Ribosomal Synthesis of N-methylated peptides

Natural peptide products isolated from various organisms often contain N-methylated backbones. Such a modification of backbone of the peptide changes its conformational rigidity. This modification improves the biological properties of the peptide, such as improved target affinity, proteolytic stability or membrane permeability. Therefore synthesis of N-methylated peptide libraries is valuable in screening for drug-like peptides suitable for therapeutic uses. Protein synthesis using recombinant elements (PURE) and Flexizyme were used in order to reassign specific codons to N-methyl amino acids. mRNA-dependent translation system enable us to make our desired peptides with N-methyl amino acids. This technology is a convenient tool for the construction of N-methyl peptide libraries. Using Flexizyme in order to make library of N-methyl peptides requires significant amount of tRNA. Therefore developing a simple and rapid method for purification of specific tRNA from fully modified E. coli total tRNA would be advantageous Here we reported a new technique in purification of individual tRNAs using fluorous affinity tag. From total tRNA, desired tRNA could be charged with related amino acid and tagged with fluorous molecule through reductive amination

The human body at cellular resolution: the NIH Human Biomolecular Atlas Program

Abstract: Transformative technologies are enabling the construction of three-dimensional maps of tissues with unprecedented spatial and molecular resolution. Over the next seven years, the NIH Common Fund Human Biomolecular Atlas Program (HuBMAP) intends to develop a widely accessible framework for comprehensively mapping the human body at single-cell resolution by supporting technology development, data acquisition, and detailed spatial mapping. HuBMAP will integrate its efforts with other funding agencies, programs, consortia, and the biomedical research community at large towards the shared vision of a comprehensive, accessible three-dimensional molecular and cellular atlas of the human body, in health and under various disease conditions

Design, synthesis, and application of OB2C combinatorial peptide and peptidomimetic libraries.

The "one-bead two-compound" (OB2C) combinatorial library is constructed on topologically segregated trifunctional bilayer beads such that each bead has a fixed cell-capturing ligand and a random library compound co-displayed on its surface and a chemical coding tag (bar code) inside the bead. An OB2C library containing thousands to millions of compounds can be synthesized and screened concurrently within a short period of time. When live cells are incubated with such OB2C libraries, every bead will be coated with a monolayer of cells. The cell membranes of the captured cells facing the bead surface are exposed to the library compounds tethered to each bead. A specific biochemical or cellular response can be detected with an appropriate reporter system. The OB2C method enables investigators to rapidly discover synthetic molecules that not only interact with cell-surface receptors but can also stimulate or inhibit downstream cell signaling. To demonstrate this powerful method, one OB2C peptide library and two OB2C peptidomimetic libraries were synthesized and screened against Molt-4 lymphoma cells to discover "death ligands." Apoptosis of the bead-bound cells was detected with immunocytochemistry using horseradish peroxidase (HRP)-conjugated anti-cleaved caspase-3 antibody and 3,3'-diaminobenzidine as a substrate. Two novel synthetic "death ligands" against Molt-4 cells were discovered using this OB2C library approach

Longitudinal fundus imaging and its genome-wide association analysis provide evidence for a human retinal aging clock

Biological age, distinct from an individual’s chronological age, has been studied extensively through predictive aging clocks. However, these clocks have limited accuracy in short time-scales. Here we trained deep learning models on fundus images from the EyePACS dataset to predict individuals’ chronological age. Our retinal aging clocking, ‘eyeAge’, predicted chronological age more accurately than other aging clocks (mean absolute error of 2.86 and 3.30 years on quality-filtered data from EyePACS and UK Biobank, respectively). Additionally, eyeAge was independent of blood marker-based measures of biological age, maintaining an all-cause mortality hazard ratio of 1.026 even when adjusted for phenotypic age. The individual-specific nature of eyeAge was reinforced via multiple GWAS hits in the UK Biobank cohort. The top GWAS locus was further validated via knockdown of the fly homolog, Alk, which slowed age-related decline in vision in flies. This study demonstrates the potential utility of a retinal aging clock for studying aging and age-related diseases and quantitatively measuring aging on very short time-scales, opening avenues for quick and actionable evaluation of gero-protective therapeutics

Rapid Discovery of Functional Small Molecule Ligands against Proteomic Targets through Library-Against-Library Screening

Identifying “druggable” targets and their corresponding therapeutic agents are two fundamental challenges in drug discovery research. The one-bead-one-compound (OBOC) combinatorial library method has been developed to discover peptides or small molecules that bind to a specific target protein or elicit a specific cellular response. The phage display cDNA expression proteome library method has been employed to identify target proteins that interact with specific compounds. Here, we combined these two high-throughput approaches, efficiently interrogated approximately 10¹³ possible molecular interactions, and identified 91 small molecule compound beads that interacted strongly with the phage library. Of 19 compounds resynthesized, 4 were cytotoxic against cancer cells; one of these compounds was found to interact with EIF5B and inhibit protein translation. As more binding pairs are confirmed and evaluated, the “library-against-library” screening approach and the resulting small molecule–protein domain interaction database may serve as a valuable tool for basic research and drug development

SPECIFICITIES OF THE IMMUNE RESPONSE DURING THE PROCESS OF WOUND HEALING IN A DIABETES MODEL IN CD26 DEFICIENT MICE

Uvod: Dipeptidil-peptidaza IV (DPP IV/CD26) multifunkcionalan je protein s značajnom proteolitičkom i kostimulacijskom ulogom čime utječe i na proliferaciju, angiogenezu, adheziju, migraciju i apoptozu stanica u procesu cijeljenja rana. Modulacijom biološke aktivnosti inkretina sudjeluje i u regulaciji koncentracije glukoze u krvi, međutim, patofiziološki procesi cijeljenja rana u dijabetesu nisu dovoljno razjašnjeni. Pretpostavka ovog istraživanja je da DPP IV/CD26 ima važnu ulogu u modulaciji imunosnog odgovora u procesu cijeljenja rana u hiperglikemiji. Cilj istraživanja bio je ispitati utječe li nedostatak DPP IV/CD26 na makroskopske i/ili mikroskopske promjene tijekom procesa cijeljenja rana kože u dijabetesu, istražiti izražaj makrofaga i limfocita T kod CD26 deficijentnih te divljeg tipa životinja s induciranim dijabetesom te ispitati promjene u aktivnosti serumske DPP IV/CD26 tijekom uspostave eksperimentalne hiperglikemije i tijekom procesa cijeljenja rana kože u C57BL/6 životinjama. Materijal i metode: CD26 deficijentnim (CD26-/-) te divljem tipu miševa (C57BL/6) induciran je model dijabetesa intraperitonealnom aplikacijom otopine streptozotocina u citratnom puferu u dozi od 50 mg/kg tijekom pet dana. Miševima s potvrđenim dijabetesom je potom na interskapularnom dijelu leđa učinjeno šest rana promjera 5 mm te su pojedine skupine pokusnih životinja žrtvovane drugog, četvrtog, sedmog, desetog te petnaestog dana. Primjenom različitih metoda analize makroskopskih i mikroskopskih uzoraka (patohistološkim, imunohistokemijskim, histomorfometrijskim i spektrofotometrijskim metodama) pratio se stupanj regeneracije pojedinih slojeva kože, stupanj proliferacije stanica bazalnog sloja epidermisa i fibroblastadermisa, izražaj limfocita T i makrofaga u vezivu koriuma oba soja ispitivanih životinja, te enzimska aktivnost serumske DPP IV/CD26 kod CD57BL/6soja tijekom procesa cijeljenja rana. Rezultati dobiveni ovim istraživanjem ukazuju na relativno uspješniji proces cijeljenja rana kože u uvjetima nedostatka DPP IV/CD26 i razvijenog dijabetesa. Stupanj regeneracije koriuma kod CD26-/- miševa statistički značajno je veći (p<0,05) u odnosu na C57BL/6 miševe kao i broj Ki67 pozitivnih stanica, što upućuje na pojačanu proliferaciju stanica veziva uz obnovu ekstracelularnog matriksa u uvjetima nedostatka CD26. Izražaj limfocita T statistički je značajno veći (p<0,05) kod divljeg tipa životinja što ukazuje da je upalna faza manje izražena kod CD26-/- životinja. Porast izražaja makrofaga bio je brži i intenzivniji u uvjetima nedostatka CD26. Aktivnost serumske DPP IV/CD26 kodC57BL/6 miševa u uvjetima hiperglikemije je statistički značajno veća (p<0,05) u usporedbi s fiziološkim uvjetima dok je tijekom procesa cijeljenja rana značajno snižena drugog i četvrtog dana od indukcije rana. Zaključak: Dobiveni rezultati potvrđuju pretpostavku kako molekula DPP IV/CD26 utječe na intenzitet i dinamiku cijeljenja rana kao i specifičnost imunosnog odgovora u uvjetima eksperimentalne hiperglikemije. Pokazano je da je kod CD26-/- miševa upalni odgovor manje izražen nego kod divljeg tipa miševa. Proces regeneracije i reparacije tkiva u uvjetima hiperglikemije uspješniji je kod nedostatka DPP IV/CD26 što potvrđuje važnost inhibitora ove molekule u terapijske svrhe kod oboljelih od dijabetesa.Introduction: Dipeptidyl peptidase IV (DPP IV/CD26) is a multifunctional protein with a significant proteolytic and costimulatory role, thus affecting the process of proliferation, angiogenesis, adhesion, migration and apoptosis of cells in the wound healing process. It is also involved in the regulation of blood glucose concentrations by modulating the biological activity of incretins. However, the pathophysiological processes of wound healing in diabetes are not sufficiently clarified. The hypothesis of this study is that DPP IV/CD26 plays an important role in modulating the immune response in the wound healing process in conditions of hyperglycemia. The aim of this study was to determine whether DPP IV/CD26 deficiency affects macroscopic and/or microscopic changes during cutaneous wound healing process in diabetes. We aimed to determine the expression of macrophages and lymphocytes T in CD26 deficient and wild-type animals with induced diabetes and to investigate changes in serum DPP IV/CD26 activity during the development of experimental hyperglycemia and during the wound healing process in C57BL/6 animals. Materials and Methods: A model of diabetes was induced in CD26 deficient (CD26-/-) and wild type (C57BL/6) mice by intraperitoneal administration of a streptozotocin solution in citrate buffer at a dose of 50 mg/kg for five days. After the induction of diabetes, six experimental wounds (5 mm in diameter) were induced on the interscapular dorsal part of animals. Groups of experimental animals were sacrificed the second, fourth, seventh, tenth and fifteenth day of wound healing. Different methods of analysis were used for macroscopic and microscopic examinations (pathohistological, imunohistochemical, histomorphometrical and spectrophotometrical methods). The degree of regeneration of different skin layers, the degree of proliferation of the basal layer of epidermis and fibroblasts of the dermis, and the expression of lymphocytes T and macrophages in wound tissues of both mice strains were determined. Likewise, serum DPP IV/CD26 activity during the wound healing process in C57BL/6 mice was analyzed. The results of this study indicate a relatively more successful skin wound healing process under conditions of DPP IV/CD26 deficiency in diabetes. The rate of tissue regeneration in CD26-/- mice was statistically significantly higher (p<0.05) than in C57BL/6 mice as well as the number of Ki67 positive cells, indicating an increase drate of cell proliferation and regeneration of extracellular matrix in conditions of CD26 deficiency. The number of lymphocytes T is statistically significantly higher (p<0.05) in wild type mice indicating that the inflammatory phase is less pronounced in CD26-/-animals. The increase in macrophage expression was faster and more intense in the absence of DPP IV/CD26. The activity of serum DPP IV / CD26 in C57BL/6 diabetic mice was statistically significantly higher (p<0.05) compared to physiological conditions while it was significantly decreased the second and fourthday of wound healing. Conclusion: Obtained results confirm the hypothesis that DPP IV/CD26 influences the intensity and dynamics of wound healing as well as the specificity of the immune response in experimental hyperglycemic conditions. It has been shown that CD26-/-mice show a less pronounced inflammation response than wild type mice. The process of tissue regeneration and reparation in hyperglycemia is more successful in conditions of DPP IV/CD26 deficiency, which confirms the importance of inhibitors of this molecule for therapeutic purposes in patients with diabetes