitiligo, which is a disease that also affects melanocytes. RESULTS: The specificity of the SOX10 assay in serum was high, with only 1% of healthy blood donors being positive. In contrast, elevated serum SOX10 was found with high frequency among vitiligo and melanoma patients. In patients with metastases, lack of SOX10 detection was associated with treatment benefit. In two responding patients, a change from SOX10 positivity to undetectable levels was seen before the response was evident clinically. CONCLUSIONS: We show for the first time that SOX10 represents a promising new serum melanoma marker for detection of early stage disease, complementing the established S100B marker. Our findings imply that SOX10 can be used to monitor responses to treatment and to assess if the treatment is of benefit at stages earlier than what is possible radiologically.

Mouse Brachyury the Second (T2) is a gene next to classical T and a candidate gene for tct.

The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation in T2 disrupts notochord development, pointing to the existence of a second T/t complex gene involved in axis development. T2 encodes a novel protein that is disrupted by an insertion in T2(Bob) mice. Sequence analysis of T2 from several t haplotypes shows that they all share the same changed stop codon, and, thus, T2 is a candidate gene for the t complex tail interaction factor. T1, T2, and the unlinked t-int are distinct and unrelated loci, and mutations in these genes do not complement one another genetically. Either their products interact in the same pathway during the genesis of the embryonic axis, or the T/t region itself is truly complex.

Effective dimension reduction for sparse functional data.

We propose a method of effective dimension reduction for functional data, emphasizing the sparse design where one observes only a few noisy and irregular measurements for some or all of the subjects. The proposed method borrows strength across the entire sample and provides a way to characterize the effective dimension reduction space, via functional cumulative slicing. Our theoretical study reveals a bias-variance trade-off associated with the regularizing truncation and decaying structures of the predictor process and the effective dimension reduction space. A simulation study and an application illustrate the superior finite-sample performance of the method.

Synthesis and single-molecule imaging of highly mobile adamantane-wheeled nanocars.

The synthesis and single-molecule imaging of two inherently fluorescent nanocars equipped with adamantane wheels is reported. The nanocars were imaged using 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as the chromophore, which was rigidly incorporated into the nanocar chassis via Sonogashira cross-coupling chemistry that permitted the synthesis of nanocars having different geometries. In particular, studied here were four- and three-wheeled nanocars with adamantane wheels. It was found that, for the four-wheeled nanocar, the percentage of moving nanocars and the diffusion constant show a significant improvement over p-carborane-wheeled nanocars with the same chassis. The three-wheeled nanocar showed only limited mobility due to its geometry. These results are consistent with a requisite wheel-like rolling motion. We furthermore developed a model that relates the percentage of moving nanocars in single-molecule experiments with the diffusion constant. The excellent agreement between the model and the new results presented here as well as previous single-molecule studies of fluorescent nanocars yields an improved understanding of motion in these molecular machines.