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Mesenchymal stromal cells are excellent candidates for regenerative medicine since they are multipotent, easy to isolate, can be expanded to obtain clinically relevant numbers and are immunoprivileged. Stable genetic modification with viral vectors can improve mesenchymal stromal cell function and enhance their therapeutic potential. However, standard viral vectors achieve sub-optimal transduction efficiency with a single infection. On the other hand, multiple transduction cycles or antibiotic-based selection methods may alter the stem cell phenotype. We hypothesized that the use of lentiviral vectors containing specific regulatory sequences may result in improved transduction efficiency. Thus, we compared two types of third generation lentiviral vectors, one of which, the pLenti7.3 vector, contains the optimized sequences for Polypurine Tract and Woodchuck Post-transcriptional Regulatory Element. We demonstrated that with the pLenti7.3 it is possible to efficiently transduce human mesenchymal stromal cells with a single transduction cycle. Additionally, we successfully showed that by using the pLenti7.3 vector it is possible to efficiently over-express different growth factors, particularly relevant for cardiac protection and differentiation, in human mesenchymal stromal cells. [ABSTRACT FROM AUTHOR]

Background Long QT Syndrome type 2 (LQT2) is caused by mutations in the KCNH2 gene that encodes for the α-subunit (hERG) of the ion channel conducting the rapid delayed rectifier potassium current (I Kr ). We have previously identified a disease causing mutation (IVS9-28A/G) in the branch point of the splicing of KCNH2 intron 9. However, the mechanism through which this mutation causes the disease is unknown. Methods and results We generated human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from fibroblasts of two IVS9-28A/G mutation carriers. IVS9-28A/G iPSC-CMs showed prolonged repolarization time, mimicking what observed at the ECG level in the same patients. The expression of the full-length ERG1a isoform resulted reduced, whereas the C-terminally truncated ERG 1aUSO isoform was upregulated in mutant iPSC-CMs, with consequent alteration of the physiological ERG 1aUSO /ERG1a ratio. Importantly, we observed an impairment of hERG trafficking to the cell membrane. The severity of the alterations in hERG expression and trafficking correlated with the clinical severity of the disease in the two patients under study. Finally, we were able to revert the trafficking defect and reduce the repolarization duration in LQT2 iPSC-CMs using the proteasome inhibitor ALLN. Conclusion Our results highlight the key role of the KCNH2 intron 9 branch point in the regulation of KCNH2 isoform expression and hERG channel function, and allow to categorize the IVS9-28A/G mutation as LQT2 class 2 mutation. These findings may result in a more personalized clinical management of IVS9-28A/G mutation carriers. [ABSTRACT FROM AUTHOR]

Aurein 2.6-COOH and aurein 3.1-COOH were studied along with their naturally occurring C-terminally amidated analogues. Circular dichroism (CD) and molecular dynamic (MD) simulations were used to study the effects of amidation on the interaction of antimicrobial peptides (AMPs) with lipid bilayers. CD measurements and MD analysis suggested that both peptide analogues were predominantly random coil and adopted low levels of $$\alpha$$ -helical structure in solution (<30 %) and in the presence of a lipid bilayer the peptides formed a stable $$\alpha$$ -helical structure. In general, amidated analogues have a greater propensity than the non-amidated peptides to form a $$\alpha$$ -helical structure. MD simulations predicted that aurein 2.6-COOH and aurein 3.1-CHOOH destabilised lipid bilayers from 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphoserine via angled bilayer penetration. They also showed that aurein 2.6-CONH $$_2$$ and aurein 3.1-CONH $$_2$$ formed a helix horizontal to the plane of an asymmetric interface. [ABSTRACT FROM AUTHOR]

Faking, the intentional distortion of answers to personality tests, is likely a complex process. In particular, participants in previous research have mentioned that they used different kind of strategies to appear more hirable, including systematically more extreme or more midpoint responses. However, quantitative evidence is still lacking. An experiment was conducted in which 327 students (173 women, 153 men, 1 not indicated; M age = 22.1 yr., SD = 2.8) were randomly assigned to two groups. Hypothetical job advertisements primed the participants into believing that the hiring company preferred a person with either a "strong" (Strong Character group) or a "well-balanced" character (Well-balanced Character group). The participants filled out 40 items that were chosen from four established questionnaires as neither socially desirable nor undesirable. The responses to these items were used to calculate two extreme response measures and one midpoint response measure. The Strong Character group used extreme scores more often than the Well-balanced Character group (and the midpoint scores less often), independently of mean differences. This suggests that fakers use more sophisticated strategies than is often assumed. [ABSTRACT FROM AUTHOR]

Maximin H5 is an anionic antimicrobial peptide from amphibians, which carries a C-terminal amide moiety, and was found to be moderately haemolytic (20%). The α-helicity of the peptide was 42% in the presence of lipid mimics of erythrocyte membranes and was found able to penetrate (10.8 mN m − 1 ) and lyse these model membranes (64 %). In contrast, the deaminated peptide exhibited lower levels of haemolysis (12%) and α-helicity (16%) along with a reduced ability to penetrate (7.8 mN m − 1 ) and lyse (55%) lipid mimics of erythrocyte membranes. Taken with molecular dynamic simulations and theoretical analysis, these data suggest that native maximin H5 primarily exerts its haemolytic action via the formation of an oblique orientated α-helical structure and tilted membrane insertion. However, the C-terminal deamination of maximin H5 induces a loss of tilted α-helical structure, which abolishes the ability of the peptide's N-terminal and C-terminal regions to H-bond and leads to a loss in haemolytic ability. Taken in combination, these observations strongly suggest that the C-terminal amide moiety carried by maximin H5 is required to stabilise the adoption of membrane interactive tilted structure by the peptide. Consistent with previous reports, these data show that the efficacy of interaction and specificity of maximin H5 for membranes can be attenuated by sequence modification and may assist in the development of variants of the peptide with the potential to serve as anti-infectives. [ABSTRACT FROM AUTHOR]

A systematic analysis of the hypothesis of the antimicrobial peptides' (AMPs) cooperative action is performed by means of full atomistic molecular dynamics simulations accompanied by circular dichroism experiments. Several AMPs from the aurein family (2.5,2.6, 3.1), have a similar sequence in the first ten amino acids, are investigated in different environments including aqueous solution, trifluoroethanol (TFE), palmitoyloleoylphosphatidylethanolamine (POPE), and palmitoyloleoylphosphatidylglycerol (POPG) lipid bilayers. It is found that the cooperative effect is stronger in aqueous solution and weaker in TFE. Moreover, in the presence of membranes, the cooperative effect plays an important role in the peptide/lipid bilayer interaction. The action of AMPs is a competition of the hydrophobic interactions between the side chains of the peptides and the hydrophobic region of lipid molecules, as well as the intra peptide interaction. The aureins 2.5-COOH and 2.6-COOH form a hydrophobic aggregate to minimize the interaction between the hydrophobic group and the water. Once that the peptides reach the water/lipid interface the hydrophobic aggregate becomes smaller and the peptides start to penetrate into the membrane. In contrast, aurein 3.1-COOH forms only a transient aggregate which disintegrates once the peptides reached the membrane, and it shows no cooperativity in membrane penetration. [ABSTRACT FROM AUTHOR]

Abstract: In this study, an amphibian antimicrobial peptide, aurein 2.3, was predicted to use oblique orientated α-helix formation in its mechanism of membrane destabilisation. Molecular dynamic (MD) simulations and circular dichroism (CD) experimental data suggested that aurein 2.3 exists in solution as unstructured monomers and folds to form predominantly α-helical structures in the presence of a dimyristoylphosphatidylcholine membrane. MD showed that the peptide was highly surface active, which supported monolayer data where the peptide induced surface pressure changes>34mNm−1. In the presence of a lipid membrane MD simulations suggested that under hydrophobic mismatch the peptide is seen to insert via oblique orientation with a phenylalanine residue (PHE3) playing a key role in the membrane interaction. There is evidence of snorkelling leucine residues leading to further membrane disruption and supporting the high level of lysis observed using calcein release assays (76%). Simulations performed at higher peptide/lipid ratio show peptide cooperativity is key to increased efficiency leading to pore-formation. [Copyright &y& Elsevier]

A novel mechanism of hybrid assembly of molecules on surfaces is proposed stemming from interactions between molecules and on-surface metal atoms which eventually got trapped inside the network pores. Based on state-of-the-art theoretical calculations, we find that the new mechanism relies on formation of molecule-metal atom pairs which, together with molecules themselves, participate in the assembly growth. Most remarkably, the dissociation of pairs is facilitated by a cooperative interaction involving many molecules. This new mechanism is illustrated on a low coverage Melamine hexagonal network on the Au( 111) surface where multiple events of gold atoms trapping via a set of so-called "gate" transitions are found by kinetic Monte Carlo simulations based on transition rates obtained using ab initio density functional theory calculations and the nudged elastic band method. Simulated STM images of gold atoms trapped in the pores of the Melamine network predict that the atoms should appear as bright spots inside Melamine hexagons. No trapping was found at large Melamine coverages, however. These predictions have been supported by preliminary STM experiments which show bright spots inside Melamine hexagons at low Melamine coverages, while empty pores are mostly observed at large coverages. Therefore, we suggest that bright spots sometimes observed in the pores of molecular assemblies on metal surfaces may be attributed to trapped substrate metal atoms. We believe that this type of mechanism could be used for delivering adatom species of desired functionality (e.g., magnetic) into the pores of hydrogen-bonded networks serving as templates for their capture. [ABSTRACT FROM AUTHOR]

Typical avian influenza A viruses do not replicate efficiently in humans. The molecular basis of host range restriction and adaptation of avian influenza A viruses to a new host species is still not completely understood. Genetic determinants of host range adaptation have been found on the polymerase complex (PB1, PB2, and PA) as well as on the nucleoprotein (NP). These four viral proteins constitute the minimal set for transcription and replication of influenza viral RNA. It is widely documented that in human cells, avian-derived influenza A viral polymerase is poorly active, but despite extensive study, the reason for this blockade is not known. We monitored the activity of influenza A viral polymerases in heterokaryons formed between avian (DF1) and human (293T) cells. We have discovered that a positive factor present in avian cells enhances the activity of the avian influenza virus polymerase. We found no evidence for the existence of an inhibitory factor for avian virus polymerase in human cells, and we suggest, instead, that the restriction of avian influenza virus polymerases in human cells is the consequence of the absence or the low expression of a compatible positive cofactor. Finally, our results strongly suggest that the well-known adaptative mutation E627K on viral protein PB2 facilitates the ability of a human positive factor to enhance replication of influenza virus in human cells. [ABSTRACT FROM AUTHOR]

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists. [ABSTRACT FROM AUTHOR]

The sheep genome harbors &ap;20 copies of endogenous retroviruses (enJSRVs) closely related to the exogenous and oncogenic Jaag-siekte sheep retrovirus (JSRV). One of the enJSRV loci, enJSS6A1, has a defect for viral exit. We report a previously uncharacterized mechanism of retroviral interference. The defect possessed by enJS56A1 is determined by its Gag protein and is transdominant over the exogenous JSRV. By electron microscopy, cells transfected by eniS56A1, with or without JSRV, show agglomerates of tightly packed intracellular particles most abundant in the perinuclear area. The defect in exit and ability to interfere with JSRV exit could be largely attributed to the presence of tryptophan, rather than arginine, at position 21 of enJS56A1 Gag; C98 and V102 also contribute to these properties. We found that enJS56A1 or similar loci containing W21, C98, and V102 are expressed in sheep endometrium. enJS56A1 is a previously unrecognized example of a naturally occurring endogenous retrovirus expressing a dominant negative Gag acting at a late step of the viral replication cycle. Understanding the late blockade exerted by enJS56A1 could unravel fundamental aspects of retroviral biology and help to devise new antiretroviral strategies. [ABSTRACT FROM AUTHOR]

Betaretroviruses of sheep include two exogenous viruses, Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV), and a group of endogenous viruses known as enJSRVs. The exogenous JSRV and ENTV are the etiological agents of ovine pulmonary adenocarcinoma (OPA) and enzootic nasal tumor (ENT), respectively. Sheep affected by OPA or ENT do not show an appreciable antibody response to JSRV or ENTV. Consequently, it is conceivable that enJSRV expression in the fetal lamb tolerizes sheep to the related exogenous viruses. In this study, possible mechanisms of interference between the sheep exogenous and endogenous betaretroviruses were investigated. In situ hybridization detected enJSRV RNAs in lymphoid cells associated with the lamina propria of the small intestine and in the thymus of sheep fetuses. Low-level expression of enJSRVs was also detected in the lungs. In addition, expression of enJSRVs was found to block entry of the exogenous JSRV, presumably via mechanisms of receptor interference. Indeed, enJSRVs, like JSRV and ENTV, were found to utilize hyaluronidase-2 as a cellular receptor. [ABSTRACT FROM AUTHOR]

IMPROVING AVAILABILITY OF MEDICINES AND MEDICAL DEVICES Shortages of medicines have been a problem in the EU and globally long before the pandemic. Moreover, EMA and ETF members have contributed to scientific and regulatory initiatives by EU bodies such as the directorate-general of the European Commission, the HERA and the EU member states to purchase and use available medicines. With the emergence of COVID-19 in early 2019, the European Union (EU) was confronted with unexpected challenges on many fronts. Finally, the high number of medicines developed and assessed for COVID-19 as opposed to previous emergencies, as well as the duration of the COVID-19 pandemic, have clearly highlighted the need for the EU network to become more agile and to better use available resources in Europe. [Extracted from the article]

BACKGROUND: Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium channel blocker, with proven efficacy in patients with sodium channel-mediated long QT syndrome type 3. Our aim was to evaluate the efficacy of mexiletine in long QT syndrome type 2 (LQT2) using cardiomyocytes derived from patient-specific human induced pluripotent stem cells, a transgenic LQT2 rabbit model, and patients with LQT2. METHODS: Heart rate-corrected field potential duration, a surrogate for QTc, was measured in human induced pluripotent stem cells from 2 patients with LQT2 (KCNH2-p.A561V, KCNH2-p.R366X) before and after mexiletine using a multiwell multi-electrode array system. Action potential duration at 90% repolarization (APD90) was evaluated in cardiomyocytes isolated from transgenic LQT2 rabbits (KCNH2-p.G628S) at baseline and after mexiletine application. Mexiletine was given to 96 patients with LQT2. Patients were defined as responders in the presence of a QTc shortening ≥40 ms. Antiarrhythmic efficacy of mexiletine was evaluated by a Poisson regression model. RESULTS: After acute treatment with mexiletine, human induced pluripotent stem cells from both patients with LQT2 showed a significant shortening of heart rate-corrected field potential duration compared with dimethyl sulfoxide control. In cardiomyocytes isolated from LQT2 rabbits, acute mexiletine significantly shortened APD90 by 113 ms, indicating a strong mexiletine-mediated shortening across different LQT2 model systems. Mexiletine was given to 96 patients with LQT2 either chronically (n=60) or after the acute oral drug test (n=36): 65% of the patients taking mexiletine only chronically and 75% of the patients who performed the acute oral test were responders. There was a significant correlation between basal QTc and ΔQTc during the test (r= -0.8; P<0.001). The oral drug test correctly predicted long-term effect in 93% of the patients. Mexiletine reduced the mean yearly event rate from 0.10 (95% CI, 0.07-0.14) to 0.04 (95% CI, 0.02-0.08), with an incidence rate ratio of 0.40 (95% CI, 0.16-0.84), reflecting a 60% reduction in the event rate (P=0.01). CONCLUSIONS: Mexiletine significantly shortens cardiac repolarization in LQT2 human induced pluripotent stem cells, in the LQT2 rabbit model, and in the majority of patients with LQT2. Furthermore, mexiletine showed antiarrhythmic efficacy. Mexiletine should therefore be considered a valid therapeutic option to be added to conventional therapies in higher-risk patients with LQT2. [ABSTRACT FROM AUTHOR]

Aims In long QT syndrome (LQTS) patients, modifier genes modulate the arrhythmic risk associated with a disease-causing mutation. Their recognition can improve risk stratification and clinical management, but their discovery represents a challenge. We tested whether a cellular-driven approach could help to identify new modifier genes and especially their mechanism of action. Methods and results We generated human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) from two patients carrying the same KCNQ1 -Y111C mutation, but presenting opposite clinical phenotypes. We showed that the phenotype of the iPSC-CMs derived from the symptomatic patient is due to impaired trafficking and increased degradation of the mutant KCNQ1 and wild-type human ether-a-go-go-related gene. In the iPSC-CMs of the asymptomatic (AS) patient, the activity of an E3 ubiquitin-protein ligase (Nedd4L) involved in channel protein degradation was reduced and resulted in a decreased arrhythmogenic substrate. Two single-nucleotide variants (SNVs) on the Myotubularin-related protein 4 (MTMR4) gene, an interactor of Nedd4L, were identified by whole-exome sequencing as potential contributors to decreased Nedd4L activity. Correction of these SNVs by CRISPR/Cas9 unmasked the LQTS phenotype in AS cells. Importantly, the same MTMR4 variants were present in 77% of AS Y111C mutation carriers of a separate cohort. Thus, genetically mediated interference with Nedd4L activation seems associated with protective effects. Conclusion Our finding represents the first demonstration of the cellular mechanism of action of a protective modifier gene in LQTS. It provides new clues for advanced risk stratification and paves the way for the design of new therapies targeting this specific molecular pathway. [ABSTRACT FROM AUTHOR]

Aims  NOS1AP single-nucleotide polymorphisms (SNPs) correlate with QT prolongation and cardiac sudden death in patients affected by long QT syndrome type 1 (LQT1). NOS1AP targets NOS1 to intracellular effectors. We hypothesize that NOS1AP SNPs cause NOS1 dysfunction and this may converge with prolonged action-potential duration (APD) to facilitate arrhythmias. Here we test (i) the effects of NOS1 inhibition and their interaction with prolonged APD in a guinea pig cardiomyocyte (GP-CMs) LQT1 model; (ii) whether pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from LQT1 patients differing for NOS1AP variants and mutation penetrance display a phenotype compatible with NOS1 deficiency. Methods and results  In GP-CMs, NOS1 was inhibited by S-Methyl-L-thiocitrulline acetate (SMTC) or Vinyl-L-NIO hydrochloride (L-VNIO); LQT1 was mimicked by I Ks blockade (JNJ303) and β-adrenergic stimulation (isoproterenol). hiPSC-CMs were obtained from symptomatic (S) and asymptomatic (AS) KCNQ1 -A341V carriers, harbouring the minor and major alleles of NOS1AP SNPs (rs16847548 and rs4657139), respectively. In GP-CMs, NOS1 inhibition prolonged APD, enhanced I CaL and I NaL, slowed Ca2+ decay, and induced delayed afterdepolarizations. Under action-potential clamp, switching to shorter APD suppressed 'transient inward current' events induced by NOS1 inhibition and reduced cytosolic Ca2+. In S (vs. AS) hiPSC-CMs, APD was longer and I CaL larger; NOS1AP and NOS1 expression and co-localization were decreased. Conclusion  The minor NOS1AP alleles are associated with NOS1 loss of function. The latter likely contributes to APD prolongation in LQT1 and converges with it to perturb Ca2+ handling. This establishes a mechanistic link between NOS1AP SNPs and aggravation of the arrhythmia phenotype in prolonged repolarization syndromes. [ABSTRACT FROM AUTHOR]

By reacting with NaCl on Au(111), the formation of hypoxanthine (HX) tetrads is demonstrated at the atomic scale in real space. These results directly demonstrate that alternative purine tetrads can be formed in both planar and non‐planar configuration, and that ionic bonding plays a crucial role for the formation and planar‐to‐stereo transformation of the tetrads, providing deeper insight for constructing artificial DNA/RNA quadruplexes. Moreover, both the tilted HXs and Na show strong charge transfer with the substrate in the non‐planar phase. The insights gained by this work also open up new routes to tune the electrostatic nature of metal–organic interfaces and design stereo‐nanostructures on surfaces. [ABSTRACT FROM AUTHOR]

By reacting with NaCl on Au(111), the formation of hypoxanthine (HX) tetrads is demonstrated at the atomic scale in real space. These results directly demonstrate that alternative purine tetrads can be formed in both planar and non‐planar configuration, and that ionic bonding plays a crucial role for the formation and planar‐to‐stereo transformation of the tetrads, providing deeper insight for constructing artificial DNA/RNA quadruplexes. Moreover, both the tilted HXs and Na show strong charge transfer with the substrate in the non‐planar phase. The insights gained by this work also open up new routes to tune the electrostatic nature of metal–organic interfaces and design stereo‐nanostructures on surfaces. By reacting with NaCl on Au(111), the formation of hypoxanthine (HX) tetrads is demonstrated at the atomic scale in real space. The results show that alternative purine tetrads can be formed in both planar and non‐planar configuration, and that ionic bonding plays a crucial role for the formation and planar‐to‐stereo transformation of the tetrads. [ABSTRACT FROM AUTHOR]

The development of human induced pluripotent stem cell (iPSC) technology has revitalized the efforts made in the last decade to exploit the potential of human embryonic stem cells (ESCs) for scientific research. In the field of cardiac arrhythmias, the possibility of generating an unlimited amount of patient-specific cardiomyocyte-like cells (iPSC-CMs) has clear advantages compared with the use of ESC-derived cardiac cells. In particular, with the introduction and implementation of the large-scale precision medicine initiative, we anticipate that the iPSC technology will play an important role in the advancement of cardiovascular research and medicine. This platform is not free from technical limitations that must be carefully taken into account; however, the utility of iPSC-CMs in disease modeling and drug testing studies is hardly questionable. Here, we summarize some of the progresses made in the field of iPSC technology applied to inherited cardiac arrhythmias, with particular emphasis on the use of iPSC-CMs for modelling the long QT syndrome and for the development of personalized drug and molecular therapies. The growing role of iPSC technology in the practice of precision medicine will also be discussed. [ABSTRACT FROM AUTHOR]

Host defence peptides (HDPs) are antimicrobial agents produced by organisms across the prokaryotic and eukaryotic kingdoms. Many prokaryotes produce HDPs, which utilise lipid and protein receptors in the membranes of bacterial competitors to facilitate their antibacterial action and thereby survive in their niche environment. As a major example, it is well established that cinnamycin and duramycins from Streptomyces have a high affinity for phosphatidylethanolamine (PE) and exhibit activity against other Gram-positive organisms, such as Bacillus. In contrast, although eukaryotic HDPs utilise membrane interactive mechanisms to facilitate their antimicrobial activity, the prevailing view has long been that these mechanisms do not involve membrane receptors. However, this view has been recently challenged by reports that a number of eukaryotic HDPs such as plant cyclotides also use PE as a receptor to promote their antimicrobial activities. Here, we review current understanding of the mechanisms that underpin the use of PE as a receptor in the antimicrobial and other biological actions of HDPs and describe medical and biotechnical uses of these peptides, which range from tumour imaging and detection to inclusion in topical microbicidal gels to prevent the sexual transmission of HIV. [ABSTRACT FROM AUTHOR]

Host defense peptides show great potential for development as new antimicrobial agents with novel mechanisms of action. However, a small number of resistance mechanisms to their action are known, and here, we report a novel bacterial resistance mechanism mediated by a lipid receptor. Maximin H5 from Bombina maxima bound anionic and zwitterionic membranes with low affinity (Kd > 225 μM) while showing a strong ability to lyse (>55%) and penetrate (π > 6.0 mN m-1) these membranes. However, the peptide bound Escherichia coli and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) membranes with higher affinity (Kd < 65 μM) and showed a very low ability for bilayer lysis (<8%) and partitioning (π > 1.0 mN m-1). Increasing levels of membrane DMPE correlated with enhanced binding by the peptide (R² = 0.96) but inversely correlated with its lytic ability (R² = 0.98). Taken with molecular dynamic simulations, these results suggest that maximin H5 possesses membranolytic activity, primarily involving bilayer insertion of its strongly hydrophobic N-terminal region. However, this region was predicted to form multiple hydrogen bonds with phosphate and ammonium groups within PE head-groups, which in concert with charge-charge interactions anchor the peptide to the surface of E. coli membranes, inhibiting its membranolytic action. [ABSTRACT FROM AUTHOR]

Retroviruses use different strategies to regulate transcription and translation and exploit the cellular machinery involved in these processes. This study shows that the signal peptide of the envelope glycoprotein (Env) of Jaagsiekte sheep retrovirus (JSRV) plays a major role in posttranscriptional viral gene expression. Expression of the JSRV Env in trans increases viral particle production by mechanisms dependent on (i) its leader sequence, (ii) an intact signal peptide cleavage site, (iii) a cis-acting RNA-responsive element located in the viral genome, (iv) Crm1, and (v) B23. The signal peptide of the JSRV Env (JSE-SP) is 80 amino acid residues in length and contains putative nuclear localization and export signals, in addition to an arginine-rich RNA binding motif. JSE-SP localizes both in the endoplasmic reticulum and in the nucleus, where it colocalizes with nucleolar markers. JSE-SP is a multifunctional protein, as it moderately enhances nuclear export of unspliced viral mRNA and considerably increases viral particle release by favoring a posttranslational step of the replication cycle. [ABSTRACT FROM AUTHOR]

Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of a contagious lung cancer of sheep known as ovine pulmonary adenocarcinoma (OPA). Expression of the JSRV envelope protein (Env) is sufficient to transform immortalized and primary fibroblasts, but the precise mechanisms of this process are not known. The cellular receptor for JSRV is hyaluronidase 2 (Hyal-2), the product of a putative tumor suppressor gene that in humans maps to a chromosomal region frequently deleted in the development of lung and breast cancers. Here we report studies to determine whether the Hyal-2-JSRV Env interaction plays a role in virus-induced transformation of rodent fibroblasts. Chimeric Env proteins between JSRV and the unrelated murine retroviruses Moloney murine leukemia virus (MMuLV) and mouse mammary tumor virus (MMTV) showed cell surface expression comparable to that of wild-type MMuLV Env and rescued infection of MMuLV particle pseudotypes. Interestingly, an MMuLV-JSRV chimera in which the putative receptor binding domain (RBD) and proline-rich region (PRR) of JSRV Env were replaced by the RBD and PRR of MMuLV induced transformation of 208F, a rodent fibroblast line. Cell lines derived from foci of MMuLV-JSRV chimera-transformed 208F cells grew in soft agar and showed Akt activation, a hallmark of JSRV-transformed rodent fibroblasts. Transformation assays performed using proteins with amino-terminal deletion mutations showed that the carboxy-terminal 141 amino acids of the transmembrane subunit (TM) were sufficient to induce cell transformation when targeted to the membrane with a myristoylation signal. Thus, the JSRV TM is necessary and sufficient to transform rodent fibroblasts. Taken together these results indicate that the interaction with Hyal-2 at least is not an essential determinant of JSRV-induced transformation of fibroblasts and that the viral TM functions essentially as an oncoprotein. [ABSTRACT FROM AUTHOR]

Using ultrahigh vacuum scanning tunneling microscopy (STM) and ab initio density functional theory, we have investigated in detail structures formed by cytosine on the Au(111) surface in clean ultrahigh vacuum conditions. In spite of the fact that the ground state of this DNA base on the surface is shown to be an ordered arrangement of cytosine one-dimensional branches (filaments), this structure has never been observed in our STM experiments. Instead, disordered structures are observed, which can be explained by only a few elementary structural motifs: filaments, five- and sixfold rings, which randomly interconnect with each other forming bent chains, T junctions, and nanocages. The latter may have trapped smaller structures inside. The formation of such an unusual assembly is explained by simple kinetic arguments as a liquid-glass transition. [ABSTRACT FROM AUTHOR]

The quartet of xanthine (X), a purine base ubiquitously distributed in most human body tissues and fluids, has been for the first time fabricated and visualized, as the first alternative purine quartet besides the known guanine (G)-quartet. The X-quartet network is demonstrated to be the most stable phase on Au(111). Unlike guanine, the fabrication of the X-quartets is not dependent on the presence of metal atoms, which makes it the first metal-free purine quartet. The X-quartet holds great promise to potentially construct artificial new DNA quadruplexes for genetic regulation and antitumor therapy. Moreover, both the X-quartet itself and the quartet networks favor homochirality, suggesting homo- chiral xanthine oligomers and the networks may have been formed as the precursors of the pristine oligonucleotides on primitive Earth. [ABSTRACT FROM AUTHOR]

Aims Calmodulin (CaM) is a small protein, encoded by three genes (CALM1-3), exerting multiple Ca2+-dependent modulatory roles. A mutation (F142L) affecting only one of the six CALM alleles is associated with long QT syndrome (LQTS) characterized by recurrent cardiac arrests. This phenotypic severity is unexpected from the predicted allelic balance. In this work, the effects of heterozygous CALM1-F142L have been investigated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from a LQTS patient carrying the F142L mutation, i.e. in the context of native allelic ratio and potential gene modifiers. Methods and Results Skin fibroblasts of the mutation carrier and two unrelated healthy subjects (controls) were reprogrammed to hiPSC and differentiated into hiPSC-CMs. Scanty IK1 expression, an hiPSC-CMs feature potentially biasing repolarization, was corrected by addition of simulated IK1 (Dynamic-Clamp). Abnormalities in repolarization ratedependency (in single cells and cell aggregates), membrane currents and intracellular Ca2+ dynamics were evaluated as putative arrhythmogenic factors. CALM1-F142L prolonged repolarization, altered its rate-dependency and its response to isoproterenol. This was associated with severe impairment of Ca2+-dependent inactivation (CDI) of ICaL, resulting in augmented inward current during the plateau phase. As a result, the repolarization of mutant cells failed to adapt to high pacing rates, a finding well reproduced by using a recent hiPSC-CM action potential model. The mutation failed to affect IKs and INaL and changed If only marginally. Intracellular Ca2+ dynamics and Ca2+ store stability were not significantly modified. Mutation-induced repolarization abnormalities were reversed by verapamil. Conclusion The main functional derangement in CALM1-F142L was prolonged repolarization with altered rate-dependency and sensitivity to β-adrenergic stimulation. Impaired CDI of ICaL underlined the electrICaL abnormality, which was sensitive to ICaL blockade. High mutation penetrance was confirmed in the presence of the native genotype, implying strong dominance of effects. [ABSTRACT FROM AUTHOR]

Background: We have previously reported that ATP treatment of M bovis-BCG infected human macrophages induces P2X7 receptor-dependent killing of intracellular mycobacteria. The mechanism mediating this bactericidal effect has not been full characterized but is known to be Ca2+-dependent and to promote the maturation and acidification of mycobacteria-containing phagosomes. In this study we demonstrate that the ATP/P2X7-mediated, mycobactericidal effect also involves the induction of cell autophagy. Results: We report that 3 mM ATP induces rapid cell autophagy in THP1 cells and monocytederived macrophages within 30 minutes post-treatment, as revealed by the expression of LC3-II bands on western blot analysis. Using Ca2+-free media and selective P2X7 agonists and antagonists, ATP-induced cell autophagy was shown to be Ca2+ and P2X7 receptor-dependent. Electron microscopy of ATP-treated, BCG-infected MDMs revealed the presence of the bacteria within characteristic double-membraned autophagosomes. Confocal analysis further confirmed that pharmacological inhibition of autophagy by wortmannin or pre-treatment of macrophages with anti-P2X7 antibody blocked ATP-induced phago-lysosomal fusion. Induction of cell autophagy with ATP was also temporally associated with a fall in intracellular mycobacterial viability, which was suppressed by treatment with wortmannin or the selective P2X7 antagonist, oxidized ATP (oATP). Conclusion: We provide the first evidence that ATP/P2X7-mediated killing of intracellular mycobacteria involves the induction of cell autophagy. The findings support the hypothesis that autophagy plays a key role in the control of mycobacterial infections. [ABSTRACT FROM AUTHOR]

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of the host germline transmitted vertically from generation to generation. It is hypothesized that some ERVs are used by the host as restriction factors to block the infection of pathogenic retroviruses. Indeed, some ERVs efficiently interfere with the replication of related exogenous retroviruses. However, data suggesting that these mechanisms have influenced the coevolution of endogenous and/or exogenous retroviruses and their hosts have been more difficult to obtain. Sheep are an interesting model system to study retrovirus-host coevolution because of the coexistence in this animal species of two exogenous (i.e., horizontally transmitted) oncogenic retroviruses, Jaagsiekte sheep retrovirus and Enzootic nasal tumor virus, with highly related and biologically active endogenous retroviruses (enJSRVs). Here, we isolated and characterized the evolutionary history and molecular virology of 27 enJSRV proviruses. enJSRVs have been integrating in the host genome for the last 5-7 million y. Two enJSRV proviruses (enJS56A1 and enJSRV-20), which entered the host genome within the last 3 million y (before and during speciation within the genus Ovis), acquired in two temporally distinct events a defective Gag polyprotein resulting in a transdominant phenotype able to block late replication steps of related exogenous retroviruses. Both transdominant proviruses became fixed in the host genome before or around sheep domestication (~ 9,000 y ago). Interestingly, a provirus escaping the transdominant enJSRVs has emerged very recently, most likely within the last 200 y. Thus, we determined sequentially distinct events during evolution that are indicative of an evolutionary antagonism between endogenous and exogenous retroviruses. This study strongly suggests that endogenization and selection of ERVs acting as restriction factors is a mechanism used by the host to fight retroviral infections. [ABSTRACT FROM AUTHOR]

OBJECTIVES : Sardinia is an island community of 1.6 million people. There are also about 3.5 million sheep and one hundred thousand cattle in which Johne's disease and Mycobacterium avium subspecies paratuberculosis infection are endemic. The present study was designed to determine what proportion of people in Sardinia attending for ileocolonoscopy with or without Crohn's disease were infected with this pathogen. METHODS : Mycobacterium avium subspecies paratuberculosis was detected by IS 900 PCR on DNA extracts of fresh intestinal mucosal biopsies as well as by isolation in culture using supplemented MGIT media followed by PCR with amplicon sequencing. RESULTS : Twenty five patients (83.3%) with Crohn's disease and 3 control patients (10.3%) were IS 900 PCR positive (p = 0.000001; Odds ratio 43.3). Mycobacterium avium subspecies paratuberculosis grew in cultures from 19 Crohn's patients (63.3%) and from 3 control patients (10.3%) (p = 0.00001; Odds ratio 14.9). All patients positive by culture had previously been positive by PCR. Mycobacterium avium subspecies paratuberculosis first appeared in the liquid cultures in a Ziehl Neelsen (ZN) staining negative form and partially reverted through a rhodamine-auramine positive staining form to the classical ZN positive form. This resulted in a stable mixed culture of all 3 forms illustrating the phenotypic versatility of these complex chronic enteric pathogens. CONCLUSIONS : Mycobacterium avium subspecies paratuberculosis was detected in the majority of Sardinian Crohn's disease patients. The finding of the organism colonizing a proportion of people without Crohn's disease is consistent with what occurs in other conditions caused by a primary bacterial pathogen in susceptible hosts. (Am J Gastroenterol 2005;100:1529–1536) [ABSTRACT FROM AUTHOR]

Background. Platinum-based chemotherapy is the mainstay of front-line treatment of patients affected by pluri-metastatic intermediate/high grade NeuroEndocrine Neoplasms (NENs). However, there are no standard second-line treatments at disease progression. Previous clinical experiences have evidenced that temozolomide (TMZ), an oral analog of dacarbazine, is active against NENs at standard doses of 150 to 200 mg/mq per day on days 1 to 5 of a 28-day cycle, even if a significant treatment-related toxicity is reported. Methods. Metastatic NENs patients were treated at the ENETS (European NeuroEndocrine Tumor Society) center of excellence of Naples (Italy), from 2014 to 2017 with a second-line alternative metronomic schedule of TMZ, 75 mg/m2per os "one week on/one week off". Toxicity was graded with NCI-CTC criteria v4.0; objective responses with RECIST v1.1 and performance status (PS) according to ECOG. Results. Twenty-six consecutive patients were treated. Median age was 65.5 years. The predominant primary organs were pancreas and lung. Grading was G2 in 11 patients, G3 in 15. More than half of patients had a PS 2 (15 vs. 11 with PS 1). The median time-on-temozolomide therapy was 12.2 months (95% CI: 11.4–19.6). No G3/G4 toxicities were registered. Complete response was obtained in 1 patient, partial response in 4, stable disease in 19 (disease control rate: 92.3%), and progressive disease in 2. The median overall survival from TMZ start was 28.3 months. PS improved in 73% of patients. Conclusions. Metronomic TMZ is a suitable treatment for G2 and G3 NENs particularly in PS 2 patients. Prospective and larger trials are needed to confirm these results. [ABSTRACT FROM AUTHOR]