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The development of advanced genetic tools is boosting microbial engineering which can potentially tackle wide-ranging challenges currently faced by our society. Here we present SURE editing, a multi-recombinase engineering rationale combining oligonucleotide recombineering with the selective capacity of antibiotic resistance via transient insertion of selector plasmids. We test this method in Mycoplasma pneumoniae, a bacterium with a very inefficient native recombination machinery. Using SURE editing, we can seamlessly generate, in a single step, a wide variety of genome modifications at high efficiencies, including the largest possible deletion of this genome (30 Kb) and the targeted complementation of essential genes in the deletion of a region of interest. Additional steps can be taken to remove the selector plasmid from the edited area, to obtain markerless or even scarless edits. Of note, SURE editing is compatible with different site-specific recombinases for mediating transient plasmid integration. This battery of selector plasmids can be used to select different edits, regardless of the target sequence, which significantly reduces the cloning load associated to genome engineering projects. Given the proven functionality in several microorganisms of the machinery behind the SURE editing logic, this method is likely to represent a valuable advance for the synthetic biology field. European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program [670216 (MYCOCHASSIS)]; Spanish Ministry of Economy, Industry and Competitiveness (MEIC) (to the EMBL partnership); Centro de Excelencia Severo Ochoa; CERCA Program from the Generalitat de Catalunya; European Union's Horizon 2020 Research; Innovation Program [634942 (MycoSynVac)]; LaCaixa Fundation [Livetherapeutics HR18-00058]; C.P.-L. acknowledges the support of ‘Programa Torres Quevedo’ grant [PTQ2020-011048] funded by MCIN/AEI/10.13039/501100011033; European Union ‘NextGenerationEU/PRTR’; M.L.-S. acknowledges the support from FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016) [CP16/00094].

Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome-reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm-associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo. We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro, ex vivo, and in vivo. To our knowledge, this is the first engineered genome-reduced bacterium that can fight against clinically relevant biofilm-associated bacterial infections., This work has been supported by the European Research Council (ERC) underthe European Union’s Horizon2020research and innovation program, undergrant agreement 670216(MYCOCHASSIS). We also acknowledge the support ofthe Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to theEMBL partnership, the Centro de Excelencia Severo Ochoa, the CERCA Programfrom the Generalitat de Catalunya, the European Union’s Horizon2020research and innovation program under grant agreement 634942 (Myco-SynVac), and the LaCaixa Fundation grant (Livetherapeutics HR18-00058).M.L.-S. acknowledges the support from FEDER project from Instituto Carlos III(ISCIII, Accion Estrategica en Salud2016) (reference CP16/00094). We alsoacknowledge the staff of CRG/UPF Proteomics Unit, which is part of the Span-ish Infrastructure for Omics Technologies (ICTS OmicsTech) unit is a memberof the ProteoRed PRB3consortium, which is supported by grant PT17/0019ofthe PE I+D+i2013-2016from the Instituto de Salud Carlos III (ISCIII) and ERDF. We also acknowledge Margarita Ecay for her assistance in the PETimages analysis and Samuel Miravet-Verde for the bioinformatic support.

RNA editing is being recognized as an important post-transcriptional mechanism that may have crucial roles in introducing genetic variation and phenotypic diversity. Despite microRNA editing recurrence, defining its biological relevance is still under extended debate. To better understand microRNA editing function and regulation we performed an exhaustive characterization of the A-to-I site-specific patterns in mir-376a-1, a mammalian microRNA which RNA editing is involved in the regulation of development and in disease. Thorough an integrative approach based on high-throughput small RNA sequencing, Sanger sequencing and computer simulations we explored mir-376a-1 editing in samples from various individuals and primate species including human placenta and macaque, gorilla, chimpanzee and human brain cortex. We observed that mir-376a-1 editing is a common phenomenon in the mature and primary microRNA molecules and it is more frequently detected in brain than in placenta. Primary mir-376a-1 is edited at three positions, -1, +4 and +44. Editing frequency estimations and in silico simulations indicated that editing was not equally recurrent along the three mir-376a-1 sites, nevertheless no epistatic interactions among them were observed. Particularly, the +4 site, located in the seed region of the mature miR-376a-5p, reached the highest editing frequency in all samples. Secondary structure predictions revealed that the +4 position was the one that conferred the highest stability to the mir-376a-1 hairpin. We suggest that molecular stability might partially explain the editing recurrence observed in certain microRNAs and that editing events conferring new functional regulatory roles in particular tissues and species could have been conserved along evolution, as it might be the case of mir-376a-1 in primate brain cortex.

Mycoplasma species share a set of features, such as lack of a cell wall, streamlined genomes, simplified metabolism, and the use of a deviant genetic code, that make them attractive approximations of what a chassis strain should ideally be. Among them, Mycoplasma pneumoniae arises as a candidate for synthetic biology projects, as it is one of the most deeply characterized bacteria. However, the historical paucity of tools for editing Mycoplasma genomes has precluded the establishment of M. pneumoniae as a suitable chassis strain. Here, we developed an oligonucleotide recombineering method for this strain based on GP35, a ssDNA recombinase originally encoded by a Bacillus subtilis-associated phage. GP35-mediated oligo recombineering is able to carry out point mutations in the M. pneumoniae genome with an efficiency as high as 2.7 × 10-2, outperforming oligo recombineering protocols developed for other bacteria. Gene deletions of different sizes showed a decreasing power trend between efficiency and the scale of the attempted edition. However, the editing rates for all modifications increased when CRISPR/Cas9 was used to counterselect nonedited cells. This allowed edited clones carrying chromosomal deletions of up to 1.8 kb to be recovered with little to no screening of survivor cells. We envision this technology as a major step toward the use of M. pneumoniae, and possibly other Mycoplasmas, as synthetic biology chassis strains. This project has received funding from the European Union’s Horizon 2020 research and innovation program under Grant 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under Grant 670216 (MYCOCHASSIS) and the FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016) (reference CP16/00094). We also acknowledge support of the Spanish Ministry of Science and Innovation, to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme/Generalitat de Catalunya. Finally, we would like to thank Dr. Sarah A. Head for critical manuscript revision.

Nucleotide variants in microRNA regions have been associated with disease; nevertheless, few studies still have addressed the allele-dependent effect of these changes. We studied microRNA genetic variation in human populations and found that while low-frequency variants accumulate indistinctly in microRNA regions, the mature and seed regions tend to be depleted of high-frequency variants, probably as a result of purifying selection. Comparison of pairwise population fixation indexes among regions showed that the seed had higher population fixation indexes than the other regions, suggesting the existence of local adaptation in the seed region. We further performed functional studies of three microRNA variants associated with cancer (rs2910164:C > G in MIR146A, rs11614913:C > T in MIR196A2, and rs3746444:A > G in both MIR499A and MIR499B). We found differences in the expression between alleles and in the regulation of several genes involved in cancer, such as TP53, KIT, CDH1, CLH, and TERT, which may result in changes in regulatory networks related to tumorigenesis. Furthermore, luciferase-based assays showed that MIR499A could be regulating the cadherin CDH1 and the cell adhesion molecule CLH1 in an allele-dependent fashion. A better understanding of the effect of microRNA variants associated with disease could be key in our way to a more personalized medicine., Contract grant sponsors: Spanish National Institute for Bioinformatics (www.inab.org); “Ministerio de Ciencia e Innovación de España” (BFU2010-18477, CGL2009-09013 and FPI-MINECO); European Union Seventh Framework Programme (PIOF-GA-2009-236836 and PIRSES-GA-2013-612583); “Direcció general de Recerca de la Generalitat de Catalunya; Agencia de Gestió d'Ajuts Universitaris de Recerca” (2014SGR-866, 2014SGR-1311); “Ministerio de Educación, Cultura y Deporte de España” (FPU-MEC, FPU13/06813); FEDER European Regional Development Fund “A way to build Europe”; Ministerio de Economia y Competitividad, Spain (grant no. BFU2013-43726-P).

Synthetic biology seeks to envision living cells as a matter of engineering. However, increasing evidence suggests that the genetic load imposed by the incorporation of synthetic devices in a living organism introduces a sort of unpredictability in the design process. As a result, individual part characterization is not enough to predict the behavior of designed circuits and thus, a costly trial-error process is eventually required. In this work, we provide a new theoretical framework for the predictive treatment of the genetic load. We mathematically and experimentally demonstrate that dependences among genes follow a quantitatively predictable behavior. Our theory predicts the observed reduction of the expression of a given synthetic gene when an extra genetic load is introduced in the circuit. The theory also explains that such dependence qualitatively differs when the extra load is added either by transcriptional or translational modifications. We finally show that the limitation of the cellular resources for gene expression leads to a mathematical formulation that converges to an expression analogous to the Ohm's law for electric circuits. Similitudes and divergences with this law are outlined. Our work provides a suitable framework with predictive character for the design process of complex genetic devices in synthetic biology., Fundación Botín, Banco de Santander through its Santander Universities Global Division [BES-2010-038940]; ERC SYNCOM [291294]; Spanish Ministry of Economy and Competitiveness [MINECO SAF2014-59284-R and FEDER]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [MINECO SAF2014-59284-R and FEDER].

Objective: Clinical progression in lacunar strokes (LS) is an unpredictable and fearful complication. Endothelial dysfunction (ED) is believed to be the first step in the pathophysiology of LS therefore we aimed to analyze the association of three markers of ED: albuminuria, von Willebrand factor (vWF), and oxidized LDL cholesterol (ox-LDL) with LS progression. Methods: From December 2007 to December 2010, 127 LS patients admitted within 6 h of symptom onset were prospectively assessed. Progression was defined as initial NIHSS score worsening ≥4 points within the first 72 h. Analysis of vWF and ox-LDL was done at admission. Albuminuria was measured in the first morning spot urine. Association between 3 biomarkers and progression was tested using logistic regression analysis. Other clinical variables of interest were also studied. Discriminative power was analyzed with a receiver operator curve. Results: Twenty-two patients (17.3%) progressed. Progression was associated with worse outcome at 90 days. Albuminuria and ox-LDL were associated in univariate analysis; vWF was not. Adjusted OR were: ox-LDL [OR: 1.03; 95% CI: 1.01–1.07, p = 0.019], albuminuria [OR: 2.07; 95% CI: 1.04–4.13, p = 0.039]. Association was linear without a cut-off point. Clinical variables were not associated with progression. The model including albuminuria and ox-LDL had a good predictive value [AUC: 0.80 [0.70–0.89)]. Conclusions: Albuminuria and ox-LDL levels are independently associated with higher risk of progression in LS. The lack of reliable clinical predictors makes biomarker research a priority to improve progression detection in this subtype of ischemic strokes. © 2011 Elsevier Ireland Ltd. All rights reserved.

Hepatic stellate cells (HSCs) participate in the regulation of hepatic microcirculation and have receptors for many vasoconstrictor factors. It is unknown whether HSCs have receptors for circulating vasodilators such as atrial natriuretic peptide (ANP). This study investigated the presence of ANP receptors in human HSCs and whether ANP antagonizes the effects of endothelin-1 in these cells. ANP receptors were assessed by binding and cross-linking studies, reverse-transcriptase polymerase chain reaction (PCR), and measuring intracellular cyclic guanosine monophosphate concentration. Intracellular calcium concentration ([Ca(2+)](i)) and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method. Binding and cross-linking affinity experiments showed the existence of ANP receptors in human HSCs. PCR products with the expected length were obtained for guanylate cyclase A receptor, the physiological receptor of ANP, both in quiescent and activated human cells. ANP induced a dose-dependent increase in intracellular cyclic guanosine monophosphate concentration and blunted the increase in [Ca(2+)](i) elicited by endothelin-1. Most importantly, ANP markedly reduced cell contraction induced by endothelin-1. HSCs isolated from rats with carbon tetrachloride-induced cirrhosis showed a higher number of ANP receptors compared with HSCs isolated from normal rats, indicating that in vivo activation of HSCs is associated with an up-regulation of ANP receptors. These results indicate that human HSCs have receptors for ANP, the activation of which reduces the effects of endothelin-1 on [Ca(2+)](i) and cell contraction. ANP could participate in regulating the contractility of HSCs by antagonizing the effect of vasoconstrictors.

Background/Aims: Voltage-operated calcium channels are essential for the regulation of vascular tone and are potential targets for vasodilating agents. They regulate calcium entry and thereby cell contraction in vascular cell types. Hepatic stellate cells in the activated phenotype have contractile properties and could participate in the regulation of sinusoidal blood flow. Thus, this study was aimed at investigating the presence of voltage-operated calcium channels in human hepatic stellate cells activated in culture and the effects of their stimulation on intracellular calcium concentration ([Ca 2+ ] i ) and cell contractility. Methods: Binding studies using [ 3 H]-nitrendipine were performed to demonstrate the presence of voltage-operated calcium channels. Voltage-operated calcium channels were stimulated by causing cell membrane depolarization either by electrical field stimulation or extracellular high potassium. [Ca 2+ ] i and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method with an epifluorescence microscope coupled to a charge-coupled device-imaging system. Results: Binding studies demonstrated the existence of voltage-operated calcium channels in human activated hepatic stellate cells (7.1±1.4×10 4 sites/cell with a Kd of 2.1±0.1 nM). Both electrical field stimulation and potassium chloride-induced cell depolarization resulted in a marked and prolonged increase in [Ca 2+ ] i followed by intense cell contraction. The degree of cell contraction correlated with the intensity of calcium peaks. Removal of extracellular calcium or preincubation of cells with nitrendipine, a specific antagonist of voltage-operated calcium channels, completely blocked the effects on [Ca 2+ ] i and cell contraction, whereas preincubation of cells with BayK-8644, a specific agonist of voltage-operated calcium channels, increased calcium peaks and contraction. Conclusion: Activated human hepatic stellate cells have a large number of voltage-operated calcium channels, the activation of which is associated with an increase in [Ca 2+ ] i followed by marked cell contraction. Voltage-operated calcium channels probably play an important role in the regulation of activated hepatic stellate cells contractility.

Gallego, Alicia et al., microRNAs are crucial post-transcriptional regulators of gene expression involved in a wide range of biological processes. Although microRNAs are highly conserved among species, the functional implications of existing lineage-specific changes and their role in determining differences between humans and other great apes have not been specifically addressed. We analyzed the recent evolutionary history of 1,595 human microRNAs by looking at their intra- and inter-species variation in great apes using high-coverage sequenced genomes of 82 individuals including gorillas, orangutans, bonobos, chimpanzees and humans. We explored the strength of purifying selection among microRNA regions and found that the seed and mature regions are under similar and stronger constraint than the precursor region. We further constructed a comprehensive catalogue of microRNA species-specific nucleotide substitutions among great apes and, for the first time, investigated the biological relevance that human-specific changes in microRNAs may have had in great ape evolution. Expression and functional analyses of four microRNAs (miR-299-3p, miR-503-3p, miR-508-3p and miR-541-3p) revealed that lineage-specific nucleotide substitutions and changes in the length of these microRNAs alter their expression as well as the repertoires of target genes and regulatory networks. We suggest that the studied molecular changes could have modified crucial microRNA functions shaping phenotypes that, ultimately, became human-specific. Our work provides a frame to study the impact that regulatory changes may have in the recent evolution of our species., This work was funded by Spanish National Institute for Bioinformatics (www.inab.org); “Ministerio de Ciencia e Innovación de España” [grant numbers BFU2012-38236, BFU2010-18477, BFU2009-06974, CGL2009-09013 and FPI-MINECO to ITL]; “Direcció General de Recerca de la Generalitat de Catalunya” [2009SGR-1101, 2014SGR-866 and SGR2014-1311]; European Union Seventh Framework Programme [grant number PIOF-GA-2009-236836 and PIRSES-GA-2013-612583]; “Ministerio de Educación, Cultura y Deporte de España” [grant FPU-MEC to AG]; FEDER European Regional Development Fund “A way to build Europe”. Three authors of this work were employed by the funding organizations of either the “Parc Zoológic de Barcelona” or the Copenhagen Zoo. The funder “Parc Zoológic de Barcelona” provided support in the form of salaries for authors HFB and TA and the funder Copenhagen Zoo for author CH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. The unique role of “Parc Zoológic de Barcelona” and Copenhagen Zoo was to provide support in the form of salaries for authors HFB, TA and CH. These funders did not have any additional role in the study design, data collection and analysis, decisionto publish, or preparation of the manuscript.

Background/Aims : Hepatic stellate cells (HSCs) are perisinusoidal pericytes which have receptors for vasoactive factors, such as endothelin-1, which can regulate cell contractility in an autocrine manner. It is unknown whether human HSCs have receptors for and are able to synthesize the vasodilator peptide adrenomedullin (ADM), a peptide produced by most contractile cells. Methods and results : Stimulation of HSCs with ADM resulted in a dose-dependent raise in cAMP concentration (radioimmunoassay) and markedly blunted the endothelin-induced increase in [Ca 2+ ] i and cell contraction, as assessed in cells loaded with fura-2 using a morphometric method. The existence of the receptor CRLR for ADM and their associated proteins RAMP-1 and RAMP-2 was demonstrated by reverse transcriptase–polymerase chain reaction (RT–PCR). Moreover, activated human HSCs spontaneously secreted ADM in the culture medium in a time-dependent manner. ADM secretion was markedly enhanced by tumour necrosis factor-α and interleukin-1 β . Specific mRNA for ADM (RT–PCR and Northern blot) was detected in HSCs and increased after incubation of cells with cytokines. Conclusions : Human HSCs have functional receptors for ADM, the stimulation of which blunts the contractile effect of endothelin-1. Cultured human HSCs secrete ADM in baseline conditions. This secretion is markedly increased by cytokines. These results suggest that ADM can regulate HSCs' contractility in an autocrine manner.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are perisinusoidal cells believed to participate in the regulation of hepatic blood flow because of their contractile properties and presence of receptors for several vasoactive factors. It is unknown whether HSCs have receptors for vasopressin, one of the most potent endogenous vasoconstrictors. This study investigated the existence of receptors for and the effects of arginine vasopressin (AVP) on cultured human HSCs. METHODS: intracellular calcium concentration ([Ca2+]i) and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method with an epifluorescence microscope coupled to a CCD imaging system (Photometrics, Tucson, AZ). AVP-specific binding was measured with [3H]AVP. Mitogen-activated protein kinase (MAPk) activity and DNA synthesis were measured by in vitro phosphorylation of myelin basic protein and [3H]thymidine incorporation, respectively. Parallel experiments were performed in vascular smooth muscle cells. RESULTS: AVP elicited a dose-dependent increase in [Ca2+]i and contraction of HSCs. Moreover, AVP increased MAPk activity, DNA synthesis, and cell number. These effects were similar to those observed in vascular smooth muscle cells and were blocked by a V1 receptor antagonist. The existence of V1 receptors was further confirmed by binding studies. CONCLUSIONS: Human HSCs have V1-vasopressin receptors that induce effects similar to those observed in vascular smooth muscle cells. AVP may play a role in the regulation of HSC function. (Gastroenterology 1997 Aug;113(2):615-24)