Putative Players Research Articles

An in Vitro triple screen model for human mineralocorticoid receptor activity

The mineralocorticoid receptor (MR, NR3C2) mediates ion and water homeostasis in epithelial cells of the distal nephron and other tissues. Aldosterone, the prototypical mineralocorticoid, regulates electrolyte and fluid balance. Cortisol binds to MR with equal affinity to aldosterone, but many MR-expressing tissues inactivate cortisol to cortisone via 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2). Dysregulated MR activation contributes to direct cardiovascular tissue insults. Besides aldosterone and cortisol, a variety of MR agonists and/or HSD11B2 inhibitors are putative players in the pathophysiology of low-renin hypertension (LRH), and cardiovascular and metabolic pathology. We developed an in vitro human MR (hMR) model, to facilitate screening for MR agonists, antagonists, and HSD11B2 inhibitors. The CV1 monkey kidney cells were transduced with lentivirus to stably express hMR and an MR-responsive gaussia luciferase gene. Clonal populations of MR-expressing cells (CV1-MRluc) were further transduced to express HSD11B2 (CV1-MRluc-HSD11B2). CV1-MRluc and CV1-MRluc-HSD11B2 cells were treated with aldosterone, cortisol, 11-deoxycorticosterone (DOC), 18-hydroxycorticosterone (18OHB), 18-hydroxycortisol (18OHF), 18-oxocortisol (18oxoF), progesterone, or 17-hydroxyprogesterone (17OHP). In CV1-MRLuc cells, aldosterone and DOC displayed similar potency (EC50: 0.45 nM and 0.30 nM) and maximal response (31- and 23-fold increase from baseline) on hMR; 18oxoF and 18OHB displayed lower potency (19.6 nM and 56.0 nM, respectively) but similar maximal hMR activation (25- and 27-fold increase, respectively); cortisol and corticosterone exhibited higher maximal responses (73- and 52-fold, respectively); 18OHF showed no MR activation. Progesterone and 17OHP inhibited aldosterone-mediated MR activation. In the MRluc-HSD11B2 model, the EC50 of cortisol for MR activation increased from 20 nM (CV1-MRLuc) to ∼2000 nM, while the EC50 for aldosterone remained unchanged. The addition of 18β-glycyrrhetinic acid (18β-GA), a HSD11B2 inhibitor, restored the potency of cortisol back to ∼70 nM in CV1-hMRLuc-HSD11B2 cells. Together, these two cell models will facilitate the discovery of novel MR-modulators, informing MR-mediated pathophysiology mechanisms and drug development efforts.

Replisome Proximal Protein Associations and Dynamic Proteomic Changes at Stalled Replication Forks

DNA replication is a fundamental cellular process that ensures the transfer of genetic information during cell division. Genome duplication takes place in S phase and requires a dynamic and highly coordinated recruitment of multiple proteins at replication forks. Various genotoxic stressors lead to fork instability and collapse, hence the need for DNA repair pathways. By identifying the multitude of protein interactions implicated in those events, we can better grasp the complex and dynamic molecular mechanisms that facilitate DNA replication and repair. Proximity-dependent biotin identification was used to identify associations with 17 proteins within four core replication components, namely the CDC45/MCM2-7/GINS helicase that unwinds DNA, the DNA polymerases, replication protein A subunits, and histone chaperones needed to disassemble and reassemble chromatin. We further investigated the impact of genotoxic stress on these interactions. This analysis revealed a vast proximity association network with 108 nuclear proteins further modulated in the presence of hydroxyurea; 45 being enriched and 63 depleted. Interestingly, hydroxyurea treatment also caused a redistribution of associations with 11 interactors, meaning that the replisome is dynamically reorganized when stressed. The analysis identified several poorly characterized proteins, thereby uncovering new putative players in the cellular response to DNA replication arrest. It also provides a new comprehensive proteomic framework to understand how cells respond to obstacles during DNA replication.

Effect of ammonium/nitrate ratio on microalgae continuous cultures: Species-specificity of nutrient uptake and modelling perspectives

Microalgae-based wastewater treatment is promising in view of circularity as they can uptake most of the nitrogen sources usually present in waste streams: ammonium, nitrite and nitrate as well as some organic species. However, regulation pathways are not fully understood yet. This complexity is a challenge for accurate modelling of these processes, necessary for the design and optimization of wastewater treatment systems. It is generally observed in microalgae that the presence of ammonium inhibits the assimilation of other nitrogen compounds. This was confirmed in this work for Synechocystis sp. PCC6803, even in acclimated continuous cultures. A different uptake regulation was instead observed for the species Auxenochlorella protothecoides, able to uptake nitrate even when high concentrations of ammonium are present. From a modelling perspective, the Droop model was implemented to account for the exploitation of both ammonium and nitrate: a switch function was used to describe the ammonium inhibition, but this was applicable for Synechocystis only. For A. protothecoides, a changing nutrient uptake rate is needed, which was found to be function of the dissolved nitrogen concentration. A bioinformatic analysis was performed to infer putative players involved in nitrogen uptake, accounting for the different behavior of the two taxonomic groups.

FRI161 An in Vitro Triple Screen Model for Human Mineralocorticoid Receptor Activity

Abstract Disclosure: H. Liu: None. S.M. Konzen: None. A. Coy: None. J. Rege: None. C.E. Gomez-Sanchez: None. W.E. Rainey: None. A.F. Turcu: None. Background: The mineralocorticoid receptor (MR, NR3C2) mediates ion and water homeostasis in epithelial cells of the distal nephron. MR is expressed in a variety of other tissues, including gastrointestinal tract, exocrine glands, central nervous system, heart, and adipose tissue. Aldosterone, the prototypical mineralocorticoid, regulates electrolyte and fluid balance. Cortisol binds to MR with equal affinity to aldosterone. In humans, cortisol concentrations far exceed those of aldosterone, but many MR-expressing tissues inactivate cortisol to cortisone via 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2). Dysregulated MR activation contributes to direct cardiovascular tissue insults. Besides aldosterone and cortisol, a variety of MR agonists and/or HSD11B2 inhibitors are putative players in the pathophysiology of low-renin hypertension (LRH), and cardiovascular and metabolic pathology. Objective: To develop an in vitro human MR (hMR) model, to facilitate screening for MR agonists, antagonists, and HSD11B2 inhibitors. Methods: The CV1 monkey kidney cells were transduced with lentivirus to stably express hMR and an MR-responsive gaussian luciferase gene. Clonal populations of MR-expressing cells (CV1-MRluc) were further transduced to express HSD11B2 (CV1-MRluc-HSD11B2). CV1-MRluc and CV1-MRluc-HSD11B2 cells were treated with aldosterone, cortisol, 11-deoxycorticosterone (DOC), 18-hydroxycorticosterone (18OHB), 18-hydroxycortisol (18OHF), 18-oxocortisol (18oxoF), progesterone, or 17-hydroxyprogesterone (17OHP). Cells were also treated with serum from patients with LRH (N=3) or primary hypertension (PH, N=3). Luminescence was measured in conditioned medium using coelenterazine. Results: In CV1-MRLuc cells, aldosterone and DOC displayed similar potency (EC50: 0.45nM and 0.30nM) and maximal response (31- and 23-fold increase from baseline) on hMR; 18oxoF and 18OHB displayed lower potency (19.6nM and 56.0nM, respectively) but similar maximal hMR activation (25- and 27-fold increase, respectively); cortisol and corticosterone exhibited higher maximal responses (73- and 52-fold, respectively); 18OHF showed no MR activation. Progesterone and 17OHP inhibited aldosterone-mediated MR activation. In the MRluc-HSD11B2 model, the EC50 of cortisol for MR activation increased from 20nM (CV1-MRLuc) to 850nM, while the EC50 for aldosterone remained unchanged. Serum from patients with both LRH and PH led to variable degrees of luminescence in the CV1-MRLuc cells, but not in the CV1-MRluc-HSD11B2 cells, suggesting a cortisol-mediated MR activation in these patients, possibly via glycyrrhetinic acid-like factors. Conclusions: Together, these two cell models will facilitate the discovery of novel MR-modulators, informing MR-mediated pathophysiology mechanisms and drug development efforts. Presentation Date: Friday, June 16, 2023

High Cerebrospinal Fluid CX3CL1 Levels in Alzheimer's Disease Patients but Not in Non-Alzheimer's Disease Dementia.

Alzheimer’s disease (AD) is the most common form of cognitive decline worldwide, occurring in about 10% of people older than 65 years. The well-known hallmarks of AD are extracellular aggregates of amyloid β (Aβ) and intracellular neurofibrillary tangles (NFTs) of tau protein. The evidence that Aβ overproduction leads to AD has paved the way for the AD pathogenesis amyloid cascade hypothesis, which proposes that the neuronal damage is sustained by Aβ overproduction. Consistently, AD cerebrospinal fluid (CSF) biomarkers used in clinical practice, including Aβ 1–42, Aβ 1–40, Aβ 42/40 ratio, and pTau, are related to the amyloid hypothesis. Recently, it was suggested that the Aβ deposition cascade cannot fully disclose AD pathogenesis, with other putative players being involved in the pathophysiology of the disease. Among all, one of the most studied factors is inflammation in the brain. Hence, biomarkers of inflammation and microglia activation have also been proposed to identify AD. Among them, CX3 chemokine ligand 1 (CX3CL1) has taken center stage. This transmembrane protein, also known as fractalkine (FKN), is normally expressed in neurons, featuring an N-terminal chemokine domain and an extended mucin-like stalk, following a short intra-cytoplasmatic domain. The molecule exists in both membrane-bound and soluble forms. It is accepted that the soluble and membrane-bound forms of FKN evoke differential signaling within the CNS. Given the link between CX3XL1 and microglial activation, it has been suggested that CX3CL1 signaling disruption could play a part in the pathogenesis of AD. Furthermore, a role for chemokine as a biomarker has been proposed. However, the findings collected are controversial. The current study aimed to describe the cerebrospinal fluid (CSF) levels of CX3XL1 and classical biomarkers in AD patients.

Abstract P1028: The Epicardium As The Conductor Of Cardiovascular Cells In Cardiac Regeneration

Regenerative cardiovascular medicine is emerging as a promising therapeutic alternative to heart transplantation in patients suffering from chronic heart failure. Although promising, challenges to cell therapy for cardiac repair remain, including cell survival, maturation, graft size, revascularisation, and immunogenicity. It has recently been shown that co-transplantation of human embryonic stem cell (hESC)-derived epicardial cells (EPI) with hESC-derived cardiomyocytes (CM) improves cardiac repair processes with respect to engraftment, cell maturation, and graft and host vascularisation. One plausible explanation for the observed benefits of co-transplantation is paracrine-mediated effects. While RNA sequencing data has been used to identify putative players in hESC-EPIs, little is known about their involvement in mediating hESC-CM maturation and revascularization, in particular in communication with endothelial cells (EC). Here, the role of paracrine signalling in hESC-EPI-mediated promotion of hESC-CM maturation and angiogenesis is being investigated with an emphasis on the involvement of extracellular vesicles (EVs). hESC-EPI-EVs improved responsiveness to pacing and coordination of contraction in vitro in hESC-CM cell culture and engineered heart tissues (EHTs). Moreover, hESC-EPI-EVs and hESC-EPI supernatant excluding EVs independently promoted tube formation in in vitro gel-based angiogenesis assays. hESC-EPI supernatant excluding EVs also promoted EC proliferation in MTS assays and EC migration in wound closure assays. Characterisation of the hESC-EPI-EV-cargo on a protein level revealed candidate factors involved in cardiac repair processes. Together with hESC-EPI-EV miRNA-sequencing and hESC-EPI gene expression data key paracrine signalling pathways and candidate factors to manipulate them are being identified and investigated in in vitro angiogenesis and CM maturation assays. Promising angiogenesis promoting candidates are being validated in vivo using yolk-sac membrane and chorioallantoic membrane assays. Identification of the paracrine signalling pathways involved would make it possible to effectively address selected cardiac repair processes, resulting in new approaches to therapy.

Negative self-evaluation induced by acute stress indexed using facial EMG

Maladaptive stress responses are a key feature of several psychiatric disorders, but findings of stress effects on social behavior are inconsistent. Using a within-subject design, we investigated, in 35 healthy participants, the effects of acute stress on psychophysiological and behavioral responses during a simulated online social interaction task. Participants were exposed to established stress and non-stress exposure procedures in two separate sessions. During the task, participants liked or disliked pictures of other putative players and, similarly, saw their own picture being judged by others. After stress exposure, corrugator muscle activity (frowning) was significantly increased when participants saw their own picture while anticipating feedback from others. Consistently, zygomatic muscle activity (smiling) for self-evaluation was lower after stress than in the non-stress session. We found self-report of stress to be a significant predictor of corrugator activity in both sessions, indicating that higher levels of subjective stress overall were accompanied by increased negative self-evaluation. Surprisingly, no stress effects were found on behavioral measures of other-evaluation (i.e., percentage of dislikes to others), but corrugator response significantly predicted the percentage of dislikes during the stress session only. Overall, our findings suggest that stress increases negative self-evaluation as indexed by elevated corrugator activity. Furthermore, stress might sharpen the consistency between corrugator activity and negative evaluation of others. Our results indicate that negative self-evaluation might be a useful therapeutic target in patients with stress-related psychiatric disorders. In this context, facial muscle activity may be an adequate biomarker for identifying stress-related differences in self-evaluation.

Abstract PS5-29: Insights into the molecular underpinnings of the mevalonate pathway-YAP/TAZ-driven anti-HER2 therapy resistance in HER2+ breast cancer (BC)

Abstract Background. We recently reported that the biosynthetic mevalonate (MVA) pathway that produces cholesterol and isoprenoid intermediates, regulates the YAP/TAZ (Y/T) transcriptional co-activators to promote resistance to treatment regimens that effectively inhibit HER2 signaling in HER2+ BC. We further showed that the mTORC1 complex and survivin protein, which contribute to HER2-elicited oncogenic signaling in HER2-driven BC cells, are alternatively activated by the MVA pathway-Y/T axis in anti-HER2 therapy resistant cell models. Of note, other recent reports also showed that increased Y/T activity enables resistant cells to proliferate when other oncogenic pathways (e.g., RAS, EGFR, and FGFR) were effectively inhibited. Here, we sought to further determine the molecular underpinnings of MVA-Y/T axis-driven anti-HER2 therapy resistance to discover novel therapeutic targets and predictive biomarkers for HER2+ BC.Methods. SKBR3 HER2+ BC parental (P) cells and their lapatinib plus trastuzumab (LT) resistant (LTR) derivatives with sustained HER2 inhibition were treated with the MVA pathway inhibitor simvastatin (Sim), with or without the MVA metabolite to rescue Sim’s inhibitory effects. P and LTR cells were also transfected with control or combined Y/T siRNAs. The transcriptomes of all treatment groups were assessed by RNA-seq. Integrative bioinformatics analyses were used to identify differentially expressed (DE) genes and gene sets with functional annotations in LTR vs. P cells upon different interventions.Results. We found that cell cycle and cell proliferation processes were among the top common DE molecular signatures preferentially downregulated (DN) in LTR vs. P cells by both Sim and Y/T knockdown (KD). The top common genes preferentially DN in LTR vs. P cells include the cell cycle regulatory genes CDCA3 and ERCC6L, and the nucleotide metabolism genes TYMS and RRM2. Interestingly, 20% of the genes preferentially DN in LTR vs. P cells by Sim or Y/T KD were predicted to be direct Y/T transcriptional targets based on previously reported ChIP-seq data (PMID: 26258633). Of the Y/T-dependent genes, a significant enrichment of Sim-repressed genes was observed in both P and LTR cells (P = 1.2e-115 and P = 5.5e-138, respectively). The proportion of these enriched genes was higher in LTR vs. P cells (61% vs. 29%). Of note, we found that the global inhibited genes in LTR cells upon Sim or Y/T KD were significantly enriched for the genes DN by short-term LT treatment in P cells (P < 2.2e-16). Likewise, of the genes nominated as putative molecular players in the MVA pathway-Y/T-mediated resistance, BIRC5 (survivin), CDC6, KIF2C, RRM2, and TYMS were recently also reported to be DN in HER2+ tumors treated with neo-adjuvant LT in the PAMELA trial (NCT01973660), a finding that is in line with what we observed in our P cells treated with short-term LT. Conclusions. Upon acquisition of resistance to sustained HER2 inhibition, the MVA pathway-Y/T axis takes over the regulation of pro-proliferative transcriptional programs that are generally downstream of HER2 signaling in treatment-naïve HER2+ BC. The MVA pathway-Y/T axis leads to Y/T-driven transcriptional reprogramming, an emerging mechanism of therapy resistance to anti-HER2 and other targeted therapies that warrants further investigation. The identification of multiple cell cycle related processes as putative targets of the MVA pathway-Y/T axis presents additional targetable vulnerabilities and implies that inhibitors of Y/T and cell cycle checkpoints may help circumvent anti-HER2 resistance in the clinical setting. Citation Format: Vidyalakshmi Sethunath, Xiaoyong Fu, Pamela L Luna, Sarmistha Nanda, Martin Shea, Jamunarani Veeraraghavan, Carmine De Angelis, Huizhong Hu, Chad Shaw, Mothaffar Rimawi, C. Kent Osborne, Rachel Schiff. Insights into the molecular underpinnings of the mevalonate pathway-YAP/TAZ-driven anti-HER2 therapy resistance in HER2+ breast cancer (BC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-29.

In Vivo Pre-Instructed HSCs Robustly Execute Asymmetric Cell Divisions In Vitro.

Hematopoietic stem cells (HSCs) are responsible for life-long production of all mature blood cells. Under homeostasis, HSCs in their native bone marrow niches are believed to undergo asymmetric cell divisions (ACDs), with one daughter cell maintaining HSC identity and the other committing to differentiate into various mature blood cell types. Due to the lack of key niche signals, in vitro HSCs differentiate rapidly, making it challenging to capture and study ACD. To overcome this bottleneck, in this study, we used interferon alpha (IFNα) treatment to ”pre-instruct” HSC fate directly in their native niche, and then systematically studied the fate of dividing HSCs in vitro at the single cell level via time-lapse analysis, as well as multigene and protein expression analysis. Triggering HSCs’ exit from dormancy via IFNα was found to significantly increase the frequency of asynchronous divisions in paired daughter cells (PDCs). Using single-cell gene expression analyses, we identified 12 asymmetrically expressed genes in PDCs. Subsequent immunocytochemistry analysis showed that at least three of the candidates, i.e., Glut1, JAM3 and HK2, were asymmetrically distributed in PDCs. Functional validation of these observations by colony formation assays highlighted the implication of asymmetric distribution of these markers as hallmarks of HSCs, for example, to reliably discriminate committed and self-renewing daughter cells in dividing HSCs. Our data provided evidence for the importance of in vivo instructions in guiding HSC fate, especially ACD, and shed light on putative molecular players involved in this process. Understanding the mechanisms of cell fate decision making should enable the development of improved HSC expansion protocols for therapeutic applications.

Controlled sampling of ribosomally active protistan diversity in sediment-surface layers identifies putative players in the marine carbon sink

Marine sediments are one of the largest carbon reservoir on Earth, yet the microbial communities, especially the eukaryotes, that drive these ecosystems are poorly characterised. Here, we report implementation of a sampling system that enables injection of reagents into sediments at depth, allowing for preservation of RNA in situ. Using the RNA templates recovered, we investigate the ‘ribosomally active’ eukaryotic diversity present in sediments close to the water/sediment interface. We demonstrate that in situ preservation leads to recovery of a significantly altered community profile. Using SSU rRNA amplicon sequencing, we investigated the community structure in these environments, demonstrating a wide diversity and high relative abundance of stramenopiles and alveolates, specifically: Bacillariophyta (diatoms), labyrinthulomycetes and ciliates. The identification of abundant diatom rRNA molecules is consistent with microscopy-based studies, but demonstrates that these algae can also be exported to the sediment as active cells as opposed to dead forms. We also observe many groups that include, or branch close to, osmotrophic–saprotrophic protists (e.g. labyrinthulomycetes and Pseudofungi), microbes likely to be important for detrital decomposition. The sequence data also included a diversity of abundant amplicon-types that branch close to the Fonticula slime moulds. Taken together, our data identifies additional roles for eukaryotic microbes in the marine carbon cycle; where putative osmotrophic–saprotrophic protists represent a significant active microbial-constituent of the upper sediment layer.

Alterations of perineuronal nets in the dorsolateral prefrontal cortex of neuropsychiatric patients

BackgroundAlterations in the structure and physiology of interneurons in the prefrontal cortex (PFC) are important factors in the etiopathology of different psychiatric disorders. Among the interneuronal subpopulations, parvalbumin (PV) expressing cells appear to be specially affected. Interestingly, during development and adulthood the connectivity of these interneurons is regulated by the presence of perineuronal nets (PNNs), specialized regions of the extracellular matrix, which are frequently surrounding PV expressing neurons. Previous reports have found anomalies in the density of PNNs in the PFC of schizophrenic patients. However, although some studies have described alterations in PNNs in some extracortical regions of bipolar disorder patients, there are no studies focusing on the prefrontocortical PNNs of bipolar or major depression patients. For this reason, we have analyzed the density of PNNs in post-mortem sections of the dorsolateral PFC (DLPFC) from the Stanley Neuropathology Consortium, which includes controls, schizophrenia, bipolar and major depression patients.ResultsWe have not observed differences in the distribution of PV+ cells or PNNs, or in the percentage of PV+ interneurons surrounded by PNNs. The density of PV+ interneurons was similar in all the experimental groups, but there was a significantly lower density of PNNs in the DLPFC of bipolar disorder patients and a tendency towards a decrease in schizophrenic patients. No differences were found when evaluating the density of PV+ cells surrounded by PNNs. Interestingly, when assessing the influence of demographic data, we found an inverse correlation between the density of PNNs and the presence of psychosis.ConclusionsThe present results point to prefrontocortical PNNs and their role in the regulation of neuronal plasticity as putative players in the etiopathology of bipolar disorder and schizophrenia. Our findings also suggest a link between these specialized regions of the extracellular matrix and the presence of psychosis.

Exploring the functional role of an ancient mitochondrial fatty acid synthesis pathway

Fatty acid synthesis plays a crucial role in the cellular energetic landscape. Cytoplasmic Fatty Acid Synthesis (FAS) generates fatty acid intermediates that are used to store energy, build cellular structures such as membranes, and act as precursors for the biosynthesis of hormones. In addition to the cytoplasmic fatty acid synthesis pathway, eukaryotic cells have maintained a second, highly conserved mitochondrial fatty acid synthesis pathway (mtFAS), reminiscent of prokaryotic fatty acid synthesis pathways. Here, we explore the functional role of this ancient mitochondrial fatty acid synthesis pathway. Historically, a major function of mtFAS was considered to be the production of lipoic acid which is an essential co‐factor for key mitochondrial, metabolic enzymes such as pyruvate dehydrogenase. However, recently our lab has shown that mtFAS also plays a critical role in mitochondrial biogenesis by coupling Acetyl‐CoA levels with iron‐sulfur biogenesis as well as electron transport chain complex activation by promoting late‐stage assembly (Van Vranken et al., 2016; Van Vranken et al., 2018). These findings expanded the functional roles of the so far underappreciated mtFAS pathway into its broader role in the regulation of mitochondrial biogenesis and metabolic function. The identification of human patients with mutations in mtFAS genes further highlights the importance of the mtFAS pathway and the need to understand all aspects of its functions. However, many facets, especially regarding its own regulation, are still unknown. Here we present putative players in the regulation of the mtFAS pathway and their impact on cellular function.Support or Funding InformationCardiovascular Training Grant, University of Utah, 5T32HL007576‐32, Howard Hughes Medical InstituteThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deciphering the role of cyanobacteria in water resistome: Hypothesis justifying the antibiotic resistance (phenotype and genotype) in Planktothrix genus

The importance of environmental microorganisms in the emergence and dissemination of antibiotic resistance is an undeniable fact. However, cyanobacteria are not seen yet as putative players in the dynamic of environmental resistome, despite their ubiquity in water environments, where they are exposed to antibiotic pollution and in straight contact with native and pathogenic bacteria harboring antibiotic resistance genes (ARGs). In this work we evaluated the susceptibility of 8 strains of Planktothrix agardhii (from surface freshwaters reservoirs) and 8 strains of Planktothrix mougeotii (from a wastewater treatment plant) to several classes of antibiotics, using a microplate dilution method previously described by us. We also search for ARGs in those strains by molecular methods. None of the 16 tested strains were susceptible to trimethoprim, nalidixic acid and norfloxacin, from 0.0015–1.6 mg/L, but all were susceptible to streptomycin, gentamicin, kanamycin, ceftazidime and ceftriaxone. The minimum inhibitory concentrations (MICs) ranged between 0.05–0.8 mg/L for the aminoglycosides and 0.4–1.6 mg/L for the two β‑lactams. Major differences were found in the susceptibility to amoxicillin and tetracycline, with P. agardhii being susceptible (MIC of 0.05 mg/L and 0.4 mg/L, respectively) and P. mougeotii not susceptible. These distinct responses might be due to differences between species. However, the lower susceptibility of wastewater strains suggests that antibiotic resistance phenotype of cyanobacteria is related with their habitat. The failure to detect acquired genes conferring resistance to trimethoprim/quinolones, strongly supports the hypothesis that cyanobacteria are intrinsically resistant to these antibiotics. Interestingly, we detected a class-1-type integron and a sul1 gene in 3 strains of both P. agardhii and P. mougeotii, which supports the possibility of cyanobacteria to acquire and transfer antibiotic resistance determinants. In conclusion, the identification of ARGs and related integrons, as well as the reduced susceptibility to some antibiotics, suggests that cyanobacteria may play a role on environmental resistome.

Commentary on Paper "Drug-Induced HSP90 Inhibition Alleviates Pain in Monoarthritic Rats and Alters the Expression of New Putative Pain Players at the DRG"

Commentary on Paper "Drug-Induced HSP90 Inhibition Alleviates Pain in Monoarthritic Rats and Alters the Expression of New Putative Pain Players at the DRG"

Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly.

HIV-1 is a retrovirus replicating within cells by reverse transcribing its genomic RNA (gRNA) into DNA. Within cells, virus assembly requires the structural Gag proteins with few accessory proteins, notably the viral infectivity factor (Vif) and two copies of gRNA as well as cellular factors to converge to the plasma membrane. In this process, the nucleocapsid (NC) domain of Gag binds to the packaging signal of gRNA which consists of a series of stem-loops (SL1-SL3) ensuring gRNA selection and packaging into virions. Interestingly, mutating NC activates a late-occurring reverse transcription (RT) step in producer cells, leading to the release of DNA-containing HIV-1 particles. In order to decipher the molecular mechanism regulating this late RT, we explored the role of several key partners of NC, such as Vif, gRNA and the cellular cytidine deaminase APOBEC3G that restricts HIV-1 infection by targeting the RT. By studying combinations of deletions of these putative players, we revealed that NC, SL1-SL3 and in lesser extent Vif, but not APOBEC3G, interplay regulates the late RT.

Quantitative differential proteomics of yeast extracellular matrix: there is more to it than meets the eye.

BackgroundSaccharomyces cerevisiae multicellular communities are sustained by a scaffolding extracellular matrix, which provides spatial organization, and nutrient and water availability, and ensures group survival. According to this tissue-like biology, the yeast extracellular matrix (yECM) is analogous to the higher Eukaryotes counterpart for its polysaccharide and proteinaceous nature. Few works focused on yeast biofilms, identifying the flocculin Flo11 and several members of the HSP70 in the extracellular space. Molecular composition of the yECM, is therefore mostly unknown. The homologue of yeast Gup1 protein in high Eukaryotes (HHATL) acts as a regulator of Hedgehog signal secretion, therefore interfering in morphogenesis and cell-cell communication through the ECM, which mediates but is also regulated by this signalling pathway. In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation.MethodsS. cerevisiae W303-1A wt strain and gup1∆ mutant were used as previously described to generate biofilm-like mats in YPDa from which the yECM proteome was extracted. The proteome from extracellular medium from batch liquid growing cultures was used as control for yECM-only secreted proteins. Proteins were separated by SDS-PAGE and 2DE. Identification was performed by HPLC, LC-MS/MS and MALDI-TOF/TOF. The protein expression comparison between the two strains was done by DIGE, and analysed by DeCyder Extended Data Analysis that included Principal Component Analysis and Hierarchical Cluster Analysis. ResultsThe proteome of S. cerevisiae yECM from biofilm-like mats was purified and analysed by Nano LC-MS/MS, 2D Difference Gel Electrophoresis (DIGE), and MALDI-TOF/TOF. Two strains were compared, wild type and the mutant defective in GUP1. As controls for the identification of the yECM-only proteins, the proteome from liquid batch cultures was also identified. Proteins were grouped into distinct functional classes, mostly Metabolism, Protein Fate/Remodelling and Cell Rescue and Defence mechanisms, standing out the presence of heat shock chaperones, metalloproteinases, broad signalling cross-talkers and other putative signalling proteins. The data has been deposited to the ProteomeXchange with identifier PXD001133.ConclusionsyECM, as the mammalian counterpart, emerges as highly proteinaceous. As in higher Eukaryotes ECM, numerous proteins that could allow dynamic remodelling, and signalling events to occur in/and via yECM were identified. Importantly, large sets of enzymes encompassing full antagonistic metabolic pathways, suggest that mats develop into two metabolically distinct populations, suggesting that either extensive moonlighting or actual metabolism occurs extracellularly. The gup1∆ showed abnormally loose ECM texture. Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance.

Presynaptic Mechanisms of l-DOPA-Induced Dyskinesia: The Findings, the Debate, and the Therapeutic Implications.

The dopamine (DA) precursor l-DOPA has been the most effective treatment for Parkinson’s disease (PD) for over 40 years. However, the response to this treatment changes with disease progression, and most patients develop dyskinesias (abnormal involuntary movements) and motor fluctuations within a few years of l-DOPA therapy. There is wide consensus that these motor complications depend on both pre- and post-synaptic disturbances of nigrostriatal DA transmission. Several presynaptic mechanisms converge to generate large DA swings in the brain concomitant with the peaks-and-troughs of plasma l-DOPA levels, while post-synaptic changes engender abnormal functional responses in dopaminoceptive neurons. While this general picture is well-accepted, the relative contribution of different factors remains a matter of debate. A particularly animated debate has been growing around putative players on the presynaptic side of the cascade. To what extent do presynaptic disturbances in DA transmission depend on deficiency/dysfunction of the DA transporter, aberrant release of DA from serotonin neurons, or gliovascular mechanisms? And does noradrenaline (which is synthetized from DA) play a role? This review article will summarize key findings, controversies, and pending questions regarding the presynaptic mechanisms of l-DOPA-induced dyskinesia. Intriguingly, the debate around these mechanisms has spurred research into previously unexplored facets of brain plasticity that have far-reaching implications to the treatment of neuropsychiatric disease.

Structural determinants of the hydrogen peroxide permeability of aquaporins

Aquaporins (AQP) conduct small, uncharged molecules, such as water (orthodox AQPs), ammonia (aquaammoniaporins) or glycerol (aquaglyceroporins). The physiological functions of AQPs are involved in osmotic volume regulation or the transport of biochemical precursors and metabolic waste products. The recent identification of hydrogen peroxide (H₂O₂) as a permeant of certain AQPs suggests additional roles in mitigating oxidative stress or enabling paracrine H₂O₂ signalling. Yet, an analysis of the structural requirements of the H₂O₂ permeability of AQPs is missing. We subjected a representative set of wild-type and mutant AQPs to a newly established quantitative phenotypic assay. We confirmed high H₂O₂ permeability of the human aquaammoniaporin AQP8 and found intermediate H₂O₂ permeability of the prototypical orthodox water channel AQP1 from the rat. Differences from an earlier report showing an absence of H₂O₂ permeability of human AQP1 can be explained by expression levels. By generating point mutations in the selectivity filter of rat orthodox aquaporin AQP1, we established a correlation of H₂O₂ permeability primarily with water permeability and secondarily with the pore diameter. Even the narrowest pore of the test set (i.e. rat orthodox aquaporin AQP1 H180F with a pore diameter smaller than that of natural orthodox AQPs) conducted water and H₂O₂. We further found that H₂O₂ permeability of the aquaglyceroporin from the malaria parasite Plasmodium falciparum was lower despite its wider pore diameter. The data suggest that all water-permeable AQPs are H₂O₂ channels, yet H₂O₂ permeability varies with the isoform. Thus, generally, AQPs must be considered as putative players in situations of oxidative stress (e.g. in Plasmodium-infected red blood cells, immune cells, the cardiovascular system or cells expressing AQP8 in their mitochondria).

Isolation and structural identification of glycopolymers of Bifidobacterium bifidum BIM B-733D as putative players in pathogenesis of autoimmune thyroid diseases.

Bifidobacterium bifidum 791 (commercially available as B. bifidum BIM B-733D) cell-surface biopolymers (BPs) interact selectively with human serum thyroid peroxidase (TPO) and thyroglobulin (Tg) autoantibodies (anti TPO and anti Tg, respectively). BPanti-TPO and BPanti-Tg were isolated from the soluble fraction of B. bifidum BIM B-733D by affinity chromatography with anti-TPO or anti-Tg, respectively. Homogeneity of affinity eluates (AEanti-TPO and AEanti-Tg) was tested by size exclusion chromatography. For each AE, the elution profiles generated on the basis of absorbance at 280 nm do not conform to ELISA data for functional activity characteristic of BPs. Moreover, high functional activity was detected in chromatographic fractions that had significantly different molecular weights and no absorbance at 280 nm, which suggests a non-protein (carbohydrate) nature of BPanti-TPO and BPanti-Tg. The semi-preparative size exclusion chromatography of AEanti-TPO and AEanti-Tg with detection by refractometer gave 5,000-7,000 Da fractions containing substances that interact selectively with either anti TPO (BPanti-TPO) or anti-Tg (BPanti-Tg) according to ELISA data. Analysis by two-dimensional NMR spectroscopy including a 1H, 13C-heteronuclear single-quantum coherence experiment indicated that both substances are linear α-1,6-glucans. For the first time, an immunological similarity (molecular mimicry) of glycopolymers of B. bifidum BIM B-733D and human thyroid proteins, TPO and Tg, was shown. On the whole, our data point to a possible role of bifidobacteria in the pathogenesis of autoimmune thyroid diseases (ATD). The main requirements for triggering/acceleration or prevention/abrogation of ATD by bifidobacteria through molecular mimicry mechanism are hypothesised to be (1) genetic predisposition to ATD and (2) intestinal epithelium penetration by α-1,6-glucan.

Wolbachia in the Malpighian Tubules: Evolutionary Dead‐End or Adaptation?

Facultative endosymbionts, such as Wolbachia, perpetuate by vertical transmission mostly through colonization of the germline during embryogenesis. The remaining Wolbachia inside the embryo are internalized in progenitor cells of the somatic tissue. This perpetuation strategy triggers a cyclic bacterial bottleneck across host generations. However, throughout the host's life history (Drosophila, for example), some somatic tissues such as the Malpighian tubules (MTs) show large numbers of Wolbachia. It is assumed that Wolbachia present in the progenitor cells of the MTs are confined to this somatic tissue, implicitly considering MTs as an evolutionary dead-end for these bacteria. Nevertheless, the fact that bacteria can survive and proliferate inside MTs suggests a different fate as they may access the host's reproductive system and persist in the host population through vertical transmission. Indeed, based on the particular physiological and developmental characteristics of MT, as well as of Wolbachia, we argue the bacteria present in the MTs may constitute a secondary pool of vertically transmitted bacteria. Moreover, somatic pools of Wolbachia capable of reaching the gonads and insure vertical transmission may also provide an interesting element to the elucidation of horizontal transmission mechanisms. Finally, we also speculate that somatic pools of Wolbachia may play an important role in host fitness, namely during viral infections. In brief, we argue that the somatic pools of Wolbachia, with special emphasis on the MT subset, deserve experimental attention as putative players in the physiology and evolution of both bacteria and hosts.