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Fluorescent probes for bacterial detection can be obtained by conjugating antimicrobial peptides with fluorescent dyes. However, little is known about the effect of the conjugation site and linker chemistry on staining efficiency. We synthesized three conjugates of the antimicrobial peptide ubiquicidin with the environmentally sensitive fluorophore Nile Red that differed by the attachment site and the chemical composition of the linker. We showed that incorporating fluorophore as a minimalistic non-natural amino acid resulted in a superior probe compared with the typically used bioconjugation approaches. The new peptide-based probe named UNR-1 displayed red fluorescence and enabled robust wash-free staining of Gram-positive and Gram-negative bacteria. The probe exhibited selectivity over mammalian cells and enabled rapid fluorescence detection of bacteria by fluorescence microscopy and flow cytometry in an add-and-read format. Our results may foster the development of next-generation fluorescent AMPs for clinical laboratory diagnostics and medical imaging.

Autism Spectrum Disorders (ASD) are neurodevelopmental disorders whose diagnosis relies on deficient social interaction and communication together with repetitive behaviours. Multiple studies have highlighted the potential of oxytocin (OT) to ameliorate behavioural abnormalities in animal models and subjects with ASD. Clinical trials, however, yielded disappointing results. Our study aimed at assessing the behavioural effects of different regimens of OT administration in the Oprm1 null mouse model of ASD. We assessed the effects of intranasal OT injected once at different doses (0.15, 0.3, and 0.6 IU) and time points (5, 15, and 30 min) following administration, or chronically, on ASD-related behaviours (social interaction and preference, stereotypies, anxiety, nociception) in Oprm1 +/+ and Oprm1 -/- mice. We then tested whether pairing intranasal OT injection with social experience would influence its outcome on ASD-like symptoms, and measured gene expression in the reward/social circuit. Acute intranasal OT at 0.3 IU improved social behaviour in Oprm1 -/- mice 5 min after administration, with limited effects on non-social behaviours. Chronic (8-17 days) OT maintained rescuing effects in Oprm1 null mice but was deleterious in wild-type mice. Finally, improvements in the social behaviour of Oprm1 -/- mice were greater and longer lasting when OT was administered in a social context. Under these conditions, the expression of OT and vasopressin receptor genes, as well as marker genes of striatal projection neurons, was suppressed. We detected no sex difference in OT effects. Our results highlight the importance of considering dosage and social context when evaluating the effects of OT treatment in ASD., (© 2024. The Author(s).)

Fluorogenic dimers with polarity-sensitive folding are powerful probes for live-cell bioimaging. They switch on their fluorescence only after interacting with their targets, thus leading to a high signal-to-noise ratio in wash-free bioimaging. We previously reported the first near-infrared fluorogenic dimers derived from cyanine 5.5 dyes for the optical detection of G protein-coupled receptors. Owing to their hydrophobic character, these dimers are prone to form nonspecific interactions with proteins such as albumin and with the lipid bilayer of the cell membrane resulting in a residual background fluorescence in complex biological media. Herein, we report the rational design of new fluorogenic dimers derived from cyanine 5. By modulating the chemical structure of the cyanine units, we discovered that the two asymmetric cyanine 5.25 dyes were able to form intramolecular H-aggregates and self-quenched in aqueous media. Moreover, the resulting original dimeric probes enabled a significant reduction of the nonspecific interactions with bovine serum albumin and lipid bilayers compared with the first generation of cyanine 5.5 dimers. Finally, the optimized asymmetric fluorogenic dimer was grafted to carbetocin for the specific imaging of the oxytocin receptor under no-wash conditions directly in cell culture media, notably improving the signal-to-background ratio compared with the previous generation of cyanine 5.5 dimers.

To fill the need for environmentally sensitive fluorescent unnatural amino acids able to operate in the red region of the spectrum, we have designed and synthesized Alared, a red solvatochromic and fluorogenic amino acid derived from the Nile Red chromophore. The new unnatural amino acid can be easily integrated into bioactive peptides using classical solid-phase peptide synthesis. The fluorescence quantum yield and the emission maximum of Alared-labeled peptides vary in a broad range depending on the peptide's environment, making Alared a powerful reporter of biomolecular interactions. Due to its red-shifted absorption and emission spectra, Alared-labeled peptides could be followed in living cells with minimal interference from cellular autofluorescence. Using ratiometric fluorescence microscopy, we were able to track the fate of the Alared-labeled peptide agonists of the apelin G protein-coupled receptor upon receptor activation and internalization. Due to its color-shifting environmentally sensitive emission, Alared allowed for distinguishing the fractions of peptides that are specifically bound to the receptor or unspecifically bound to different cellular membranes., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Herein, we describe a catalyst-free thia-Diels-Alder cycloaddition for the chemoselective labeling of fully deprotected phosphonodithioester-peptides in solution with fluorophores functionalized with an exocyclic diene. The reaction was optimized on the model tripeptide 1 containing a lysine residue, which enabled its rapid and straightforward labeling with three different fluorophores (fluorescein, lissamine rhodamine B, and squaraine) in very mild conditions (H 2 O/ i PrOH, 37 °C, 1 h). The reaction was then successfully applied to the chemoselective labeling of fully deprotected apelin-13 with squaraine dye. The resulting fluorescent ligand 18 exhibited a high affinity (0.17 ± 0.03 nM) for apelinR. It enabled the development of time-resolved FRET-based competition assays for high-throughput screening and drug discovery. Thanks to its fluorogenic properties, ligand 18 was also successfully involved in the live-cell optical imaging of apelinR in no-wash conditions.

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.

The local lipid microenvironment of transmembrane receptors is an essential factor in G protein coupled receptor (GPCR) signaling. However, tools are currently missing for studying endogenously expressed GPCRs in primary cells and tissues. Here, we introduce fluorescent environment-sensitive GPCR ligands for probing the microenvironment of the receptor in living cells using fluorescence microscopy under no-wash conditions. We designed and synthesized antagonist ligands of the oxytocin receptor (OTR) by conjugating a high-affinity nonpeptidic OTR ligand PF-3274167 to the environment-sensitive fluorescent dye Nile Red. The length of the polar PEG spacer between the pharmacophore and the fluorophore was adjusted to lower the nonspecific interactions of the probe while preserving a strong fluorogenic response. We demonstrated that the new probes embed into the lipid bilayer in the vicinity of the receptor and convey information about the local polarity and the lipid order via the wavelength-shifting emission of the Nile Red fluorophore.

Hydrophobins are small secreted cysteine-rich proteins exclusively found in fungi. They are able to self-assemble in single molecular layers at hydrophobic-hydrophilic interfaces and can therefore be directly involved in establishment of fungi in their habitat. The genomes of filamentous mycotrophic fungi Trichoderma encode a rich diversity of hydrophobins, which are divided in several groups based on their structure and evolution. Here we describe a new member of class II hydrophobins, HFB7, that has a taxonomically restricted occurrence in Harzianum and Virens clades of Trichoderma. Evolutionary analysis reveals that HFB7 proteins form a separate clade distinct from other Trichoderma class II hydrophobins and that genes encoding them evolve under positive selection pressure. Homology modelling of HFB7 structure in comparison to T. reesei HFB2 reveals that the two large hydrophobic patches on the surface of the protein are remarkably conserved between the two hydrophobins despite significant difference in their primary structures. Expression of hfb7 gene in T. virens increases at interactions with other fungi and a plant and in response to a diversity of abiotic stress conditions, and is also upregulated during formation of aerial mycelium in a standing liquid culture. This upregulation significantly exceeds that of expression of hfb7 under a strong constitutive promoter, and T. virens strains overexpressing hfb7 thus display only changes in traits characterized by low hfb7 expression, i.e. faster growth in submerged liquid culture. The hfb7 gene is not expressed in conidia. Our data allow to conclude that this protein is involved in defence of Trichoderma against a diversity of stress factors related to the oxidative stress. Moreover, HFB7 likely helps in the establishment of the fungus in wetlands or other conditions related to high humidity., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Patient-centered access to mental health describes the fit between patient needs and resources of the system. To date, little data are available to guide implementation of services to women veterans, an underrepresented minority within Department of Veteran Affairs (VA) health care. The current study examines access to mental health care among women veterans, and identifies gender-related indicators of perceived access to mental health care., Methods: A population-based sample of 6287 women veterans using VA primary care services participated in a survey of past year perceived need for mental health care, mental health utilization, and gender-related mental health care experiences. Subjective rating of how well mental health care met their needs was used as an indicator of perceived access., Results: Half of all women reported perceived mental health need; 84.3% of those women received care. Nearly all mental health users (90.9%) used VA services, although only about half (48.8%) reported that their mental health care met their needs completely or very well. Gender related experiences (availability of female providers, women-only treatment settings, women-only treatment groups, and gender-related comfort) were each associated with 2-fold increased odds of perceived access, and associations remained after adjusting for ease of getting care., Conclusions: Women VA users demonstrate very good objective access to mental health services. Desire for, and access to specialized mental health services for women varies across the population and are important aspects of shared decision making in referral and treatment planning for women using VA primary care.

Background: Military sexual trauma (MST) is the Veteran Health Administration's (VHA) term for sexual assault and/or sexual harassment that occurs during military service. The experience of MST is associated with a variety of mental health conditions. Preliminary research suggests that MST may be associated with homelessness among female Veterans, although to date MST has not been examined in a national study of both female and male homeless Veterans., Objective: To estimate the prevalence of MST, examine the association between MST and mental health conditions, and describe mental health utilization among homeless women and men., Design and Participants: National, cross-sectional study of 126,598 homeless Veterans who used VHA outpatient care in fiscal year 2010., Main Measures: All variables were obtained from VHA administrative databases, including MST screening status, ICD-9-CM codes to determine mental health diagnoses, and VHA utilization., Key Results: Of homeless Veterans in VHA, 39.7 % of females and 3.3 % of males experienced MST. Homeless Veterans who experienced MST demonstrated a significantly higher likelihood of almost all mental health conditions examined as compared to other homeless women and men, including depression, posttraumatic stress disorder, other anxiety disorders, substance use disorders, bipolar disorders, personality disorders, suicide, and, among men only, schizophrenia and psychotic disorders. Nearly all homeless Veterans had at least one mental health visit and Veterans who experienced MST utilized significantly more mental health visits compared to Veterans who did not experience MST., Conclusions: A substantial proportion of homeless Veterans using VHA services have experienced MST, and those who experienced MST had increased odds of mental health diagnoses. Homeless Veterans who had experienced MST had higher intensity of mental health care utilization and high rates of MST-related mental health care. This study highlights the importance of trauma-informed care among homeless Veterans and the success of VHA homeless programs in providing mental health care to homeless Veterans.