Antimicrobial Activity and Phytochemical Characterization of Baccharis concava Pers., a Native Plant of the Central Chilean Coast.

Few sclerophyllous plants from the central coast of Chile have been systematically studied. This work describes the phytochemical composition and antimicrobial properties of Baccharis concava Pers. (sin. B. macraei), a shrub found in the first line and near the Pacific coast. B. concava has been traditionally used by indigenous inhabitants of today's central Chile for its medicinal properties. Few reports exist regarding the phytochemistry characterization and biological activities of B. concava. A hydroalcoholic extract of B. concava was prepared from leaves and small branches. Qualitative phytochemical characterization indicated the presence of alkaloids, steroids, terpenoids, flavonoids, phenolic, and tannin compounds. The antimicrobial activity of this extract was assessed in a panel of microorganisms including Gram-positive bacteria, Gram-negative bacteria, and pathogenic yeasts. The extract displayed an important antimicrobial effect against Gram-positive bacteria, Candida albicans, and Cryptococcus neoformans but not against Gram-negatives, for which an intact Lipopolysaccharide is apparently the determinant of resistance to B. concava extracts. The hydroalcoholic extract was then fractionated through a Sephadex LH-20/methanol-ethyl acetate column. Afterward, the fractions were pooled according to a similar pattern visualized by TLC/UV analysis. Fractions obtained by this criterion were assessed for their antimicrobial activity against Staphylococcus aureus. The fraction presenting the most antimicrobial activity was HPLC-ESI-MS/MS, obtaining molecules related to caffeoylquinic acid, dicaffeoylquinic acid, and quercetin, among others. In conclusion, the extracts of B. concava showed strong antimicrobial activity, probably due to the presence of metabolites derived from phenolic acids, such as caffeoylquinic acid, and flavonoids, such as quercetin, which in turn could be responsible for helping with wound healing. In addition, the development of antimicrobial therapies based on the molecules found in B. concava could help to combat infection caused by pathogenic yeasts and Gram-positive bacteria, without affecting the Gram-negative microbiota.

Sex-Specific Control of Human Heart Maturation by the Progesterone Receptor.

BACKGROUND: Despite in-depth knowledge of the molecular mechanisms controlling embryonic heart development, little is known about the signals governing postnatal maturation of the human heart. METHODS: Single-nucleus RNA sequencing of 54 140 nuclei from 9 human donors was used to profile transcriptional changes in diverse cardiac cell types during maturation from fetal stages to adulthood. Bulk RNA sequencing and the Assay for Transposase-Accessible Chromatin using sequencing were used to further validate transcriptional changes and to profile alterations in the chromatin accessibility landscape in purified cardiomyocyte nuclei from 21 human donors. Functional validation studies of sex steroids implicated in cardiac maturation were performed in human pluripotent stem cell-derived cardiac organoids and mice. RESULTS: Our data identify the progesterone receptor as a key mediator of sex-dependent transcriptional programs during cardiomyocyte maturation. Functional validation studies in human cardiac organoids and mice demonstrate that the progesterone receptor drives sex-specific metabolic programs and maturation of cardiac contractile properties. CONCLUSIONS: These data provide a blueprint for understanding human heart maturation in both sexes and reveal an important role for the progesterone receptor in human heart development.

Low molecular weight sulfated chitosan efficiently reduces infection capacity of porcine circovirus type 2 (PCV2) in PK15 cells.

BACKGROUND: Porcine circovirus type 2 (PCV2)-associated diseases are a major problem for the swine industry worldwide. In addition to vaccines, the availability of antiviral polymers provides an efficient and safe option for reducing the impact of these diseases. By virtue of their molecular weight and repetitious structure, polymers possess properties not found in small-molecule drugs. In this perspective, we focus on chitosan, a ubiquitous biopolymer, that adjusts the molecular weight and sulfated-mediated functionality can act as an efficient antiviral polymer by mimicking PCV2-cell receptor interactions. METHODS: Sulfated chitosan (Chi-S) polymers of two molecular weights were synthesized and characterized by FTIR, SEM-EDS and elemental analysis. The Chi-S solutions were tested against PCV2 infection in PK15 cells in vitro and antiviral activity was evaluated by measuring the PCV2 DNA copy number, TCID50 and capsid protein expression, upon application of different molecular weights, sulfate functionalization, and concentrations of polymer. In addition, to explore the mode of action of the Chi-S against PCV2 infection, experiments were designed to elucidate whether the antiviral activity of the Chi-S would be influenced by when it was added to the cells, relative to the time and stage of viral infection. RESULTS: Chi-S significantly reduced genomic copies, TCID50 titers and capsid protein of PCV2, showing specific antiviral effects depending on its molecular weight, concentration, and chemical functionalization. Assays designed to explore the mode of action of the low molecular weight Chi-S revealed that it exerted antiviral activity through impeding viral attachment and penetration into cells. CONCLUSIONS: These findings help better understanding the interactions of PCV2 and porcine cells and reinforce the idea that sulfated polymers, such as Chi-S, represent a promising candidates for use in antiviral therapies against PCV2-associated diseases. Further studies in swine are warranted.

Biological Effects of Quinolones: A Family of Broad-Spectrum Antimicrobial Agents.

Broad antibacterial spectrum, high oral bioavailability and excellent tissue penetration combined with safety and few, yet rare, unwanted effects, have made the quinolones class of antimicrobials one of the most used in inpatients and outpatients. Initially discovered during the search for improved chloroquine-derivative molecules with increased anti-malarial activity, today the quinolones, intended as antimicrobials, comprehend four generations that progressively have been extending antimicrobial spectrum and clinical use. The quinolone class of antimicrobials exerts its antimicrobial actions through inhibiting DNA gyrase and Topoisomerase IV that in turn inhibits synthesis of DNA and RNA. Good distribution through different tissues and organs to treat Gram-positive and Gram-negative bacteria have made quinolones a good choice to treat disease in both humans and animals. The extensive use of quinolones, in both human health and in the veterinary field, has induced a rise of resistance and menace with leaving the quinolones family ineffective to treat infections. This review revises the evolution of quinolones structures, biological activity, and the clinical importance of this evolving family. Next, updated information regarding the mechanism of antimicrobial activity is revised. The veterinary use of quinolones in animal productions is also considered for its environmental role in spreading resistance. Finally, considerations for the use of quinolones in human and veterinary medicine are discussed.

Cysteine auxotrophy drives reduced susceptibility to quinolones and paraquat by inducing the expression of efflux-pump systems and detoxifying enzymes in S. Typhimurium.

Currently, Salmonella enterica serovar Typhimurium (S. Typhimurium), is a major global public health problem, which has caused food-borne illnesses in many countries. Today, with the extensive use of antimicrobials, antimicrobial resistance is increasing at a serious rate in S. Typhimurium isolates. The present study sought the role of cysteine (Cys) auxotrophy on the resistance to quinolones and paraquat in S. Typhimurium. Cys auxotrophy was achieved by deleting either the cysDNC, cysJIH or cysQ loci. Deletion of these loci resulted in loss of susceptibility against nalidixic acid, levofloxacin, ciprofloxacin (CIP) and paraquat. Further studies with cysJIH mutant indicated increased expression of multi-antibiotic resistance genes marA and ramA, and consequently increased expression of efflux-pump systems. The cysJIH mutant presented a smaller increase of reactive oxygen species (ROS) in presence of paraquat or CIP. Expression of katG and sodA (expressing for a catalase and a superoxide dismutase, respectively) genes was increased in presence of paraquat in the cysJIH mutant; while expression of the superoxide dismutase gene sodB was decreased. These results indicate that deletion of cysDNC, cysJIH or cysQ genes of S. Typhimurium renders Cys auxotrophy along with decreased susceptibility in response to quinolone and paraquat. Overexpression of efflux-pump systems AcrB-TolC and SmvA-OmpD and antioxidant enzymes KatG and SodA could explain the mechanisms of antimicrobial resistance in the Cys auxotrophic mutants.

A PCR-RFLP assay for discrimination of Echinococcus granulosus sensu stricto and Taenia spp. in dogs stool.

In order to develop a method of identification and discrimination of Echinococcus granulosus sensu stricto from faecal samples of dogs infected with taeniid eggs (Echinococcus spp., Taenia spp.), a combined strategy of Polymerase Chain Reaction (PCR) and Restriction Fragments of Lenght Polymorphisms (RFLP) was proposed. Initially, a pair of primers was designed to amplify a fragment of the 12 Subunit of ribosomal RNA gene (12SrRNA) from mitochondrial DNA. The amplified product was digested by SspI restriction enzyme, which in E. granulosus kept the intact fragment of 160 basis pairs (bp), while in Taenia spp. produced two fragments (62 bp and another of 98 bp). The method was tested using positive controls of DNA, in faecal samples experimentally contaminated with eggs of E. granulosus and Taenia spp. and in dogs naturally infected. In all of them, reproducible results were obtained and the primers were specific to amplify only Taeniidae DNA. The sensitivity of the technique was tested, achieving amplification of DNA extractions with a single egg. In conclusion, the technique developed was optimal and easy to identify patent infections by E. granulosus s.s., constituting a possible alternative for epidemiological studies in dogs, especially in endemic areas where this infection occurs.

DNA extraction in Echinococcus granulosus and Taenia spp. eggs in dogs stool samples applying thermal shock.

The extraction of DNA in taeniid eggs shows complications attached to the composition of stool samples and the high resistance of eggs to degradation. The objective of this study was to test a method of DNA extraction in taeniid eggs by applying a thermal shock to facilitate the chemical-enzymatic degradation of these elements. A group of six tubes containing 1 ml of dog stool sample was spiked with eggs of Echinococcus granulosus and another group of six with Taenia pisiformis. Samples were floated with supersaturated sugar solution and centrifuged. The upper portion of each tube (500 µl) was aspirated and deposited in 1.5 ml tubes. Three tubes from each group were incubated at -20 °C and then at 90 °C, the remaining three from each group, incubated at room temperature. Proteinase K and lysis buffer were added to each tube and incubated for 12 h at 58 °C. The lysis effect was evaluated by microscopy at 3, 6 and 12 h and integrity by electrophoresis in 1% agarose gels. With the same experimental scheme, the thermal shock effect was evaluated in extractions of 1, 2, 3 and 4 eggs of each species and the DNA was quantified. Additionally, the protocol was applied in samples of 4 dogs diagnosed with natural infection by Taeniidae worms. Finally, all the extractions were tested by PCR amplification. Both E. granulosus and T. pisiformis eggs showed a similar response in the tests. In samples without treatment, the lysis effect was poor and showed no differences over time, but in those subjected to thermal shock, eggs degradation increased with time. In both treatments, there was no DNA loss integrity. The protocol applied to limited amounts of eggs yielded PCR products in 100% of the samples exposed to thermal shock, allowing PCR amplifications up to 1 egg. In non-exposed samples, the results were not replicable. However, DNA quantification showed low values in both treatments. In turn, DNA extractions with thermal shock in infected dog samples finally yielded PCR amplifications in 100%. It was concluded that thermal shock facilitates the DNA extraction for molecular analysis in taeniid eggs. The technique is effective extracting DNA even from a single egg and also to analyze natural infections samples with a relatively simple implementation.

Xylose Improves Antibiotic Activity of Chloramphenicol and Tetracycline against K. pneumoniae and A. baumannii in a Murine Model of Skin Infection.

Increased resistance to antimicrobials in clinically important bacteria has been widely reported. The major mechanism causing multidrug resistance (MDR) is mediated by efflux pumps, proteins located in the cytoplasmic membrane to exclude antimicrobial drug. Some efflux pumps recognize and expel a variety of unrelated antimicrobial agents, while other efflux pumps can expel only one specific class of antibiotics. Previously, we have reported that xylose decreases the efflux-mediated antimicrobial resistance in Salmonella typhimurium, Pseudomonas aeruginosa, and Acinetobacter baumannii in vitro. In this work, we assessed the effectiveness of combining xylose with antibiotics to kill resistant Acinetobacter baumannii and Klebsiella pneumoniae in a murine model of skin infection. Skin infections were established by seeding 109 bacteria onto eroded skin of mice. Mice treated with the antibiotic alone or with a mixture of glucose and antibiotics or xylose and antibiotics were compared to a control group that was infected but received no further treatment. We observed that the mixtures xylose-tetracycline and xylose-chloramphenicol produced a decrease of at least 10 times viable Acinetobacter baumannii and Klebsiella pneumoniae recovered from infected skin, compared with mice treated with the antibiotic alone. Our results show that xylose improves the antibiotic activity of tetracycline and chloramphenicol against efflux-mediated resistance Acinetobacter baumannii and Klebsiella pneumoniae, in a murine model of skin infection. We envision these combined formulations as an efficient treatment of skin infections with bacteria presenting efflux-mediated resistance, in both humans and animals.

SPI-9 of Salmonella enterica serovar Typhi is constituted by an operon positively regulated by RpoS and contributes to adherence to epithelial cells in culture.

The genomic island 9 (SPI-9) from Salmonella enterica serovar Typhi (S. Typhi) carries three ORFs (STY2876, STY2877, STY2878) presenting 98 % identity with a type 1 secretory apparatus (T1SS), and a single ORF (STY2875) similar to a large RTX-like protein exhibiting repeated Ig domains. BapA, the Salmonella enterica serovar Enteritidis orthologous to S. Typhi STY2875, has been associated with biofilm formation, and is described as a virulence factor in mice. Preliminary in silico analyses revealed that S. Typhi STY2875 ORF has a 600 bp deletion compared with S. Enteritidis bapA, suggesting that S. Typhi STY2875 might be non-functional. At present, SPI-9 has not been studied in S. Typhi. We found that the genes constituting SPI-9 are arranged in an operon whose promoter was up-regulated in high osmolarity and low pH in a RpoS-dependent manner. All the proteins encoded by S. Typhi SPI-9 were located at the membrane fraction, consistent with their putative role as T1SS. Furthermore, SPI-9 contributed to adherence of S. Typhi to epithelial cells when bacteria were grown under high osmolarity or low pH. Under the test conditions, S. Typhi SPI-9 did not participate in biofilm formation. SPI-9 is functional in S. Typhi and encodes an adhesin induced under conditions normally found in the intestine, such as high osmolarity. Hence, this is an example of a locus that might be designated a pseudogene by computational approaches but not by direct biological assays.

A conditionally lethal mutant of Salmonella Typhimurium induces a protective response in mice.

Here we present the design of a conditionally lethal mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium) which growth depends on tetracycline (Tet). Four mutants of S. Typhimurium, with Tet-conditional growth, were created by inserting the tetRA cassette. Three of the mutants presented a conditional-lethal phenotype in vitro. One mutant in the yabB gene remained conditional inside cells and did not persisted after 24 h in cell cultures. The capacity of S. Typhimurium yabB::tetRA to invade deep organs was investigated in intraperitoneally (IP) infected mice fed with or without chlortetracycline (CTet), a Tet analog with lower antibiotic activity. The yabB::tetRA mutant was undetectable in liver or spleen of animals under normal diet, while in mice under diet including CTet, yabB::tetRA invaded at a level comparable to the WT in mice under normal diet. Moreover, yabB::tetRA produced a strong humoral-immunoresponse after one IP immunization with 10(6) bacteria, measured as serum reactivity against S. Typhimurium whole cell extract. By contrast, oral immunization with 10(6) bacteria was weaker and variable on inducing antibodies. Consistently, IP infected mice were fully protected in a challenge with 10(4) oral S. Typhimurium, while protection was partial in orally immunized mice. Our data indicate that S. Typhimurium yabB::tetRA is a conditionally attenuated strain capable of inducing a protective response in mice in non-permissive conditions.

RpoS integrates CRP, Fis, and PhoP signaling pathways to control Salmonella Typhi hlyE expression.

BACKGROUND: SPI-18 is a pathogenicity island found in some Salmonella enterica serovars, including S. Typhi. SPI-18 harbors two ORFs organized into an operon, hlyE and taiA genes, both implicated in virulence. Regarding the hlyE regulation in S. Typhi, it has been reported that RpoS participates as transcriptional up-regulator under low pH and high osmolarity. In addition, CRP down-regulates hlyE expression during exponential growth. Previously, it has been suggested that there is another factor related to catabolite repression, different from CRP, involved in the down-regulation of hlyE. Moreover, PhoP-dependent hlyE up-regulation has been reported in bacteria cultured simultaneously under low pH and low concentration of Mg2+. Nevertheless, the relative contribution of each environmental signal is not completely clear. In this work we aimed to better understand the regulation of hlyE in S. Typhi and the integration of different environmental signals through global regulators. RESULTS: We found that Fis participates as a CRP-independent glucose-dependent down-regulator of hlyE. Also, Fis and CRP seem to exert the repression over hlyE through down-regulating rpoS. Moreover, PhoP up-regulates hlyE expression via rpoS under low pH and low Mg2+ conditions. CONCLUSIONS: All these results together show that, at least under the tested conditions, RpoS is the central regulator in the hlyE regulatory network, integrating multiple environmental signals and global regulators.

Parasitic findings on threatened pudu deer from Central Chile accounts first genetic characterization of lice parasitizing P. puda in Chile and the first molecular report of Taenia hydatigena metacestodes in this species.

Southern pudu (Pudu puda) is a threatened endemic deer of the temperate forests of Chile. In recent years pudu populations rates have decreased mainly due to anthropogenic causes including forest loss and landscape fragmentation. In this context, the parasitic fauna of Chilean pudu has been scarcely investigated. The aim of this study was to determine the parasitic status of rescued pudu n = 13 from its natural habitat in Central Chile (Maule region) during March 2022 and June 2023 by applying morphological, histopathological, and molecular analyses. As result, we report the presence of transmission of parasites from dogs to pudus as showed by the presence of metacestodes of the parasite Taenia hydatigena on omentum, liver, and pleura of pudus during postmortem examinations, being the first molecular report on the presence of this parasite on Chilean pudu. Meanwhile, ectoparasite examinations determined the presence of chewing and sucking lice on pudu exemplars here analysed. Molecular and phylogenetic analysis of lice revealed new insights on Bovicola and Anoplura lice parasitizing P. puda in Chile, equally being the first genetic characterization of lice parasitizing pudu exemplars in Chile. In addition, parasite loads of lice and metacestodes were analysed. However, no statistically significance was observed when comparing environmental and individual traits influence on parasite load variation. Overall, the study area is the northern limit of habitat distribution of this specie in Chile and we here provide novel information on pudu deer parasites, thus making a useful and valuable contribution to the parasitological knowledge on this threatened species.

Design of a New Vaccine Prototype against Porcine Circovirus Type 2 (PCV2), M. hyopneumoniae and M. hyorhinis Based on Multiple Antigens Microencapsulation with Sulfated Chitosan.

This work evaluated in vivo an experimental-multivalent-vaccine (EMV) based on three Porcine Respiratory Complex (PRC)-associated antigens: Porcine Circovirus Type 2 (PCV2), M. hyopneumoniae (Mhyop) and M. hyorhinis (Mhyor), microencapsulated with sulfated chitosan (M- ChS + PRC-antigens), postulating chitosan sulphate (ChS) as a mimetic of the heparan sulfate receptor used by these pathogens for cell invasion. The EMV was evaluated physicochemically by SEM (Scanning-Electron-Microscopy), EDS (Energy-Dispersive-Spectroscopy), Pdi (Polydispersity-Index) and zeta potential. Twenty weaned pigs, distributed in four groups, were evaluated for 12 weeks. The groups 1 through 4 were as follows: 1-EMV intramuscular-route (IM), 2-EMV oral-nasal-route (O/N), 3-Placebo O/N (M-ChS without antigens), 4-Commercial-vaccine PCV2-Mhyop. qPCR was used to evaluate viral/bacterial load from serum, nasal and bronchial swab and from inguinal lymphoid samples. Specific humoral immunity was evaluated by ELISA. M-ChS + PRC-antigens measured between 1.3-10 µm and presented low Pdi and negative zeta potential, probably due to S (4.26%). Importantly, the 1-EMV protected 90% of challenged animals against PCV2 and Mhyop and 100% against Mhyor. A significant increase in antibody was observed for Mhyor (1-EMV and 2-EMV) and Mhyop (2-EMV), compared with 4-Commercial-vaccine. No difference in antibody levels between 1-EMV and 4-Commercial-vaccine for PCV2-Mhyop was observed. Conclusion: The results demonstrated the effectiveness of the first EMV with M-ChS + PRC-antigens in pigs, which were challenged with Mhyor, PCV2 and Mhyop, evidencing high protection for Mhyor, which has no commercial vaccine available.

Comparative transcriptomic analysis of chemoreceptors in two sympatric scarab beetles, Hylamorpha elegans and Brachysternus prasinus.

Chemoreception through odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs) represents the functions of key proteins in the chemical ecology of insects. Recent studies have identified chemoreceptors in coleopterans, facilitating the evolutionary analysis of not only ORs but also IRs and GRs. Thus, Cerambycidae, Tenebrionidae and Curculionidae have received increased attention. However, knowledge of the chemoreceptors from Scarabaeidae is still limited, particularly for those that are sympatric. Considering the roles of chemoreceptors, this analysis could shed light on evolutionary processes in the context of sympatry. Therefore, the aim of this study was to identify and compare the repertoires of ORs, GRs and IRs between two sympatric scarab beetles, Hylamorpha elegans and Brachysternus prasinus. Here, construction of the antennal transcriptomes of both scarab beetle species and analyses of their phylogeny, molecular evolution and relative expression were performed. Thus, 119 new candidate chemoreceptors were identified for the first time, including 17 transcripts for B. prasinus (1 GR, 3 IRs and 13 ORs) and 102 for H. elegans (22 GRs, 14 IRs and 66 ORs). Orthologs between the two scarab beetle species were found, revealing specific expansions as well as absence in some clades. Purifying selection appears to have occurred on H. elegans and B. prasinus ORs. Further efforts will be focused on target identification to characterize kairomone and/or pheromone receptors.

Biological evolution and human cognition are analogous information processing systems.

The mechanisms that govern biological evolution and human cognition are analogous, as both follow the same principles of natural information processing systems. In this article, we describe the following five principles that provide an analogy between biological evolution and human cognition: (a) Randomness as Genesis Principle and (b) Borrowing and Reorganizing Principle, which indicate how natural information processing systems obtain information; (c) Narrow Limits of Change Principle and (d) Information Store Principle, which indicate how information is processed and stored; and (e) Environmental Organizing and Linking Principle, which indicate how stored information is used to generate actions appropriate to an environment. In human cognition, these analogs only apply to cognitive processes associated with biologically secondary knowledge, the knowledge typically taught in educational institutions. Based on these five principles, cognitive load theory researchers have provided diverse prescriptions to optimize instructional activities and materials. We conclude by discussing general instructional implications and future research directions based on this analogy.

Dexamethasone enhances 1alpha,25-dihydroxyvitamin D3 effects by increasing vitamin D receptor transcription.

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner.

The carbon source influences the efflux pump-mediated antimicrobial resistance in clinically important Gram-negative bacteria.

OBJECTIVES: Multidrug efflux pumps are proteins known to play an important role in resistance in bacteria. These proteins are located in the inner membrane (IM), together with many other proteins, including inducible permeases that participate in the uptake of non-phosphotransferase system (PTS) carbohydrates (i.e. carbohydrates uptaken by mechanisms other than the PTS). However, lipid bilayer space in the IM is limited. Therefore, we examined whether the overexpression of unrelated IM proteins is able to interfere with the efflux-mediated resistance mechanism, consequently increasing the susceptibility towards different antimicrobial compounds. METHODS: We cultured bacteria under different conditions that increase the synthesis of unrelated IM proteins, either by using a non-PTS carbohydrate as the sole carbon source or by artificially overexpressing IM proteins, prior to determining the resistance to different antimicrobial compounds by disc diffusion assays. RESULTS: We observed that efflux-pump-mediated resistance is affected by the carbon source in all the strains tested, exhibiting increased susceptibility when a non-PTS carbohydrate was used as the sole carbon source. Moreover, when we artificially overexpressed an unrelated IM protein, we also observed decreased efflux-mediated resistance. CONCLUSIONS: These results strongly suggest that overexpression of IM proteins, by using a non-PTS carbohydrate as the sole carbon source, or by artificially introducing a high number of copies of an unrelated IM protein, competes with the antibiotic efflux systems, thereby decreasing the efflux-mediated resistance to different antimicrobial compounds. This sort of competition arises because of the limited available space in the bacterial IM, or by an unknown mechanism.

Human pluripotent stem cell-derived macrophages host Mycobacterium abscessus infection.

Human macrophages are a natural host of many mycobacterium species, including Mycobacterium abscessus (M. abscessus), an emerging pathogen affecting immunocompromised and cystic fibrosis patients with few available treatments. The search for an effective treatment is hindered by the lack of a tractable in vitro intracellular infection model. Here, we established a reliable model for M. abscessus infection using human pluripotent stem cell-derived macrophages (hPSC-macrophages). hPSC differentiation permitted reproducible generation of functional macrophages that were highly susceptible to M. abscessus infection. Electron microscopy demonstrated that M. abscessus was present in the hPSC-macrophage vacuoles. RNA sequencing analysis revealed a time-dependent host cell response, with differing gene and protein expression patterns post-infection. Engineered tdTOMATO-expressing hPSC-macrophages with GFP-expressing mycobacteria enabled rapid image-based high-throughput analysis of intracellular infection and quantitative assessment of antibiotic efficacy. Our study describes the first to our knowledge hPSC-based model for M. abscessus infection, representing a novel and accessible system for studying pathogen-host interaction and drug discovery.

Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure.

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.

Biochemical characterization of nuclear receptors for vitamin D3 and glucocorticoids in prostate stroma cell microenvironment.

The disruption of stromal cell signals in prostate tissue microenvironment influences the development of prostate cancer to androgen independence. 1α,25-Dihydroxyvitamin D(3) (1,25D(3)) and glucocorticoids, either alone or in combination, have been investigated as alternatives for the treatment of advanced prostate cancers that fails androgen therapies. The effects of glucocorticoids are mediated by the intracellular glucocorticoid receptor (GR). Similarly, the effect of 1,25D(3) is mediated by the 1,25D(3) nuclear receptor (VDR). In this study, fibroblasts from benign- (BAS) and carcinoma-associated stroma (CAS) were isolated from human prostates to characterize VDR and GR function as transcription factors in prostate stroma. The VDR-mediated transcriptional activity assessed using the CYP24-luciferase reporter was limited to 3-fold induction by 1,25D(3) in 9 out of 13 CAS (70%), as compared to >10-fold induction in the BAS clinical sample pair. Expression of His-tagged VDR (Ad-his-VDR) failed to recover the low transcriptional activity of the luciferase reporter in 7 out of 9 CAS. Interestingly, expression of Ad-his-VDR successfully recovered receptor-mediated induction in 2 out of the 9 CAS analyzed, suggesting that changes in the receptor protein itself was responsible for decreased response and resistance to 1,25D(3) action. Conversely, VDR-mediated transcriptional activity was more efficient in 4 out of 13 CAS (30%), as compared to the BAS sample pair. Consistent with the reduced response to 1,25D(3) observed in CAS, chromatin immunoprecipitation (ChIP) assays indicated decreased recruitment of coactivators SRC-1/CBP, without major changes in the recruitment of VDR to the CYP24 promoter. In addition, we observed that GR-mediated transcriptional activity was also altered in CAS, as compared to BAS. Disruption of coactivators SRC-1/CBP recruitment may promote hormone resistance in CaP, and highlights the relevance of molecular diagnosis and drug design in tumor cell microenvironment.