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Introduction Faculty and residents strive for appropriate autonomy and entrustment. Initial direct supervision of clinical care gradually shifts to increasing levels of resident independence over time. Faculty members are inconsistent in resident supervision leading to missed opportunities for resident independence. Methods Family medicine faculty workshop participants completed teaching style self-evaluations prior to discussion of clinical examples with excessive or insufficient autonomy. Participants reviewed real resident feedback examples to increase insight into teaching styles. Participants were presented with cases to discuss varying degrees of resident autonomy and entrustment. Learners committed to one specific behavior to calibrate the degree of autonomy they provide. Results Of the faculty, 113 members participated in the workshop with the majority (98%) finding the workshop relevant in helping them to identify strategies for reflecting on their degree of autonomy allowed and to look for appropriate situations for enhancing their resident entrustment. Discussion This interactive workshop provided clear ways for addressing the issue of independence versus control in supervision of patient care. It provided a feedback mechanism for educators who provide too much or too little autonomy for the best resident learning. Additionally, this conversation encouraged participants to engage in self-reflection on the autonomy given to their resident.

Delivering nucleic acid therapeutics across cell membranes is a significant challenge. Cell-penetrating peptides (CPPs) containing arginine (R), tryptophan (W), and histidine (H) show promise for siRNA delivery. To improve siRNA delivery and silence a model STAT3 gene, we hypothesized that oleyl acylation to CPPs, specifically (WRH) n , would enhance STAT3 silencing efficiency in breast and ovarian cancer cells. Using Fmoc/tBu solid-phase peptide chemistry, we synthesized, purified, and characterized the oleyl-conjugated (WRH) n (n = 1-4) peptides. The peptide/siRNA complexes were non-cytotoxic at N/P 40 (~20 μM) against MDA-MB-231, MCF-7, SK-OV-3, and HEK-293 cells after 72 h incubation. All peptide/siRNA complexes showed serum stability at N/P ≥ 40. The synthesized conjugates, with a diameter of <100 nm, formed nano-complexes with siRNA and exhibited a stable range of zeta potential values (13-18 mV at N/P = 40). Confocal microscopy and flow cytometry analysis provided qualitative and quantitative evidence of a successful cellular internalization of siRNA. The peptides oleyl-(WRH) 3 and oleyl-(WRH) 4 showed ~60% and ~75% cellular uptake of siRNA, respectively, in both MDA-MB-231 and SK-OV-3 cells. Western blot analysis of oleyl-(WRH) 4 demonstrated effective silencing of the STAT-3 gene, with ~75% silencing in MDA-MB-231 cells and ~45% in SK-OV-3 cells.

Antimicrobial peptides (AMPs) are being explored as a potential strategy to combat antibiotic resistance due to their ability to reduce susceptibility to antibiotics. This study explored whether the [R 4 W 4 ] peptide mode of action is bacteriostatic or bactericidal using modified two-fold serial dilution and evaluating the synergism between gentamicin and [R 4 W 4 ] against Escherichia coli ( E. coli ) and methicillin-resistant Staphylococcus aureus (MRSA) by a checkered board assay. [R 4 W 4 ] exhibited bactericidal activity against bacterial isolates (MBC/MIC ≤ 4), with a synergistic effect with gentamicin against E. coli (FICI = 0.3) but not against MRSA (FICI = 0.75). Moreover, we investigated the mechanism of action of [R 4 W 4 ] against MRSA by applying biophysical assays to evaluate zeta potential, cytoplasmic membrane depolarization, and lipoteichoic acid (LTA) binding affinity. [R 4 W 4 ] at a 16 mg/mL concentration stabilized the zeta potential of MRSA -31 ± 0.88 mV to -8.37 mV. Also, [R 4 W 4 ] at 2 × MIC and 16 × MIC revealed a membrane perturbation process associated with concentration-dependent effects. Lastly, in the presence of BODIPY-TR-cadaverine (BC) fluorescence dyes, [R 4 W 4 ] exhibited binding affinity to LTA comparable with melittin, the positive control. In addition, the antibacterial activity of [R 4 W 4 ] against MRSA remained unchanged in the absence and presence of LTA, with an MIC of 8 µg/mL. Therefore, the [R 4 W 4 ] mechanism of action is deemed bactericidal, involving interaction with bacterial cell membranes, causing concentration-dependent membrane perturbation. Additionally, after 30 serial passages, there was a modest increment of MRSA strains resistant to [R 4 W 4 ] and a change in antibacterial effectiveness MIC [R 4 W 4 ] and vancomycin by 8 and 4 folds with a slight change in Levofloxacin MIC 1 to 2 µg/mL. These data suggest that [R 4 W 4 ] warrants further consideration as a potential AMP.

The emergence of multidrug-resistant (MDR) strains causes severe problems in the treatment of microbial infections owing to limited treatment options. Antimicrobial peptides (AMPs) are drawing considerable attention as promising antibiotic alternative candidates to combat MDR bacterial and fungal infections. Herein, we present a series of small amphiphilic membrane-active cyclic peptides composed, in part, of various nongenetically encoded hydrophilic and hydrophobic amino acids. Notably, lead cyclic peptides 3b and 4b showed broad-spectrum activity against drug-resistant Gram-positive (MIC = 1.5-6.2 µg/mL) and Gram-negative (MIC = 12.5-25 µg/mL) bacteria, and fungi (MIC = 3.1-12.5 µg/mL). Furthermore, lead peptides displayed substantial antibiofilm action comparable to standard antibiotics. Hemolysis (HC 50 = 230 µg/mL) and cytotoxicity (>70 % cell viability against four different mammalian cells at 100 µg/mL) assay results demonstrated the selective lethal action of 3b against microbes over mammalian cells. A calcein dye leakage experiment substantiated the membranolytic effect of 3b and 4b, which was further confirmed by scanning electron microscopy. The behavior of 3b and 4b in aqueous solution and interaction with phospholipid bilayers were assessed by employing nuclear magnetic resonance (NMR) spectroscopy in conjunction with molecular dynamics (MD) simulations, providing a solid structural basis for understanding their membranolytic action. Moreover, 3b exhibited stability in human blood plasma (t 1/2 = 13 h) and demonstrated no signs of resistance development against antibiotic-resistant S. aureus and E. coli. These findings underscore the potential of these newly designed amphiphilic cyclic peptides as promising anti-infective agents, especially against Gram-positive bacteria., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The burden of breast cancer, the second leading cause of death worldwide, is increasing at an alarming rate. Cuscuta, used in traditional medicine for different ailments, including cancer, is known for containing phytochemicals that exhibit anticancer activity; however, the bioactivities of proteins from this plant remain unexplored. This study aimed to screen the cytotoxic potential of proteins from the crude herbal product of Cuscuta epithymum(L.) (CE) harvested from the host plants Alhagi maurorum and Medicago sativa., Methods: The proteins from CE were extracted using a salting-out method, followed by fractionation with a gel filtration chromatography column. Gel-free shotgun proteomics was subsequently performed for protein characterization. The viability assay using MTT was applied to deduce the cytotoxic potential of proteins against MCF-7 breast cancer cells, with further exploration of the effect of treatment on the expression of the apoptotic mediator BCL2-associated X protein (BAX) and B-cell lymphoma protein 2 (BCL-2) proteins, using western blotting to strengthen the findings from the in vitro viability assay., Results: The crude proteins (CP) of CE were separated into four protein peaks (P1, P2, P3, and P4) by gel filtration chromatography. The evaluation of potency showed a dose-dependent decline in the MCF-7 cell line after CP, P1, P2, and P3 treatment with the respective IC 50 values of 33.8, 43.1, 34.5, and 28.6 µg/ml. The percent viability of the cells decreased significantly upon treatment with 50 µg/ml CP, P1, P2, and P3 (P < 0.001). Western-blot analysis revealed upregulation of proapoptotic protein BAX in the cells treated with CP, P3 (P < 0.01), and P2 (P < 0.05); however, the antiapoptotic protein, BCL-2 was downregulated in the cells treated with CP and P3 (P < 0.01), but no significant change was detected in P2 treated cells. The observed cytotoxic effects of proteins in the CP, P1, P2, and P3 from the in vitro viability assay and western blot depicted the bioactivity potential of CE proteins. The database search revealed the identities of functionally important proteins, including nonspecific lipid transfer protein, superoxide dismutase, carboxypeptidase, RNase H domain containing protein, and polyribonucleotide nucleotidyltransferase, which have been previously reported from other plants to exhibit anticancer activity., Conclusion: This study indicated the cytotoxic activity of Cuscuta proteins against breast cancer MCF-7 cells and will be utilized for future investigations on the mechanistic effect of active proteins. The survey of CE proteins provided substantial data to encourage further exploration of biological activities exhibited by proteins in Cuscuta., (© 2024. The Author(s).)

Breast cancer remains a significant global health concern, emphasizing the critical need for effective treatment strategies, especially targeted therapies. This systematic review summarizes the findings from in vitro and in vivo studies regarding the therapeutic potential of exosomes as drug delivery platforms in the field of breast cancer treatment. A comprehensive search was conducted across bibliographic datasets, including Web of Science, PubMed, and Scopus, using relevant queries from several related published articles and the Medical Subject Headings Database. Then, all morphological, biomechanical, histopathological, and cellular-molecular outcomes were systematically collected. A total of 30 studies were identified based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. These studies underwent assessment using the Systematic Review Centre for Laboratory Animal Experimentation risk of bias assessment tool. The results indicate that exosomes exhibit promise as effective drug delivery platforms, capable of hindering cancer cell viability, proliferation, migration, and angiogenesis. However, a comprehensive assessment is challenging due to some studies deviating from guidelines and having incomplete methodology. Addressing these, future studies should detail methodologies, optimize dosing, and enhance exosome production. Standardization in reporting, consistent protocols, and exploration of alternative sources are crucial., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

With the first reports on the possibility of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas)9 surfacing in 2005, the enthusiasm for protein silencing via nucleic acid delivery experienced a resurgence following a period of diminished enthusiasm due to challenges in delivering small interfering RNAs (siRNA), especially in vivo. However, delivering the components necessary for this approach into the nucleus is challenging, maybe even more than the cytoplasmic delivery of siRNA. We previously reported the birth of peptide/lipid-associated nucleic acids (PLANAs) for siRNA delivery. This project was designed to investigate the efficiency of these nanoparticles for in vitro delivery of CRISPR/Cas9 ribonucleoproteins. Our initial experiments indicated higher toxicity for PLANAs with the more efficient reverse transfection method. Therefore, polyethylene glycol (PEG) was added to the composition for PEGylation of the nanoparticles by partially replacing two of the lipid components with the PEG-conjugated counterparts. The results indicated a more significant reduction in the toxicity of the nanoparticle, less compromise in encapsulation efficiency and more PEGylation of the surface of the nanoparticles using DOPE-PEG2000 at 50 % replacement of the naïve lipid. The cell internalization and transfection efficiency showed a comparable efficiency for the PEGylated and non-PEGylated PLANAs and the commercially available Lipofectamine™ CRISPRMAX™. Next Generation Sequencing of the cloned cells showed a variety of indels in the transfected cell population. Overall, our results indicate the efficiency and safety of PEGylated PLANAs for in vitro transfection with CRISPR/Cas9 ribonucleoproteins. PEGylation has been studied extensively for in vivo delivery, and PEGylated PLANAs will be candidates for future in vivo studies., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

In our ongoing quest to design effective antimicrobial peptides (AMPs), this study aimed to elucidate the mechanisms governing cyclic amphiphilic AMPs and their interactions with membranes. The objective was to discern the nature of these interactions and understand how peptide sequence and structure influence antimicrobial activity. We introduced modifications into the established cyclic AMP peptide, [W 4 R 4 ], incorporating an extra aromatic hydrophobic residue (W), a positively charged residue (R), or the unique 2,5-diketopiperazine (DKP). This study systematically explored the structure-activity relationships (SARs) of a series of cyclic peptides derived from the [W 4 R 4 ] scaffold, including the first synthesis and evaluation of [W 4 R 4 (DKP)]. Structural, dynamic, hydrophobic, and membrane-binding properties of four cyclic peptides ([W 4 R 4 ], [W 5 R 4 ], [W 4 R 5 ], [W 4 R 4 (DKP)]) were explored using molecular dynamics simulations within a DOPC/DOPG lipid bilayer that mimics the bacterial membrane. The results revealed distinct SARs linking antimicrobial activity to parameters such as conformational plasticity, immersion depth in the bilayer, and population of the membrane binding mode. Notably, [W 4 R 5 ] exhibited an optimal "activity/binding to the bacterial membrane" pattern. This multidisciplinary approach efficiently decoded finely regulated SAR profiles, laying a foundation for the rational design of novel antimicrobial peptides.

The field of Alzheimer's disease (AD) has witnessed recent breakthroughs in the development of disease-modifying biologics and diagnostic markers. While immunotherapeutic interventions have provided much-awaited solutions, nucleic acid-based tools represent other avenues of intervention; however, these approaches are costly and invasive, and they have serious side effects. Previously, we have shown in AD animal models that tolfenamic acid (TA) can lower the expression of AD-related genes and their products and subsequently reduce pathological burden and improve cognition. Using TA as a scaffold and the zinc finger domain of SP1 as a pharmacophore, we developed safer and more potent brain-penetrating analogs that interfere with sequence-specific DNA binding at transcription start sites and predominantly modulate the expression of SP1 target genes. More importantly, the proteome of treated cells displayed ~75% of the downregulated products as SP1 targets. Specific levels of SP1-driven genes and AD biomarkers such as amyloid precursor protein (APP) and Tau proteins were also decreased as part of this targeted systemic response. These small molecules, therefore, offer a viable alternative to achieving desired therapeutic outcomes by interfering with both amyloid and Tau pathways with limited off-target systemic changes.

Fungal infections remain a significant concern for human health. The emergence of microbial resistance, the improper use of antimicrobial drugs, and the need for fewer toxic antifungal treatments in immunocompromised patients have sparked substantial interest in antifungal research. Cyclic peptides, classified as antifungal peptides, have been in development as potential antifungal agents since 1948. In recent years, there has been growing attention from the scientific community to explore cyclic peptides as a promising strategy for combating antifungal infections caused by pathogenic fungi. The identification of antifungal cyclic peptides from various sources has been possible due to the widespread interest in peptide research in recent decades. It is increasingly important to evaluate narrow- to broad-spectrum antifungal activity and the mode of action of synthetic and natural cyclic peptides for both synthesized and extracted peptides. This short review aims to highlight some of the antifungal cyclic peptides isolated from bacteria, fungi, and plants. This brief review is not intended to present an exhaustive catalog of all known antifungal cyclic peptides but rather seeks to showcase selected cyclic peptides with antifungal properties that have been isolated from bacteria, fungi, plants, and synthetic sources. The addition of commercially available cyclic antifungal peptides serves to corroborate the notion that cyclic peptides can serve as a valuable source for the development of antifungal drugs. Additionally, this review discusses the potential future of utilizing combinations of antifungal peptides from different sources. The review underscores the need for the further exploration of the novel antifungal therapeutic applications of these abundant and diverse cyclic peptides.

The biological significance of benzopyran-4-ones as cytotoxic agents against multi-drug resistant cancer cell lines and isoxazoles as anti-inflammatory agents in cellular assays prompted us to design and synthesize their hybrid compounds and explore their antiproliferative activity against a panel of six cancer cell lines and two normal cell lines. Compounds 5a - d displayed significant antiproliferative activities against all the cancer cell lines tested, and IC 50 values were in the range of 5.2-22.2 μM against MDA-MB-231 cancer cells, while they were minimally cytotoxic to the HEK-293 and LLC-PK1 normal cell lines. The IC 50 values of 5a - d against normal HEK-293 cells were in the range of 102.4-293.2 μM. Compound 5a was screened for kinase inhibitory activity, proteolytic human serum stability, and apoptotic activity. The compound was found inactive towards different kinases, while it completely degraded after 2 h of incubation with human serum. At 5 μM concentration, it induced apoptosis in MDA-MB-231 by 50.8%. Overall, these findings suggest that new benzopyran-4-one-isoxazole hybrid compounds, particularly 5a - d , are selective anticancer agents, potentially safe for human cells, and could be synthesized at low cost. Additionally, Compound 5a exhibits potential anticancer activity mediated via inhibition of cancer cell proliferation and induction of apoptosis.

The entry of proteins through the cell membrane is challenging, thus limiting their use as potential therapeutics. Seven cell-penetrating peptides, designed in our laboratory, were evaluated for the delivery of proteins. Fmoc solid-phase peptide synthesis was utilized for the synthesis of seven cyclic or hybrid cyclic-linear amphiphilic peptides composed of hydrophobic (tryptophan (W) or 3,3-diphenylalanine (Dip) and positively-charged arginine (R) residues, such as [WR] 4 , [WR] 9 , [WWRR] 4 , [WWRR] 5 , [(RW) 5 K](RW) 5 , [R 5 K]W 7 , and [DipR] 5 . Confocal microscopy was used to screen the peptides as a protein delivery system of model cargo proteins, green and red fluorescein proteins (GFP and RFP). Based on the confocal microscopy results, [WR] 9 and [DipR] 5 were found to be more efficient among all the peptides and were selected for further studies. [WR] 9 (1-10 µM) + protein (GFP and RFP) physical mixture did not show high cytotoxicity (>90% viability) in triple-negative breast cancer cells (MDA-MB-231) after 24 h, while [DipR] 5 (1-10 µM) physical mixture with GFP exhibited more than 81% cell viability. Confocal microscopy images revealed internalization of GFP and RFP in MDA-MB-231 cells using [WR] 9 (2-10 μM) and [DipR] 5 (1-10 µM). Fluorescence-activated cell sorting (FACS) analysis indicated that the cellular uptake of GFP was concentration-dependent in the presence of [WR] 9 in MDA-MB-231 cells after 3 h of incubation at 37 °C. The concentration-dependent uptake of GFP and RFP was also observed in the presence of [DipR 5 ] in SK-OV-3 and MDA-MB-231 cells after 3 h of incubation at 37 °C. FACS analysis indicated that the cellular uptake of GFP in the presence of [WR] 9 was partially decreased by methyl-β-cyclodextrin and nystatin as endocytosis inhibitors after 3 h of incubation in MDA-MB-231 cells, whereas nystatin and chlorpromazine as endocytosis inhibitors slightly reduced the uptake of GFP in the presence of [DipR] 5 after 3 h of incubation in MDA-MB-231. [WR] 9 was able to deliver therapeutically relevant proteins (Histone H2A) at different concentrations. These results provide insight into the use of amphiphilic cyclic peptides in the delivery of protein-related therapeutics.

RNA interference (RNAi) has drawn enormous attention as a powerful tool because of its capability to interfere with mRNA and protein production. However, designing a safe and efficient delivery system in RNAi therapeutics remains challenging. Herein, we have designed and synthesized several linear peptides containing tryptophan (W) and arginine (R) residues separated by the β-alanine (βA) spacer and attached to a lipophilic fatty acyl chain, cholesterol, or PEG. The peptide backbone sequences were: Ac-C-βA-βA-W 4 -βA-βA-R 4 -CO-NH 2 and Ac-K-βA-βA-W 4 -βA-βA-R 4 -CO-NH 2 , with only a difference in N -terminal amino acid. The cysteine side chain in the first sequence was used for the conjugation with PEG2000 and PEG550. Alternatively, the side chain of lysine in the second sequence was used for conjugation with cholesterol or oleic acid. We hypothesized that amphiphilic peptides and optimum fatty acyl chain or PEG could function as an effective siRNA carrier by complementing each structural component's self-assembly and membrane internalization properties. None of the designed peptides showed cytotoxicity up to 10 µM. Serum stability studies suggested that the newly designed peptides efficiently protected siRNA against early degradation by nucleases. Flow cytometry analysis indicated 50-90% cellular uptake of siRNA using the newly developed modified linear peptides (MLPs). Western blot results revealed more than 90% protein downregulation after targeting STAT3 in MDA-MB-231 and SKOV-3 cell lines. In summary, a new peptide class was developed to safely and efficiently deliver siRNA.

A series of small (7-12 mer) amphipathic cationic peptides were designed and synthesized to create short helical peptides with broad-range bactericidal activity and selectivity toward the bacterial cells. The analysis identified a lead 12-mer peptide 8b with broad-spectrum activity against Gram-positive (MIC = 3.1-6.2 μg/mL) and Gram-negative (MIC = 6.2-12.5 μg/mL) bacteria and selectivity toward prokaryotic versus eukaryotic cells (HC 50 = 280 μg/mL, >75% cell viability at 150 μg/mL). The rapid membranolytic action of 8b was demonstrated by a calcein dye leakage assay and confirmed using scanning electron microscopy. According to circular dichroism and NMR spectroscopy, the peptides have an irregular spatial structure in water. A lipid bilayer induced an amphipathic helix only in 12-mer peptides, including 8b . Molecular dynamics simulations provided detailed information about the interaction of 8b and its closest analogues with bacterial and mammalian membranes and revealed the roles of particular amino acids in the activity and selectivity of peptides.

Cell-impermeable and negatively charged compounds' cellular uptake across the cell membranes remains challenging. Herein, the synthesis of four linear [(WWRR) 2 , (WWRR) 3 , (WWRR) 4 , and (WWRR) 5 ] and four cyclic ([WWRR] 2 , [WWRR] 3 , [WWRR] 4 , and [WWRR] 5 ) peptides containing alternate two tryptophan (WW) and two arginine (RR) residues and their biological evaluation as molecular transporters are reported. The peptides did not show any significant cytotoxicity in different cell lines (MDA-MB-23, SK-OV-3, and HEK 293) at a concentration of 5 μM and after 3 h of incubation time. The uptake of fluorescence-labeled cargo molecules (F'-GpYEEI, F'-siRNA, and F'-3TC) in the presence of the peptides was monitored in different cell lines (SK-OV-3 and MDA-MB-231) with fluorescence-activated cell sorting. Among all the peptides, [WWRR] 5 ( C4 ) showed the highest cellular uptake of cargo molecules, indicating it can act as effective molecular transporter. Confocal microscopy in MDA-MB-231 cells showed the cellular uptake of F'-GpYEEI in the presence of C4 and the intracellular localization of fluorescence-labeled C4 ( F'-C4 ) in the cytosol. The F'-C4 cellular uptake was found to be concentration- and time-dependent, as shown by flow cytometry in MDA-MB-231 cells. Confocal microscopy and flow cytometry of F'-C4 in MDA-MB-231 cells were examined alone and in the presence of different endocytosis inhibitors (chlorpromazine, methyl-β-cyclodextrin, chloroquine, and nystatin). The data showed that the cellular uptake of F'-C4 in the presence of chlorpromazine, chloroquine, and methyl-β-cyclodextrin was reduced but not completely eliminated, indicating that both energy-independent and energy-dependent pathways contributed to the cellular uptake of F'-C4 . Similar results were obtained using the confocal microscopy of C4 and F'-GpYEEI in the presence of endocytosis inhibitors (chlorpromazine, methyl-β-cyclodextrin, chloroquine, and nystatin). These data indicate that C4 has the potential to be used as a cell-penetrating peptide and cargo transporter.

Introduction/objectives: Primary care practice-based research networks (PBRNs) participated in a point of care (POC) device study funded by by the National Institutes of Health and led by the University of Massachusetts Chan Medical School (UMass) to speed the development, validation, and commercialization of POC tests to detect SARS-CoV-2. The purposes of this study were to describe the characteristics of participating PBRNs and their respective collaborators in this device trial and describe complications challenging its execution., Methods: Semi-structured interviews were conducted with lead personnel from participating PBRNs and UMass., Results: Four PBRNs and UMass were invited to participate and 3 PBRNs and UMass participated. This device trial recruited 321 subjects in 6 months; 65 subjects from PBRNs. Each PBRN and the academic medical center site enrolled and recruited subjects differently. Main challenges identified were having adequate clinic personnel to enroll and aid in consent and questionnaire completion, frequently changing inclusion/exclusion criteria, use of the digital electronic data collection platform, and having access to a -80°C freezer to store supplies., Discussion: This trial involved numerous researchers, primary care clinic leaders and staff, and academic center sponsored program staff and attorneys resulting in a resource-intensive endeavor to enroll 65 subjects in the real-world clinical setting of primary care PBRNs with the academic medical center enrolling the rest. Multiple obstacles to standing up the study were encountered by the PBRNS., Conclusions: Primary care PBRNs rely largely on the goodwill established between academic health centers and participating practices. For future investigations involving device studies, collaborating PBRN leaders should assess whether recruitment criteria may change, obtain detailed lists of equipment needed, and/or know if the study is likely to be halted suddenly to appropriately prepare their member practices.

We designed a library of 24 cyclic peptides containing arginine (R) and tryptophan (W) residues in a sequential manner [R n W n ] ( n = 2-7) to study the impact of the hydrophilic/hydrophobic ratio, charge, and ring size on the antibacterial activity against Gram-positive and Gram-negative strains. Among peptides, 5a and 6a demonstrated the highest antimicrobial activity. In combination with 11 commercially available antibiotics, 5a and 6a showed remarkable synergism against a large panel of resistant pathogens. Hemolysis (HC 50 = 340 μg/mL) and cell viability against mammalian cells demonstrated the selective lethal action of 5a against bacteria over mammalian cells. Calcein dye leakage and scanning electron microscopy studies revealed the membranolytic effect of 5a . Moreover, the stability in human plasma ( t 1/2 = 3 h) and the negligible ability of pathogens to develop resistance further reflect the potential of 5a for further development as a peptide-based antibiotic.

Small-conductance Ca 2+ -activated potassium (K Ca 2.x) channels are gated exclusively by intracellular Ca 2+ . The activation of K Ca 2.3 channels induces hyperpolarization, which augments Ca 2+ signaling in endothelial cells. Cilia are specialized Ca 2+ signaling compartments. Here, we identified compound 4 that potentiates human K Ca 2.3 channels selectively. The subtype selectivity of compound 4 for human K Ca 2.3 over rat K Ca 2.2a channels relies on an isoleucine residue in the HA/HB helices. Positive modulation of K Ca 2.3 channels by compound 4 increased flow-induced Ca 2+ signaling and cilia length, while negative modulation by AP14145 reduced flow-induced Ca 2+ signaling and cilia length. These findings were corroborated by the increased cilia length due to the expression of Ca 2+ -hypersensitive K Ca 2.3&#95;G351D mutant channels and the reduced cilia length resulting from the expression of Ca 2+ -hyposensitive K Ca 2.3&#95;I438N channels. Collectively, we were able to associate functions of K Ca 2.3 channels and cilia, two crucial components in the flow-induced Ca 2+ signaling of endothelial cells, with potential implications in vasodilation and ciliopathic hypertension.

The activity of nucleoside and nucleotide analogs as antiviral agents requires phosphorylation by endogenous enzymes. Phosphate-substituted analogs have low bioavailability due to the presence of ionizable negatively-charged groups. To circumvent these limitations, several prodrug approaches have been proposed. Herein, we hypothesized that the conjugation or combination of the lipophilic amide bond with nucleotide-based tenofovir (TFV) ( 1 ) could improve the anti-HIV activity. During the current study, the hydroxyl group of phosphonates in TFV was conjugated with the amino group of L-alanine, L-leucine, L-valine, and glycine amino acids and other long fatty ester hydrocarbon chains to synthesize 43 derivatives. Several classes of derivatives were synthesized. The synthesized compounds were characterized by 1H NMR, IR, UV, and mass spectrometry. In addition, several of the synthesized compounds were evaluated as racemic mixtures for anti-HIV activity in vitro in a single round infection assay using TZM-bl cells at 100 ng/mL. TFV ( 1 ) was used as a positive control and inhibited HIV infection by 35%. Among all the evaluated compounds, the disubstituted heptanolyl ester alanine phosphonamidate with naphthol oleate ( 69 ), pentanolyl ester alanine phosphonamidate with phenol oleate ( 62 ), and butanolyl ester alanine phosphonamidate with naphthol oleate ( 87 ) ester conjugates of TFV were more potent than parent drug TFV with 79.0%, 76.5%, 71.5% inhibition, respectively, at 100 ng/mL. Furthermore, two fatty acyl amide conjugates of tenofovir alafenamide (TAF) were synthesized and evaluated for comparative studies with TAF and TFV conjugates. Tetradecanoyl TAF conjugate 95 inhibited HIV infection by 99.6% at 100 ng/mL and showed comparable activity to TAF (97-99% inhibition) at 10-100 ng/mL but was more potent than TAF when compared at molar concentration.

A number of 5′-O-fatty acyl derivatives of 3′-fluoro-2′,3′-dideoxythymidine (FLT, 1) were synthesized. These conjugates were evaluated for their potential as topical microbicides with anti-HIV activity against cell-free (X4 and R5), cell-associated, and multidrug-resistant viruses. Compared to FLT and 3′-azido-2′,3′-dideoxythymidine (AZT), 5′-O-(12-azidododecanoyl) (5), 5′-O-myristoyl (6), and 5′-O-(12-thioethyldodecanoyl) (8) derivatives of FLT were found to be more active against both cell-free viruses (lymphocytotropic and monocytotropic strains) with EC50 values of 0.4 μM, 1.1 μM, and <0.2 μM, respectively, as well as cell-associated virus with EC50 values of 12.6, 6.4, and 2.3 μM, respectively. Conjugates 5, 6, and 8 exhibited >4 and >30 times better antiviral index than FLT and AZT, respectively. Conjugates 5 and 8 were significantly more potent than FLT against many multidrug-resistant strains. A comparison of the anti-HIV activity with the corresponding non-hydrolyzable ether conjugates suggested that ester hydrolysis to FLT and fatty acids is critical to enable anti-HIV activity. Cellular uptake studies were conducted using fluorescent derivatives of FLT attached with 5(6)-carboxyfluorescein through either β-alanine (23) or 12-aminododecanoic acid (24) spacers. The lipophilic fluorescent analog with a long chain (24) showed more than 12 times higher cellular uptake profile than the fluorescent analog with a short chain (23). These studies further confirmed that the attachment of fatty acids improved the cellular uptake of nucleoside conjugates. In addition, 5, 6, and 8 were the least cytotoxic and did not alter vaginal cell and sperm viability compared to the positive control, a commercial topical spermicide (N-9), which significantly decreased sperm and vaginal cell viability inducing the generation of proinflammatory cytokines.

Linear and cyclic amphiphilic peptides, (W 4 KR 5 ) and [W 4 KR 5 ], were evaluated as antibacterial agents against Gram-positive and Gram-negative bacteria, including four multi-drug resistant strains and the corresponding four non-resistant strains. Cyclic peptide [W 4 KR 5 ] showed higher antibacterial activity than the linear (W 4 KR 5 ) counterpart. Cyclic [W 4 KR 5 ] was subjected to combination (physical mixture or covalent conjugation) with meropenem as a model antibiotic to study the impact of the combination on antimicrobial activity. A physical mixture of meropenem and [W 4 KR 5 ] showed synergistic antibacterial activity against Gram-negative P. aeruginosa (ATCC BAA-1744) and P. aeruginosa (ATCC 27883) strains. [W 4 KR 5 ] was further subjected to extensive antibacterial studies against additional 10 bacteria strains, showing significant antibacterial efficacy against Gram-positive bacteria strains. Combinations studies of [W 4 KR 5 ] with an additional 9 commercially available antibiotics showed significant enhancement in antibacterial activity for all tested combinations, especially with tetracycline, tobramycin, levofloxacin, clindamycin, daptomycin, polymyxin, kanamycin, and vancomycin. Time-kill kinetics assay and flow cytometry results exhibited that [W 4 KR 5 ] had a time-dependent synergistic effect and membrane disruption property. These data indicate that [W 4 KR 5 ] improves the antibacterial activity, presumably by facilitating the internalization of antibiotics and their interaction with the intracellular targets. This study introduces a potential strategy for treating multidrug-resistant pathogens by combining [W 4 KR 5 ] and a variety of classical antibiotics to improve the antibacterial effectiveness., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rakesh K. Tiwari, Keykavous Parang reports equipment, drugs, or supplies was provided by AJK Biopharmaceutical. Eman H. M. Mohammed, Sandeep Lohan, Rakesh K. Tiwari, Keykavous Parang has patent Synthetic antimicrobial peptides pending to AJK Biopharmaceutical., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)