Knowledge about clonal diversity and selection is critical to understand multiple myeloma (MM) pathogenesis, chemoresistance and progression. If targeted therapy becomes reality, identification and ...monitoring of intraclonal plasma cell (PC) heterogeneity would become increasingly demanded. Here we investigated the kinetics of intraclonal heterogeneity among 116 MM patients using 23-marker multidimensional flow cytometry (MFC) and principal component analysis, at diagnosis and during minimal residual disease (MRD) monitoring. Distinct phenotypic subclones were observed in 35/116 (30%) newly diagnosed MM patients. In 10/35 patients, persistent MRD was detected after 9 induction cycles, and longitudinal comparison of patient-paired diagnostic vs MRD samples unraveled phenotypic clonal tiding after therapy in half (5/10) of the patients. After demonstrating selection of distinct phenotypic subsets by therapeutic pressure, we investigated whether distinct fluorescence-activated cell-sorted PC subclones had different clonogenic and cytogenetic profiles. In half (5/10) of the patients analyzed, distinct phenotypic subclones showed different clonogenic potential when co-cultured with stromal cells, and in 6/11 cases distinct phenotypic subclones displayed unique cytogenetic profiles by interphase fluorescence in situ hybridization, including selective del(17p13). Collectively, we unravel potential therapeutic selection of preexisting diagnostic phenotypic subclones during MRD monitoring; because phenotypically distinct PCs may show different clonogenic and cytogenetic profiles, identification and follow-up of unique phenotypic-genetic myeloma PC subclones may become relevant for tailored therapy.
Schistosomiasis is one of the most significant neglected tropical diseases, affecting around 260 million people worldwide, and Praziquantel is currently the only available drug for the treatment of ...infected persons. Thus, the search for new schistosomicidal compounds is urgent. The objective of this study was to investigate of the schistosomicidal effect of barbatic acid, a lichen metabolite, on adult worms of Schistosoma mansoni. The in vitro schistosomicidal effect was evaluated through the assessment of motility and mortality, cellular viability of the worms and ultrastructural analysis through scanning electron microscopy. To evaluate the cytotoxicity of barbatic acid, a cell viability assay was performed with human peripheral blood mononuclear cells. Barbatic acid showed a schistosomicidal effect after 3 h of exposure. At the end of 24 h the concentrations of 50–200 μM presented lethality on the worms. Motility changes were observed at sublethal concentrations. The IC50 obtained by the cell viability assay for S. mansoni was 99.43 μM. Extensive damage to the worm's tegument was observed from 25 μM. No cytotoxicity was observed on human peripheral blood mononuclear cells. This report provides data showing the schistosomicidal effect of barbatic acid on S. mansoni, causing death, motility changes and ultrastructural damage to worms. In addition, barbatic acid was shown to be non-toxic to human peripheral blood mononuclear cells at concentrations that are effective against S. mansoni.
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•Barbatic acid exhibited schistosomicidal effect against adult S. mansoni.•Motility changes were observed in S.mansoni at all concentrations after 24 h.•Tegumentary damages were caused by the barbatic acid in Schistosoma mansoni.•No cytotoxicity was observed on PBMC exposed to barbatic acid after 72 h.
Most cases of Cystic Fibrosis (CF) are diagnosed early in life. However, people with atypical CF forms pose diagnosis dilemmas, requiring laboratory support for diagnosis confirmation/exclusion.
...analysis of fresh rectal biopsies by Ussing chamber has been the best discriminant biomarker for CF diagnosis/prognosis so far. Here we aimed to evaluate different electrophysiological parameters from Ussing chamber analysis of rectal biopsies from people with CF (PwCF) to establish the one with highest correlations with clinical features as the best CF diagnosis/prognosis biomarker. We analyzed measurements of CFTR-mediated Cl
secretion in rectal biopsies from 143 individuals (∼592 biopsies), the largest cohort so far analyzed by this approach. New parameters were analyzed and compared with the previous biomarker, i.e., the IBMX (I)/Forskolin (F)/Carbachol (C)-stimulated short-circuit current (I'
). Correlations with clinical features showed that the best parameter corresponded to voltage measurements of the I/F + (I/F/CCH) response (V
), with higher correlations vs. I'
for: sweat chloride (59 vs. 52%), fecal elastase (69 vs. 55%) and lung function, measured by FEV
(27 vs. 20%). Altogether data show that V
is the most sensitive, reproducible, and robust predictive biomarker for CF diagnosis/prognosis effectively discriminating classical, atypical CF and non-CF groups.
Revealing the intracellular location of novel therapeutic agents is paramount for the understanding of their effect at the cell ultrastructure level. Here, we apply a novel correlative cryo 3D ...imaging approach to determine the intracellular fate of a designed protein-nanomaterial hybrid with antifibrotic properties that shows great promise in mitigating myocardial fibrosis. Cryo 3D structured illumination microscopy (cryo-3D-SIM) pinpoints the location and cryo soft X-ray tomography (cryo-SXT) reveals the ultrastructural environment and subcellular localization of this nanomaterial with spatial correlation accuracy down to 70 nm in whole cells. This novel high resolution 3D cryo correlative approach unambiguously locates the nanomaterial after overnight treatment within multivesicular bodies which have been associated with endosomal trafficking events by confocal microscopy. Moreover, this approach allows assessing the cellular response towards the treatment by evaluating the morphological changes induced. This is especially relevant for the future usage of nanoformulations in clinical practices. This correlative super-resolution and X-ray imaging strategy joins high specificity, by the use of fluorescence, with high spatial resolution at 30 nm (half pitch) provided by cryo-SXT in whole cells, without the need of staining or fixation, and can be of particular benefit to locate specific molecules in the native cellular environment in bio-nanomedicine.
A novel 3D cryo correlative approach locates designed therapeutic protein-nanomaterial hybrids in whole cells with high specificity and resolution. Detection of treatment-induced morphological changes, crucial for pre-clinical studies, are revealed.
A key challenge in the treatment of cancer with nanomedicine is to engineer and select nanoparticle formulations that lead to the desired selectivity between tumorigenic and non-tumorigenic cells. To ...this aim, novel designed nanomaterials, deep biochemical understanding of the mechanisms of interaction between nanomaterials and cells, and computational models are emerging as very useful tools to guide the design of efficient and selective nanotherapies. This works shows, using a combination of detailed experimental approaches and simulations, that the specific targeting of cancer cells in comparison to non-tumorigenic cells can be achieved through the custom design of multivalent nanoparticles. A theoretical model that provides simple yet quantitative predictions to tune the nanoparticles targeting and cytotoxic properties by their degree of functionalization is developed. As a case study, a system that included a targeting agent and a drug and is amenable to controlled experimental manipulation and theoretical analysis is used. This study shows how at defined functionalization levels multivalent nanoparticles can selectively kill tumor cells, while barely affecting non-tumorigenic cells. This work opens a way to the rational design of multifunctionalized nanoparticles with defined targeting and cytotoxic properties for practical applications.
The YEAst Search for Transcriptional Regulators And Consensus Tracking (YEASTRACT) information system (http://www.yeastract.com) was developed to support the analysis of transcription regulatory ...associations in Saccharomyces cerevisiae. Last updated in June 2010, this database contains over 48 200 regulatory associations between transcription factors (TFs) and target genes, including 298 specific DNA-binding sites for 110 characterized TFs. All regulatory associations stored in the database were revisited and detailed information on the experimental evidences that sustain those associations was added and classified as direct or indirect evidences. The inclusion of this new data, gathered in response to the requests of YEASTRACT users, allows the user to restrict its queries to subsets of the data based on the existence or not of experimental evidences for the direct action of the TFs in the promoter region of their target genes. Another new feature of this release is the availability of all data through a machine readable web-service interface. Users are no longer restricted to the set of available queries made available through the existing web interface, and can use the web service interface to query, retrieve and exploit the YEASTRACT data using their own implementation of additional functionalities. The YEASTRACT information system is further complemented with several computational tools that facilitate the use of the curated data when answering a number of important biological questions. Since its first release in 2006, YEASTRACT has been extensively used by hundreds of researchers from all over the world. We expect that by making the new data and services available, the system will continue to be instrumental for yeast biologists and systems biology researchers.
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•Divaricatic acid exhibits toxicity against B. glabrata embryos and adult snails.•Malformations and delay in embryonic development were observed.•Divaricatic acid showed lethal ...effects against cercariae of Schistosoma mansoni.•Divaticatic acid was not toxic to Artemia salina.
In this study, the molluscicidal and antiparasitic activities of divaricatic acid was evaluated, targeting the mollusc Biomphalaria glabrata and cercariae of the helminth Schistosoma mansoni. In addition, the environmental toxicity of divaricatic acid was assessed by bioassay using the microcrustacean Artemia salina. Divaricatic acid showed high toxicity against both adult snails (5μg/mL) and embryos (20μg/mL after 6h of exposure). Similar activity was observed in Schistosoma mansoni cercariae after only a short exposure time (10μg/mL after 30min of exposure). The divaricatic acid did not show toxicity in the acute test using Artemia salina at concentrations equal to or below 200μg/mL. The divaricatic acid proved to be a promising substance for the elimination of the snail Biomphalaria glabrata, an intermediate host of schistosomiasis, as well as the cercariae of the pathogen, while being non-toxic to the Artemia salina at the same concentrations. This is the first experimental observation of the molluscicidal and cercaricide activity of divaricatic acid.
Myocardial fibroblast activation coupled with extracellular matrix production is a pathological signature of myocardial fibrosis and is governed mainly by transforming growth factor TGFβ-Smad2/3 ...signaling. Targeting the ubiquitous TGFβ leads to cellular homeostasis deregulation with adverse consequences. We previously showed the anti-fibrotic effects upon downregulation of 90-kDa heat shock protein (Hsp90), a chaperone that associates to the TGFβ signaling cascade. In the present study, we use a fluorescent-labeled Hsp90 protein inhibitor (CTPR390–488) with specific Hsp90 binding properties to reduce myocardial pro-fibrotic events in vitro and in vivo. The mechanism of action involves the disruption of TGFβRI-Hsp90 complex, resulting in a decrease in TGFβ signaling and reduction in extracellular matrix collagen. In vivo, decreased myocardial collagen deposition was observed upon CTPR390–488 treatment in a pro-fibrotic mouse model. This is the first study demonstrating the ability of an engineered Hsp90 protein inhibitor to block collagen expression, reduce the motility of myocardial TGFβ-activated fibroblasts and ameliorate angiotensin-II induced cardiac myocardial fibrosis in vivo.
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•-We propose the interaction between CTPR390–488 inhibitor and Hsp90 chaperone preserving Hsp90 ATPase activity•-Collagen expression, collagen deposition and fibroblast motility are reduced in myocardial fibroblasts upon CTPR390–488 addition or Hsp90aa1 deletion.•Angiotensin II infused mice presents increased myocardial fibrosis that is reduced in CTPR390–488 treated mice or Hsp90aa1 KO mice.•-CTPR390–488 plasma concentrations increases in angiotensin II infused WT mice.
As highly effective CFTR modulator therapies (HEMT) emerge, there is an unmet need to find effective drugs for people with CF (PwCF) with ultra-rare mutations who are too few for classical clinical ...trials and for whom there are no drug discovery programs. Therefore, biomarkers reliably predicting the benefit from CFTR modulator therapies are essential to find effective drugs for PwCF through personalized approaches termed theranostics. Here, we assess CFTR basal function and the individual responses to CFTR modulators in primary human nasal epithelial (pHNE) cells from PwCF carrying rare mutations and compare these measurements with those in native rectal biopsies and intestinal organoids, respectively, in the same individual. The basal function in pHNEs shows good correlation with CFTR basal function in rectal biopsies. In parallel, CFTR rescue in pHNEs by CFTR modulators correlates to that in intestinal organoids. Altogether, results show that pHNEs are a bona fide theranostic model to assess CFTR rescue by CFTR modulator drugs, in particular for PwCF and rare mutations.
•Direct capture of monoclonal antibodies from a serum culture medium by PBA chromatography.•Final purity was improved by designing a washing step to remove weakly bound impurities.•The biological ...activity was enhanced by optimising the elution buffer composition.•PBA chromatography generated yields comparable to protein A chromatography.
In this work, phenylboronic acid (PBA) was thoroughly investigated as a synthetic ligand for the purification of an immunoglobulin G (IgG) from a clarified cell supernatant from Chinese Hamster Ovary (CHO) cell cultures. In particular, the study was focused on the development of a washing step and in the optimization of the elution step using a serum containing supernatant. From the different conditions tested, best recoveries – 99% – and purifications – protein purity of 81% and a purification factor of 16 out of a maximum of 20 – were achieved using 100mM d-sorbitol in 10mM Tris–HCl as washing buffer and 0.5M d-sorbitol with 150mM NaCl in 10mM Tris–HCl as elution buffer. The purification outcome was also compared with protein A chromatography that revealed a recovery of 99%, 87% protein purity and 29 out of a maximum of 33 purification factor. Following the main purification, purified IgG was characterized in terms of isoelectric point, size and activity. In the end, a proof of concept was performed using two different mAbs from serum-free CHO cell cultures.
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