In Vitro Properties and Pharmacokinetics of Temporarily PEGylated Onc72 Prodrugs.

The favorable properties of antimicrobial peptides (AMPs) to rapidly kill pathogens are often limited by unfavorable pharmacokinetics due to fast degradation and renal clearance rates. Here, a prodrug strategy linking proline-rich AMP Onc72 to polyethylene glycol (PEGs) with average molecular weights of 5 and 20 kDa via a peptide linker containing a protease cleavage site is tested for the first time in vivo. Onc72 is released from these 5k- and 20k-prodrugs in mouse serum with half-life times (t1/2 ) of 8 and 14 h, respectively. Importantly, PEGylation protects Onc72 from proteolytic degradation providing a prolonged release of Onc72, balancing the degradation of free Onc72, and leading to relatively stable Onc72 concentrations and high antibacterial activities. The prodrugs are not hemolytic on human erythrocytes and show only slight cytotoxic effects on human cell lines indicating promising safety margins. When administered subcutaneously to female CD-1 mice, the prodrugs elimination t1/2 are 66 min and ≈5.5 h, respectively, compared to 43 min of free Onc72. The maximal Onc72 plasma levels are obtained ≈1 and ≈8 h postadministration, respectively. In conclusion, the prodrugs provide extended elimination t1/2 and a constant release of Onc72 in mice, potentially limiting adverse effects and increasing efficacy.

Gas Phase Reactivity of [Mo6X14]2- Dianions (X = Cl - I).

We investigate collision-induced dissociation (CID) of [Mo6X14]2- (X = Cl, Br, I) and the reactivity of fragment ions of these precursors with background gases. Ion mobility measurements and theoretical calculations provide structural information for some of the observed ions. Sequential losses of MoX2 units dominate the dissociation pathways of [Mo6Cl14]2-. Meanwhile, loss of X radicals is the main channel for X = Br and I. Ion mobility measurements and computational investigations indicate minor structural changes in the octahedral Mo6 unit for [Mo6Im]- (m = 6-13) fragments. We observe that mass spectra obtained using CID substantially vary among mass spectrometers: Specifically, ions with molecular formula [Mo6Xm(O2)n]- (X = Br and I) are observed as dominant species produced through reactions with O2 in several mass spectrometers, but also adduct free fragment ions were observed in other instruments, depending on the background conditions. Ion-trap fragmentation combined with theoretical investigations indicates that spontaneous losses of X radicals occur upon binding of O2 to [Mo6Im]- fragments (m ≤ 12). Theoretical investigations indicate that both oxygen atoms are bound to the vacant sites of the Mo6 units. This study opens up a new vista to generate and study a large variety of hexanuclear Mo6Xm(O2)n anions.

Evaluating Peptide Fragment Ion Detection Using Traveling Wave Ion Mobility Spectrometry with Signal-Enhanced MSE (SEMSE).

The inherent poor sampling of fragment ions in time-of-flight mass analyzers was recently improved for data-dependent acquisition (DDA) by considering their drift times in traveling wave ion mobility spectrometry (TWIMS). Here, we extend this TWIMS-DDA approach to the data-independent acquisition (DIA) mode MSE to improve the signal intensities of fragment ions by providing improved ion beam sampling efficiency, which we termed therefore signal-enhanced MSE (SEMSE). The theoretical expectation that SEMSE improves the number of identified peptides, the number of quantifiable peptides, and the lower limit of quantitation in wideband DIA was evaluated on an electrospray ionisation-ion mobility spectrometry-quadrupole-time-of-flight-MS (ESI-IMS-Q-TOF-MS) (Synapt G2-Si) in comparison to five established TWIMS-DDA and TWIMS-MSE methods with respect to the number of peptide identifications, the spectral quality of supporting peptide spectra matches, and (most importantly) fragment ion signal sensitivity. A comparison of the fragment signals clearly indicated that SEMSE provides 6.8- to 11.5-fold larger peak areas than established MSE techniques. While this clearly shows the advantages of SEMSE, the inherent limitations of the current software tools do not allow using all benefits in routine analyses. As the simultaneous fragmentation of co-eluting peptides limited peptide identification, DDA and MSE data sets were integrated using Skyline.

Sensitive and specific serological ELISA for the detection of SARS-CoV-2 infections.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the worldwide coronavirus disease 2019 (COVID-19) pandemic. Serological assays for the detection of SARS-CoV-2 infections are important to understand the immune response in patients and to obtain epidemiological data about the number of infected people, especially to identify asymptomatic persons not aware of a past infection. METHODS: We recombinantly produced SARS-CoV-2 nucleocapsid (N)-protein in Escherichia coli. We used the purified protein to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV-2 specific antibodies. This ELISA method was optimized and validated with serum samples collected from 113 patients with RT-PCR-confirmed SARS-CoV-2 infections including hospitalized COVID-19 patients and 1500 control sera mostly collected before 2015 with different clinical background. RESULTS: The optimized N-protein-ELISA provided a sensitivity of 89.7% (n = 68) for samples collected from patients with confirmed SARS-CoV-2 infections and mild to severe symptoms more than 14 days after symptom onset or a positive PCR test. The antibody levels remained low for serum samples collected in the first six days (n = 23) and increased in the second week (n = 22) post symptom onset or PCR confirmation. At this early phase, the ELISA provided a sensitivity of 39.1% and 86.4%, respectively, reflecting the time of an IgG immune response against pathogens. The assay specificity was 99.3% (n = 1500; 95% CI 0.995-0.999). Serum samples from persons with confirmed antibody titers against human immunodeficiency viruses 1/2, parvovirus B19, hepatitis A/B virus, cytomegalovirus, Epstein Barr virus, and herpes simplex virus were tested negative. CONCLUSIONS: We conclude that the N-protein-based ELISA developed here is well suited for the sensitive and specific serological detection of SARS-CoV-2 specific IgG antibodies in human serum for symptomatic infections. It may also prove useful to identify previous SARS-CoV-2 infections in vaccinated people, as all currently approved vaccines rely on the SARS-CoV-2 spike (S-) protein.

Identification of Disease-Associated Cryptococcal Proteins Reactive With Serum IgG From Cryptococcal Meningitis Patients.

Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from C. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Rα-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anti-cryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines pre-existing anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons.

Sex-dependent dynamics of metabolism in primary mouse hepatocytes.

The liver is one of the most sexually dimorphic organs. The hepatic metabolic pathways that are subject to sexual dimorphism include xenobiotic, amino acid and lipid metabolism. Non-alcoholic fatty liver disease and hepatocellular carcinoma are among diseases with sex-dependent prevalence, progression and outcome. Although male and female livers differ in their abilities to metabolize foreign compounds, including drugs, sex-dependent treatment and pharmacological dynamics are rarely applied in all relevant cases. Therefore, it is important to consider hepatic sexual dimorphism when developing new treatment strategies and to understand the underlying mechanisms in model systems. We isolated primary hepatocytes from male and female C57BL6/N mice and examined the sex-dependent transcriptome, proteome and extracellular metabolome parameters in the course of culturing them for 96 h. The sex-specific gene expression of the general xenobiotic pathway altered and the female-specific expression of Cyp2b13 and Cyp2b9 was significantly reduced during culture. Sex-dependent differences of several signaling pathways increased, including genes related to serotonin and melatonin degradation. Furthermore, the ratios of male and female gene expression were inversed for other pathways, such as amino acid degradation, beta-oxidation, androgen signaling and hepatic steatosis. Because the primary hepatocytes were cultivated without the influence of known regulators of sexual dimorphism, these results suggest currently unknown modulatory mechanisms of sexual dimorphism in vitro. The large sex-dependent differences in the regulation and dynamics of drug metabolism observed during cultivation can have an immense influence on the evaluation of pharmacodynamic processes when conducting initial preclinical trials to investigate potential new drugs.

Hybrid Peptides Based on α-Aminoxy Acids as Antimicrobial and Anticancer Foldamers.

α-Aminoxy peptides represent an interesting group of peptidomimetics with high proteolytic stability and the ability to fold into specific, predictable secondary structures. Here, we present a series of hybrid peptides consisting of α-aminoxy acids and α-amino acids with cationic and aromatic, hydrophobic side chains in an alternating manner synthesized using an efficient protocol that combines solution- and solid-phase synthesis. 2D ROESY experiments with a representative hexamer suggested the presence of a 7/8 helical conformation in solution. Biological evaluation revealed a significant impact of the peptide chain length and the N-terminal cap on the antimicrobial and anticancer properties of this series of hybrid peptides. The Fmoc-capped peptide 6e displayed the most potent antimicrobial activity against a panel of Gram-negative and Gram-positive bacterial strains (e. g. against E. Coli: MIC=8 mg/L; S. aureus: MIC=4 mg/L).

Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1.

BACKGROUND: Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. METHODS: The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. RESULTS: The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. CONCLUSIONS: The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.

Correction: The anti-tumorigenic activity of A2M-A lesson from the naked mole-rat.

[This corrects the article DOI: 10.1371/journal.pone.0189514.].

The anti-tumorigenic activity of A2M-A lesson from the naked mole-rat.

Cancer resistance is a major cause for longevity of the naked mole-rat. Recent liver transcriptome analysis in this animal compared to wild-derived mice revealed higher expression of alpha2-macroglobulin (A2M) and cell adhesion molecules, which contribute to the naked mole-rat's cancer resistance. Notably, A2M is known to dramatically decrease with age in humans. We hypothesize that this might facilitate tumour development. Here we found that A2M modulates tumour cell adhesion, migration and growth by inhibition of tumour promoting signalling pathways, e.g. PI3K / AKT, SMAD and up-regulated PTEN via down-regulation of miR-21, in vitro and in tumour xenografts. A2M increases the expression of CD29 and CD44 but did not evoke EMT. Transcriptome analysis of A2M-treated tumour cells, xenografts and mouse liver demonstrated a multifaceted regulation of tumour promoting signalling pathways indicating a less tumorigenic environment mediated by A2M. By virtue of these multiple actions the naturally occurring A2M has strong potential as a novel therapeutic agent.

The diadinoxanthin diatoxanthin cycle induces structural rearrangements of the isolated FCP antenna complexes of the pennate diatom Phaeodactylum tricornutum.

The study investigated the influence of the xanthophyll cycle pigments diadinoxanthin (DD) and diatoxanthin (Dt) on the spectroscopic characteristics, structure and protein composition of isolated fucoxanthin chlorophyll protein (FCP) complexes of the pennate diatom Phaeodactylum tricornutum. 77 K fluorescence emission spectra revealed that Dt-containing FCP complexes showed a characteristic long wavelength fluorescence emission at 700 nm at a pH-value of 5 whereas DD-enriched FCPs retained the typical 680 nm fluorescence emission maximum of isolated FCPs. The 700 nm emission in Dt-containing FCPs indicates an aggregation of antenna complexes and is a typical feature of the quenching site Q1 in recent models for non-photochemical fluorescence quenching (NPQ). A comparable long-wavelength fluorescence emission was found in FCP complexes prepared with either triton X-100 or n-dodecyl ß-D-maltoside as detergent. A treatment of the FCP complexes at low pH-values in the presence of a high concentration of Mg(2+) ions showed that the extent of FCP aggregation which leads to the 700 nm fluorescence emission is different from the macro-aggregation of antenna complexes in higher plants. Protein analyses by mass spectrometry showed that the protein composition of the DD- and Dt-enriched FCP complexes was comparable. However, the Lhcf6 and Lhcr1 polypeptides were only found in Dt-enriched FCPs isolated with dodecyl maltoside whereas the Lhcf17 protein was only detected in DD-enriched FCPs prepared with triton. With respect to low pH-induced antenna aggregation it is important that the Lhcx1 protein was found in both DD- and Dt-enriched FCPs, albeit with only two peptides with confident scores.

Quantitative proteomics by fluorescent labeling of cysteine residues using a set of two cyanine-based or three rhodamine-based dyes.

Despite all remarkable progress in gel-based proteomics in recent years, there is still need to further improve quantification by decreasing the detection limits and increasing the dynamic range. These criteria are achieved best by fluorescent dyes that specifically stain the proteins either by adsorption after gel electrophoresis (in-gel staining) or covalent coupling prior to gel electrophoresis (in-solution staining). Here we report a multiplex analysis of protein samples using maleimide-activated cyanine-based (Cy3 and Cy5) and rhodamine-based dyes (Dy505, Dy535, and Dy635) to permanently label all thiol-groups of cysteine-containing proteins. The detection limits in SDS-PAGE were about 10 ng per band and even 2 ng for BSA due to its high content of cysteine residues. Thus only 5 microg protein of a mouse brain homogenate were analyzed by 2-DE. Both cyanine- and rhodamine-based dyes also stained proteins that did not contain cysteines, probably by reaction with amino groups. This side reactivity did not limit the method and might even extend its general use to proteins missing cysteine residues, but at a lower sensitivity. The dynamic range was more than two orders of magnitude in SDS-PAGE and the Dy-fluorophores did not alter the mobility of the tested proteins. Thus, a mixture of Dy505-, Dy555-, and Dy635-labeled Escherichia coli lysates were separated by 2-DE in a single gel and the three spot patterns relatively quantified.