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Protein poly‐ and oligophosphorylation are recently discovered post‐translational modifications that remain poorly characterized due to (1) the difficulty of extracting endogenously polyphosphorylated species without degradation and (2) the absence of synthetic and analytical tools to prepare and characterize poly‐ and oligophosphorylated species in biochemical contexts. Herein, we report a methodology for the selective oligophosphorylation of peptides with monodisperse phosphate chain lengths (Pn=3–6). A library of oligophosphorimidazolide (oligoP‐imidazolide) reagents featuring benzyl and o‐nitrophenylethyl protecting groups was synthesized in moderate‐to‐good yields (65–93 %). These oligoP‐imidazolide reagents enabled the selective and simultaneous conjugation of multiple phosphate units to phosphoryl nucleophiles, circumventing tedious iterative processes. The generalizability of this approach is illustrated by a substrate scope study that includes several biologically relevant phosphopeptide sequences, culminating in the synthesis of >60 examples of peptide oligophosphates (Pn=2–6). Moreover, we report the preparation of oligoP‐diimidazolides (Pn=3–5) and discuss their application in generating unique condensed phosphate‐peptide conjugates. We also demonstrate that human phospho‐ubiquitin (pS65‐Ub) is amenable to functionalization by our reagents. Overall, we envision the methods described here will enable future studies that characterize these newly discovered but poorly understood phosphorylation modes. [ABSTRACT FROM AUTHOR]

The development of therapies followed a generalized approach for a long time, assuming that a single treatment could effectively address various patient populations. However, recent breakthroughs have revealed the limitations of this one-size-fits-all paradigm. More recently, the field of therapeutics has witnessed a shift toward other modules, including cell therapies, high molecular weight remedies, personalized medicines, and gene therapies. Such advancements in therapeutic modules have the potential to revolutionize healthcare and pave the way for medicines that are more efficient and with minimal side effects. Cell therapies have gained considerable attention in regenerative medicine. Stem cell-based therapies, for instance, hold promise for tissue repair and regeneration, with ongoing research focusing on enhancing their efficacy and safety. High molecular weight drugs like peptides and proteins emerged as promising therapeutics because of their high specificity and diverse biological functions. Engineered peptides and proteins are developed for targeted drug delivery, immunotherapy, and disease-modulation. In personalized medicine, tailored treatments to individuals based on specific genetic profiling, lifestyle, biomarkers, and disease characteristics are all implemented. Clinicians have tailored treatments to optimize outcomes and minimize adverse effects, using targeted therapies based on specific mutations, yielding remarkable results. Gene therapies have revolutionized the treatment of genetic disorders by directly targeting the underlying genetic abnormalities. Innovative techniques, such as CRISPR-Cas9 have allowed precise gene editing, opening up possibilities for curing previously incurable conditions. In conclusion, advancements in therapeutic modules have the potential to revolutionize healthcare and pave the way for medicines that are more efficient and with minimal side effects. [ABSTRACT FROM AUTHOR]

Optical microcavities, particularly whispering gallery mode (WGM) microcavities enhanced by plasmonic nanorods, are emerging as powerful platforms for single‐molecule sensing. However, the impact of optical forces from the plasmonic near field on analyte molecules is inadequately understood. Using a standard optoplasmonic WGM single‐molecule sensor to monitor two enzymes, both of which undergo an open‐to‐closed‐to‐open conformational transition, the work done on an enzyme by the WGM sensor as atoms of the enzyme move through the electric field gradient of the plasmonic hotspot during conformational change has been quantified. As the work done by the sensor on analyte enzymes can be modulated by varying WGM intensity, the WGM microcavity system can be used to apply free energy penalties to regulate enzyme activity at the single‐molecule level. The findings advance the understanding of optical forces in WGM single‐molecule sensing, potentially leading to the capability to precisely manipulate enzyme activity at the single‐molecule level through tailored optical modulation. [ABSTRACT FROM AUTHOR]

Implantable drug delivery systems formed upon injection offer a host of advantages, including localized drug administration, sustained release, minimized side effects, and enhanced patient compliance. Among the various techniques utilized for the development of in situ forming drug implants, solvent-induced phase inversion emerges as a particularly promising approach. However, synthetic polymer-based implants have been associated with undesirable effects arising from polymer degradation. In response to this challenge, a novel category of drug delivery systems, known as phospholipids-based phase separation gels (PPSGs), has emerged. These gels, characterized by their low initial viscosity, exhibit injectability and undergo rapid transformation into in situ implants when exposed to an aqueous environment. A typical PPSG formulation comprises biodegradable components, such as phospholipids, pharmaceutical oil, and a minimal amount of ethanol. The minimized organic solvents in the composition show good biocompatibility. And the relatively simple composition holds promise for industrial-scale manufacturing. This comprehensive review provides an overview of the principles and advancements in PPSG systems, with specific emphasis on their suitability as drug delivery systems for a wide range of active pharmaceutical ingredients (APIs), spanning from small molecules to peptides and proteins. Additionally, we explore the critical parameters and underlying principles governing the formulation of PPSG-based drug delivery strategies, offering valuable insights on optimization strategies. [ABSTRACT FROM AUTHOR]

Biosorption mediated by peptides or proteins has emerged as an efficient and sustainable alternative for selective rare earth element (REE) recovery from waste streams, and will diversify REE supply while reducing the environmental burden. REE-binding peptides/proteins can be created and identified by using the state-of-the-art approaches, including phage display, rational design, proteomics, and homology modeling, and can be optimized by protein engineering for improved functionality and desirable properties. The activity of REE-binding peptides and proteins can be harnessed by engineering robust and reusable biomaterials for the selective recovery of REEs. The escalating global demand for rare earth elements (REEs) and the overabundance of REE-containing waste require innovative technologies for REE recovery from waste to achieve a sustainable supply of REEs while reducing the environmental burden. Biosorption mediated by peptides or proteins has emerged as a promising approach for selective REE recovery. To date, multiple peptides and proteins with high REE-binding affinity and selectivity have been discovered, and various strategies are being exploited to engineer robust and reusable biosorptive materials for selective REE recovery. This review highlights recent advances in discovering and engineering peptides and proteins for REE recovery. Future research prospects and challenges are also discussed. [ABSTRACT FROM AUTHOR]

Carbonic Anhydrases (CAs) have been a target for de novo protein designers due to the simplicity of the active site and rapid rate of the reaction. The first reported mimic contained a Zn(II) bound to three histidine imidazole nitrogens and an exogenous water molecule, hence closely mimicking the native enzymes' first coordination sphere. Co(II) has served as an alternative metal to interrogate CAs due to its d7 electronic configuration for more detailed solution characterization. We present here the Co(II) substituted [Co(II)(H2O/OH−)]N(TRIL2WL23H)3n+ that behaves similarly to native Co(II) substituted human‐CAs. Like the Zn(II) analogue, the cobalt‐derivative at slightly basic pH is incapable of hydrolyzing p‐nitrophenylacetate (pNPA); however, as the pH is increased a significant activity develops, which at pH values above 10 eventually yields a catalytic efficiency that exceeds that of the [Zn(II)(OH−)]N(TRIL2WL23H)3+ peptide complex. X‐ray absorption analysis is consistent with an octahedral species at pH 7.5 that converts to a 5‐coordinate species by pH 11. UV‐vis spectroscopy can monitor this transition, giving a pKa for the conversion of 10.3. We assign this conversion to the formation of a 5‐coordinate Co(II)(Nimid)3(OH)(H2O) species. The pH dependent kinetic analysis indicates the maximal rate (kcat), and thus the catalytic efficiency (kcat/Km), follow the same pH profile as the spectroscopic conversion to the pentacoordinate species. This correlation suggests that the chemically irreversible ester hydrolysis corresponds to the rate determining process. [ABSTRACT FROM AUTHOR]

Nigella sativa, a medicinal plant with a long history of traditional use, is renowned for its wide range of pharmacological properties. Peptides and proteins extracted from Nigella sativa have shown to possess various bioactive potentials including antimicrobial activity, anticancer and anti-inflammatory, properties. This study aims to explore the bioactive potential, antimicrobial properties, and toxicity of peptides and proteins extracted from Nigella sativa for drug discovery. The peptides and proteins are extracted using various techniques including solvent extraction, enzymatic hydrolysis, and size exclusion chromatography. The extracted peptides and proteins are screened for their bioactive potential using in vitro assays such as cell viability assays, enzyme inhibition assays, and cytokine release assays. The antimicrobial properties of the peptides and proteins are evaluated using minimum inhibitory concentration (MIC) assays against various bacteria and fungi. The toxicity of the peptides and proteins is assessed using in vitro and in vivo assays. The results potentially contribute to the development of novel drugs from Nigella sativa peptides and proteins. [ABSTRACT FROM AUTHOR]

Ion–ion reactions are valuable tools in mass-spectrometry-based peptide and protein sequencing. To boost the generation of sequence-informative fragment ions from low charge-density precursors, supplemental activation methods, via vibrational and photoactivation, have become widely adopted. However, long-lived radical peptide cations undergo intramolecular hydrogen atom transfer from c-type ions to z•-type ions. Here we investigate the degree of hydrogen transfer for thousands of unique peptide cations where electron transfer dissociation (ETD) was performed and was followed by beam-type collisional activation (EThcD), resonant collisional activation (ETcaD), or concurrent infrared photoirradiation (AI-ETD). We report on the precursor charge density and the local amino acid environment surrounding bond cleavage to illustrate the effects of intramolecular hydrogen atom transfer for various precursor ions. Over 30% of fragments from EThcD spectra comprise distorted isotopic distributions, whereas over 20% of fragments from ETcaD have distorted distributions and less than 15% of fragments derived from ETD and AI-ETD reveal distorted isotopic distributions. Both ETcaD and EThcD give a relatively high degree of hydrogen migration, especially when D, G, N, S, and T residues were directly C-terminal to the cleavage site. Whereas all postactivation methods boost the number of c- and z•-type fragment ions detected, the collision-based approaches produce higher rates of hydrogen migration, yielding fewer spectral identifications when only c- and z•-type ions are considered. Understanding hydrogen rearrangement between c- and z•-type ions will facilitate better spectral interpretation. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) are an important part of the innate immune system and demonstrate promising applications in the fight against antibiotic-resistant infections due to their unique mechanism of targeting bacterial membranes. However, it is challenging to study the interactions of these peptides within lipid bilayers, making it difficult to understand their mechanisms of toxicity and selectivity. Here, we used fast photochemical oxidation of peptides, an irreversible footprinting technique that labels solvent accessible residues, and native charge detection–mass spectrometry to study AMP–lipid interactions with different lipid bilayer nanodiscs. We observed differences in the oxidation of two peptides, indolicidin and LL-37, in three distinct lipid environments, which reveal their affinity for lipid bilayers. Our findings suggest that indolicidin interacts with lipid head groups via a simple charge-driven mechanism, but LL-37 is more specific for Escherichia coli nanodiscs. These results provide complementary information on the potential modes of action and lipid selectivity of AMPs. [ABSTRACT FROM AUTHOR]

Vaccines are designed to leverage the immune system and produce long-lasting protection against specific diseases. Peptide vaccines are regarded as safe and effective way of circumventing problems such as mild allergic reactions associated with conventional vaccines. The biggest challenges associated with formulation of peptide vaccines are stability issues and conformational changes which lead to destruction of their activity when exposed to lyophilization process that may act as stressors. Lyophilization process is aimed at removal of water which involves freezing, primary drying and secondary drying. To safeguard the peptide molecules from such stresses, cryoprotectants are used to offer them viability and structural stability. This paper is an attempt to understand the physicochemical properties of peptide vaccines, mechanism of cryoprotection under the shed of water replacement, water substitution theory and cation-pi interaction theory of amino acids which aims at shielding the peptide from external environment by formation of hydrogen bonds, covalent bonds or cation-pi interaction between cryoprotectant and peptide followed by selection criteria of cryoprotectants and their utility in peptide vaccines development along with challenges and opportunities. [ABSTRACT FROM AUTHOR]

Developing ideal IR probes is essential to understand the structure and dynamics of biomolecules with time‐resolved IR spectroscopies and imaging techniques. Especially, nitrile (CN) group has recently been proposed to serve as IR probes of the local environment of proteins. Herein, we investigated the effect of a substituent on the vibrational properties of the benzonitrile. The electron‐donating and withdrawing character of p‐substituent on benzonitrile are expected to modulate the vibrational frequency, molar extinction coefficient, and vibrational lifetime of CN probe. FT‐IR revealed the positive correlation between electron‐donating character and the molar extinction coefficient of CN stretch mode. Infrared pump‐probe (IR‐PP) measurements showed that the vibrational lifetime of CN stretch mode exhibits a relatively weak correlation with the electron‐donating strength. Among the investigated samples, 4‐dimethylamino benzonitrile with the strongest electron‐donating strength shows enhanced absorption and extended vibrational lifetime. Utilizing substituent effects will be a practical strategy to improve the performance of the IR probe. [ABSTRACT FROM AUTHOR]

In microdroplets, rates of chemical or biomolecular reactions can exceed those in the bulk phase by more than a million times. As electrospray ionization-based mass spectrometry (MS) involves the formation of charged microdroplets, reaction acceleration and online MS monitoring of reaction products can readily be performed at the same time. We investigated accelerated enzymatic reactions in microdroplets and focused on the proteolytic enzyme pepsin. Electrosonic spray ionization (ESSI) was utilized for developing the ultrarapid pepsin in-spray digestion of two different proteins, cytochrome c and RocC, at low pH values. The optimization of the protein digestion aimed at achieving maximum sequence coverage for the analyzed proteins. Furthermore, carefully designed control experiments allowed us to unambiguously prove that enzymatic protein cleavage almost exclusively occurs within the spray at a millisecond time scale and not prior to microdroplet generation. [ABSTRACT FROM AUTHOR]

Peptides and proteins play an essential role in biological systems and the human body. Deficiency or dysfunction of peptides and proteins such as insulin can lead to various illnesses. The therapeutic use of peptides and proteins in some illnesses such as diabetes, cardiovascular disease, autoimmunity, and cancer, among others, is highly considered. Peptides and proteins have large molecular structures and are generally hydrophilic, and maintaining their spatial composition or tertiary configurations are necessary for their pharmacological activities. Maintaining the stability of the protein structure and composition is therefore very important and necessary to maintain functionality. There are different routes for peptides and proteins administration such as injection (subcutaneous, intravenous, and intramuscular), oral, nasal, pulmonary, rectal, and ocular routes. The oral administration route is better accepted by the patient due to safety and ease of use. However, oral administration often is unsuitable because of the physicochemical properties and macromolecular structure of proteins and the presence of gastrointestinal (GI) enzymes under an acidic environment within the stomach. Proteins and peptides undergo enzymatic degradation, tertiary structural alteration, and low absorption. This necessitates the need to use alternative oral drug delivery systems for peptides and proteins that maintain their overall structure and enhances their absorption and bioavailability. In this article, we review carrier-based oral drug delivery systems that were suitable for peptides and proteins. [ABSTRACT FROM AUTHOR]

In this article, we carried out an analysis of the main aspects related to iron metabolism, as the understanding on this subject has been greatly intensified in recent years. The understanding of this subject has been greatly increased in recent years, while a simple harmony between the processes of absorption, metabolization and distribution of this ion are already sufficient to guarantee the supply of such nutrient in our body. The identification of certain genes that encode specific proteins participating in such homeostasis has stimulated the development of several studies on the subject in the current scientific environment. This article details the main enzymes and proteins participating in the iron cycle in the human body, also identifying their applications and uses, and also highlighting the role of this nutrient in the functioning of the body [ABSTRACT FROM AUTHOR]

Surfactants play critical roles in various applications including food, pharmaceuticals, cosmetics, mineral processing, wastewater treatment, etc. However, traditional chemical-based surfactants are often non-biodegradable, toxic, and persistent, leading to environmental pollution and potential health risks. To address these challenges, bio-inspired surfactants have emerged as promising alternatives owing to their well-defined compositions, biodegradability, biocompatibility, and the potential to finely tune their functionality by altering the amino acid sequence. Here, we report a comparative study of traditional foaming agents, MIBC, DPG, and DPM, and a bio-inspired peptide surfactant, AM1, and a protein surfactant, DAMP4. Furthermore, the pH- and metal ion- responsive properties of these two biosurfactants make them an innovative class of smart materials that can further expand the scope of applications. Our findings provide new insights into the potential of applying these new biosurfactants for various applications, representing a sustainable and environmentally friendly alternative approach. [Display omitted] • Comparison of chemical-based surfactants with peptide and protein surfactants. • Investigation of dynamic surface tension, foamability, bubble size, and coalescence. • Study of biomolecule surfactants and their unique functionalities. • A glimpse of future potentials of bio-surfactants with an environmentally friendly approach. [ABSTRACT FROM AUTHOR]

Oral mucositis, a common inflammatory side effect of oncological treatments, is a disorder of the oral mucosa that can cause painful ulcerations, local motor disabilities, and an increased risk of infections. Due to the discomfort it produces and the associated health risks, it can lead to cancer treatment restrains, such as the need for dose reduction, cycle delays or abandonment. Current mucositis management has low efficiency in prevention and treatment. A topical drug application for a local action can be a more effective approach than systemic routes when addressing oral cavity pathologies. Local delivery of growth factors, antibodies, and anti-inflammatory cytokines have shown promising results. However, due to the peptide and protein nature of these novel agents, and the several anatomic, physiological and environmental challenges of the oral cavity, their local action might be limited when using traditional delivering systems. This review is an awareness of the issues and strategies in the local delivery of macromolecules for the management of oral mucositis. [ABSTRACT FROM AUTHOR]

[Display omitted] The global pharmaceutical market has recently shifted its focus from small molecule drugs to peptide, protein, and nucleic acid drugs, which now comprise a majority of the top-selling pharmaceutical products on the market. Although these biologics often offer improved drug specificity, new mechanisms of action, and/or enhanced efficacy, they also present new challenges, including an increased potential for degradation and a need for frequent administration via more invasive administration routes, which can limit patient access, patient adherence, and ultimately the clinical impact of these drugs. Controlled-release systems have the potential to mitigate these challenges by offering superior control over in vivo drug levels, localizing these drugs to tissues of interest (e.g., tumors), and reducing administration frequency. Unfortunately, adapting controlled-release devices to release biologics has proven difficult due to the poor stability of biologics. In this review, we summarize the current state of controlled-release peptides and proteins, discuss existing techniques used to stabilize these drugs through encapsulation, storage, and in vivo release, and provide perspective on the most promising opportunities for the clinical translation of controlled-release peptides and proteins. [ABSTRACT FROM AUTHOR]

Objective:We aimed to investigate the effects of thyroid hormone withdrawal on N-terminal prohormone forms of atrial natriuretic peptide (NT-proANP) and brain natriuretic peptide (NT-proBNP) during radioiodine therapy in female patients with differentiated thyroid cancer (DTC). Methods:Serum concentrations of thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), NT-proANP and NT-proBNP were measured in 51 female patients with DTC (48.7 ± 4.2 years) at three time-points: day of radioiodine therapy (t1 – under acute hypothyroidism), 5 days after radioiodine (t2 – under acute hypothyroidism) and 3 months after radioiodine (t3 – under TSH suppression). Thirty healthy euthyroid women served as controls (42.8 ± 5.6 years). Results:At t1/t2/t3, median NT-proANP was 5.2/1.7/487 pmol/L vs. 297.7 pmol/L in control group (p < 0.001), median NT-proBNP was 50.1/36.5/79.5 pmol/L vs. 64.5 pmol/L (p < 0.001) and median NT-proANP/NT-proBNP ratios was 0.20/0.18/4.81 vs. 4.14 (p < 0.001). In acute hypothyroidism, FT3 levels were positively correlated with NT-proANP (r = 0.38,p = 0.005), NT-proANP/NT-proBNP ratios (r = 0.47,p = 0.001), heart rate (r = 0.39,p = 0.005), and negatively with mean arterial blood pressure (r = −0.58,p < 0.001). Conclusions:Our results indicate that NT-proANP reflects more accurately direct thyroid hormone effects than NT-proBNP. Thyroid hormone-dependent hemodynamic effects seem to be overlapped on the direct stimulatory effect of thyroid hormones on NT-proANP secretion by cardiac myocytes. [ABSTRACT FROM PUBLISHER]

Mutations in the Leucine Rich Repeat Protein Kinase 2 gene (LRRK2) are genetic predispositions for Parkinson's Disease, of which the G2019S (GS) missense mutation is the most common. GS-LRRK2 has a hyperactive kinase, and although numerous drug discovery programs have targeted the LRRK2 kinase, few have reached clinical trials. We recently reported on the discovery of a novel LRRK2 kinase inhibitor chemotype, 1 H -pyrazole biaryl sulfonamides. Although both potent and selective GS-LRRK2 inhibitors, 1 H -pyrazole biaryl sulfonamides are incapable of crossing the blood-brain barrier. Retaining the core 1 H -pyrazole and focusing our efforts on a phenylsulfonamide bioisosteric replacement, we report the discovery and preliminary development of azaspirocyclic 1 H -3,4,5-trisubstituted pyrazoles as potent and selective (>2000-fold) GS-LRRK2 kinase inhibitors capable of entering rodent brain. The compounds disclosed here present an excellent starting point for the development of more brain penetrant compounds. [Display omitted] • HIGHLIGHTS: Discovery of Azaspirocyclic 1 H- 3,4,5 - Trisubstitued Pyrazoles as Novel G2019S-LRRK2 Selective Kinase Inhibitors. • Mutations in the LRRK2 gene are the most common genetic causes of Parkinson's Disease. • G2019S is the most common inherited LRRK2 mutation and increases kinase activity. • We identified highly kinome and G2019S-selective inhibitors with in vivo efficacy. • Azaspirocyclic bioisostere of phenylsulfonamide enables improved PK properties. [ABSTRACT FROM AUTHOR]

Molecular translational self-diffusion, a measure of diffusive motion, provides information on the effective molecular hydrodynamic radius, as well as information on the properties of media or solution through which the molecule diffuses. Protein translational diffusion measured by pulsed-field gradient nuclear magnetic resonance (PFG-NMR) has seen increased application in structure and interaction studies, as structural changes or protein-protein interactions are often accompanied by alteration of their effective hydrodynamic radii. Unlike the analysis of complex mixtures by PFG-NMR, for monitoring changes of protein translational diffusion under various conditions, such as different stages of folding/unfolding, a partial region of the spectrum or even a single resonance is sufficient. We report translational diffusion coefficients measured by PFG-NMR with a modified stimulated echo (STE) sequence where band-selective pulses are employed for all three H RF pulses. Compared with conventional non-selective sequence, e.g. the BPP-LED sequence, the advantage of this modified band-selective excitation short transient (BEST) version of STE (BEST-STE) sequence is multi-fold, namely: (1) potential sensitivity gain as in generalized BEST-based sequences, (2) water suppression is no longer required as the magnetization of solvent water is not perturbed during the measurement, and (3) dynamic range problems due to the presence of intense resonances from molecules other than the protein or peptide of interest, such as non-deuterated detergent micelles, are avoided. [ABSTRACT FROM AUTHOR]

Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in 'functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP). [ABSTRACT FROM AUTHOR]

Inhaled therapeutic aerosols continue to be an important treatment for asthma and pulmonary diseases. A variety of dosage forms are employed for different indications and demographics including pressurized or propellant-driven metered dose inhalers, dry powder inhalers, and nebulizers/nebules. Research and development in this field has shown remarkable innovation in the past decade. Important new drug products for the treatment of asthma, chronic obstructive pulmonary disease, cystic fibrosis, diabetes, and a range of neurological disorders have been developed. New devices in each of the dosage form categories also have been developed, and new formulation technologies have been adopted. Unlike many other dosage forms, as new inhaled products appeared few of the existing products were converted to generic form. This may be explained by the formulation and device complexity, the implications for degree of difficulty in obtaining regulatory approval, and the prevalence of intellectual property in the field. After the setback of the initial approval and subsequent withdrawal of the Exubera®-inhaled insulin, there appeared to be reluctance to consider the pulmonary route of administration for systemically acting agents, particularly peptides and proteins. However, recent product development activities and approvals suggest that attitudes may be changing in favor of systemic delivery following inhaled aerosol administration. The new inhaled drug technologies seem to be driving reconsideration of therapeutic categories for indications that were of interest at the inception of modern inhaled drug therapy in the past century. We should embrace the opportunity to use new drugs and technologies to go back to the future! © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1165-1172, 2013 [ABSTRACT FROM AUTHOR]

Clinical Proteomics biomarker discovery programs lead to the selection of putative new biomarkers of human pathologies. Following an initial discovery phase, validation of these candidates in larger populations is a major task that recently started relying upon the use of mass spectrometry approaches, especially in cases where classical immune-detection methods were lacking. Thanks to highly sensitive spectrometers, adapted measurement methods like selective reaction monitoring (SRM) and various pre-fractionation methods, the quantitative detection of protein/peptide biomarkers in low concentrations is now feasible from complex biological fluids. This possibility leads to the use of similar methodologies in clinical biology laboratories, within a new proteomic field that we shall name 'Clinical Chemistry Proteomics' (CCP). Such evolution of Clinical Proteomics adds important constraints with regards to the in vitro diagnostic (IVD) application. As measured values of analytes will be used to diagnose, follow-up and adapt patient treatment on a routine basis; medical utility, robustness, reference materials and clinical feasibility are among the new issues of CCP to consider. [ABSTRACT FROM AUTHOR]

Surface treatment of biological tissue sections improves detection of peptides and proteins for mass spectrometry imaging. However, liquid surface treatments can result in diffusion of surface analytes and fragile tissue sections can be easily damaged by typical washing solvents. Here, we present a new surface washing procedure for mass spectrometry imaging. This procedure uses solvent wetted fiber-free paper to enable local washing of tissue sections for mass spectrometry imaging and tissue profiling experiments. In addition, the method allows fragile tissues that cannot be treated by conventional washing techniques to be analyzed by mass spectrometry imaging. [ABSTRACT FROM AUTHOR]

Abstract: Acetonitrile, which is a by-product of acrylonitrile synthesis, is the commonly used solvent in ion-pair reversed phase chromatography. In consequence of the decreasing demand for acrylonitrile due to the financial crisis, a worldwide shortage of acetonitrile is observed. Therefore, the aim of this study was to establish ion-pair reversed phase chromatographic assays using alternative eluents for acetonitrile and to decrease costs incurred hereby. We compared the performance of ion-pair reversed phase chromatography using acetonitrile with the alternative eluents methanol, ethanol and n-propanol, using monolithic reversed phase C5 as well as C18 chromatography columns. We used triethylammonium acetate (TEAA) and tetrabutylammonium sulfate (TBA) as representative cationic ion-pair reagents and trifluoroacetic acid (TFA) as representative anionic ion-pair reagent. For covering a large field of applications, we fractionated representative low, middle and high-molecular weight biomolecules, in particular dinucleoside polyphosphates, peptides, proteins and tryptic digested human serum albumin. Whereas the chromatographic characteristics of both methanol and n-propanol were partly insufficient, ethanol was characterised equally or partly even better in the matter of elution strength and separation quality compared to the eluent water–acetonitrile. In conclusion, ethanol is an appropriate alternative for acetonitrile in ion-pair reversed phase chromatography of biomolecules. [Copyright &y& Elsevier]

Calcium is the major intracellular messenger that triggers smooth muscle contraction. The study of calcium-binding proteins, such as calmodulin and its downstream effectors, reveals critical regulation of smooth muscle contraction by protein kinases and phosphatases. Moreover, the small GTP-binding protein RhoA and its downstream effector protein, Rho-kinase, have been shown to play a novel role in the regulation of smooth muscle contraction. Studies have shown that the activation of Rho-kinase is involved in the development of endothelial dysfunction, inflammation, restenosis, and increased vascular tone in a number of cardiovascular disorders. Because inhibitors of this pathway promote vasodilation independent of the mechanism that increases vasoconstrictor tone, it is our hypothesis that Rho-kinase is constitutively active in regulating vasoconstrictor tone in the pulmonary and systemic vascular beds. Studies in the literature suggest that the RhoA/Rho-kinase pathway has an important role in the pathogenesis of pulmonary hypertension. [ABSTRACT FROM AUTHOR]

Abstract: Size-exclusion chromatography (SEC) coupled with online laser light-scattering (LS) and refractive index (RI) detection provides an excellent approach to determine the molecular weights (Mw) of proteins by the “two-detector” approach. Mw is determined only at the maximum of a peak, using either peak heights or area ratio from the two detectors. However, proper calibration of the SEC/LS/RI system is critical to obtain high precision. Today, an essential part of any analysis is to evaluate the uncertainty associated with the method. Basically, it is possible to distinguish between factors related to signal nature, precision and those due to signal processing. Given the signal of interest is the peak height or area ratio from two detectors, the signal ratio uncertainty was calculated using the random propagation of error formula. In this case, the effect of signal correlation was evaluated to avoid the uncertainty overestimation. In the second case, the sources of uncertainty affecting analytical measurement were estimated with the information from the precision assessment. For this, two designs with two-factor fully nested were followed for each method. Finally, the contributions from various uncertainty sources related with calibration are also analysed in detail. There are in fact only three main sources of measurement uncertainty: intermediate precision, calibration and repeatability. Of these, method precision is always the greatest, regardless of approach. For all proteins and peptides studied, the Mw calculated using both methods are close to the theoretical results, independently of the design, but the contributions of individual terms to combined uncertainty depend on both the design and method used. For example, the combined uncertainty varied between 223 and 813.2Da for carbonic anhydrase, although higher values were found for human insulin and ovalbumin dimer. Other considerations that can have a significant impact on the results are discussed. The reproducibility of the two methods versus that based on ASTRA software used as reference method was performed using the concordance correlation coefficient. The methods’ reproducibility depends on the permitted losses in precision and accuracy. [Copyright &y& Elsevier]

Analysis of biologically active substances is particularly important in the pharmaceutical and biomedical area. For separation of polar compounds or complex mixtures by normal (NP) or reversed phase liquid chromatography (RP-HPLC) and/or electromigration techniques, it is necessary to apply a new generation of packings and columns with strictly defined properties. It is connected to the definition of chromatographic behavior and determination of compounds that are described by its structure, as well as chemical and physical properties. One of the factors playing a predominant role in the separation process is the interaction between analyte and stationary phases. During recent years a large variety of stationary phases have become available and have been also applied in routine and research chromatographic separations. Bonded phases are in widespread use and popular because of the great number of available packing materials, allowing the solution of a scale of different separation problems. At the present time analytical methods cannot be restricted only to the so-called “black box.” To meet the requirements of modern analytical techniques, strong demands are put on further, deeper understanding of the essence of the separation process, among other things, on the basis of conclusions about interactions between solute and stationary phase surfaces. For surface characterization different physicochemical methods such as CP/MAS, NMR, FT IR, DCS, chromatography, etc., have been described. The resolution, as reflected by efficiency, selectivity, and retention patterns on these materials, has been demonstrated. The effect of structure of stationary phases on retention of a model series of test analytes has been proved and numerically expressed by means of the Quantitative Structure-Retention Relationship (QSSR). The main aim of this paper is to present possibilities of determining different biologically active compounds (e.g., vitamins, steroids, nucleosides, peptides) in complex chromatographic methods (sample preparation, final analysis, validation) using new generation of stationary phases, columns, and chips divides. Special attention is dedicated to the advantage of packing materials imitating natural membranes because of the possible examination of the interaction between drug &LongLeftRightArrow; membrane. They can permit to the design of new pharmaceuticals and observation of processes taking place on the border of phases without interfering in natural systems. [ABSTRACT FROM AUTHOR]

Abstract: Exhaustive efforts have been made toward the administration of drugs, via alternative routes, that are poorly absorbed after the oral administration. The vagina as a route of drug delivery has been known since ancient times. In recent years, the vaginal route has been rediscovered as a potential route for systemic delivery of peptides and other therapeutically important macromolecules. However, successful delivery of drugs through the vagina remains a challenge, primarily due to the poor absorption across the vaginal epithelium. The rate and extent of drug absorption after intravaginal administration may vary depending on formulation factors, vaginal physiology, age of the patient and menstrual cycle. Suppositories, creams, gels, tablets and vaginal rings are commonly used vaginal drug delivery systems. The purpose of this communication is to provide the reader with a summary of advances made in the field of vaginal drug delivery. This report, therefore, summarizes various vaginal drug delivery systems with an introduction to vaginal physiology and factors affecting drug absorption from the vaginal route. [Copyright &y& Elsevier]

Capillary electrochromatography (CEC) is a separation method that has gained increasing attention in recent years. It combines the advantages of high performance liquid chromatography (HPLC) selectivity with the high efficiency of electrophoresis. Due to its promise of high peak capacities, CEC appears to be promising for resolution of complex mixtures, such as those generated during protein and peptide mapping. However, direct transfer of HPLC and capillary zone electrophoresis (CZE) methods to CEC is not trivial due to the unique requirements for CEC in terms of stationary phase and operating conditions. In this paper, we discuss the various approaches undertaken for the CEC separation of peptides and proteins. [ABSTRACT FROM AUTHOR]

Electrostatic interactions have a determining role in the conformational and dynamic behavior of polyelectrolyte molecules. In this study, anionic polyelectrolyte molecules, poly(glutamic acid) (PGA) and poly(aspartic acid) (PASA), in a water solution with the most commonly used K+ or Na+ counterions, were investigated using atomistic molecular dynamics (MD) simulations. We performed a comparison of seven popular force fields, namely AMBER99SB-ILDN, AMBER14SB, AMBER-FB15, CHARMM22*, CHARMM27, CHARMM36m and OPLS-AA/L, both with their native parameters and using two common corrections for overbinding of ions, the non-bonded fix (NBFIX), and electronic continuum corrections (ECC). These corrections were originally introduced to correct for the often-reported problem concerning the overbinding of ions to the charged groups of polyelectrolytes. In this work, a comparison of the simulation results with existing experimental data revealed several differences between the investigated force fields. The data from these simulations and comparisons with previous experimental data were then used to determine the limitations and strengths of these force fields in the context of the structural and dynamic properties of anionic polyamino acids. Physical properties, such as molecular sizes, local structure, and dynamics, were studied using two types of common counterions, namely potassium and sodium. The results show that, in some cases, both the macroion size and dynamics depend strongly on the models (parameters) for the counterions due to strong overbinding of the ions and charged side chain groups. The local structures and dynamics are more sensitive to dihedral angle parameterization, resulting in a preference for defined monomer conformations and the type of correction used. We also provide recommendations based on the results. [ABSTRACT FROM AUTHOR]

Chemical reactions can be performed to covalently modify specific residues in proteins. When applied to native enzymes, these chemical modifications can greatly expand the available set of building blocks for the development of biocatalysts. Nucleophilic canonical amino acid sidechains are the most readily accessible targets for such endeavors. A rich history of attempts to design enhanced or novel enzymes, from various protein scaffolds, has paved the way for a rapidly developing field with growing scientific, industrial, and biomedical applications. A major challenge is to devise reactions that are compatible with native proteins and can selectively modify specific residues. Cysteine, lysine, N-terminus, and carboxylate residues comprise the most widespread naturally occurring targets for enzyme modifications. In this review, chemical methods for selective modification of enzymes will be discussed, alongside with examples of reported applications. We aim to highlight the potential of such strategies to enhance enzyme function and create novel semisynthetic biocatalysts, as well as provide a perspective in a fast-evolving topic. [ABSTRACT FROM AUTHOR]

Interfaces can strongly accelerate or inhibit protein aggregation, destabilizing proteins that are stable in solution or, conversely, stabilizing proteins that are aggregation-prone. Although this behaviour is well-known, our understanding of the molecular mechanisms underlying surface-induced protein aggregation is still largely incomplete. A major challenge is represented by the high number of physico-chemical parameters involved, which are highly specific to the considered combination of protein, surface properties, and solution conditions. The key aspect determining the role of interfaces is the relative propensity of the protein to aggregate at the surface with respect to bulk. In this review, we discuss the multiple molecular determinants that regulate this balance. We summarize current experimental techniques aimed at characterizing protein aggregation at interfaces, and highlight the need to complement experimental analysis with theoretical modelling. In particular, we illustrate how chemical kinetic analysis can be combined with experimental methods to provide insights into the molecular mechanisms underlying surface-induced protein aggregation, under both stagnant and agitation conditions. We summarize recent progress in the study of important amyloids systems, focusing on selected relevant interfaces. Unlabelled Image • Interactions between proteins and surfaces can promote or inhibit amyloids formation. • We discuss the molecular mechanisms underlying this effect. • A major challenge is the high specificity to the combination of protein, surface properties, and solution conditions. • We summarize current experimental techniques to characterize protein aggregation at interfaces. • We illustrate how chemical kinetic analysis can be combined with experimental methods to provide mechanistic insights. [ABSTRACT FROM AUTHOR]

• D-2-allylglycine embedded imidazolium-bridged monolithic column was fabricated. • The highly cross-linked column exhibits RPLC/HILIC mixed-mode mechanisms. • The highest column efficiency of the column reaches 571,000 plates/m for amides. • The column was successfully applied for separation of peptides and proteins. The development of multifarious stationary phases is still a growing demand so as to solve the tasks of ever evolving actual applications. Herein, with D-2-allylglycine hydrochloride (AG·HCl) as the hydrophilic monomer, diene ionic liquid 1-allyl-3-vinylimidazolium bromide (AVI·Br) and polyhedral oligomeric silsesquioxane methacryl substituted (POSS-MA) as the dual crosslinkers, the highly cross-linked imidazolium-bridged POSS-AVI-AG hybrid monolithic column was fabricated via the "one-pot" free radical copolymerization. The AG·HCl embedded POSS-AVI-AG column displays typical reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography mixed-mode retention mechanisms. Both hydrophobic phenols, alkylbenzenes, aromatic amines and hydrophilic nucleosides/nucleic acid bases, amides and thioureas were successfully separated with high column efficiencies (up to 571,000 plates/m for amides), outperforming our previously reported AVI·Br modified POSS-AVI column. Moreover, the column was also explored for the separation of cytochrome c tryptic digests and egg white protein extraction. All these results demonstrate that the POSS-AVI-AG column has a good potential in separation of both small molecules and complex biological samples with multiple mechanisms. [ABSTRACT FROM AUTHOR]