Search

Ameloblastin is a protein in biomineralization of tooth enamel. However recent results indicate that this is probably not its only role in an organism. Enamel matrix formation represents a complex process enabled via specific crosslinking of two proteins – the most abundant amelogenin and the ameloblastin (AMBN). The human AMBN (hAMBN) gene possesses 13 protein coding exons with alternatively spliced transcripts and the longest isoform about 447 amino acid residues. It has been described that AMBN molecules in vitro assemble into oligomers via a sequence encoded by exon 5. Enamel is formed by the processing of enamel proteins by two specific proteases - enamelysin (MMP-20) and kallikrein 4 (KLK-4). The scaffold made of AMEL and non-amelogenin proteins is cleaved and removed from the developed tooth enamel. The hAMBN is expressed in two isoforms (ISO I and II), which could lead to their different utilization determined by distinct proteolytic profiles. In this study, we compared proteolytic profiles of both isoforms of hAMBN expressed in E. coli after proteolysis by MMP-20, KLK-4, and their 1:2 mixture. Proteolysis products were analysed and cleavage sites were identified by mass spectrometry. The proteolytic profiles of two AMBN isoforms showed different results, although we have to determine that the analysed AMBN was not post-translationally modified as expressed in prokaryotic cells. These results may lead to the suggestion of potentially divergent roles of AMBN isoforms cleavage products in various cell signalling pathways such as calcium buffering or signalling cascades.

Immunoproteomic analysis was applied to study the immunoreactivity of serum samples collected at different time points from a laboratory assistant accidentally infected with highly virulent strain of Francisella tularensis subsp. tularensis. Immunoblotting showed that the spectrum of F. tularensis antigens recognized specifically by immune sera remained with the exception for 1 antigen stable for up to 16 years after infection. Using immunoproteomics approach 10 immunoreactive antigens were successfully identified. Several new immunogenic F. tularensis proteins were described for the first time. [ABSTRACT FROM AUTHOR]

Michael Hubalek, Christine Brantner, Christian MarthBrustgesundheitzentrum Tirol, Department of Obstetrics and Gynecology, Innsbruck Medical University, Innsbruck, AustriaAbstract: Pertuzumab, a humanized monoclonal antibody to the HER2 receptor, represents a promising new anti-HER2 agent with a novel mechanism of action targeting the inhibition of HER2 dimerization. Nonclinical and clinical data to date indicate that pertuzumab provides a broader HER2 blockade through the inhibition of HER2 heterodimerization. In preclinical experiments, pertuzumab has demonstrated superior antitumor effects when combined with other anti-HER2 treatments such as trastuzumab, compared to when used as monotherapy. Trastuzumab and pertuzumab monoclonal antibodies bind to distinct epitopes on the HER2 receptor without competing with each other, resulting in distinctive mechanisms for disrupting HER2 signaling. These mechanisms are complementary and result in augmented therapeutic efficacy when pertuzumab and trastuzumab are given in combination. Clinically, pertuzumab may have optimal therapeutic effects when given to patients with HER2-positive cancers, in combination with trastuzumab. This observation is supported by recent clinical trials in the metastatic as well as neoadjuvant setting. Intravenous pertuzumab had an acceptable tolerability profile when added to trastuzumab and chemotherapy. This overview will review recent advances in the clinical development of this HER2-targeted therapy.Keywords: HER2, breast cancer, pertuzumab, trastuzumab

Abstract Background Current studies on adherence to endocrine therapy in breast cancer patients suffer from methodological limitations due to a lack of well-validated methods for assessing adherence. There is no gold standard for measuring adherence. The aim of our study was to compare four different approaches for evaluating adherence to anastrozole therapy for breast cancer with regard to concordance between methods. Methods Outpatients with early breast cancer treated with anastrozole completed the multi-method assessment of adherence. We implemented a self-report scale (the Simplified Medication Adherence Questionnaire), physician- ratings, refill records and determination of anastrozole serum concentration. Results Comparison of the four approaches using Spearman rank correlation revealed poor concordance across all methods reflecting weak correlations of 0.2-0.4. Considering this data incomparability across methods, we still observed high adherence rates of 78%-98% across measures. Conclusion Our findings contribute to the growing body of knowledge on the impact that methodological aspects exert on the results of adherence measurement in breast cancer patients receiving endocrine treatment. Our findings suggest that the development and validation of instruments specific to patients receiving endocrine agents is imperative in order to arrive at a more accurate assessment and to subsequently obtain more precise estimates of adherence rates in this patient population.

Abstract Background In advanced breast cancer, multiple sequential lines of treatments are frequently applied. Pegylated liposomal doxorubicin (PLD) has a favourable toxicity profile and can be used in first or higher lines of therapy. PLD has demonstrated response activity even after prior anthracycline exposure. Methods 129 consecutive patients with advanced breast cancer, of whom the majority had been massively pretreated, received PLD as monotherapy within licensed approval, for which efficacy and toxicities were documented. Results In a routine therapy setting, PLD was administered in a slightly reduced dose (median, 40 mg/m2 per cycle). Response rate (complete and partial remission) was 26%, and stable disease was observed in 19% of patients. Progression-free (PFS) and overall survival (OS) were 5.8 months and 14.2 months, respectively. There was no difference in terms of response and PFS, no matter if patients had already received anthracycline treatment. Interestingly, PFS proved similar regardless whether PLD was administered as palliative therapy in first, second or third line. Furthermore, PFS and OS were similar in patients with response or stable disease, underscoring the view that disease stabilization is associated with a profound clinical benefit. The most common side effects reported were palmar-plantar erythrodysesthesia (17%), exanthema (14%) and mucositis (12%). Conclusions Efficacy and toxicity data in these "real life" patients permit the conclusion that PLD is a valuable option in the treatment of advanced breast cancer even in heavily pretreated patients.

Abstract Background Both BALB/c mice and common voles (Microtus arvalis) are considered highly susceptible to tularemia. However, the common vole is reported to harbour Francisella tularensis in European habitats as well as to survive longer with chronic shedding of the bacterium. The purpose of the present study was to compare the response of these two rodents to a wild Francisella tularensis subsp. holarctica strain infection. Methods Rodents were evaluated for differences in the total antioxidant capacity derived from low-molecular-weight antioxidants, biochemistry including lipid metabolism, tissue bacterial burdens and histopathology following experimental intraperitoneal infection with 160 colony forming units (CFU) pro toto. Results Bacterial burdens in common voles started to develop later post-exposure and amounted to lower levels than in BALB/c mice. Elevation of liver function enzymes was more pronounced in mice than common voles and there were marked differences in lipid metabolism in the course of tularemia in these two species. Hypertriglyceridemia and hypercholesterolemia developed in mice, while physiologically higher levels of triglycerides and cholesterol showed a decreasing tendency in common voles. On the other hand, the total plasma antioxidant capacity gradually dropped to 81.5% in mice on day 5 post-infection, while it increased to 130% on day 6 post-infection in common voles. Significant correlations between tissue bacterial burdens and several biochemical parameters were found. Conclusion As differences in lipid metabolism and the total antioxidant capacity of highly susceptible rodent species were demonstrated, the role of triglycerides, cholesterol and antioxidants in tularemic sepsis should be further investigated.

In mycobacteria, σ A is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes. Our knowledge about this factor in mycobacteria is limited. Here, we performed an unbiased search for interacting partners of Mycobacterium smegmatis σ A . The search revealed a number of proteins; prominent among them was MoaB2. The σ A -MoaB2 interaction was validated and characterized by several approaches, revealing that it likely does not require RNAP and is specific, as alternative σ factors ( e.g. , closely related σ B ) do not interact with MoaB2. The structure of MoaB2 was solved by X-ray crystallography. By immunoprecipitation and nuclear magnetic resonance, the unique, unstructured N-terminal domain of σ A was identified to play a role in the σ A -MoaB2 interaction. Functional experiments then showed that MoaB2 inhibits σ A -dependent (but not σ B -dependent) transcription and may increase the stability of σ A in the cell. We propose that MoaB2, by sequestering σ A , has a potential to modulate gene expression. In summary, this study has uncovered a new binding partner of mycobacterial σ A , paving the way for future investigation of this phenomenon.IMPORTANCEMycobacteria cause serious human diseases such as tuberculosis and leprosy. The mycobacterial transcription machinery is unique, containing transcription factors such as RbpA, CarD, and the RNA polymerase (RNAP) core-interacting small RNA Ms1. Here, we extend our knowledge of the mycobacterial transcription apparatus by identifying MoaB2 as an interacting partner of σ A , the primary sigma factor, and characterize its effects on transcription and σ A stability. This information expands our knowledge of interacting partners of subunits of mycobacterial RNAP, providing opportunities for future development of antimycobacterial compounds., Competing Interests: The authors declare no conflict of interest.

Mycobacterial HelD is a transcription factor that recycles stalled RNAP by dissociating it from nucleic acids and, if present, from the antibiotic rifampicin. The rescued RNAP, however, must disengage from HelD to participate in subsequent rounds of transcription. The mechanism of release is unknown. We show that HelD from Mycobacterium smegmatis forms a complex with RNAP associated with the primary sigma factor σ A and transcription factor RbpA but not CarD. We solve several structures of RNAP-σ A -RbpA-HelD without and with promoter DNA. These snapshots capture HelD during transcription initiation, describing mechanistic aspects of HelD release from RNAP and its protective effect against rifampicin. Biochemical evidence supports these findings, defines the role of ATP binding and hydrolysis by HelD in the process, and confirms the rifampicin-protective effect of HelD. Collectively, these results show that when HelD is present during transcription initiation, the process is protected from rifampicin until the last possible moment., (© 2024. The Author(s).)

In this study, we present a high-resolution dataset and bioinformatic analysis of the proteome of Bacillus subtilis 168 trp+ (BSB1) during germination and spore outgrowth. Samples were collected at 14 different time points (ranging from 0 to 130 min) in three biological replicates after spore inoculation into germination medium. A total of 2191 proteins were identified and categorized based on their expression kinetics. We observed four distinct clusters that were analyzed for functional categories and KEGG pathways annotations. The examination of newly synthesized proteins between successive time points revealed significant changes, particularly within the first 50 min. The dataset provides an information base that can be used for modeling purposes and inspire the design of new experiments., (© 2024 The Author(s). PROTEOMICS published by Wiley‐VCH GmbH.)

For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles., Competing Interests: MČ, JS, JP, AP, LP, ZB, MH, TR No competing interests declared, (© 2024, Častorálová, Sýs, Prchal et al.)

σ factors are considered as positive regulators of gene expression. Here we reveal the opposite, inhibitory role of these proteins. We used a combination of molecular biology methods and computational modeling to analyze the regulatory activity of the extracytoplasmic σ E factor from Streptomyces coelicolor. The direct activator/repressor function of σ E was then explored by experimental analysis of selected promoter regions in vivo. Additionally, the σ E interactome was defined. Taken together, the results characterize σ E , its regulation, regulon, and suggest its direct inhibitory function (as a repressor) in gene expression, a phenomenon that may be common also to other σ factors and organisms., (© 2024. The Author(s).)

The inherent diversity of approaches in proteomics research has led to a wide range of software solutions for data analysis. These software solutions encompass multiple tools, each employing different algorithms for various tasks such as peptide-spectrum matching, protein inference, quantification, statistical analysis, and visualization. To enable an unbiased comparison of commonly used bottom-up label-free proteomics workflows, we introduce WOMBAT-P, a versatile platform designed for automated benchmarking and comparison. WOMBAT-P simplifies the processing of public data by utilizing the sample and data relationship format for proteomics (SDRF-Proteomics) as input. This feature streamlines the analysis of annotated local or public ProteomeXchange data sets, promoting efficient comparisons among diverse outputs. Through an evaluation using experimental ground truth data and a realistic biological data set, we uncover significant disparities and a limited overlap in the quantified proteins. WOMBAT-P not only enables rapid execution and seamless comparison of workflows but also provides valuable insights into the capabilities of different software solutions. These benchmarking metrics are a valuable resource for researchers in selecting the most suitable workflow for their specific data sets. The modular architecture of WOMBAT-P promotes extensibility and customization. The software is available at https://github.com/wombat-p/WOMBAT-Pipelines.

Cocaine- and amphetamine-regulated transcript peptide (CARTp) is an anorexigenic neuropeptide whose receptor is undisclosed. Previously, we reported the specific binding of CART(61-102) to pheochromocytoma PC12 cells, where CART(61-102) affinity and the number of binding sites per cell corresponded to ligand-receptor binding. Recently, Yosten et al. designated orphan GPR160 as the CARTp receptor, because the GPR160 antibody abolished neuropathic pain and anorexigenic effects induced by CART(55-102) and exogenous CART(55-102) coimmunoprecipitated with GPR160 in KATOIII cells. As no direct evidence that CARTp is a ligand for GPR160 has been described, we decided to verify this hypothesis by testing CARTp affinity to the GPR160 receptor. We investigated the GPR160 expression in PC12 cells since it is cell line known to specifically bind CARTp. Moreover, we examined the specific CARTp binding in THP1 cells, with high endogenous GPR160 expression and GPR160-transfected cell lines U2OS and U-251 MG. In PC12 cells, the GPR160 antibody did not compete for specific binding with 125 I-CART(61-102) or with 125 I-CART(55-102), and GPR160 mRNA expression and GPR160 immunoreactivity were not detected. Moreover, THP1 cells did not show any 125 I-CART(61-102) or 125 I-CART(55-102) specific binding despite GPR160 detection by fluorescent immunocytochemistry (ICC). Finally, no 125 I-CART(61-102) or 125 I-CART(55-102) specific binding in the GPR160-transfected cell lines U2OS and U-251 MG, selected due to their negligible endogenous expression of GPR160, was detected, despite the detection of GPR160 by fluorescent ICC. Our binding studies clearly demonstrated that GPR160 cannot be a receptor for CARTp. Further studies are needed to identify true CARTp receptors., Competing Interests: Declaration of competing interest None., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

A typical bottom-up proteomic workflow comprises sample digestion with trypsin, separation of the hydrolysate using reversed-phase HPLC, and detection of peptides via electrospray ionization (ESI) tandem mass spectrometry. Despite the advantages and wide usage of protein identification and quantification, the procedure has limitations. Some domains or parts of the proteins may remain inadequately described due to inefficient detection of certain peptides. This study presents an alternative approach based on sample acetylation and mass spectrometry with atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). These ionizations allowed for improved detection of acetylated peptides obtained via chymotrypsin or glutamyl peptidase I (Glu-C) digestion. APCI and APPI spectra of acetylated peptides often provided sequence information already at the full scan level, while fragmentation spectra of protonated molecules and sodium adducts were easy to interpret. As demonstrated for bovine serum albumin, acetylation improved proteomic analysis. Compared to ESI, gas-phase ionizations APCI and APPI made it possible to detect more peptides and provide better sequence coverages in most cases. Importantly, APCI and APPI detected many peptides which passed unnoticed in the ESI source. Therefore, analytical methods based on chymotrypsin or Glu-C digestion, acetylation, and APPI or APCI provide data complementary to classical bottom-up proteomics.

Pollen germination and subsequent pollen tube elongation are essential for successful land plant reproduction. These processes are achieved through well-documented activation of membrane trafficking and cell metabolism. Despite this, our knowledge of the dynamics of cellular phospholipids remains scarce. Here we present the turnover of the glycerolipid composition during the establishment of cell polarity and elongation processes in tobacco pollen and show the lipid composition of pollen plasma membrane-enriched fraction for the first time. To achieve this, we have combined several techniques, such as lipidomics, plasma membrane isolation, and live-cell microscopy, and performed a study with different time points during the pollen germination and pollen tube growth. Our results showed that tobacco pollen tubes undergo substantial changes in their whole-cell lipid composition during the pollen germination and growth, finding differences in most of the glycerolipids analyzed. Notably, while lysophospholipid levels decrease during germination and growth, phosphatidic acid increases significantly at cell polarity establishment and continues with similar abundance in cell elongation. We corroborated these findings by measuring several phospholipase activities in situ. We also observed that lysophospholipids and phosphatidic acid are more abundant in the plasma membrane-enriched fraction than that in the whole cell. Our results support the important role for the phosphatidic acid in the establishment and maintenance of cellular polarity in tobacco pollen tubes and indicate that plasma membrane lysophospholipids may be involved in pollen germination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Serrano, Pejchar, Soukupová, Hubálek and Potocký.)

Sturgeons are ancient fish exhibiting unique genome plasticity and a high tendency to produce spontaneously autopolyploid genome states. The temperature profiles of the rivers in which sturgeon live and reproduce have been severely altered by human intervention, and the effect of global warming is expected to cause further temperature shifts, which may be detrimental for early developmental stages with narrow windows of thermal tolerance. The comparison of the performance of diploid and autopolyploid sturgeon kept at unfavourable temperatures contributes to scientific knowledge of the effects of polyploid genome states on organisms and can shed light on the ability of polyploids to cope with human-induced alterations to natural conditions. Using the sterlet Acipenser ruthenus as a model species, we carried out conventional artificial fertilization, as well as the induction of the second polar body retention (SPBR), of the first mitotic division suppression (FMDS) and of the second polar body retention followed by the first mitotic division suppression (SPBR+FMDS). Two experiments were conducted to evaluate the effect of polyploidy on two basic performance parameters, survival and growth. In Experiment 1, fish belonging to untreated, SPBR-, FMDS- and SPBR+FMDS-induced groups were kept at 10, 16 and 20°C from the neurula stage until the end of endogenous feeding. In Experiment 2, larvae from the untreated and SPBR-induced groups were reared at 10, 16 and 20°C after their endogenous feeding transition for 3 weeks. Based on our findings, we report that the embryos, prelarvae and larvae of triploid A. ruthenus do not differ from diploids in their ability to survive, grow and develop under suboptimal temperature conditions, while the survival of tetraploids was significantly reduced even at the optimal temperature and even more so at temperatures far from the optimum. This was also the case in the 2n/4n mosaics observed in FMDS-induced group. Thus, we assume that in tetraploid and 2n/4n individuals, the limits of thermal tolerance are closer to the optimum than in diploids. We also conclude that the hexaploid genome state is probably lethal in A. ruthenus since none of the hexaploids or 3n/6n mosaics arising from the SPBR+FMDS induction survived the prelarval period., (© 2022 Fisheries Society of the British Isles.)

Hepatitis B virus uses e antigen (HBe), which is dispensable for virus infectivity, to modulate host immune responses and achieve viral persistence in human hepatocytes. The HBe precursor (p25) is directed to the endoplasmic reticulum (ER), where cleavage of the signal peptide (sp) gives rise to the first processing product, p22. P22 can be retro-translocated back to the cytosol or enter the secretory pathway and undergo a second cleavage event, resulting in secreted p17 (HBe). Here, we report that translocation of p25 to the ER is promoted by translocon-associated protein complex. We have found that p25 is not completely translocated into the ER; a fraction of p25 is phosphorylated and remains in the cytoplasm and nucleus. Within the p25 sp sequence, we have identified three cysteine residues that control the efficiency of sp cleavage and contribute to proper subcellular distribution of the precore pool., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Here we developed a powerful tool for comprehensive data collection and mapping of molecular and elemental signatures in the Melanoma-bearing Libechov Minipig (MeLiM) model. The combination of different mass spectrometric methods allowed for detail investigation of specific melanoma markers and elements and their spatial distribution in tissue sections. MALDI-MSI combined with HPLC-MS/MS analyses resulted in identification of seven specific proteins, S100A12, CD163, MMP-2, galectin-1, tenascin, resistin and PCNA that were presented in the melanoma signatures. Furthermore, the ICP-MS method allowed for spatial detection of zinc, calcium, copper, and iron elements linked with the allocation of the specific binding proteins., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Glyoxal-linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC click reaction of 4-azidophenylglyoxal or a Sonogashira reaction of 4-bromophenylglyoxal with 5-ethynyl-dUMP or -dUTP. The triphosphates were used as substrates for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The glyoxal-linked nucleotides reacted with arginine-containing peptides to form stable imizadolone-linked conjugates. This reactive glyoxal modification in DNA was used for efficient bioconjugations and crosslinking with Arg-containing peptides or proteins (e. g., histones) and was found to be more reactive than previously reported 1,3-diketone-linked DNA probes., (© 2022 Wiley-VCH GmbH.)

Galectin-3 plays a crucial role in cancerogenesis; its targeting is a prospective pathway in cancer diagnostics and therapy. Multivalent presentation of glycans was shown to strongly increase the affinity of glycoconjugates to galectin-3. Further strengthening of interaction with galectin-3 may be accomplished using artificial glycomimetics with apt aryl substitutions. We established a new, as yet undescribed chemoenzymatic method to produce selective C-3-substituted N,N'-diacetyllactosamine glycomimetics and coupled them to human serum albumin. From a library of enzymes, only β-N-acetylhexosaminidase from Talaromyces flavus was able to efficiently synthesize the C-3-propargylated disaccharide. Various aryl residues were attached to the functionalized N,N'-diacetyllactosamine via click chemistry to assess the impact of the aromatic substitution. In ELISA-type assays with galectin-3, free glycomimetics exhibited up to 43-fold stronger inhibitory potency to Gal-3 than the lactose standard. Coupling to human serum albumin afforded multivalent neo-glycoproteins with up to 4209-fold increased inhibitory potency per glycan compared to the monovalent lactose standard. Surface plasmon resonance brought further information on the kinetics of galectin-3 inhibition. The potential of prepared neo-glycoproteins to target galectin-3 was demonstrated on colorectal adenocarcinoma DLD-1 cells. We investigated the uptake of neo-glycoproteins into cells and observed limited non-specific transport into the cytoplasm. Therefore, neo-glycoproteins primarily act as efficient scavengers of exogenous galectin-3 of cancer cells, inhibiting its interaction with the cell surface, and protecting T-lymphocytes against galectin-3-induced apoptosis. The present neo-glycoproteins combine the advantage of a straightforward synthesis, selectivity, non-toxicity, and high efficiency for targeting exogenous galectin-3, with possible application in the immunomodulatory treatment of galectin-3-overexpressing cancers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones)., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Flow cytometry is an effective and widely used tool for determination of ploidy in fish, but it is not always possible to access the fresh samples for analysis. We investigated the potential for extended storage of fish tissue with sterlet and tench as representative species of Chondrostei and Teleostei, using blood and fin of subadult/adult specimens and tail of larvae. Thirteen procedures for extending storage, selected for rapidity and simplicity in both field and laboratory conditions, were tested for each tissue sample. Flow cytometry was applied to fresh tissue immediately after sampling and to tissue subjected to experimental protocols, always along with species-specific standard, after 1, 5, and 10 days storage at 0-4°C or freezing at -80°C. The fluorochrome 4',6-diamidine-2'-phenylindole dihydrochloride was used with excitation/emission maximum 358/461 nm. Based on the measurability of stored samples, evaluation of directly measured coefficients of variation of their DNA peaks and the changes in fluorescence intensity compared to fresh tissue, optimal procedures for extended storage of the selected tissue types of the model species are suggested. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry., (© 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.)

The formation of a G-quadruplex motif in the promoter region of the c-MYC protooncogene prevents its expression. Accordingly, G-quadruplex stabilization by a suitable ligand may be a viable approach for anticancer therapy. In our study, we used the 4-(4-methylpiperazin-1-yl)aniline molecule, previously identified as a fragment library screen hit, as a template for the SAR-guided design of a new small library of clickable fragments and subjected them to click reactions, including kinetic target-guided synthesis in the presence of a G-quadruplex forming oligonucleotide Pu24. We tested the clickable fragments and products of click reactions for their G-quadruplex stabilizing activity and determined their mode of binding to the c-MYC G-quadruplex by NMR spectroscopy. The enhanced stabilizing potency of click products in biology assays (FRET, Polymerase extension assay) matched the increased yields of in situ click reactions. In conclusion, we identified the newly synthesized click products of bis-amino derivatives of 4-(4-methylpiperazin-1-yl)aniline as potent stabilizers of c-MYC G-quadruplex, and their further evolution may lead to the development of an efficient tool for cancer treatment., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Hepatitis B virus (HBV) core protein (HBc) plays many roles in the HBV life cycle, such as regulation of transcription, RNA encapsidation, reverse transcription, and viral release. To accomplish these functions, HBc interacts with many host proteins and undergoes different post-translational modifications (PTMs). One of the most common PTMs is ubiquitination, which was shown to change the function, stability, and intracellular localization of different viral proteins, but the role of HBc ubiquitination in the HBV life cycle remains unknown. Here, we found that HBc protein is post-translationally modified through K29-linked ubiquitination. We performed a series of co-immunoprecipitation experiments with wild-type HBc, lysine to arginine HBc mutants and wild-type ubiquitin, single lysine to arginine ubiquitin mutants, or single ubiquitin-accepting lysine constructs. We observed that HBc protein could be modified by ubiquitination in transfected as well as infected hepatoma cells. In addition, ubiquitination predominantly occurred on HBc lysine 7 and the preferred ubiquitin chain linkage was through ubiquitin-K29. Mass spectrometry (MS) analyses detected ubiquitin protein ligase E3 component N-recognin 5 (UBR5) as a potential E3 ubiquitin ligase involved in K29-linked ubiquitination. These findings emphasize that ubiquitination of HBc may play an important role in HBV life cycle.