Search

Abstract Trypanosomes are protozoan parasites that cycle between insect and mammalian hosts and are the causative agent of sleeping sickness. Here, we describe the changes of pseudouridine (Ψ) modification on rRNA in the two life stages of the parasite using four different genome-wide approaches. CRISPR-Cas9 knock-outs of all four snoRNAs guiding Ψ on helix 69 (H69) of the large rRNA subunit were lethal. A single knock-out of a snoRNA guiding Ψ530 on H69 altered the composition of the 80S monosome. These changes specifically affected the translation of only a subset of proteins. This study correlates a single site Ψ modification with changes in ribosomal protein stoichiometry, supported by a high-resolution cryo-EM structure. We propose that alteration in rRNA modifications could generate ribosomes preferentially translating state-beneficial proteins.

Summary: Leishmania is the causative agent of cutaneous and visceral diseases affecting millions of individuals worldwide. Pseudouridine (Ψ), the most abundant modification on rRNA, changes during the parasite life cycle. Alterations in the level of a specific Ψ in helix 69 (H69) affected ribosome function. To decipher the molecular mechanism of this phenotype, we determine the structure of ribosomes lacking the single Ψ and its parental strain at ∼2.4–3 Å resolution using cryo-EM. Our findings demonstrate the significance of a single Ψ on H69 to its structure and the importance for its interactions with helix 44 and specific tRNAs. Our study suggests that rRNA modification affects translation of mRNAs carrying codon bias due to selective accommodation of tRNAs by the ribosome. Based on the high-resolution structures, we propose a mechanism explaining how the ribosome selects specific tRNAs.

Abstract Dilated cardiomyopathy (DCM) is a primary myocardial disease, leading to heart failure and excessive risk of sudden cardiac death with rather poorly understood pathophysiology. In 2015, Parvari's group identified a recessive mutation in the autophagy regulator, PLEKHM2 gene, in a family with severe recessive DCM and left ventricular non-compaction (LVNC). Fibroblasts isolated from these patients exhibited abnormal subcellular distribution of endosomes, Golgi apparatus, lysosomes and had impaired autophagy flux. To better understand the effect of mutated PLEKHM2 on cardiac tissue, we generated and characterized induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) from two patients and a healthy control from the same family. The patient iPSC-CMs showed low expression levels of genes encoding for contractile functional proteins (α and β-myosin heavy chains and 2v and 2a-myosin light chains), structural proteins integral to heart contraction (Troponin C, T and I) and proteins participating in Ca2+ pumping action (SERCA2 and Calsequestrin 2) compared to their levels in control iPSC-derived CMs. Furthermore, the sarcomeres of the patient iPSC-CMs were less oriented and aligned compared to control cells and generated slowly beating foci with lower intracellular calcium amplitude and abnormal calcium transient kinetics, measured by IonOptix system and MuscleMotion software. Autophagy in patient’s iPSC-CMs was impaired as determined from a decrease in the accumulation of autophagosomes in response to chloroquine and rapamycin treatment, compared to control iPSC-CMs. Impairment in autophagy together with the deficiency in the expression of NKX2.5, MHC, MLC, Troponins and CASQ2 genes, which are related to contraction-relaxation coupling and intracellular Ca2+ signaling, may contribute to the defective function of the patient CMs and possibly affect cell maturation and cardiac failure with time.

Abstract Transforming long-term conflicts into peaceful intergroup relations is one of the most difficult challenges for humanity. Such meaningful social changes are often driven by young people. But do young people living in contexts of long-term conflicts believe that change is even possible? In a series of six studies (N total = 119,671) over two decades and across two unrelated intractable conflicts in Israel/Palestine and Cyprus, we found that younger (compared to older) generations from both respective rival groups have less hope for peace, and consequently less conciliatory attitudes. We also show that this gradual improvement of peace-promoting emotions and attitudes with increasing age can be experimentally accelerated in young people through a virtual reality-based aging simulation. These findings provide a new perspective on the fundamental question of why long-term conflicts are so difficult to resolve and highlight the importance of instilling hope in young generations to advance peace processes.

Abstract After myocardial infarction (MI), the heart's reparative response to the ischemic insult and the related loss of cardiomyocytes involves cardiac fibrosis, in which the damaged tissue is replaced with a fibrous scar. Although the scar is essential to prevent ventricular wall rupture in the infarction zone, it expands over time to remote, non-infarct areas, significantly increasing the extent of fibrosis and markedly altering cardiac structure. Cardiac function in this scenario deteriorates, thereby increasing the probability of heart failure and the risk of death. Recent works have suggested that the matricellular protein periostin, known to be involved in fibrosis, is a candidate therapeutic target for the regulation of MI-induced fibrosis and remodeling. Different strategies for the genetic manipulation of periostin have been proposed previously, yet those works did not properly address the time dependency between periostin activity and cardiac fibrosis. Our study aimed to fill that gap in knowledge and fully elucidate the explicit timing of cellular periostin upregulation in the infarcted heart to enable the safer and more effective post-MI targeting of periostin-producing cells. Surgical MI was performed in C57BL/6J and BALB/c mice by ligation of the left anterior descending coronary artery. Flow cytometry analyses of cells derived from the infarcted hearts and quantitative real-time PCR of the total cellular RNA revealed that periostin expression increased during days 2–7 and peaked on day 7 post-infarct, regardless of mouse strain. The established timeline for cellular periostin expression in the post-MI heart is a significant milestone toward the development of optimal periostin-targeted gene therapy.

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

Successful gene therapy requires the development of suitable carriers for the selective and efficient delivery of genes to specific target cells, with minimal toxicity. In this work, we present a non-viral vector for gene delivery composed of biocompatible materials, CaCl2, plasmid DNA and the semi-synthetic anionic biopolymer alginate sulfate (AlgS), which spontaneously co-assembled to form nanoparticles (NPs). The NPs were characterized with a slightly anionic surface charge (Zeta potential [ζ] = −14 mV), an average size of 270 nm, and their suspension was stable for several days with no aggregation. X-ray photoelectron spectroscopy (XPS) validated their ternary composition, and it elucidated the molecular interactions among Ca2+, the plasmid DNA, and the AlgS. Efficient cellular uptake (>80%), associated with potent GFP gene expression (22%–35%), was observed across multiple cell types: primary rat neonatal cardiac fibroblasts, human breast cancer cell line, and human hepatocellular carcinoma cells. The uptake mechanism of the NPs was studied using imaging flow cytometry and shown to be via active, clathrin-mediated endocytosis, as chemical inhibition of this pathway significantly reduced EGFP expression. The NPs were cytocompatible and did not activate the T lymphocytes in human peripheral blood mononuclear cells. Proof of concept for the efficacy of these NPs as a carrier in cancer gene therapy was demonstrated for Diphtheria Toxin Fragment A (DT-A), resulting in abrogation of protein synthesis and cell death in the human breast cancer cell line. Collectively, our results show that the developed AlgS-Ca2+-plasmid DNA (pDNA) NPs may be used as an effective non-viral carrier for pDNA. Keywords: polymeric nanoparticles, plasmid DNA, gene therapy, calcium, alginate sulfate

Cardiac tissue engineering has emerged as a promising strategy to treat infarcted cardiac tissues by replacing the injured region with an ex vivo fabricated functional cardiac patch. Nevertheless, integration of the transplanted patch with the host tissue is still a burden, limiting its clinical application. Here, a bi-functional, 3D bio-printed cardiac patch (CP) design is proposed, composed of a cell-laden compartment at its core and an extracellular vesicle (EV)-laden compartment at its shell for better integration of the CP with the host tissue. Alginate-based bioink solutions were developed for each compartment and characterized rheologically, examined for printability and their effect on residing cells or EVs. The resulting 3D bio-printed CP was examined for its mechanical stiffness, showing an elastic modulus between 4–5 kPa at day 1 post-printing, suitable for transplantation. Affinity binding of EVs to alginate sulfate (AlgS) was validated, exhibiting dissociation constant values similar to those of EVs with heparin. The incorporation of AlgS-EVs complexes within the shell bioink sustained EV release from the CP, with 88% cumulative release compared with 92% without AlgS by day 4. AlgS also prolonged the release profile by an additional 2 days, lasting 11 days overall. This CP design comprises great potential at promoting more efficient patch assimilation with the host.

Autophagy serves as a master regulator of cellular homeostasis. Hence, expectedly autophagic dysfunction has been documented in many diseases such as cancer, neurodegeneration and cardiovascular disorders. A novel homozygous mutation in PLEKHM2 gene (mPLEKHM2) resulted in dilated cardiomyopathy with left ventricular noncompaction (DCM-LVNC), probably as result of impaired autophagy due to disruption of lysosomal movement assisted by PLEKHM2. Here we report a generation of three iPSC lines, four clones originated from two patients with homozygous mPLEKHM2 and two from a heterozygote sibling. All generated lines highly expressed pluripotency markers, spontaneously differentiated into three germ layers, retained the mutation after reprogramming and displayed normal karyotypes.

The nucleolus is a subnuclear compartment whose primary function is the biogenesis of ribosomal subunits. Certain viral infections affect the morphology and composition of the nucleolar compartment and influence ribosomal RNA (rRNA) transcription and maturation. However, no description of nucleolar morphology and function during infection with Kaposi’s sarcoma-associated herpesvirus (KSHV) is available to date. Using immunofluorescence microscopy, we documented extensive destruction of the nuclear and nucleolar architecture during the lytic reactivation of KSHV. This was manifested by the redistribution of key nucleolar proteins, including the rRNA transcription factor UBF. Distinct delocalization patterns were evident; certain nucleolar proteins remained together whereas others dissociated, implying that nucleolar proteins undergo nonrandom programmed dispersion. Significantly, the redistribution of UBF was dependent on viral DNA replication or late viral gene expression. No significant changes in pre-rRNA levels and no accumulation of pre-rRNA intermediates were found by RT-qPCR and Northern blot analysis. Furthermore, fluorescent in situ hybridization (FISH), combined with immunofluorescence, revealed an overlap between Fibrillarin and internal transcribed spacer 1 (ITS1), which represents the primary product of the pre-rRNA, suggesting that the processing of rRNA proceeds during lytic reactivation. Finally, small changes in the levels of pseudouridylation (Ψ) and 2′-O-methylation (Nm) were documented across the rRNA; however, none were localized to the functional domain. Taken together, our results suggest that despite dramatic changes in the nucleolar organization, rRNA transcription and processing persist during lytic reactivation of KSHV. Whether the observed nucleolar alterations favor productive infection or signify cellular anti-viral responses remains to be determined.

Summary: The parasite Trypanosoma brucei is the causative agent of sleeping sickness and cycles between insect and mammalian hosts. The parasite appears to lack conventional transcriptional regulation of protein coding genes, and mRNAs are processed from polycistronic transcripts by the concerted action of trans-splicing and polyadenylation. Regulation of mRNA function is mediated mainly by RNA binding proteins affecting mRNA stability and translation. In this study, we describe the identification of 62 non-coding (nc) RNAs that are developmentally regulated and/or respond to stress. We characterized two novel anti-sense RNA regulators (TBsRNA-33 and 37) that originate from the rRNA loci, associate with ribosomes and polyribosomes, and interact in vivo with distinct mRNA species to regulate translation. Thus, this study suggests for the first-time anti-sense RNA regulators as an additional layer for controlling gene expression in these parasites.

Background: Despite intensive research, regeneration of articular cartilage largely remains an unresolved medical concern as the clinically available modalities still suffer from long-term inconsistent data, relatively high failure rates and high prices of more promising approaches, such as cell therapy. In the present study, we aimed to evaluate the feasibility and long-term efficacy of a bilayered injectable acellular affinity-binding alginate hydrogel in a large animal model of osteochondral defects. Methods: The affinity-binding alginate hydrogel is designed for presentation and slow release of chondrogenic and osteogenic inducers (transforming growth factor-β1 and bone morphogenic protein 4, respectively) in two distinct and separate hydrogel layers. The hydrogel was injected into the osteochondral defects created in the femoral medial condyle in mini-pigs, and various outcomes were evaluated after 6 months. Results: Macroscopical and histological assessment of the defects treated with growth factor affinity-bound hydrogel showed effective reconstruction of articular cartilage layer, with major features of hyaline tissue, such as a glossy surface and cellular organisation, associated with marked deposition of proteoglycans and type II collagen. Microcomputed tomography showed incomplete bone formation in both treatment groups, which was nevertheless augmented by the presence of affinity-bound growth factors. Importantly, the physical nature of the applied hydrogel ensured its shear resistance, seamless integration and topographical matching to the surroundings and opposing articulating surface. Conclusions: The treatment with acellular injectable growth factor–loaded affinity-binding alginate hydrogel resulted in effective tissue restoration with major hallmarks of hyaline cartilage, shown in large animal model after 6-month follow-up. The translational potential of this article: This proof-of-concept study in a clinically relevant large animal model showed promising potential of an injectable acellular growth factor–loaded affinity-binding alginate hydrogel for effective repair and regeneration of articular hyaline cartilage, representing a strong candidate for future clinical development. Keywords: Affinity-binding alginate, Bone morphogenic protein 4, Hyaline cartilage, Osteochondral defect, Transforming growth factor-β1

Heart failure (HF) after myocardial infarction (MI) due to blockage of coronary arteries is a major public health issue. MI results in massive loss of cardiac muscle due to ischemia. Unfortunately, the adult mammalian myocardium presents a low regenerative potential, leading to two main responses to injury: fibrotic scar formation and hypertrophic remodeling. To date, complete heart transplantation remains the only clinical option to restore heart function. In the last two decades, tissue engineering has emerged as a promising approach to promote cardiac regeneration. Tissue engineering aims to target processes associated with MI, including cardiomyogenesis, modulation of extracellular matrix (ECM) remodeling, and fibrosis. Tissue engineering dogmas suggest the utilization and combination of two key components: bioactive molecules and biomaterials. This chapter will present current therapeutic applications of biomaterials in cardiac regeneration and the challenges still faced ahead. The following biomaterial-based approaches will be discussed: Nano-carriers for cardiac regeneration-inducing biomolecules; corresponding matrices for their controlled release; injectable hydrogels for cell delivery and cardiac patches. The concept of combining cardiac patches with controlled release matrices will be introduced, presenting a promising strategy to promote endogenous cardiac regeneration.

It is assumed that the sequencing of ribosomes better reflects the active microbial community than the sequencing of the ribosomal RNA encoding genes. Yet, many studies exploring microbial communities in various environments, ranging from the human gut to deep oceans, questioned the validity of this paradigm due to the discrepancies between the DNA and RNA based communities. Here, we focus on an often neglected key step in the analysis, the reverse transcription (RT) reaction. Previous studies showed that RT may introduce biases when expressed genes and ribosmal rRNA are quantified, yet its effect on microbial diversity and community composition was never tested. High throughput sequencing of ribosomal RNA is a valuable tool to understand microbial communities as it better describes the active population than DNA analysis. However, the necessary step of RT may introduce biases that have so far been poorly described. In this manuscript, we compare three RT enzymes, commonly used in soil microbiology, in two temperature modes to determine a potential source of bias due to non-standardized RT conditions. In our comparisons, we have observed up to six fold differences in bacterial class abundance. A temperature induced bias can be partially explained by G-C content of the affected bacterial groups, thus pointing toward a need for higher reaction temperatures. However, another source of bias was due to enzyme processivity differences. This bias is potentially hard to overcome and thus mitigating it might require the use of one enzyme for the sake of cross-study comparison.

A critical step in the cellular stress response is transient activation of the RNA-dependent protein kinase PKR by double-helical RNA, resulting in down-regulation of protein synthesis through phosphorylation of the α chain of translation initiation factor eIF2, a major PKR substrate. However, intragenic elements of 100–200 nucleotides in length within primary transcripts of cellular genes, exemplified by the tumor necrosis factor (TNF)-α gene and fetal and adult globin genes, are capable of forming RNA structures that potently activate PKR and thereby strongly enhance mRNA splicing efficiency. By inducing nuclear eIF2α phosphorylation, these PKR activator elements enable highly efficient early spliceosome assembly yet do not impair translation of the mature spliced mRNA. The TNF-α RNA activator of PKR folds into a compact pseudoknot that is highly conserved within the phylogeny. Upon excision of β-globin first intron, the RNA activator of PKR, located in exon 1, is silenced through strand displacement by a short sequence within exon 2, restricting thereby the ability to activate PKR to the splicing process without impeding subsequent synthesis of β-globin essential for survival. This activator/silencer mechanism likewise controls splicing of α-globin pre-mRNA, but the exonic locations of PKR activator and silencer sequences are reversed, demonstrating evolutionary flexibility. Impaired splicing efficiency may underlie numerous human β-thalassemia mutations that map to the β-globin RNA activator of PKR or its silencer. Even where such mutations change the encoded amino acid sequence during subsequent translation, they carry the potential of first impairing PKR-dependent mRNA splicing or shutoff of PKR activation needed for optimal translation.

Background. Cell therapy for heart disease has been proven safe and efficacious, despite poor cell retention in the injected area. Improving cell retention is hypothesized to increase the treatment effect. In the present study, human adipose-derived stromal cells (ASCs) were delivered in an in situ forming alginate hydrogel following acute myocardial infarction (AMI) in rats. Methods. ASCs were transduced with luciferase and tested for ASC phenotype. AMI was inducted in nude rats, with subsequent injection of saline (controls), 1 × 106 ASCs in saline or 1 × 106 ASCs in 1% (w/v) alginate hydrogel. ASCs were tracked by bioluminescence and functional measurements were assessed by magnetic resonance imaging (MRI) and 82rubidium positron emission tomography (PET). Results. ASCs in both saline and alginate hydrogel significantly increased the ejection fraction (7.2% and 7.8% at 14 days and 7.2% and 8.0% at 28 days, resp.). After 28 days, there was a tendency for decreased infarct area and increased perfusion, compared to controls. No significant differences were observed between ASCs in saline or alginate hydrogel, in terms of retention and functional salvage. Conclusion. ASCs improved the myocardial function after AMI, but administration in the alginate hydrogel did not further improve retention of the cells or myocardial function.

Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite Trypanosoma brucei, exosome secretion is induced by stress that affects trans-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes. The exosomes are formed in multivesicular bodies (MVB) utilizing the endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, Vps36, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonstrates that exosomes are delivered to trypanosome cells and can change their migration. Exosomes are used to transmit stress signals for communication between parasites.

Emotional barriers have been found to play a critical role in forming attitudes and behaviors in conflict and peace-making. A major effect of such affective barriers is cognitive freezing, which reduces openness to new information and opportunities to conflict resolution. In the current research, we examined the hypothesis that hope and fear have opposite effects on information processing in such contexts. A time-lagged correlational study with 222 Israeli-Jews was conducted using a new computerized information processing simulator. Results revealed that when faced with an opportunity for peace, long-term hope was associated with acquiring information in favor of accepting the opportunity, whereas fear was associated with acquiring information that was biased towards rejecting the opportunity. Results also showed that both emotions were not associated with the amount of information gathered by participants. Findings have both theoretical and practical implications regarding the differential roles of hope and fear in identifying opportunities for, and promoting, conflict resolution.

Angiogenesis is considered integral to the reparative process after ischemic injury. The αvβ3 integrin is a critical modulator of angiogenesis and highly expressed in activated endothelial cells. 68Ga-NODAGA-E[(cRGDyK)]2 (RGD) is a positron-emission-tomography (PET) ligand targeted towards αvβ3 integrin. The aim was to present data for the uptake of RGD and correlate it with histology and to further illustrate the differences in angiogenesis due to porcine adipose-derived mesenchymal stromal cell (pASC) or saline treatment in minipigs after induction of myocardial infarction (MI). Three minipigs were treated with direct intra-myocardial injection of pASCs and two minipigs with saline. MI was confirmed by 82Rubidium (82Rb) dipyridamole stress PET. Mean Standardized Uptake Values (SUVmean) of RGD were higher in the infarct compared to non-infarct area one week and one month after MI in both pASC-treated (SUVmean: 1.23 vs. 0.88 and 1.02 vs. 0.86, p < 0.05 for both) and non-pASC-treated minipigs (SUVmean: 1.44 vs. 1.07 and 1.26 vs. 1.04, p < 0.05 for both). However, there was no difference in RGD uptake, ejection fractions, coronary flow reserves or capillary density in histology between the two groups. In summary, indications of angiogenesis were present in the infarcted myocardium. However, no differences between pASC-treated and non-pASC-treated minipigs could be demonstrated.

Tissue engineering holds the promise of providing new solutions for heart transplant shortages and pediatric heart transplantation. The aim of this study was to evaluate the ability of a peritoneal-generated, tissue-engineered cardiac patch to replace damaged myocardium in a heterotopic heart transplant model. Fetal cardiac cells (1 × 10 6 /scaffold) from syngeneic Lewis rats were seeded into highly porous alginate scaffolds. The cell constructs were cultured in vitro for 4 days and then they were implanted into the rat peritoneal cavity for 1 week. During this time the peritoneal-implanted patches were vascularized and populated with myofibroblasts. They were harvested and their performance in an infrarenal heterotopic abdominal heart transplantation model was examined ( n = 15). After transplantation and before reperfusion of the donor heart, a 5-mm left ( n = 6) or right ( n = 9) ventriculotomy was performed and the patch was sutured onto the donor heart to repair the defect. Echocardiographical studies carried out 1–2 weeks after transplantation showed normal LV function in seven of the eight hearts studied. After 1 month, visual examination of the grafted patch revealed no aneurysmal dilatation. Microscopic examination revealed, in most of the cardiac patches, a complete disappearance of the scaffold and its replacement by a consistent tissue composed of myofibroblasts embedded in collagen bundles. The cardiac patch was enriched with a relatively large number of infiltrating blood vessels. In conclusion, cardiac patches generated in the peritoneum were developed into consistent tissue patches with properties to seal and correct myocardial defects. Our study also offers a viable rat model for screening and evaluating new concepts in cardiac reconstruction and engineering.