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The skin has an important role in regulating homeostasis and protecting the body from endogenous and exogenous microenvironments. Although 3D models for drug discovery have been extensively studied, there is a growing demand for more advanced 3D skin models to enhance skin research. The use of these advanced skin models holds promise across domains such as cosmetics, skin disease treatments, and toxicity testing of new therapeutics. Recent advances include the development of skin-on-a-chip, spheroids, reconstructed skin, organoids, and computational approaches, including quantitative structure-activity relationship (QSAR) and quantitative structure-property relationship (QSPR) research. These innovations are bridging the gap between traditional 2D and advanced 3D models, moving progress from research to clinical applications. In this review, we highlight in vitro and computational skin models with advanced drug discovery for skin-related applications., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Palliative care (PC) is a health care specialty that is focused on the holistic care of individuals with serious illness. It requires interprofessional collaboration and expertise to meet the physical, psychosocial, social, cultural, and spiritual needs of patients experiencing serious illness and their families. The interprofessional team (IPT) is most often composed of Advanced Practice Providers (APPs) [including Nurse Practitioners (NPs), Clinical Nurse Specialists (CNSs), and Physician Associates/Assistants (PAs)], Chaplains, Registered Nurses (RNs), Doctors of Medicine and Doctors of Osteopathic Medicine (MDs/DOs), Registered Pharmacists (RPhs), Social Workers (SWs) and other professions depending on site of care, the age of the patient, and the illness. The United States has specialty palliative care (SPC) IPT members who have completed advanced education and training and obtained specialty certification. However, there is currently no interprofessional consensus education and training resulting in interprofessional variability of definitions of education and requirements for academic preparation into the specialty. This article offers the results of an online review and survey of the current availability of SPC education in the United States which includes certificate programs, residencies, fellowships, and immersion programs available to each profession. The purpose of this review is to unify the available information regarding SPC programs, providing a succinct, yet thorough, overview of the SPC educational landscape. It emphasizes the length of time, cost, and delivery method for IPT members in choosing programs.

Vaccine development against group A Streptococcus (GAS) has gained traction in the last decade, fuelled by recognition of the significant worldwide burden of the disease. Several vaccine candidates are currently being evaluated in preclinical and early clinical studies. Here, we investigate two conjugate vaccine candidates that have shown promise in mouse models of infection. Two antigens, the J8 peptide from the conserved C-terminal end of the M protein, and the group A carbohydrate lacking N -acetylglucosamine side chain (ΔGAC) were each conjugated to arginine deiminase (ADI), an anchorless surface protein from GAS. Both conjugate vaccine candidates combined with alum adjuvant were tested in a non-human primate (NHP) model of pharyngeal infection. High antibody titres were detected against J8 and ADI antigens, while high background antibody titres in NHP sera hindered accurate quantification of ΔGAC-specific antibodies. The severity of pharyngitis and tonsillitis signs, as well as the level of GAS colonisation, showed no significant differences in NHPs immunised with either conjugate vaccine candidate compared to NHPs in the negative control group.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system has been a major technological breakthrough that has brought revolutionary changes to genome editing for therapeutic and diagnostic purposes and precision medicine. With the advent of the CRISPR/Cas9 system, one of the critical limiting factors has been the safe and efficient delivery of this system to cells or tissues of interest. Several approaches have been investigated to find delivery systems that can attain tissue-targeted delivery, lowering the chances of off-target editing. While viral vectors have shown promise for in vitro, in vivo and ex vivo delivery of CRISPR/Cas9, their further clinical applications have been restricted due to shortcomings including limited cargo packaging capacity, difficulties with large-scale production, immunogenicity and insertional mutagenesis. Rapid progress in nonviral delivery vectors, including the use of lipid, polymer, peptides, and inorganic nanoparticle-based delivery systems, has established nonviral delivery approaches as a viable alternative to viral vectors. This review will introduce the molecular mechanisms of the CRISPR/Cas9 gene editing system, current strategies for delivering CRISPR/Cas9-based tools, an overview of strategies for overcoming off-target genome editing, and approaches for improving genome targeting and tissue targeting. We will also highlight current developments and recent clinical trials for the delivery of CRISPR/Cas9. Finally, future directions for overcoming the limitations and adaptation of this technology for clinical trials will be discussed., (© 2023. Controlled Release Society.)

The Hospice and Palliative Nursing Association established the triannual research agenda to ( a ) provide focus for researchers to conduct meaningful scientific and quality improvement initiatives and inform evidence-based practice, ( b ) guide organizational funding, and ( c ) illustrate to other stakeholders the importance of nursing research foci. HPNA Research Agendas are developed to give direction for future research to continue advancing expert care in serious illness and ensure equitable delivery of hospice and palliative care., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 by The Hospice and Palliative Nurses Association. All rights reserved.)

Sortase A (SrtA) is an enzyme which attaches proteins, including virulence factors, to bacterial cell walls. It is a potential target for developing anti-virulence agents against pathogenic and antimicrobial resistant bacteria. This study aimed to engineer 𝛽-lactoglobulin protein nanoparticles (PNPs) for encapsulating safe and inexpensive natural SrtA inhibitors (SrtAIs; trans -chalcone (TC), curcumin (CUR), quercetin (QC), and berberine (BR)) to improve their poor aqueous dispersibility, to screen for synergy with antimicrobial peptides (AMPs), and to reduce the cost, dose, and toxicity of AMPs. Minimum inhibitory concentration (MIC), checkerboard synergy, and cell viability assays were performed for SrtAI PNPs against Gram-positive (methicillin-sensitive and -resistant S. aureus ) and Gram-negative ( E. coli , P. aeruginosa ) bacteria alone and combined with leading AMPs (pexiganan, indolicidin, and a mastoparan derivative). Each SrtAI PNP inhibited Gram-positive (MIC: 62.5-125 µg/mL) and Gram-negative (MIC: 31.3-500 µg/mL) bacterial growth. TC PNPs with pexiganan demonstrated synergy against each bacteria, while BR PNPs with pexiganan or indolicidin provided synergy towards S. aureus . Each SrtAI PNP inhibited SrtA (IC 50 : 25.0-81.8 µg/mL), and did not affect HEK-293 cell viability at their MIC or optimal synergistic concentrations with AMPs. Overall, this study provides a safe nanoplatform for enhancing antimicrobial synergy to develop treatments for superbug infections.

This study aimed to develop synergistic therapies to treat superbug infections through the encapsulation of sortase A inhibitors (SrtAIs; trans -chalcone (TC), curcumin (CUR), quercetin (QC), or berberine chloride (BR)) into MCM-41 mesoporous silica nanoparticles (MSNs) or a phosphonate-modified analogue (MCM-41-PO 3 - ) to overcome their poor aqueous solubility. A resazurin-modified minimum inhibitory concentration (MIC) and checkerboard assays, to measure SrtAI synergy in combination with leading antimicrobial peptides (AMPs; pexiganan (PEX), indolicidin (INDO), and [I5, R8] mastoparan (MASTO)), were determined against methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus , Escherichia coli , and Pseudomonas aeruginosa . The results demonstrated that the MCM-41 and MCM-41-PO 3 - formulations significantly improved the aqueous solubility of each SrtAI. The MICs for SrtAI/MCM-41-PO 3 - formulations were lower compared to the SrtAI/MCM-41 formulations against tested bacterial strains, except for the cases of BR/MCM-41 and QC/MCM-41 against P. aeruginosa . Furthermore, the following combinations demonstrated synergy: PEX with TC/MCM-41 (against all strains) or TC/MCM-41-PO 3 - (against all strains except P. aeruginosa ); PEX with BR/MCM-41 or BR/MCM-41-PO 3 - (against MSSA and MRSA); INDO with QC/MCM-41 or QC/MCM-41-PO 3 - (against MRSA); and MASTO with CUR/MCM-41 (against E. coli ). These combinations also reduced each components' toxicity against human embryonic kidney cells. In conclusion, MCM-41 MSNs provide a platform to enhance SrtAI solubility and demonstrated antimicrobial synergy with AMPs and reduced toxicity, providing novel superbug treatment opportunities.

Objectives: To investigate the relationship between magnetization transfer (MT) imaging and tissue macromolecules in high-grade serous ovarian cancer (HGSOC) and whether MT ratio (MTR) changes following neoadjuvant chemotherapy (NACT)., Methods: This was a prospective observational study. 12 HGSOC patients were imaged before treatment. MTR was compared to quantified tissue histology and immunohistochemistry. For a subset of patients ( n = 5), MT imaging was repeated after NACT. The Shapiro-Wilk test was used to assess for normality of data and Spearman's rank-order or Pearson's correlation tests were then used to compare MTR with tissue quantifications. The Wilcoxon signed-rank test was used to assess for changes in MTR after treatment., Results: Treatment-naïve tumour MTR was 21.9 ± 3.1% (mean ± S.D.). MTR had a positive correlation with cellularity, rho = 0.56 ( p < 0.05) and a negative correlation with tumour volume, ρ = -0.72 ( p = 0.01). MTR did not correlate with the extracellular proteins, collagen IV or laminin ( p = 0.40 and p = 0.90). For those patients imaged before and after NACT, an increase in MTR was observed in each case with mean MTR 20.6 ± 3.1% (median 21.1) pre-treatment and 25.6 ± 3.4% (median 26.5) post-treatment ( p = 0.06)., Conclusion: In treatment-naïve HGSOC, MTR is associated with cellularity, possibly reflecting intracellular macromolecular concentration. MT may also detect the HGSOC response to NACT, however larger studies are required to validate this finding., Advances in Knowledge: MTR in HGSOC is influenced by cellularity. This may be applied to assess for cell changes following treatment., Competing Interests: Competing interests: The authors have no conflicts of interest to declare., (© 2022 The Authors. Published by the British Institute of Radiology.)

Abstract: Africa continues to be disproportionately affected by HIV/AIDS. Poor access to medical care, limited access to antiretroviral drugs, and lack of supportive care for persons who are symptomatic lead to disease progression. Community-based palliative care services are effective in providing supportive services throughout the course of the illness, but palliative care services are limited in Africa. This article presents a theoretical approach to developing community-based palliative care services to bridge this gap., (Copyright © 2022 InterVarsity Christian Fellowship.)

Increasing antimicrobial resistance is a major global health concern. Conventional antibiotics apply selection pressures, which promote the accumulation of resistant microbes. Anti-virulence strategies, in contrast, are less potent antimicrobials, but are less likely to select for resistance, can be combined with existing antibiotics to improve their activity, and in some cases can overcome antimicrobial resistance towards other antimicrobials. Sortase A inhibitors (SrtAIs) represent an exciting example of this class; however, many reported examples demonstrate poor water solubility, which complicates their biological assessment and activity. This includes reports that use antimicrobial concentrations of organic solvents or conditions that fail to solubilise these compounds for minimal inhibitory concentration (MIC) assessments. Herein, we report the first study to optimise screening processes for a library of prospective SrtAIs (trans-chalcone (TC), berberine (BR), curcumin (CUR), and quercetin (QC)), including comparative assessment of the effects of various co-solvent concentrations, along with comparative assessment of their antimicrobial activities against multiple disease relevant bacterial strains (methicillin-sensitive and resistant S. aureus, E. coli, and P. aeruginosa), inhibition of the sortase A enzyme, and toxicity towards mammalian cells (HEK-293), using these optimised conditions. Optimal solubility with minimal effect on bacterial viability was observed in the presence of 5% (v/v) dimethyl sulfoxide (DMSO)-Mueller-Hinton Broth. Three antimicrobial susceptibility tests (broth microdilution, agar dilution, and disk diffusion) were assessed for their ability to accurately determine minimal inhibitory concentration (MIC) data for each SrtAI. Broth microdilution and agar dilution were both effective; however, the broth microdilution assay required the addition of a colorimetric metabolic indicator (resazurin) to enable simple and reliable MIC determination due to the development of precipitants over time. In contrast, disk diffusion did not provide reliable zone of inhibition data. Identical MIC data was observed with methicillin-sensitive and -resistant S. aureus (MRSA; ATCC43300), with lower potency activity against E. coli and P. aeruginosa. Under these conditions, TC and CUR demonstrated significant toxicity towards human embryonic kidney (HEK-293) cells, with QC showing less toxicity and BR limited-to-no toxicity at its MIC. Overall, the findings of this work provide optimised processes, which will prove useful for the study of other poorly soluble antimicrobial agents and SrtAIs. The obtained data suggests that BR should be considered in preference to the other SrtAIs for the development of new antimicrobial formulations, based on its superior antimicrobial and SrtA inhibition potency, and greatly reduced toxicity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Virulence factor, sortase A (SrtA), has crucial roles in the pathogenesis of Gram-positive superbugs. SrtA is a bacterial cell membrane enzyme that anchors crucial virulence factors to the cell wall surface of Gram-positive bacteria. SrtA is not necessary for bacterial growth and viability and is conveniently accessible in the cell membrane; therefore, it is an ideal target for antivirulence drug development. In this review, we focus on antimicrobial resistance (AMR)-expressing bacteria and SrtA as a potential target for overcoming AMR. The mechanism of action of SrtA and its inhibition by various types of inhibitors, such as synthetic small molecules, peptides, and natural products, are provided. Future SrtA research perspectives for alternative drug development to antibiotics are also proposed., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Neutralising antibodies (NAbs), caused by past adeno-associated virus (AAV) infection, represent a critical challenge for AAV-mediated gene therapy, with even low NAb titres capable of inhibiting gene transfer, however in protein-rich environments such as the vitreous it is expected that other constituents could also interact with the transduction process. Inhibition of AAV2/2, AAV2/5, AAV2/6 and AAV2/8 transduction by human vitreous humour (VH) obtained from 80 post-mortem eye cups was investigated in this report, with clinically relevant vitreous dilutions as low as 1:2. Unexpectedly, the highest prevalence of inhibition of transduction was observed against AAV2/6, with 66% of tested samples displaying neutralisation at a 1:2 VH dilution. Only two samples showed inhibition of AAV2/8, indicating this serotype is an attractive vector for use in non-vitrectomised eyes of unscreened individuals. Levels of anti-AAV NAbs observed in the VH were much lower than previously observed in serum of a similar Australian population. Among ten tested eye cup pairs, we observed only small variation in anti-AAV NAbs levels between the left and right eye cups. Interaction with 1:2 diluted VH had an augmentation effect on AAV2/8 transduction (p = 0.004), a phenomenon which was not due to albumin or transferrin and which, if developed, might benefit the use of AAV2/8 in clinical settings.

Glucagon-like peptide-1 GLP-1 is a gut-derived peptide secreted from pancreatic β-cells that reduces blood glucose levels and body weight; however, native GLP-1 (GLP-1(7-36)-NH 2 and GLP-1(7-37)) have short in vivo circulation half-lives (∼2 min) due to proteolytic degradation and rapid renal clearance due to its low molecular weight (MW; 3297.7 Da). This study aimed to improve the proteolytic stability and delivery properties of glucagon-like peptide-1 (GLP-1) through modifications that form nanostructures. For this purpose, N- ( NtG ) and C-terminal ( CtG ), and Lys26 side chain ( K26G ) alkyne-modified GLP-1 analogues were conjugated to an azide-modified lipidic peptide ( L ) to give N-L , C-L , and K-26-L , respectively; or CtG was conjugated with a fibrilizing self-assembling peptide (SAP) (AEAEAKAK) 3 to yield C-S , using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). N-L demonstrated the best serum stability ( t 1/2 > 48 h) compared to K-26-L (44 h), C-L (20 h), C-S (27 h), and the parental GLP-1(7-36;A8G)-NH 2 ( A8G ) (19 h) peptides. Each conjugate demonstrated subnanomolar hGLP-1RA potency, and none demonstrated toxicity toward PC-3 cells at concentrations up to 1 μM. Each analogue was observed by transmission electron microscopy to form fibrils in solution. K-26-L demonstrated among the best human serum stability ( t 48 pM) to 1/2 = 44 h) and similar hGLP-1RA potency (EC 50 48 pM) to C-S . In conclusion, this study provided an alternative to lipid modification, i.e., fibrillizing peptides, that could improve pharmacokinetic parameters of GLP-1.

This study aimed to develop and optimize chemistries to produce alkyne-modified glucagon-like peptide-1(7-36)-amide (GLP-1(7-36)-NH 2 ) libraries, which could be rapidly and efficiently conjugated to other components and screened to identify compounds with the best drug delivery properties, as potential treatments for type 2 diabetes or obesity. For this purpose, the Lys26 (K26) side-chain, and the amino (N)- and carboxy (C)-termini of a dipeptidyl peptidase 4 (DPPIV)-resistant GLP-1 sequence (GLP-1(7-36;A8G)-NH 2 ), were modified with an alkyne (4-pentynoic acid or propiolic acid). These analogs were characterized with respect to human GLP-1 receptor (hGLP-1R) agonist activity, effects on cell viability and human serum stability, revealing that these modifications maintained low (N-terminal; EC 50 1.5 × 10 -9 M) to subnanomolar (C-terminal and K26, ∼4 × 10 -10 M) agonist activity toward hGLP-1, had no effect on cell viability, and for the N-terminal and K26 modifications, increased human serum proteolytic stability ( t 1/2 > 24 h). Copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction conditions were investigated using the C-terminal modified GLP-1 analog and an azide-modified model lipid peptide, with respect to the effects of altering the azide/alkyne ratio, cosolvents, temperature, reducing agents, Cu(I)-stabilizing ligand, copper source, and the concentrations of reagents/reactants, in order to identify general conditions that provide fast reactions and high yields. A 1:2 azide-alkyne (lipid:GLP-1 peptide) and 4:1 sodium ascorbate/copper sulfate molar ratio in 65% v/v DMSO-water at room temperature, in the absence of Cu(I)-stabilizing ligands (THPTA or l-histidine) and buffers (phosphate, pH 7), provided the best yields. This work reports a library of characterized GLP-1 analogs and chemistries for their attachment to other species, providing useful tools to improve GLP-1 delivery and pharmacology (e.g., through conjugation to other species that lower blood glucose, increase the duration of action, or enable delivery via a nonparenteral route).

Subunit vaccines composed of protein antigens covalently attached to Toll-like receptor (TLR) agonists elicit superior immune responses compared to mixtures of antigens and TLR agonists. Among different conjugation approaches, enzyme-mediated ligation is one of the few that provides an opportunity for the generation of homogeneous, molecularly defined products in which protein antigens are maintained with native structures, which is most critical to elicit protective immune responses upon vaccination. Four highly conserved protein antigens from Group A Streptococcus (GAS) have the potential to be safe and efficacious vaccine candidates. After a TLR2 agonist fibroblast-stimulating lipopeptide-1 (FSL-1) was successfully attached onto each antigen using sortase A and techniques for their purification were developed, a combination vaccine containing interleukin 8 (IL-8) protease ( Streptococcus pyogenes cell envelope proteinase [SpyCEP]), Group A Streptococcal C5a peptidase (SCPA), anchorless virulence factor arginine deiminase (ADI), and trigger factor (TF)-TLR2 conjugates was produced. This combination was assessed for immunity in mice and compared with mixtures of the four antigens with FSL-1 or alum. High titer antigen-specific IgG antibodies were detected from all vaccine groups, with antibodies elicited from FSL-1 conjugates around 10-fold higher compared to the FSL-1 mixture group. Furthermore, the FSL-1 conjugates afforded a more balanced T H 1/T H 2 immune response than the alum-adjuvanted group, suggesting that this combination vaccine represents a promising candidate for the prevention of GAS diseases. Thus, we established a conjugation platform that allows for the production of defined, site-specific antigen-adjuvant conjugates, which maintain the native three-dimensional structure of antigens and can be potentially applied to a variety of protein antigens.

Aim: To develop albendazole (ABZ)-loaded bombesin(6-14) (BBN(6-14)) functionalized liposomes for targeting GRPR to enhance delivery to cancer cells. Materials & methods: ABZ-loaded liposomes were formulated using supercritical CO 2 technology; functionalized with a GRPR-targeted lipid-anchored BBN(6-14) peptide; and evaluated for effects on cell viability, particle size and targeted cell uptake. Results: BBN(6-14)-coated ABZ liposomes decreased cell viability compared with nonfunctionalized ABZ liposomes. The level of GRPR expression positively correlated with intracellular uptake and decreased cell viability. The reduced cell viability, higher cell uptake and GRPR expression were observed in the order PC-3 > Caco-2 > HepG2 cells. Conclusion: BBN(6-14)-functionalized ABZ liposomes showed enhanced reduction in cell viability compared with nonfunctionalized ABZ liposomes.

Protein antigens are, in general, weakly immunogenic, and therefore require co-delivery with adjuvants to stimulate potent immune responses. The fusion of (poly)peptide antigens to immunostimulatory adjuvants (e.g. Toll-like receptor (TLR) agonists) has been demonstrated to greatly improve vaccine potency compared to mixtures of antigen and adjuvant. Chemical approaches, to enable the rapid, site-specific and high-yielding linkage of TLR2 ligands to recombinant protein antigens, have been previously optimized. These approaches require the use of denaturing conditions to ensure high reaction yields, which limits their application, as maintenance of native protein folding is necessary to elicit antibodies against conformational epitopes. Here, this work aimed to optimize an alternative method, to ensure the efficient bioconjugation of TLR2 ligands onto folded protein antigens. An enzyme-mediated approach, using Staphylococcus aureus sortase A (or a penta mutant with enhanced efficiency), was optimized for reaction yield and time, as well as enzyme type and amount. This approach enabled the site-specific conjugation of the TLR2-agonist fibroblast-stimulating lipopeptide-1 (FSL-1) onto a model group A Streptococcus (GAS) recombinant polytope antigen under conditions that maintain protein folding, yielding a homogeneous, molecularly-defined product, with ligation yields as high as 90%. Following intramuscular (IM) administration of the ligation product to humanized plasminogen AlbPLG1 mice, high-titer, antigen-specific IgG antibodies were observed, which conferred protection against subcutaneous challenge with GAS strain 5448. In comparison, mixtures of the GAS antigen with aluminum hydroxide or FSL-1 failed to provide protection, with the FSL-1 mixture yielding ~1000-fold lower antigen-specific IgG antibody titers, and the mixture with alum yielding a Th2-biased response compared to the more balanced Th1/Th2 responses observed with the FSL-1 conjugate. Overall, a FSL-1 bioconjugation method for the efficient production of antigen-TLR2 agonist conjugates, which maintain protein folding, was produced, with broad utility for the development of self-adjuvanting vaccines against subunit protein antigens., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Self-adjuvanting vaccines, consisting of recombinant protein antigens and covalently attached Toll-like receptor (TLR) agonists, have the ability to simultaneously and efficiently deliver antigen and TLR adjuvant to antigen presenting cells (APCs). Here, an enzyme-mediated ligation approach was used to overcome difficulties in producing homogeneous, molecularly defined self-adjuvanting vaccine products under native conditions. This process was optimized to allow the incorporation of the lipopeptide TLR2 agonist fibroblast-stimulating lipopeptide (FSL)-1 onto the N- or C-termini of recombinant protein antigens, employing the enzyme Staphylococcus aureus sortase A (SrtAsa) penta mutant. In addition, because SrtAsa-mediated ligations are reversible, a tryptophan zipper derived sequence was introduced into both reactants, which was demonstrated to improve ligation efficiency through the formation of a β-hairpin structure that hinders the reverse reaction. Finally, it was demonstrated that N- or C-terminal conjugation, and the incorporation of the β-hairpin structure, did not affect the TLR2-agonist activities of protein antigen TLR agonist conjugates. Overall, this SrtAsa-mediated ligation platform enabled production of antigen TLR2 agonist conjugates with enhanced ligation efficiency, with the conjugates demonstrating potent TLR2 signaling activation (EC 50 <1nM)., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Biotic interactions through diffusible and volatile organic compounds (VOCs) are frequent in nature. Soil bacteria are well-known producers of a wide range of volatile compounds (both organic and inorganic) with various biologically relevant activities. Since the last decade, they have been identified as natural biocontrol agents. Volatiles are airborne chemicals, which when released by bacteria, can trigger plant responses such as defence and growth promotion. In this study, we tested whether diffusible and volatile organic compounds (VOCs) produced by soil bacterial isolates exert anti-oomycete and plant growth-promoting effects. We also investigated the effects of inoculation with VOC-producing bacteria on the growth and development of Capsicum annuum and Arabidopsis thaliana seedlings. Our results demonstrate that organic VOCs emitted by bacterial antagonists negatively influence mycelial growth of the soil-borne phytopathogenic oomycete Phytophthora capsici by 35% in vitro. The bacteria showed plant growth promoting effects by stimulating biomass production, primary root growth and root hair development. Additionally, we provide evidence to suggest that these activities were deployed by the emission of either diffusible organic compounds or VOCs. Bacterial VOC profiles were obtained through solid phase microextraction (SPME) and analysis by gas chromatography coupled with mass spectrometry (GC-MS). This elucidated the main volatiles emitted by the isolates, which covered a wide range of aldehydes, alcohols, esters, carboxylic acids, and ketones. Collectively, twenty-five VOCs were identified to be produced by three bacteria; some being species-specific. Our data show that bacterial volatiles inhibits P. capsici in vitro and modulate both plant growth promotion and root system development. These results confirm the significance of soil bacteria and highlights that ways of harnessing them to improve plant growth, and as a biocontrol agent for soil-borne oomycetes through their volatile emissions deserve further investigation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The development of polymer-based nanoparticulate delivery systems for siRNA is important for the clinical success of gene therapy. However, there are some major drawbacks that need to be overcome. Short interfering RNA (siRNA) has been investigated as a potential therapeutic drug to silence disease-associated genes, but its usage is limited due to the lack of effective and safe nanocarriers. In this study, DOPE-PEI, a nanoparticle consisting of the fusogenic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) conjugated with low-molecular-weight, 600 Da, branched polyethylenimine (PEI) was produced and optimized for siRNA delivery. This delivery system was modified with other components such as 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000] (DOPE-PEG2K), DOPE-PEG3.4K-bombesin and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine/1,2-dioleoyl-3-trimethylammonium-propane (DOPE/DOTAP) and tested on PC-3 cells. The conjugation of DOPE to PEI polymer (DOPE-PEI) improved the efficiency of PEI to deliver siRNA into the cytosol and knockdown genes, but demonstrated high toxicity. The addition of DOPE-PEG2K reduced cellular toxicity by masking the surface positive charge of the DOPE-PEI/siRNA complex, with the incorporation of a gastrin-releasing peptide receptor (GRPR) targeting peptide and DOPE/DOTAP components improving the cellular uptake of siRNA into targeted cells and the siRNA knockdown efficiency.

Type 2 diabetes mellitus (T2DM) is increasing in global prevalence and is associated with serious health problems (e.g., cardiovascular disease). Various treatment options are available for T2DM, including the incretin hormone glucagon-like peptide-1 (GLP-1). GLP-1 is a therapeutic peptide secreted from the intestines following food intake, which stimulates the secretion of insulin from the pancreas. The native GLP-1 has a very short plasma half-life, owning to renal clearance and degradation by the enzyme dipeptidyl peptidase-4. To overcome this issue, various GLP-1 agonists with increased resistance to proteolytic degradation and reduced renal clearance have been developed, with several currently marketed. Strategies, such as controlled release delivery systems, methods to reduce renal clearance (e.g., PEGylation and conjugation to antibodies), and methods to improve proteolytic stability (e.g., stapling, cyclization, and glycosylation) provide means to further improve the ability of GLP-1 analogs. These will be discussed in this literature review.

Liposomes are promising delivery vehicles and offer the added drawcard of being able to be made functional to target tissues such as cardiac muscle and cancerous cells. Current methods to manufacture liposomes need to be improved and supercritical fluid (SCF) technologies may offer a solution. Herein, the dispersibility of six different phospholipids (PLs) was determined in supercritical carbon dioxide (scCO 2 ). 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) showed the highest post-processing dispersibility, while 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) showed no dispersibility in scCO 2 at the assessed experimental conditions. The zetasizer results showed that the SCF conditions at 37 °C, 250 bar and 200 RPM for 60 min provided nanoparticles with the narrowest polydispersity index (PDI) and a spherical shape as shown by cryo-transmission electron microscopy (Cryo-TEM). The mean diameter of liposomes using the SCF method for DSPC-PEGylated and DOPC-PEGylated liposomes was 98.3 ± 3.3 nm and 124.5 ± 4.1 nm, while using the thin film method it was 153.6 ± 4.5 nm and 131.3 ± 3.4 nm, respectively. A size-based stability evaluation of the scCO 2 -prepared liposomes stored at different temperatures (25 °C, 4 °C and -20 °C) was compared to that of the thin film method over a period of 3 months. The current study provides a possible green alternative SCF method to preparing liposomes that is less laborious, time saving, and a low energy process., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aim: To develop a peptide/phospholipid hybrid system for gastrin-releasing peptide receptor (GRPR)-targeted delivery of pDNA or siRNA. Materials & methods: A multifunctional GRPR-targeted peptide R 9 -K(GALA)-BBN(6-14) was combined with a phospholipid oligonucleotide delivery system (1:1 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine and 1,2-dioleoyl-3-trimethylammonium-propane) and evaluated for pDNA and siRNA delivery in terms of complex size, toxicity, receptor-targeted delivery and gene expression or knockdown efficiency. Results: By combining peptide and phospholipid delivery systems, synergistic improvements in gene expression and knockdown were observed when compared with either system alone. The optimized formulation demonstrated high levels of EGFP expression and EGFP knockdown, GRPR-targeted delivery, enhanced endosomal release and minimal toxicity. Conclusion: The peptide/phospholipid hybrid system provides efficient GRPR-targeted DNA/siRNA delivery.

Group A Streptococcus (GAS) infections account for an estimated 500,000 deaths every year. This bacterial pathogen is responsible for a variety of mild and life-threatening infections and the triggering of chronic autoimmune sequelae. Pharyngitis caused by group A Streptococcus (GAS), but not asymptomatic GAS carriage, is a prerequisite for acute rheumatic fever (ARF). Repeated bouts of ARF may trigger rheumatic heart disease (RHD), a major cause of heart failure and stroke accounting for 275,000 deaths annually. A vaccine that prevents pharyngitis would markedly reduce morbidity and mortality from ARF and RHD. Nonhuman primates (NHPs) have been utilized to model GAS diseases, and experimentally infected rhesus macaques develop pharyngitis. Here we use an NHP model of GAS pharyngitis to evaluate the efficacy of an experimental vaccine, Combo5 (arginine deiminase [ADI], C5a peptidase [SCPA], streptolysin O [SLO], interleukin-8 [IL-8] protease [SpyCEP], and trigger factor [TF]), specifically designed to exclude GAS components potentially linked to autoimmune complications. Antibody responses against all Combo5 antigens were detected in NHP serum, and immunized NHPs showed a reduction in pharyngitis and tonsillitis compared to controls. Our work establishes the NHP model as a gold standard for the assessment of GAS vaccines. IMPORTANCE GAS-related diseases disproportionally affect disadvantaged populations (e.g., indigenous populations), and development of a vaccine has been neglected. A recent strong advocacy campaign driven by the World Health Organization and the International Vaccine Institute has highlighted the urgent need for a GAS vaccine. One significant obstacle in GAS vaccine development is the lack of a widely used animal model to assess vaccine efficacy. Researchers in the field use a wide range of murine models of infection and in vitro assays, sometimes yielding conflicting results. Here we present the nonhuman primate pharyngeal infection model as a tool to assess vaccine-induced protection against colonization and clinical symptoms of pharyngitis and tonsillitis. We have tested the efficacy of an experimental vaccine candidate with promising results. We believe that the utilization of this valuable tool by the GAS vaccine research community could significantly accelerate the realization of a safe and effective GAS vaccine for humans., (Copyright © 2019 Rivera-Hernandez et al.)

Gene therapy has the potential to treat both acquired and inherited genetic diseases. Generally, two types of gene delivery vectors are used - viral vectors and non-viral vectors. Non-viral gene delivery systems have attracted significant interest (e.g. 115 gene therapies approved for clinical trials in 2018; clinicaltrials.gov) due to their lower toxicity, lack of immunogenicity and ease of production compared to viral vectors. To achieve the goal of maximal therapeutic efficacy with minimal adverse effects, the cell-specific targeting of non-viral gene delivery systems has attracted research interest. Targeting through cell surface receptors; the enhanced permeability and retention effect, or pH differences are potential means to target genes to specific organs, tissues, or cells. As for targeting moieties, receptorspecific ligand peptides, antibodies, aptamers and affibodies have been incorporated into synthetic nonviral gene delivery vectors to fulfill the requirement of active targeting. This review provides an overview of different potential targets and targeting moieties to target specific gene delivery systems., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Glucagon-like peptide-1 (GLP-1) is secreted by intestinal L-cells following food intake, and plays an important role in glucose homeostasis due to its stimulation of glucose-dependent insulin secretion. Further, GLP-1 is also associated with protective effects on pancreatic β-cells and the cardiovascular system, decreased appetite, and weight loss, making GLP-1 derivatives an exciting treatment for type 2 diabetes and obesity. Despite these benefits, wild-type GLP-1 exhibits a short circulation time due to its poor metabolic stability and rapid renal clearance, and must be administered by injection, making it a poor therapeutic agent. Many strategies have been used to improve the circulation time of GLP-1 (e.g., mutations, unnatural amino acids, depot formulations, use of exendin-4 sequences, and fusions with high-molecular-weight proteins or polymers), with its therapeutic utility further improved by adding agonist activity for gastric inhibitory peptide and glucagon receptors. This minireview focuses on strategies that have been used to improve the pharmacokinetics of GLP-1 and provides an overview of GLP-1-based therapeutics in the pipeline., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Traditional vaccines derived from attenuated or inactivated pathogens are effective at inducing antibody-based protective immune responses but tend to be highly reactogenic, causing notable adverse effects. Vaccines with superior safety profiles can be produced by subunit approaches, utilizing molecularly defined antigens (e.g., proteins and polysaccharides). These antigens, however, often elicit poor immunological responses, necessitating the use of adjuvants. Immunostimulatory adjuvants have the capacity to activate antigen presenting cells directly through specific receptors (e.g., Toll-like receptors (TLRs)), resulting in enhanced presentation of antigens as well as the secretion of proinflammatory chemokines and cytokines. Consequently, innate immune responses are amplified and adaptive immunity is generated. Recently, site-specific conjugation of such immunostimulatory adjuvants (e.g., TLR ligands) onto defined antigens has shown superior efficacy over unconjugated mixtures, suggesting that the development of chemically characterized immunostimulatory adjuvants and optimized approaches for their conjugation with antigens may provide a better opportunity for the development of potent, novel vaccines. This review briefly summarizes various TLR agonists utilized as immunostimulatory adjuvants and focuses on the development of techniques (e.g., recombinant, synthetic, and semisynthetic) for generating adjuvant-antigen fusion vaccines incorporating peptide or protein antigens.

The development of non-viral gene delivery systems, with the capacity to overcome most of the biological barriers facing gene delivery, is challenging. We have developed peptide-based, multicomponent, non-viral delivery systems, incorporating: a bombesin peptide ligand (BBN(6-14)), to selectively target the gastrin releasing peptide receptor (GRPR); oligoarginine peptides (hexa- (R 6 ) and nona-arginine (R 9 )), for plasmid DNA (pDNA) condensation; and GALA, to facilitate endosome escape. The uptake and endosome escape efficiency of bombesin/oligoarginine and bombesin/oligoarginine/GALA fusion peptides for oligonucleotide delivery was evaluated in terms of their complex size, cellular uptake, endosome escape, and cellular toxicity. Complex size and cell uptake studies demonstrated that the nona-arginine/bombesin delivery system was more efficient at condensing and delivering pDNA into PC-3 prostate cancer cells compared to the hexa-arginine/bombesin delivery system. Further, competition with free bombesin peptide, and comparative uptake studies in Caco-2 cells, which express GRPR at a lower level, suggested that GRPR contributes to the targeted uptake of this system. The addition of GALA into the nona-arginine/bombesin-based system further increased the pDNA cellular uptake at all tested N/P ratios; facilitated endosomal pDNA release; and had limited effects on cell viability. In conclusion, the delivery system combining BBN(6-14) with nona-arginine and GALA had optimal characteristics for the delivery of pDNA into the GRPR overexpressing cell line PC-3., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Supercritical fluid (SCF) technology offers a potential green alternative to organic solvent-based methods for drug formulation. Albendazole (ABZ) has promising anticancer activity when formulated to increase its cellular uptake. Herein, a static volume method was used to determine the solubility of ABZ in supercritical carbon dioxide (scCO 2 ) for the future development of such ABZ formulations. The solubility of ABZ in scCO 2 (250 bar, 37 °C) was approximately 12 mg/100 mL. The extent of dissolution was measured at various time points to determine when saturation solubility occurred, which was demonstrated from 9 h. In order to determine if scCO 2 processing induced ABZ polymorphism, DSC/TGA, FTIR and XRD were used, which demonstrated no change in its solid state. Following this, ABZ loaded liposomes were manufactured using SCF technology. The liposomes diameter was 167.2 ± 5.3 nm as determined by Zetasizer, and confirmed by cryo-transmission electron microscopy. In conclusion, scCO 2 was used successfully to solubilize ABZ, and to manufacture liposomes of nano-sized range. This study provides insight into use of green technology for future ABZ liposomal formulation without the need for organic solvents.

The development of carriers for the delivery of oligonucleotide therapeutics is essential for the successful translation of gene therapies to the clinic. In the present study, a delivery system, which combines the fusogenic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) with a well-defined synthetic multifunctional peptide, was produced and optimized for gene delivery, with the aim to develop an efficient gene delivery platform for breast cancer cells. For this purpose, a breast cancer-specific cell targeting peptide (CTP) was incorporated into our leading peptide-based gene delivery system (to generate DEN-K(GALA)-TAT-K(STR)-CTP) to improve its cell-specific internalization, and investigated in combination with a formulation approach (DOPE/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)). DEN-K(GALA)-TAT-K(STR)-CTP alone efficiently complexed with DNA or siRNA, and promoted efficient cellular uptake, but low levels of gene expression. By adding the formulation approach, synergistic improvements in gene expression and silencing were observed compared to the peptide or formulation approaches alone. Of significance, this current system demonstrated more efficient gene knockdown when compared to the leading commercial siRNA delivery agent Lipofectamine® RNAiMAX. The utility of this system was demonstrated through the delivery of BCL2 (B-cell lymphoma 2) siRNA to MCF-7 cells, which led to near complete knockdown of the Bcl-2 protein, and inhibition of MCF-7 cell migration in a wound healing assay. The present peptide/lipid hybrid system is an excellent candidate for the delivery of DNA or siRNA into breast cancer cells., Statement of Significance: The identification of safe and effective delivery systems for DNA and siRNA is of great importance. Herein, we developed a well-defined, multifunctional and cell-specific lipidic peptide DEN-K(GALA)-TAT-K(STR)-CTP as a breast cancer cell targeted gene delivery vector. When combined with a lipid component (DOTAP/DOPE), the peptide/lipid hybrid system demonstrated higher gene expression or knockdown levels compared to the peptide or lipid approach alone when used to deliver pDNA or siRNA respectively, indicating synergistic enhancement of gene delivery efficiency. Importantly, this delivery strategy achieved greater knockdown of the Bcl-2 protein when compared to the leading commercial siRNA delivery system Lipofectamine® RNAiMAX, suggesting its potential utility for the targeted treatment of Bcl-2 overexpressing breast cancers., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Traditional vaccination approaches (e.g. live attenuated or killed microorganisms) are among the most effective means to prevent the spread of infectious diseases. These approaches, nevertheless, have failed to yield successful vaccines against many important pathogens. To overcome this problem, methods have been developed to identify microbial components, against which protective immune responses can be elicited. Subunit antigens identified by these approaches enable the production of defined vaccines, with improved safety profiles. However, they are generally poorly immunogenic, necessitating their administration with potent immunostimulatory adjuvants. Since few safe and effective adjuvants are currently used in vaccines approved for human use, with those available displaying poor potency, or an inability to stimulate the types of immune responses required for vaccines against specific diseases (e.g. cytotoxic lymphocytes (CTLs) to treat cancers), the development of new vaccines will be aided by the availability of characterized platforms of new adjuvants, improving our capacity to rationally select adjuvants for different applications. One such approach, involves the addition of microbial components (pathogen-associated molecular patterns; PAMPs), that can stimulate strong immune responses, into subunit vaccine formulations. The conjugation of PAMPs to subunit antigens provides a means to greatly increase vaccine potency, by targeting immunostimulation and antigen to the same antigen presenting cell. Thus, methods that enable the efficient, and inexpensive production of antigen-adjuvant fusions represent an exciting mean to improve immunity towards subunit antigens. Herein we review four protein-based adjuvants (flagellin, bacterial lipoproteins, the extra domain A of fibronectin (EDA), and heat shock proteins (Hsps)), which can be genetically fused to antigens to enable recombinant production of antigen-adjuvant fusion proteins, with a focus on their mechanisms of action, structural or sequence requirements for activity, sequence modifications to enhance their activity or simplify production, adverse effects, and examples of vaccines in preclinical or human clinical trials., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aim: To develop a new synthetic peptide-based nanoparticulate siRNA delivery system., Materials & Methods: DEN-K(GALA)-TAT-K(STR) was generated by incorporating stearic acid into a multicomponent peptide (DEN-K(GALA)-TAT), containing a cationic poly-L-lysine dendron, an endosome-disrupting peptide GALA and a cell-penetrating peptide TAT(48-60). Its physicochemical characteristics, size, toxicity, cellular uptake and gene knockdown activity of the peptide/siRNA complexes were studied., Results: DEN-K(GALA)-TAT-K(STR) exhibited a pH-responsive behavior, which assists with endosomal escape. When siRNA was delivered by DEN-K(GALA)-TAT-K(STR), it showed a significantly enhanced cellular uptake, compared with the nonlipidic peptide. This system also displayed enhanced knockdown efficiency and reduced cytotoxicity over the widely used delivery system branched 25-kDa polyethyleneimine., Conclusion: Our stearylated multicomponent delivery system has great potential as an efficient siRNA delivery vector.

Background: Healthcare services can be difficult to access, particularly for low-income or underinsured women. One way of overcoming the barriers to quality, patient-centered care is through the use of the Medical Home Model (MHM). The MHM is a cost-effective approach to care that improves patient outcomes and improves access., Aim: The purpose of this article is to discuss barriers to healthcare, with an emphasis on reducing healthcare disparities for women., Method: Extant, contemporary literature has been reviewed and synthesized to develop this theoretical paper on the benefits of using the MHM to reduce disparities in the provision of healthcare for women., Conclusions: The MHM provides an example of how healthcare can be provided in a more coordinated and effective manner. Extension of this model into the area of women's health may be one way to reduce barriers to quality, accessible care for women.

Gastrin releasing peptide (GRP) receptor (GRPR), a bombesin family receptor, is overexpressed in many cancers including breast, prostate, pancreatic and lung. The targeting of therapeutics to GRPR can be achieved using the full-length (14 amino acid) GRP analogue Bombesin (BBN) or the truncated BBN(6-14) sequence, both of which bind GRPR with high affinity and specificity. In this study, we have investigated the level of GRPR expression in various cancerous (Caco-2, HeLa, LNCap, MDA-MB-231, and PC-3) and non-cancerous (WPMY-1) cell lines using a western blotting approach. Such information is currently lacking in the literature, and is therefore of importance for the in vitro assessment of GRPR targeted therapeutics. Of the cell lines assessed, the PC-3 (prostate cancer) and Caco-2 (colon cancer) cell lines demonstrated the highest and lowest levels of GRPR expression respectively. Using this information, we further investigated the cellular uptake of carboxyfluorescein-labelled BBN and BBN(6-14) peptides by flow cytometry and confocal microscopy using cell lines that express GRPR (Caco-2, HeLa, PC-3). The uptake of each of these peptides was similar, suggesting that the shorter BBN(6-14) peptide is sufficient for GRPR targeting. Further, the uptake of these peptides could be inhibited by competition with unlabelled BBN peptides, suggesting their cellular uptake is GRPR-mediated, while the level of BBN uptake (as measured by flow cytometry) was found to be directly proportional to the level of GRPR expression. Overall, the information obtained from these studies provides useful information for the in vitro assessment of GRPR targeted therapeutics., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The purpose of this focus group study was to explore graduate students' clinical experiences with vulnerable populations, perceived barriers to care, and ethical issues related to caring for disenfranchised groups. Furthermore, based on their experiences, the students were asked to share suggestions for curricular changes that could enhance care for vulnerable populations through interdisciplinary collaboration and multidisciplinary projects. The responses of the participants add to what is known about the care of vulnerable populations, offering a first-hand description of students' preparation for work with vulnerable populations and the interdisciplinary team.

Unlabelled: Group A Streptococcus (GAS) is an important human pathogen responsible for both superficial infections and invasive diseases. Autoimmune sequelae may occur upon repeated infection. For this reason, development of a vaccine against GAS represents a major challenge, since certain GAS components may trigger autoimmunity. We formulated three combination vaccines containing the following: (i) streptolysin O (SLO), interleukin 8 (IL-8) protease (Streptococcus pyogenes cell envelope proteinase [SpyCEP]), group A streptococcal C5a peptidase (SCPA), arginine deiminase (ADI), and trigger factor (TF); (ii) the conserved M-protein-derived J8 peptide conjugated to ADI; and (iii) group A carbohydrate lacking the N-acetylglucosamine side chain conjugated to ADI. We compared these combination vaccines to a "gold standard" for immunogenicity, full-length M1 protein. Vaccines were adjuvanted with alum, and mice were immunized on days 0, 21, and 28. On day 42, mice were challenged via cutaneous or subcutaneous routes. High-titer antigen-specific antibody responses with bactericidal activity were detected in mouse serum samples for all vaccine candidates. In comparison with sham-immunized mice, all vaccines afforded protection against cutaneous challenge. However, only full-length M1 protein provided protection in the subcutaneous invasive disease model., Importance: This set of experiments demonstrates the inherent variability of mouse models for the characterization of GAS vaccine candidate protective efficacy. Such variability poses an important challenge for GAS vaccine development, as advancement of candidates to human clinical trials requires strong evidence of efficacy. This study highlights the need for an open discussion within the field regarding standardization of animal models for GAS vaccine development., (Copyright © 2016 Rivera-Hernandez et al.)

Aim: Endosome escape is essential for developing effective nonviral gene delivery systems. Herein, three endosome-disrupting peptides (HA2(1-20), GALA and KALA) were incorporated into a multicomponent oligonucleotide delivery system to identify which peptide imparted the most favorable endosome escape and toxicity profile., Materials & Methods: Copper (I)-catalyzed azide-alkyne cycloaddition was used to construct multicomponent delivery vectors. The systems were evaluated for size, toxicity, cellular uptake and endosome escape activity., Results: Each system condensed plasmid DNA to form nanosized particles. The highest cellular uptake and endosome escape were associated with GALA and KALA containing systems, with KALA incorporation correlating with greater toxicity., Conclusion: GALA was selected as the most promising endosome-disrupting peptide for incorporation into the nanosized oligonucleotide delivery system.

Conjugation of multiple peptides by their N-termini is a promising technique to produce branched multiantigenic vaccines. We established a double conjugation strategy that combines a mercapto-acryloyl Michael addition and a copper-catalysed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction to synthesise self-adjuvanting branched multiantigenic vaccine candidates. These vaccine candidates aim to treat cervical cancer and include two HPV-16 derived epitopes and a novel self-adjuvanting moiety. This is the first report of mercapto-acryloyl conjugation applied to the hetero conjugation of two unprotected peptides by their N-termini followed by a CuAAC reaction to conjugate a novel synthetic lipoalkyne self-adjuvanting moiety. In vivo experiments showed that the most promising vaccine candidate completely eradicated tumours in 46% of the mice (6 out of 13 mice).

Human papillomaviruses (HPVs) are associated with various cancers, with HPV16 linked to more than half of cervical cancer cases. Vaccines to prevent HPV infection and cancer development have proven effective, but are not useful in individuals with prior HPV exposure. Treatment vaccines to eradicate or control HPV-associated lesions are therefore desirable for these patients. Herein we describe the development of a process to enable the production of semisynthetic vaccines based on the site-specific attachment of synthetic bacterial lipid analogs (e.g., Pam2Cys) to a non-oncogenic mutant HPV16 E7 protein to generate molecularly defined vaccines. Many cytotoxic lymphocyte (CTL) epitopes from E7 are delivered by this approach; potentially ensuring that large numbers of immunized individuals can generate CTLs to clear HPV infected cells. Delivery of this construct reduced the growth of HPV16-associated tumors in a TC1 mouse model, the effects of which were better than the potent CTL epitope HPV16 E7(44-57) administered with Montanide ISA51 adjuvant., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Multicomponent gene delivery systems incorporating cell-penetrating peptides (CPP) from the human neurturin protein (NRTN-30, NRTN(132-161); NRTN-17, NRTN(145-161)) and a poly-l-lysine (PLL) dendron, were synthesized and characterized for plasmid DNA (pDNA) delivery. Acetylated NRTN peptides (Ac-CPP) and peptides conjugated to a PLL dendron (DEN-CPP) efficiently condensed and stabilized pDNA. Complexes between pDNA and DEN-CPP formed smaller and more stable nanoparticles. Flow cytometry experiments showed that pDNA-DEN-CPPs were taken up more efficiently into HeLa cells. There was also no significant difference between NRTN-30 and NRTN-17 for pDNA uptake, indicating that the truncated peptide alone is sufficient as a CPP for pDNA delivery., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Vaccination has a proven record as one of the most effective medical approaches to prevent the spread of infectious diseases. Traditional vaccine approaches involve the administration of whole killed or weakened microorganisms to stimulate protective immune responses. Such approaches deliver many microbial components, some of which contribute to protective immunity, and assist in guiding the type of immune response that is elicited. Despite their impeccable record, these approaches have failed to yield vaccines for many important infectious organisms. This has prompted a move towards more defined vaccines ('subunit vaccines'), where individual protective components are administered. This unit provides an overview of the components that are used for the development of modern vaccines including: an introduction to different vaccine types (whole organism, protein/peptide, polysaccharide, conjugate, and DNA vaccines); techniques for identifying subunit antigens; vaccine delivery systems; and immunostimulatory agents ('adjuvants'), which are fundamental for the development of effective subunit vaccines., (Copyright © 2015 John Wiley & Sons, Inc.)

Gene therapy requires safe and effective vectors to deliver genes to their target site of action. Non-viral gene delivery systems have attracted growing attention due to their low toxicity, low immunogenicity and ease of production compared to viral vectors. Most non-viral gene delivery systems enter cells via endocytic pathways, and their escape from endosomes is therefore crucial for successful transfection. Several reagents have been developed to promote endosomal escape, including peptides, polymers and lipids. Among these, endosome-disrupting peptides have been used in many studies, and have proven to be one of the most promising approaches to overcome endosomal entrapment and lysosomal degradation. This review provides an up-to-date summary of strategies for enhancing endosomal escape, with a focus on the modification of endosome-disrupting peptides to further increase the efficient delivery of oligonucleotides.

The non-human primates (NHPs) model in biomedical research has contributed to the study of human infectious, autoimmune, oncogenic, and neurological diseases. This review focuses on the importance of NHP models in vaccine development for tuberculosis, pertussis, Dengue, group A streptococcus (Streptococcus pyogenes) infection, HIV infection, and certain diseases in the elderly (influenza, for example). From understanding disease pathogenesis and mechanisms of protection, to assessing vaccine safety and efficacy, we discuss selected cases where the importance of the use of NHP models is highlighted.

Aim: Utilize lipopeptide vaccine delivery system to develop a vaccine candidate against Group A Streptococcus., Materials & Methods: Lipopeptides synthesized by solid-phase peptide synthesis-bearing carboxyl (C)-terminal and amino (N)-terminal Group A Streptococcus peptide epitopes. Nanoparticles formed were evaluated in vivo., Results: Immune responses were induced in mice without additional adjuvant. We demonstrated for the first time that incorporation of the C-terminal epitope significantly enhanced the N-terminal epitope-specific antibody response and correlated with forming smaller nanoparticles. Antigen-presenting cells had increased uptake and maturation by smaller, more immunogenic nanoparticles. Antibodies raised by vaccination recognized isolates., Conclusion: Demonstrated the lipopeptidic nanoparticles to induce an immune response which can be influenced by the combined effect of epitope choice and size.

Despite burgeoning evidence on the positive effects of perinatal loss support groups, some communities still lack such services. Those wishing to begin a support group may find the task daunting, especially if there is little support for their efforts. In this article, the authors discuss the development of a pregnancy loss support group, which included a community assessment, launching a pilot program, and providing training for facilitators. Other practical considerations will also be reviewed, such as finding a location for the group and securing funding for advertising.

Subunit vaccines offer a means to produce safer, more defined vaccines compared to traditional whole microorganism approaches. Subunit antigens, however, exhibit weak immunity, which is normally overcome through coadministration with adjuvants. Enhanced vaccine properties (e.g., improved potency) can be obtained by linking antigen and adjuvant, as observed for synthetic peptide antigens and Toll-like receptor 2 (TLR2) ligands. As few protective peptide antigens have been reported, compared to protein antigens, we sought to extend the utility of this approach to recombinant proteins, while ensuring that conjugation reactions yielded a single, molecularly defined product. Herein we describe the development and optimization of techniques that enable the efficient, site-specific attachment of three synthetic TLR2 ligands (lipid core peptide (LCP), Pam2Cys, and Pam3Cys) onto engineered protein antigens, permitting the selection of optimal TLR2 agonists during the vaccine development process. Using this approach, broadly protective (J14) and population targeted (seven M protein N-terminal antigens) multiantigenic vaccines against group A streptococcus (GAS; Streptococcus pyogenes) were produced and observed to self-assemble in PBS to yield nanoparticules (69, 101, and 123 nm, respectively). All nanoparticle formulations exhibited self-adjuvanting properties, with rapid, persistent, antigen-specific IgG antibody responses elicited toward each antigen in subcutaneously immunized C57BL/6J mice. These antibodies were demonstrated to strongly bind to the cell surface of five GAS serotypes that are not represented by vaccine M protein N-terminal antigens, are among the top 20 circulating strains in developed countries, and are associated with clinical disease, suggesting that these vaccines may elicit broadly protective immune responses.