Evolutionary History of Tissue Kallikreins

The gene family of human kallikrein-related peptidases (KLKs) encodes proteins with diverse and pleiotropic functions in normal physiology as well as in disease states. Currently, the most widely known KLK is KLK3 or prostate-specific antigen (PSA) that has applications in clinical diagnosis and monitoring of prostate cancer. The KLK gene family encompasses the largest contiguous cluster of serine proteases in humans which is not interrupted by non-KLK genes. This exceptional and unique characteristic of KLKs makes them ideal for evolutionary studies aiming to infer the direction and timing of gene duplication events. Previous studies on the evolution of KLKs were restricted to mammals and the emergence of KLKs was suggested about 150 million years ago (mya). In order to elucidate the evolutionary history of KLKs, we performed comprehensive phylogenetic analyses of KLK homologous proteins in multiple genomes including those that have been completed recently. Interestingly, we were able to identify novel reptilian, avian and amphibian KLK members which allowed us to trace the emergence of KLKs 330 mya. We suggest that a series of duplication and mutation events gave rise to the KLK gene family. The prominent feature of the KLK family is that it consists of tandemly and uninterruptedly arrayed genes in all species under investigation. The chromosomal co-localization in a single cluster distinguishes KLKs from trypsin and other trypsin-like proteases which are spread in different genetic loci. All the defining features of the KLKs were further found to be conserved in the novel KLK protein sequences. The study of this unique family will further assist in selecting new model organisms for functional studies of proteolytic pathways involving KLKs

Improvements, Variations and Biomedical Applications of the Michaelis–Arbuzov Reaction

Compounds bearing the phosphorus–carbon (P–C) bond have important pharmacological, biochemical, and toxicological properties. Historically, the most notable reaction for the formation of the P–C bond is the Michaelis–Arbuzov reaction, first described in 1898. The classical Michaelis–Arbuzov reaction entails a reaction between an alkyl halide and a trialkyl phosphite to yield a dialkylalkylphosphonate. Nonetheless, deviations from the classical mechanisms and new modifications have appeared that allowed the expansion of the library of reactants and consequently the chemical space of the yielded products. These involve the use of Lewis acid catalysts, green methods, ultrasound, microwave, photochemically-assisted reactions, aryne-based reactions, etc. Here, a detailed presentation of the Michaelis–Arbuzov reaction and its developments and applications in the synthesis of biomedically important agents is provided. Certain examples of such applications include the development of alkylphosphonofluoridates as serine hydrolase inhibitors and activity-based probes, and the P–C containing antiviral and anticancer agents

Activity-Based Probes for Proteases Pave the Way to Theranostic Applications

Proteases are important enzymes in health and disease. Their activities are regulated at multiple levels. In fact, proteases are synthesized as inactive proenzymes (zymogens) that are activated by proteolytic removal of their pro-peptide sequence and can remain active or their activity can be attenuated by complex formation with specific endogenous inhibitors or by limited proteolysis or degradation. Consequently, quite often, only a fraction of the protease molecules is in the active/functional form, thus, the abundance of a protease is not always linearly proportional to the (patho)physiological function(s). Therefore, assays to determine the active forms of proteases are needed, not only in research but also in molecular diagnosis and therapy. Activity-based probes (ABPs) are chemical entities that bind covalently to the active enzyme/protease. ABPs carry a detection tag to enable localization and quantification of specific enzymatic/proteolytic activities with applications in molecular imaging and diagnosis. Moreover, ABPs act as suicide inhibitors of proteases, which can be exploited for delineation of the functional role(s) of a given protease in (patho) biological context and as potential therapeutics. In this sense, ABPs represent new theranostic agents. We outline recent developments pertaining to ABPs for proteases with potential therapeutic applications, with the aim to highlight their importance in theranostics

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accompanied by an increase in secreted hK10 protein concentration, was observed for a subset of breast, ovarian, and prostate tumor cell lines after 5-aza-2 -dC. Genomic sequencing of sodium-bisulfi te-treated DNA demonstrated that CpG sites within the KLK10 gene exon 3 were highly methylated. Hypermethylation of exon 3 CpG regions was also detected in primary ovarian cancers. Conclusion: These data suggest that CpG island hypermethylation plays an important role in the downregulation of kallikrein 10 mRNA and protein expression, but it cannot explain the pattern of expression of this gene in all cell lines or tissue tested

Uptake and permeability studies of BBB-targeting immunoliposomes using the hCMEC/D3 cell line

The targeting potential of OX-26-decorated immunoliposomes was investigated, using the human brain endothelial cell line hCMEC/D3 as a model of the blood-brain barrier (BBB). Immuno-nanoliposomes were prepared by the biotin/streptavidin ligation strategy, and their uptake by hCMEC/D3 cells and permeability through cell monolayers was studied. In order to elucidate the mechanisms of uptake, pH-sensitive fluorescence signal of HPTS was used, while transport was measured using double labeled immunoliposomes (with aqueous and lipid membrane fluorescent tags). PEGylated and non-specific-IgG-decorated liposomes were studied under identical conditions, as controls. CHO-K1 cells (which do not overexpress the transferrin receptor) were studied in some cases for comparative purposes. Experimental results reveal that hCMEC/D3 cells are good models for in vitro screening of BBB-targeting nanoparticulate drug delivery systems. Uptake and transcytosis of immunoliposome-associated dyes by cell monolayers was substantially higher compared to those of control liposomes. HPTS-entrapping OX-26-immunoliposome uptake indicated lysosomal localization and receptor-mediated mechanism. The ratio of aqueous/lipid label transport is affected by pre-incubation with antibody, or use of high lipid doses, suggesting that vesicles are transported intact after lysosome saturation. Co-decoration with a second ligand slightly decreases OX-26-decorated vesicle uptake, but not transcytosis, proving that the biotin–streptavidin technique can be applied for the generation of dual-targeting nanoliposomes

Multi-Omics for Biomarker Discovery and Target Validation in Biofluids for Amyotrophic Lateral Sclerosis Diagnosis

Amyotrophic lateral sclerosis (ALS) is a rare but usually fatal neurodegenerative disease characterized by motor neuron degeneration in the brain and the spinal cord. Two forms are recognized, the familial that accounts for 5-10% and the sporadic that accounts for the rest. New studies suggest that ALS is a highly heterogeneous disease, and this diversity is a major reason for the lack of successful therapeutic treatments. Indeed, only two drugs (riluzole and edaravone) have been approved that provide a limited improvement in the quality of life. Presently, the diagnosis of ALS is based on clinical examination and lag period from the onset of symptoms to the final diagnosis is similar to 12 months. Therefore, the discovery of robust molecular biomarkers that can assist in the diagnosis is of major importance. DNA sequencing to identify pathogenic gene variants can be applied in the cases of familial ALS. However, it is not a routinely used diagnostic procedure and most importantly, it cannot be applied in the diagnosis of sporadic ALS. In this expert review, the current approaches in identification of new ALS biomarkers are discussed. The advent of various multi-omics biotechnology platforms, including miRNomics, proteomics, metabolomics, metallomics, volatolomics, and viromics, has assisted in the identification of new biomarkers. The biofluids are the most preferable material for the analysis of potential biomarkers (such as proteins and cell-free miRNAs), since they are easily obtained. In the near future, the biofluid-based biomarkers will be indispensable to classify different ALS subtypes and understand the molecular heterogeneity of the disease

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accompanied by an increase in secreted hK10 protein concentration, was observed for a subset of breast, ovarian, and prostate tumor cell lines after 5-aza-2 -dC. Genomic sequencing of sodium-bisulfi te-treated DNA demonstrated that CpG sites within the KLK10 gene exon 3 were highly methylated. Hypermethylation of exon 3 CpG regions was also detected in primary ovarian cancers. Conclusion: These data suggest that CpG island hypermethylation plays an important role in the downregulation of kallikrein 10 mRNA and protein expression, but it cannot explain the pattern of expression of this gene in all cell lines or tissue tested