Acid freezes uricolysis in addition to glycolysis: Utility of fluoride-EDTA-citrate tube in the measurement of uric acid for patients administered rasburicase

Background In samples from patients administered rasburicase, ex vivo uricolysis leads to spuriously low uric acid results. The manufacturer’s recommendation of storing the sample in ice-water until analysis, however, does not fully arrest uricolysis. Since uricase activity is affected by pH and metal chelators, we assessed uricolysis inhibition in sodium fluoride-ethylenediaminetetraacetic acid (EDTA)-citrate sample tube (FC Mix tube, Greiner) used primarily for plasma glucose. Method A serum pool was spiked with rasburicase and uric acid measured at 15, 45, 90, 150, 240 and 1080 min in a lithium heparin tube in ice-water, plain tube at room temperature (RT), EDTA tube at RT, FC Mix tube in ice-water, FC Mix tube at RT and FC Mix tube at RT prepared by dissolving FC Mix in serum. Results The rate of urate decay was lowest in the FC Mix tube independent of temperature, then lithium heparin tube in ice-water, then EDTA tube at RT and highest in the plain tube at RT. Uric acid concentrations in the prepared FC Mix tube at RT and heparin tube in ice-water were, respectively, 98.2% and 93.8% of control values at 90 min, 97.1% and 89.3% of control values at 4 h, and remained higher in the prepared FC Mix tube at all time points. Conclusion NaF-EDTA-citrate mixture largely arrested rasburicase mediated ex vivo uricolysis without the need for sample cooling. We propose that sample tubes containing NaF-EDTA-citrate be used for the measurement of uric acid in patients administered rasburicase.

Design of novel oocyte activation methods: The role of zinc

Oocyte activation occurs at the time of fertilization and is a series of cellular events initiated by intracellular Ca2+ increases. Consequently, oocytes are alleviated from their arrested state in meiotic metaphase II (MII), allowing for the completion of meiosis. Oocyte activation is also an essential step for somatic cell nuclear transfer (SCNT) and an important tool to overcome clinical infertility. Traditional artificial activation methods aim to mimic the intracellular Ca2+ changes which occur during fertilization. Recent studies emphasize the importance of cytoplasmic Zn2+ on oocyte maturation and the completion of meiosis, thus suggesting artificial oocyte activation approaches that are centered around the concentration of available Zn2+in oocytes. Depletion of intracellular Zn2+ in oocytes with heavy metal chelators leads to successful oocyte activation in the absence of cellular Ca2+ changes, indicating that successful oocyte activation does not always depends on intracellular Ca2+ increases. Current findings lead to new approaches to artificially activate mammalian oocytes by reducing available Zn2+ contents, and the approaches improve the outcome of oocyte activation when combined with existing Ca2+ based oocyte activation methods. Here, we review the important role of Ca2+ and Zn2+ in mammalian oocyte activation and development of novel oocyte activation approaches based on Zn2+ availability.

A high-throughput fluorescence polarization assay to discover inhibitors of arenavirus and coronavirus exoribonucleases

Viral exoribonucleases are uncommon in the world of RNA viruses. To date, this activity has been identified only in the Arenaviridae and the Coronaviridae families. These exoribonucleases play important but different roles in both families: for mammarenaviruses the exoribonuclease is involved in the suppression of the host immune response whereas for coronaviruses, exoribonuclease is both involved in a proofreading mechanism ensuring the genetic stability of viral genomes and participating to evasion of the host innate immunity. Because of their key roles, they constitute attractive targets for drug development. Here we present a high-throughput assay using fluorescence polarization to assess the viral exoribonuclease activity and its inhibition. We validate the assay using three different viral enzymes from SARS-CoV-2, lymphocytic choriomeningitis and Machupo viruses. The method is sensitive, robust, amenable to miniaturization (384 well plates) and allowed us to validate the proof-of-concept of the assay by screening a small focused compounds library (23 metal chelators). We also determined the IC50 of one inhibitor common to the three viruses.HighlightsArenaviridae and Coronaviridae viral families share an exoribonuclease activity of common evolutionary originArenaviridae and Coronaviridae exoribonuclease is an attractive target for drug developmentWe present a high-throughput assay in 384 well-plates for the screening of inhibitors using fluorescence polarizationWe validated the assay by screening of a focused library of 23 metal chelators against SARS-CoV-2, Lymphocytic Choriomeningitis virus and Machupo virus exoribonucleasesWe determined the IC50 by fluorescence polarization of one inhibitor common to the three viruses.

The Multiple Potential Biomarkers for Predicting Immunotherapy Response—Finding the Needle in the Haystack

Immune checkpoint inhibitors (ICIs) are being increasingly utilised in a variety of advanced malignancies. Despite promising outcomes in certain patients, the majority will not derive benefit and are at risk of potentially serious immune-related adverse events (irAEs). The development of predictive biomarkers is therefore critical to personalise treatments and improve outcomes. A number of biomarkers have shown promising results, including from tumour (programmed cell death ligand 1 (PD-L1), tumour mutational burden (TMB), stimulator of interferon genes (STING) and apoptosis-associated speck-like protein containing a CARD (ASC)), from blood (peripheral blood mononuclear cells (PBMCs), circulating tumour DNA (ctDNA), exosomes, cytokines and metal chelators) and finally the microbiome.

Involvement of MexS and MexEF-OprN in Resistance to Toxic Ion Chelators in Pseudomonas putida KT2440

Bacteria must be able to cope with harsh environments to survive. In Gram-negative bacteria like Pseudomonas species, resistance-nodulation-division (RND) transporters contribute to this task by pumping toxic compounds out of cells. Previously, we found that the RND system TtgABC of Pseudomonas putida KT2440 confers resistance to toxic metal chelators of the bipyridyl group. Here, we report that the incubation of a ttgB mutant in medium containing 2,2’-bipyridyl generated revertant strains able to grow in the presence of this compound. This trait was related to alterations in the pp_2827 locus (homolog of mexS in Pseudomonas aeruginosa). The deletion and complementation of pp_2827 confirmed the importance of the locus for the revertant phenotype. Furthermore, alteration in the pp_2827 locus stimulated expression of the mexEF-oprN operon encoding an RND efflux pump. Deletion and complementation of mexF confirmed that the latter system can compensate the growth defect of the ttgB mutant in the presence of 2,2’-bipyridyl. To our knowledge, this is the first report on a role of pp_2827 (mexS) in the regulation of mexEF-oprN in P. putida KT2440. The results expand the information about the significance of MexEF-OprN in the stress response of P. putida KT2440 and the mechanisms for coping with bipyridyl toxicity.

Recent Advancements in Pathogenesis, Diagnostics and Treatment of Alzheimer’s Disease

Background: The only conclusive way to diagnose Alzheimer’s is to carry out brain autopsy of the patient’s brain tissue and ascertain whether the subject had Alzheimer’s or any other form of dementia. However, due to the non-feasibility of such methods, to diagnose and conclude the conditions, medical practitioners use tests that examine a patient’s mental ability. Objective: Accurate diagnosis at an early stage is the need of the hour for initiation of therapy. The cause for most Alzheimer’s cases still remains unknown except where genetic distinctions have been observed. Thus, a standard drug regimen ensues in every Alzheimer’s patient, irrespective of the cause, which may not always be beneficial in halting or reversing the disease progression. To provide a better life to such patients by suppressing existing symptoms, early diagnosis, curative therapy, site-specific delivery of drugs, and application of hyphenated methods like artificial intelligence need to be brought into the main field of Alzheimer’s therapeutics. Methods: In this review, we have compiled existing hypotheses to explain the cause of the disease, and highlighted gene therapy, immunotherapy, peptidomimetics, metal chelators, probiotics and quantum dots as advancements in the existing strategies to manage Alzheimer’s. Conclusion: Biomarkers, brain-imaging, and theranostics, along with artificial intelligence, are understood to be the future of the management of Alzheimer’s.

Interaction of 2,6,7-Trihydroxy-Xanthene-3-Ones with Iron and Copper, and Biological Effect of the Most Active Derivative on Breast Cancer Cells and Erythrocytes

Metal chelators can be potentially employed in the treatment of various diseases, ranging from metal overload to neoplastic conditions. Some xanthene derivatives were previously reported to complex metals. Thus, in a search for a novel iron or copper chelator, a series of 9-(substituted phenyl)-2,6,7-trihydroxy-xanthene-3-ones was tested using a competitive spectrophotometric approach. The most promising compound was evaluated in biological models (breast adenocarcinoma cell lines and erythrocytes). In general, substitution of the benzene ring in position 9 had a relatively low effect on the chelation. Only the trifluoromethyl substitution resulted in stronger chelation, probably via a positive effect on solvation. All compounds chelated iron, but their copper-chelating effect was only minimal, since it was no longer observed under highly competitive conditions. Interestingly, all compounds reduced both iron and copper. Additional experiments showed that the trifluoromethyl derivative protected erythrocytes and even cancer cells against excess copper. In summary, the tested compounds are iron chelators, which are also capable of reducing iron/copper, but the copper-reducing effect is not associated with increased copper toxicity.

Recent advances and current perspectives in treatment of Alzheimer’s disease

Dementia is a disorder which is associated with disruption of cerebral neurons, resulting in its characteristic symptomatology. Acetylcholine neurotransmitter is found to be significant for processing memory and learning. However it is diminished in both concentration and function in patients with Alzheimer disease. Nootropics are the drugs which is used to improve memory and learning by acting as AChEI (Acetyl cholineesterase inhibitors). Cognitive enhancers include drugs interacting with receptors (e.g. NMDA receptor antagonist: memantine), Enzymes (e.g. AChE inhibitors: tacrine, donepezil, galantamine), Antioxidants (e.g. resveratrol, curcumin, and acetyl-L-carnitine), Metal chelators (e.g. calcium and zinc chelator: DP-b99), Vaccines, Monoclonal antibodies (e.g. A beta-Amyloid: solanezumab under Phase III clinical trial). Apart from the pharmacological approaches, supplementation of a healthy diet and healthy physical & mental lifestyle impact cognitive research in the future. There is no remedy for AD. Contemporary treatments just relive the behaviourial symptoms.Treatment centers around making a superior personal satisfaction for the individuals with Alzheimer infection. As of late, undifferentiated cell innovation (stem cell technology), and Nanotechnology has given new bits of knowledge into the treatment of Alzheimer's disease. In this review, we talk about current indicative medicines and future difficulties for new potential illness altering treatments.