Use of natural peptide TP4 as a food preservative prevents contamination by fungal pathogens.

Molecules of natural origin often possess useful biological activities. For instance, the natural peptide Tilapia Piscidin 4 (TP4) exhibits potent antimicrobial activity against a broad spectrum of pathogens. In this study, we explored the potential application of TP4 as a food preservative, asking whether it can prevent spoilage due to microbial contamination. A preliminary in silico analysis indicated that TP4 should interact strongly with fungal cell membrane components. Hence, we tested the activity of TP4 toward Candida albicans within fruit juice and found that the addition of TP4 could abolish fungal growth. We further determined that the peptide acts via a membranolytic mechanism and displays concentration-dependent killing efficiency. In addition, we showed that TP4 inhibited growth of Rhizopus oryzae in whole fruit (tomato) samples. Based on these findings, we conclude that TP4 should be further evaluated as a potentially safe and green solution to prevent food spoilage.

Optimization of sequence and chiral content enhances therapeutic potential of tilapia piscidin peptides.

Because antimicrobial peptides (AMPs) often exhibit broad-spectrum bactericidal potency, we sought to develop peptide-based antimicrobials for potential clinical use against drug-resistant pathogens. To accomplish this goal, we first optimized the amino acid sequence of a broad-spectrum AMP known as Tilapia Piscidin 4 (TP4). Then, we used the optimized sequence to create a pair of heterochiral variants (TP4-α and TP4-ß) with different percentages of D-enantiomers, as poly-L peptides often exhibit poor pharmacokinetic profiles. The conformations of the peptide pair exhibited inverted chirality according to CD and NMR spectroscopic analyses. Both heterochiral peptides displayed enhanced stability and low hemolysis activities. Irrespective of their different d-enantiomer contents, both heterochiral peptides exhibited bactericidal activities in the presence of human serum or physiological enzymes. However, the peptide with higher d-amino acid content (TP4-ß) caused better bacterial clearance when tested in mice infected with NDM-1 K. pneumoniae. In addition, we observed a relatively higher hydrogen bonding affinity in a simulation of the interaction between TP4-ß and a model bacterial membrane. In sum, our results demonstrate that the current design strategy may be applicable for development of new molecules with enhanced stability and in vivo antimicrobial activity.

Sequential rearrangement and stereochemical reorganization to design an antimicrobial peptide with enhanced stability.

Antimicrobial peptides (AMPs) are natural molecules that function within the innate immune system to counteract pathogenic invasion and minimize the detrimental consequences of infection. However, utilizing these molecules for medical applications has been challenging. In this study, we selected a model AMP with poor stability, Tilapia Piscidin 4 (TP4), and modified its sequence and chirality (TP4-γ) to improve its potential for clinical application. The strategy of chirality inversion was inspired by the cereulide peptide, which has a DDLL enantiomer pattern and exhibits exceptional stability. Sequential substitution of key residues and selective chirality inversion yielded a less toxic peptide with enhanced stability and notable antimicrobial activity. In addition to its superior stability profile and antimicrobial activity, TP4-γ treatment reduced the level of LPS-induced nitric oxide (NO) release in a macrophage cell line. This reduction in NO release may reflect anti-inflammatory properties, as NO is widely known to promote inflammatory processes. Hence, our heterochiral peptide construct shows a more suitable pharmacokinetic profile than its parental compound, and further studies are warranted to develop the molecule for potential clinical application.

Rational Design of Stapled Antimicrobial Peptides to Enhance Stability and In Vivo Potency against Polymicrobial Sepsis.

In this work, we sought to develop a TP4-based stapled peptide that can be used to counter polymicrobial sepsis. First, we segregated the TP4 sequence into hydrophobic and cationic/hydrophilic zones and substituted the preferred residue, lysine, as the sole cationic amino acid. These modifications minimized the intensity of cationic or hydrophobic characteristics within small segments. Then, we incorporated single or multiple staples into the peptide chain, bracketing the cationic/hydrophilic segments to improve pharmacological suitability. Using this approach, we were able to develop an AMP with low toxicity and notable in vivo efficacy. IMPORTANCE In our in vitro studies, one dual stapled peptide out of the series of candidates (TP4-3: FIIXKKSXGLFKKKAGAXKKKXIKK) showed significant activity, low toxicity, and high stability (in 50% human serum). When tested in cecal ligation and puncture (CLP) mouse models of polymicrobial sepsis, TP4-3 improved survival (87.5% on day 7). Furthermore, TP4-3 enhanced the activity of meropenem against polymicrobial sepsis (100% survival on day 7) compared to meropenem alone (37.5% survival on day 7). Molecules such as TP4-3 may be well suited for a wide variety of clinical applications.

Strategic modification of low-activity natural antimicrobial peptides confers antibacterial potential in vitro and in vivo.

Antimicrobial peptides (AMPs) show great promise for clinical applications, but the utility of naturally occurring AMPs is often limited by their stability. Here, we used a rational design approach to improve the characteristics of a pair of inactive peptides, tilapia piscidin 1 and 2 (TP1 and TP2). From each starting peptide, we generated a series of novel derivatives by substituting residues to adjust cationic charge density, percent hydrophobicity and hydrophilicity/hydrophobicity coefficients. This approach yielded a novel peptide, TP2-5 (KKCIAKAILKKAKKLLKKLVNP), that exhibits significant bactericidal potency, low cytotoxicity and high stability. The designed peptide further showed antibiofilm activity, rapid antibacterial action and a low capacity to induce bacterial resistance. Importantly, we also demonstrated that TP2-5 can protect mice in a Vibrio vulnificus-infected wound model. Therefore, our peptide modification strategy successfully generated a novel AMP with high potential for future clinical application.

Development of Bactericidal Peptides against Multidrug-Resistant Acinetobacter baumannii with Enhanced Stability and Low Toxicity.

Pathogenic superbugs are the root cause of untreatable complex infections with limited or no treatment options. These infections are becoming more common as clinical antibiotics have lost their effectiveness over time. Therefore, the development of novel antibacterial agents is urgently needed to counter these microbes. Antimicrobial peptides (AMPs) are a viable treatment option due to their bactericidal potency against multiple microbial classes. AMPs are naturally selected physiological microbicidal agents that are found in all forms of organisms. In the present study, we developed two tilapia piscidin 2 (TP2)-based AMPs for antimicrobial application. Unlike the parent peptide, the redesigned peptides showed significant antimicrobial activity against multidrug-resistant bacterial species. These peptides also showed minimal cytotoxicity. In addition, they were significantly active in the presence of physiological salts, 50% human serum and elevated temperature. The designed peptides also showed synergistic activity when combined with clinical antibiotics. The current approach demonstrates a fruitful strategy for developing potential AMPs for antimicrobial application. Such AMPs have potential for progression to further trials and drug development investigations.

Antimicrobial effects of syndiotactic polypeptides.

We present design and antibacterial studies of stereochemically diversified antimicrobial peptides against multidrug-resistant bacterial pathogens. Syndiotactic polypeptides are polymers of alternating L and D amino acids with LDLD or DLDL backbone stereochemical sequence, which can form stable gramicidin like helical conformations. We designed, synthesized and characterized eight model molecular systems with varied electrostatic fingerprints, modulated through calibrated sequence positioning. Six out of eight model systems showed very impressive antimicrobial activity against three difficult to treat bacterial species, Gentamicin resistant MRSA, E. coli and Mycobacterium. More importantly, the designed LDLD peptides were equally potent in serum, an important drawback of poly L peptide sequences due to enzyme mediated degradation and ion sensitivity. Further, we tested the activity of the designed peptides against drug-resistant clinical isolates of Staphylococcus aureus and Escherichia coli. Molecular dynamics simulation studies suggest formation of an assembly of individual peptides, preceding the membrane interaction and deformation. The activity estimates are comparable with the available peptide based antimicrobials, and are also highly specific and less toxic as per standard estimates. Incorporation of D amino-acids can significantly expand the peptide design space, which can in turn manifest in future biomaterial designs, especially antimicrobials.

Nurturing professionalism in medical schools. A systematic scoping review of training curricula between 1990-2019.

Introduction: Professionalism is an evolving, socioculturally informed multidimensional construct that influences doctor-patient relationships, patient satisfaction and care outcomes. However, despite its clinical significance there is little consistency in how professionalism is nurtured amongst medical students. To address this gap a systemic scoping review of nurturing professionalism in medical schools, is proposed.Methods: Levac's framework and the PRISMA-P 2015 checklist underpinned a 6-stage systematic review protocol. Concurrent use of Braun and Clarke's approach to thematic analysis and directed content analysis was used to identify the key elements in nurturing professionalism.Results: 13921 abstracts were identified from six databases, 854 full-text articles reviewed, and 162 full-text included articles were included. The 4 themes identified through thematic analysis are consistent with findings of the directed content analysis. These were the definition of professionalism, the approaches, content, barriers and enablers to teaching professionalism.Conclusion: Informed by a viable definition of professionalism and clear milestones nurturing professionalism nurturing professionalism begins with culturally appropriate training in clinical competence, humanistic qualities and reflective capacity. This process requires effective evaluations of professional identity formation, and the impact of the learning environment underlining the need for longitudinal assessments of the training process.

Educational roles as a continuum of mentoring's role in medicine - a systematic review and thematic analysis of educational studies from 2000 to 2018.

BACKGROUND: Recent studies have gone to great lengths to differentiate mentoring from teaching, tutoring, role modelling, coaching and supervision in efforts to better understand mentoring processes. This review seeks to evaluate the notion that teaching, tutoring, role modelling, coaching and supervision may in fact all be part of the mentoring process. To evaluate this theory, this review scrutinizes current literature on teaching, tutoring, role modelling, coaching and supervision to evaluate their commonalities with prevailing concepts of novice mentoring. METHODS: A three staged approach is adopted to evaluate this premise. Stage one involves four systematic reviews on one-to-one learning interactions in teaching, tutoring, role modelling, coaching and supervision within Internal Medicine, published between 1st January 2000 and 31st December 2018. Braun and Clarke's (2006) approach to thematic analysis was used to identify key elements within these approaches and facilitate comparisons between them. Stage two provides an updated view of one-to-one mentoring between a senior physician and a medical student or junior doctor to contextualise the discussion. Stage three infuses mentoring into the findings delineated in stage one. RESULTS: Seventeen thousand four hundred ninety-nine citations were reviewed, 235 full-text articles were reviewed, and 104 articles were thematically analysed. Four themes were identified - characteristics, processes, nature of relationship, and problems faced in each of the four educational roles. CONCLUSIONS: Role modelling, teaching and tutoring, coaching and supervision lie within a mentoring spectrum of increasingly structured interactions, assisted by assessments, feedback and personalised support that culminate with a mentoring approach. Still requiring validation, these findings necessitate a reconceptualization of mentoring and changes to mentor training programs and how mentoring is assessed and supported.

Topological effects on the designability and bactericidal potency of antimicrobial peptides.

New ideas and methods are being developed to generate highly designable small functional protein folds beyond the confines of natural structures, from secondary to quaternary level. Highly designable folds can have multiple sequence solutions, which are thermodynamically and kinetically stable. We have previously described how short syndiotactic helices can be exceptionally stable energetically, and how they can be used as a template for designing antibacterial agents. In this work, we have designed four syndiotactic, single turn, amphipathic; cationic 7-mer peptides which are the sequence and structural subset of earlier published 12-mer sequences. We examined the stability of the designed structures and its effects on the biological activity of such short peptide sequences. This was achieved by making objective comparisons between 12-mer and 7-mer sequences in terms of their antibacterial activity. Further, we investigated the mechanistic origins of clearly different bactericidal potency of single (7-mer) and double (12-mer) turn syndiotactic helices using molecular dynamics simulations. Our results suggest that conformationally constrained stable short double turn peptide scaffolds are highly designable, whereas single turn structures are more likely to be disordered. The stability of the designed peptide structure provides a platform for inclusion of multiple sequence variables and defined electrostatic fingerprints. Therefore, a stable peptide scaffold along with pre-defined electrostatic signatures can together be utilized for the design of novel antimicrobial peptides.

Peptide based antimicrobials: Design strategies and therapeutic potential.

Therapeutic activity of antibiotics is noteworthy, as they are used in the treatment of microbial infections. Regardless of their utility, there has been a steep decrease in the number of drug candidates due to antibiotic resistance, an inevitable consequence of noncompliance with the full therapeutic regimen. A variety of resistant species like MDR (Multi-Drug Resistant), XDR (Extensively Drug-Resistant) and PDR (Pan Drug-Resistant) species have evolved, but discovery pipeline has already shown signs of getting dried up. Therefore, the need for newer antibiotics is of utmost priority to combat the microbial infections of future times. Peptides have some interesting features like minimal side effect, high tolerability and selectivity towards specific targets, which would help them successfully comply with the stringent safety standards set for clinical trials. In this review, we attempt to present the state of the art in the discovery of peptide-based antimicrobials from a design perspective.

Effect of tacticity-derived topological constraints in bactericidal peptides.

Topology is a key element in structure-activity relationship estimation while designing physiologically-active molecular constructs. Peptides may be a preferred choice for therapeutics, principally due to their biocompatibility, low toxicity and predictable metabolism. Peptide design only guarantees functional group constitution by opting specific amino acid sequence, and not their spatial orientation to bind and incite physiological response on chosen targets. This is principally because peptide conformation is subject to external flux, due to the isotactic stereochemistry of the peptide chain. Stereochemical engineering of the peptide main chain offers the possibility of multiplying the structural space of a typical sequence to many orders of magnitude, and limiting the otherwise fluxional non-specific functional group dispensation in space by offering greater conformational rigidity. We put to test, this conceptual possibility already established in theoretical models, by designing amphipathic peptide systems and experimenting with them on Gram-positive, Gram-negative and antibiotic-resistant bacteria. The unusual conformational rigidity and stability of syndiotactic peptides enable them to retain the designed electrostatic environment, while they encounter the membrane surface. All the six designed systems exhibited bactericidal activity, pointing to the utility and specificity of stereo-engineered peptide systems for therapeutic applications. Overall, we hope that this work provides important insights and useful directives in designing novel peptide systems with antimicrobial activity, by expanding the design space, incorporating D-amino acid as an additional design variable.

Symmetry-Directed Self-Organization in Peptide Nanoassemblies through Aromatic π-π Interactions.

Almost all biological systems are assemblies of one or more biomolecules from nano- to macrodimensions. Unlike inorganic molecules, peptide systems attune with the conceptual framework of aggregation models when forming nanoassemblies. Three significant recent theoretical models have indicated that nucleation, end-to-end association, and geometry of growth are determined primarily by the size and electrostatics of the individual basic building blocks. In this study, we tested six model systems, differentially modulating the prominence of three design variables, namely, aromatic π-π interactions, local electrostatics, and overall symmetry of the basic building unit. Our results indicate that the crucial design elements in a peptide-based nanoassembly are (a) a stable extended π-π interaction network, (b) size, and (c) overall symmetry of the basic building blocks. The six model systems represent all of the design variables in the best manner possible, considering the complexity of a biomolecule. The results provide important directives in deciding the morphology and crystallinity of peptide nanoassemblies.

Mapping the Geometric Evolution of Protein Folding Motor.

Polypeptide chain has an invariant main-chain and a variant side-chain sequence. How the side-chain sequence determines fold in terms of its chemical constitution has been scrutinized extensively and verified periodically. However, a focussed investigation on the directive effect of side-chain geometry may provide important insights supplementing existing algorithms in mapping the geometrical evolution of protein chains and its structural preferences. Geometrically, folding of protein structure may be envisaged as the evolution of its geometric variables: ϕ, and ψ dihedral angles of polypeptide main-chain directed by χ1, and χ2 of side chain. In this work, protein molecule is metaphorically modelled as a machine with 4 rotors ϕ, ψ, χ1 and χ2, with its evolution to the functional fold is directed by combinations of its rotor directions. We observe that differential rotor motions lead to different secondary structure formations and the combinatorial pattern is unique and consistent for particular secondary structure type. Further, we found that combination of rotor geometries of each amino acid is unique which partly explains how different amino acid sequence combinations have unique structural evolution and functional adaptation. Quantification of these amino acid rotor preferences, resulted in the generation of 3 substitution matrices, which later on plugged in the BLAST tool, for evaluating their efficiency in aligning sequences. We have employed BLOSUM62 and PAM30 as standard for primary evaluation. Generation of substitution matrices is a logical extension of the conceptual framework we attempted to build during the development of this work. Optimization of matrices following the conventional routines and possible application with biologically relevant data sets are beyond the scope of this manuscript, though it is a part of the larger project design.

bPE toolkit: toolkit for computational protein engineering.

We present a computational toolkit consisting of five utility tools, for performing basic operations on a protein structure file in PDB format. The toolkit consists of five different programs which can be integrated as part of a pipeline for computational protein structure characterization or as a standalone analysis package. The programs include tools for chirality check for amino acids (ProChiral), contact map generation (CoMa), data redundancy (DaRe), hydrogen bond potential energy (HyPE) and electrostatic interaction energy (EsInE). All programs in the toolkit can be accessed and downloaded through the following link: http://www.iitg.ac.in/bpetoolkit/.