Influence of selenium on the transfer of cadmium along the food chain

Zaradi številnih človeških dejavnosti iz leta v leto narašča količina težkih kovin v okolju, kar predstavlja vedno večjo grožnjo zdravju ljudi in živali. V zadnjih desetletjih je bilo predvsem zaskrbljujoče naraščanje količine kadmija (Cd) v zemlji in sedimentih. Kadmij je težka kovina, ki se zelo učinkovito prenaša skozi prehranjevalno verijo. Najpogosteje gre za prenos iz zemlje v rastlino in nato v živali in človeka. Kadmij je strupen ter povzroča številne neželene učinke. Namen tega raziskovalnega dela je bil zato ugotoviti ali lahko selen (Se), ki je esencialen element tako za živali kot ljudi, preko biofortifikacije rastlin izniči oz. ublaži učinke strupenosti Cd v prehranjevalni verigi. Poskuse smo izvedli na zeleni solati (Lactuca sativa L.) ter na polžih španski lazar (Arion vulgaris). Pri solati smo ugotavljali vplive Cd preko spremljanja rasti, fizioloških in biokemijskih parametrov, kot so fotokemična učinkovitost fotosistema II (FS II) ter analiza fotosinteznih pigmentov. Pri polžih, hranjenih s kontaminirano solato s Cd z dodatkom Se ali brez Se, pa smo spremljali akumulacijo Cd v prebavni žlezi s pomočjo metode ICP-MS (masna spektrometrija z induktivno sklopljeno plazmo). Ugotovili smo, da biofortifikacija solate s Se ni zmanjšala privzema Cd iz zemlje v solato in iz solate v polže. Kadmij je pri visokih koncentracijah ter pri kombinaciji več težkih kovin negativno vplival na fotosintezo solate. Prav tako je kombinacija več težkih kovin negativno vplivala na fotosintezne pigmente. Pri polžih nismo ugotovili povezave med stopnjo rasti, prehranjevanjem ali umrljivostjo ter vsebnostjo Cd v solati. Kadmij se je večinoma kopičil v prebavni žlezi. Pri visokih koncentracijah Cd v solati je Se povečal kopičenje Cd v mišicah polžev, na kopičenje Cd v prebavni žlezi pa ni imel vpliva. Toksičnih vplivov Se na rastline in živali ni bilo.The concentration of heavy metals in the environment are increasing every year due to numerous human activities. This represents a growing threat for human and animal health, with Cd being one of the most problematic metals. Cadmium is a heavy metal, which can be transferred through the food chain very easily due to its high mobility in soil and sediments. The most frequent is transfer from soil to plants and then to animals and humans. Cadmium is toxic and causes numerous side effects. Therefore, the purpose of this research work was to find out whether biofortification of plants with Se can mitigate toxicity of Cd in plants and further in the food chain. Selenium is an essential element for both humans and animals. Experiments were carried out on green lettuce (Lactuca sativa) and Spanish slugs (Arion vulgaris). On the lettuce we determined Cd toxicity by measuring photosynthetic activity of photosystem II and the contents of photosynthetic pigments. In slugs, fed with Cd contaminated lettuce with or without Se, accumulation of Cd in digestive gland was measured with ICP-MS method. We found that biofortification of lettuce with Se did not mitigate the Cd uptake from soil to lettuce and from lettuce to slugs. The combination of several heavy metals and high levels of Cd had negative effect on photosynthesis. Also, a combination of heavy metals had a negative effect on photosynthetic pigments. In slugs we found no correlation between mortality, growth or feeding rate and Cd load in lettuce. Cadmium accumulated predominantly in digestive gland. At high concentrations of Cd in lettuce Se increased Cd accumulation in the snail muscles but had no impact on accumulation of Cd in the digestive gland. We observed no toxic effects of Se on plants or animals

Virtual Screening and Biochemical Testing of Borocycles as Immunoproteasome Inhibitors

Inhibition of the immunoproteasome (iCP) offers new opportunities in the treatment of cancer, autoimmune disorders and neurodegenerative diseases. Inspired by the success of boronic acids as proteasome inhibitors we have complied a virtual library of commercially available 5- and 6-membered borocycles and performed a structure based virtual screening against the chymotrypsin-like (β5i) subunit of the iCP. The top scored docking poses were visually inspected to select compounds for experimental testing. Six compounds with 5-membered ring and another six compounds with 6-membered ring were subjected to biochemical tests. All compounds exhibited detectable inhibitory activity at 100 µM concentration and these are the first reported cyclic boronic acid inhibitors of the iCP. Structural variations including the ring size and the substitution of the borocyles and the substitution pattern of the attached aromatic ring resulted in no major variation of the inhibitory activity. We propose that the evaluation of larger cycling boronic acid libraries is needed to fully elucidate the potential of these structures

Preventive dental care is associated with improved healthcare outcomes and reduced costs for Medicaid members with diabetes

IntroductionPreventive dental services have been associated with improved health outcomes. This study expands on previous observations by examining the relationship between oral healthcare and healthcare outcomes and costs in a publicly insured population with diabetes.MethodsUtilization of dental services, healthcare outcomes and costs were evaluated for New York State Medicaid members with a diagnosis of diabetes mellitus (DM), ages 42 to 64, who were continuously enrolled between July 1, 2012 and June 30, 2015. Utilization of dental services focused on preventive dental care (PDC) and extractions and endodontic treatment (both indicative of advanced dental infection). Data were analyzed using regression models with propensity score weighting to control for potential confounding.ResultsReceipt of PDC was associated with lower utilization rates and costs compared to members who did not access dental services. The most pronounced average cost difference was observed for inpatient admissions at $823 per year for members who had at least one PDC without extraction or endodontic treatment. Each additional PDC visit received was associated with an 11$407 per member. The need for a dental extraction or endodontic therapy was associated with relatively higher rates and costs.ConclusionsThese findings demonstrate an association between PDC and improved healthcare outcome rates and lower average costs among members with DM and suggest a general health benefit associated with the provision of preventive dental care for persons with DM

Targeting cystatin F activation enhances NK cell cytotoxicity in glioblastoma models

IntroductionGlioblastoma (GBM) is a highly invasive brain tumor with limited treatment options and poor prognosis. Natural killer (NK) cells are key effectors of antitumor immunity, capable of eliminating cancer stem-like cells. However, GBM creates an immunosuppressive microenvironment that limits NK cell function. Here, we identify cystatin F as an immunosuppressive factor involved in regulating NK cell granule-mediated cytotoxicity.MethodsWe analyzed cystatin F expression in GBM and its correlation with immune exhaustion markers. NK cell activity was compared between GBM patients and healthy donors. In vitro co-cultures of cystatin F-expressing microglial cells and glioblastoma stem-like cells were used to assess NK cell function. To block cystatin F activation from dimeric to active monomeric form, a small-molecule inhibitor of cathepsin V, the activating protease, was applied.ResultsCystatin F expression correlated with immune exhaustion and suppression markers in GBM. NK cells from patients showed reduced cytotoxicity compared to healthy donors. Co-cultures confirmed that cystatin F-expressing microglia impaired NK cell cytotoxicity, while inhibition of cathepsin V restored NK cell function in standard cytotoxicity assays, 3D spheroids, and microfluidic perfused models.DiscussionThese results indicate that cystatin F mediates NK cell suppression in GBM. Targeting its activation enhances NK cell cytotoxicity, offering a potential strategy to improve NK-based immunotherapy for glioblastoma

Leveraging ligand affinity and properties: discovery of novel benzamide-type cereblon binders for the design of PROTACs

Immunomodulatory imide drugs (IMiDs) such as thalidomide, pomalidomide, and lenalidomide are the most common cereblon (CRBN) recruiters in proteolysis-targeting chimera (PROTAC) design. However, these CRBN ligands induce the degradation of IMiD neosubstrates and are inherently unstable, degrading hydrolytically under moderate conditions. In this work, we simultaneously optimized physiochemical properties, stability, on-target affinity, and off-target neosubstrate modulation features to develop novel nonphthalimide CRBN binders. These efforts led to the discovery of conformationally locked benzamide-type derivatives that replicate the interactions of the natural CRBN degron, exhibit enhanced chemical stability, and display a favorable selectivity profile in terms of neosubstrate recruitment. The utility of the most potent ligands was demonstrated by their transformation into potent degraders of BRD4 and HDAC6 that outperform previously described reference PROTACs. Together with their significantly decreased neomorphic ligase activity on IKZF1/3 and SALL4, these ligands provide opportunities for the design of highly selective and potent chemically inert proximity-inducing compounds

Evaluation of Fragment Libraries and Their Use for the Discovery of Promising Hit Compounds on Enzyme Targets

Odkrivanje novih učinkovin je zapleten in dolgotrajen proces, ki je skozi zgodovino doživel pomemben napredek. Razvil se je od naključnih odkritij, pogosto iz naravnih proizvodov, do sodobnih pristopov, ki so bolj sistematični in temeljijo na rešetanjih kemijskih knjižnic ali racionalnem načrtovanju. V zadnjih desetletjih se je načrtovanje novih učinkovin na osnovi fragmentov izkazalo kot uspešen pristop in dobra alternativa rešetanjem visoke zmogljivosti. Začne se z rešetanjem majhnih, strukturno enostavnih spojin, ki predstavljajo dobra izhodišča za optimizacijo delovanja in povečevanje strukture do spojin vodnic. V doktorski disertaciji smo raziskali potencial rešetanja fragmentov za odkrivanje obetavnih spojin zadetkov na tarčah, ki jih preiskujemo na fakulteti. Pripravili smo knjižnico več kot 1.000 fragmentov in ovrednotili njihovo delovanje z različnimi biokemijskimi testi. Razvili smo delovni tok za temeljito vrednotenje zadetkov, s katerim lahko izločimo lažno pozitivne rezultate rešetanj. Zajema potrditev čistosti in identitete, eksperimentalno vrednotenje topnosti, reaktivnosti in redoks aktivnosti. Dodatno smo potrdili vezavo oz. delovanje obetavnih zadetkov z ortogonalno metodo. Do sedaj znani testi za določanje redoks aktivnosti niso imeli zadostne zmogljivosti ali niso bili primerni za rešetanje raznolikih kemijskih knjižnic. Z razširjenim naborom pozitivnih kontrol smo jih optimizirali in pripravili protokole, s katerimi lahko zaznavamo različne mehanizme redoks aktivnosti. Pokazali smo široko uporabnost testov, in sicer za vrednotenje spojin zadetkov, že znanih bioaktivnih spojin in kemijskih sond, za razvoj kovalentnih fragmentov in spojin z elektrofilnimi bojnimi glavami. Najbolj obetavne rezultate rešetanja knjižnice fragmentov smo dosegli na bakterijskem encimu D-alanin:D-alanin ligaza in podenoti ß5i imunoproteasoma. Poleg tega smo raziskali alternativne pristope k rešetanju fragmentov, vključno z virtualnim rešetanjem in uporabo kemijskih knjižnic kodiranih z DNA. V vseh primerih je bilo vrednotenje zadetkov ozko grlo in ključen korak za nadaljnji uspeh. S širšo analizo rezultatov rešetanja fragmentov na različnih tarčah smo ugotovili, da je treba biti pozoren na spojine s tiazolnim skeletom, ki zaradi reaktivnosti pogosto lahko izkazujejo nespecifično delovanje.The discovery of new drugs is a complex and long-term process that has made significant progress over time. It has evolved from serendipitous discoveries, often of natural products, to modern, more systematic approaches based on chemical library screening or rational design. In recent decades, fragment-based drug design has emerged as a successful approach and an alternative to high-throughput screening. It starts with screening small, structurally simple compounds as potential starting points for optimization into lead compounds. In this dissertation, we investigated the potential of fragment-based drug design for the discovery of promising hit compounds for targets under investigation by our faculty. We assembled a library of over 1,000 fragments and evaluated their activity using various biochemical assays. We have developed a workflow for thorough hit evaluation to rule out false positives from screenings. This includes confirmation of purity and identity, as well as experimental evaluation of aqueous solubility, reactivity, and redox activity. For most promising hits, we confirmed activity or target binding using an orthogonal method. Previously known assays for the determination of redox activity did not have sufficient capacity or were not suitable for testing diverse chemical libraries. Using an expanded set of positive controls, we optimized them and established protocols to detect various mechanisms of redox activity. We demonstrated the broad applicability of reactivity and redox activity assays, particularly for the evaluation of hit compounds, known bioactive compounds and chemical probes, in the development of targeted covalent fragments and compounds with electrophilic warheads. The most promising results in fragment library screening were obtained with the bacterial enzyme D-alanine:D-alanine ligase and the β5i subunit of the immunoproteasome. In addition, we investigated alternative approaches to fragment screening, including virtual screening and the use of DNA-encoded chemical libraries. In all cases, hit evaluation was a bottleneck and a critical step for further success. A broader analysis of fragment screening results on different targets revealed that it is important to pay attention to compounds with a thiazole scaffold, which can often exhibit nonspecific activity due to their reactivity