Supplying food to an ever-increasing population is one of the biggest challenges worldwide. Environmental stresses make this situation even graver. New technological approaches are imperative. Recently, genetic engineering has contributed enormously to the development of genetically modified varieties of different crops such as cotton, maize, rice, canola and soybean. Identification of stress-responsive genes and their subsequent introgression or overexpression within sensitive crop species are now being widely carried out by plant scientists. In this review, the role of biotechnology in association with genomics and its successes, endeavours, prospects and challenges in developing stress-tolerant crop cultivars are discussed. Increasing global population, urbanization and industrialization are increasing the rate of conversion of arable land into wasteland. Supplying food to an ever-increasing population is one of the biggest challenges that agriculturalists and plant scientists are currently confronting. Environmental stresses make this situation even graver. Despite the induction of several tolerance mechanisms, sensitive plants often fail to survive under environmental extremes. New technological approaches are imperative. Conventional breeding methods have a limited potential to improve plant genomes against environmental stress. Recently, genetic engineering has contributed enormously to the development of genetically modified varieties of different crops such as cotton, maize, rice, canola and soybean. The identification of stress-responsive genes and their subsequent introgression or overexpression within sensitive crop species are now being widely carried out by plant scientists. Engineering of important tolerance pathways, like antioxidant enzymes, osmolyte accumulation, membrane-localized transporters for efficient compartmentation of deleterious ions and accumulation of essential elements and resistance against pests or pathogens is also an area that has been intensively researched. In this review, the role of biotechnology and its successes, prospects and challenges in developing stress-tolerant crop cultivars are discussed. In plant biology, the transgenic approach has emerged as an important tool to adapt crops to rapidly changing environmental conditions. The use of transgenic crops has increased considerably over the past decade. The primary step before proceeding with transgenics is the identification of genes serving as key regulators of different metabolic pathways, including osmolyte synthesis, ion homeostasis through selective ion uptake, antioxidant defence system and other frontline defence pathways (Genome editing has revolutionized plant biotechnology by providing plant scientists with the option of selection or incorporation of genes of interest into desired species or cultivars . LimitatAgrobacterium-mediated transformation (Suppression Subtraction Hybridization (SSH) is an extremely powerful and widely used technique for separating cDNA or genomic DNA  pathways. C4 plants can minimize photorespiration by separating initial CO2 fixation and the Calvin cycle in different cell types, and CAM plants can fix carbon at night, and are more tolerant to drought stress due to their more efficient carbon fixation and specialized anatomical features (3 crops with enhanced photosynthetic performance either due to introgression of genes encoding C4 enzymes into C3 plants or overexpression of C3 enzymes or transcription factors (TF). Usually, C4 and CAM plants are best adapted to arid environments, because they have higher photosynthetic efficiency as well as WUE as compared with C3 plants (Capsicum annuum drought stress responsive 6 (CaDSR6) in Arabidopsis plants led to higher tolerance to drought as compared with wild type plants. SNAC1) gene controlled signalling of sucrose phosphate synthase type 2C protein phosphatases, 1-phosphatidylinositol-3-phosphate-5-kinase as well as regulatory components of ABA receptor in wheat plants under drought stress. Overall, a variety of genes contributing to drought tolerance in plants have been explored and characterized in Arabidopsis. However, few of these genes have been tested in other crops, and only under controlled or laboratory conditions instead of natural field conditions.There are three major pathways of COfeatures . Recentl3 plants . Likewis+ and Cl-) from sensitive tissues like the mesophyll (where sodium toxicity is induced by competing for K+ binding sites) and their diversion into the apoplast or vacuole  from the halophyte, Aeluropuslittoralis, in tobacco enhanced the salinity tolerance of tobacco by maintaining a suitable level of Na+ and K+/Na+ ratio (SaVHAc1) gene from Spartinaalterniflora enhanced rates of photosynthesis and cell wall expansion, improved the K+/Na+ ratio and led to a higher relative water content (RWC) in rice  were more sensitive to low temperature than their respective wild-type and MsrB3 transgenic plants. MsrB3 plays a ubiquitous role in eliminating reactive oxygen species (ROS) and methionine sulphoxide (MetO) accumulating in the endoplasmic reticulum during cold stress , a gene regulated by temperature rhythms, from Lycopersicon esculentum. Overexpression of this gene increased non-photochemical quenching, vF/mFand quantum yield, oxidizable P700, and the activity of the xanthophyll cycle and alleviated PSI and PSII photoinhibition under temperature stress. Recently, similar observations have been reported in transgenic tobacco by introgression of LeLUT1 (carotenoid epsilon-ring hydroxylase gene from tomato), which reduced ROS production and hence maintained membrane integrity , Photinia serrulata overexpressing the galactinol synthase gene (AmGSl) from a cold tolerant tree, Ammopiptanthus mongolicus, exhibited enhanced cold tolerance , have a key role in mitigating the deleterious effects induced by heat stress . For example, perature  help ourUntil recently, the genes that have been identified or introgressed in different genetically modified (GM) plants mainly relate to the regulation of the oxidative defence system. However, multiple other plant metabolic systems and activities are affected by changes in temperature, and have the potential to be tackled in transgenic crops. Moreover, a rise in ambient temperature as already visible over the last 10 years is a continuing challenge for crop productivity, creating the need to develop stress tolerant plants with heat tolerance.http://www.irac-online.org).A variety of pesticides, herbicides and fungicides are frequently used to control crop loss due to pathogen attack . Use of Synthetic pesticides, herbicides and fungicides are effective, but excessive use can generate environmental pollution, development of resistance and non-degradable residues. For example, chemical fungicides used for the treatment of plant diseases have diverse mechanisms of action involving the mitochondrial respiratory chain, inhibition of sterol biosynthesis as well as microtubule assembly, resulting in some limitations related to their toxicity and resistance in plants. A number of stress response pathways such as the cell wall integrity and high-osmolarity glycerol pathway are triggered by stimuli such as changes in osmolarity, cell wall instability and production of ROS  increased resistance to Bipolaris maydis, Fusarium graminearum, Aspergillus oryzae, Aspergillus niger and Sclerotinia sclerotiorum, thereby favouring the antifungal and antiviral activity of α-MC.Botrytis cinerea by suppressing the growth of fungi  exhibited significant inhibition of fungal growth , and when introduced into tobacco, enhanced bromoxynil resistance. Recently, CYP81A12 and CYP81A21, from a noxious weed Echinochloa phyllopogon that is resistant to the herbicides bensulphuron-methyl and penoxsulam, and developed transgenic Arabidopsis expressing either of these genes that showed enhanced herbicide resistance through the O-demethylation of herbicides. These results indicate that the characterization and understanding of molecular mechanisms and the development of resistant crops can help withstand the devastating effects of pathogens and pests and provide an important alternative to chemical dependent agriculture.Amongst the most commonly used herbicides are glyphosate and bromoxynil . Glyphosate restricts growth by reducing aromatic amino acid biosynthesis while bromoxynil prevents photosynthesis by affecting PSII activity (GS1) in wheat plants led to increased nitrogen accumulation in shoot and grains (GS1-3 led to enhanced (30 %) kernel number in maize  increased leaf expansion and improved phosphate efficiency. Overexpression of expansin genes, e.g. HvEXPB1 in barley  and cadmium (Cd) reduce the number of mitochondrial cristae leading to impaired oxidative phosphorylation. Upon binding to nucleic acids, heavy metals promote the aggregation and condensation of chromatin as well as impaired replication and transcription . The affMost heavy metal salts are hydrophilic and easily soluble in wastewater, so they are difficult to separate by physical separation methods. At low levels of heavy metals, physico-chemical methods can be ineffective or costly. Alternative methods include biosorption or bioaccumulation for the removal of heavy metals. The use of microorganisms and plants for remediation purposes is thus an effective strategy to overcome or minimize heavy metal pollution  enhanced the tolerance of A. thaliana to heavy metals and metalloids. They further reported that single-gene transgenic lines showed higher tolerance and accumulated more Cd and As than wild type plants, while dual gene transgenic lines exhibited even more tolerance and accumulated (2-fold) more Cd and As compared with single gene transformants. The elevated production of GSH and PCs resulted in accumulation and tolerance to Cd and As  showed improved tolerance to Cd by maintaining higher levels of PCs in the cytosol and vacuole. Arabidopsis thaliana overexpressing PCs1 showed higher resistance to Cd and arsenic (As) and accumulated more biomass  lyase (OASTL), a key enzyme that catalyzes cysteine formation (from sulphide and O-acetylserine) and a key limiting step in the production of GSH, showed high tolerance to heavy metals (Nicotiana tabacum plants expressing the wheat (Triticum aestivum) OASTL gene, cys1, and exposed to SO2 maintained high levels of Cys and GSH as well as higher rates of accumulation of Cu/Zn SOD transcripts.Overexpression of The advancement in transgenic approaches  could be a successful means to promote phytoextraction of toxic metalloids (Se and As) and metals, particularly Cu, Pb and Cd in the aboveground plant organs and to promote tissue up-take involving metal transporters, high production of enzymes and the production of metal-detoxifying chelators including PCs and MTs. Advances in the mechanistic basis of a transgenic approach would help provide a better understanding of the genetic basis of resistance or tolerance and hyperaccumulation of metals and metalloids, means of translocation and other environmental factors influencing phytoremediation, because these all hinder its implementation.Several intrinsic protective mechanisms are triggered in plants when they are exposed to various environmental stresses (The identification and selection of key stress responsive genes and their subsequent introgression for developing resistant crop cultivars through conventional breeding protocols are time-consuming. Plant biotechnology, despite being costly in comparison with conventional breeding, is very efficient. Several stress responsive genes have been identified and successfully introduced into other crops to create transgenic crops with enhanced stress tolerance. However, it is important to point out here that during the development of a transgenic crop variety, care is taken to introduce genes that result in enhanced tolerance to multiple stresses, specifically at the whole plant level. This requires the development of sets of markers designed to enhance stress tolerance.The advantages of biotechnology in the development of transgenic plants for efficient crop varieties are undoubtedly enormous, but their commercialization after proper field testing is still an unavoidable reality. In addition, risk assessment of transgenic plants/crops is one of the preliminary steps required before the release or use of transgenic plants. The set standard all over the world explains the risk and official registration of plants and plant products has to be under taken. In addition, the risks to the environment from the transgenic crop plants must be examined with many field tests prior to commercialization, with institutional assessments, decisions on plants or varieties and adequate management practices in place to tackle inherent risks. For decision making, risk assessment must be followed in a scientific, sound and transparent manner. There are many operational governmental regulations in many countries for the safety assessment of GM crops. Furthermore, there are some international agreements that regulate the cultivation and commercialization of transgenic plants and their derivatives. All over the world, the major objective of these regulations and risk assessment strategies is focused on protecting the environment and human/animal health. The adoption of transgenic plants entirely depends on the assessments of the risks or benefits, regulatory approval, cost and time period, commercialization as well as the economic status, requirements and values of different countries.Not applicable. P.A. conceived the idea, N.A.A., M.A. and L.W. compiled the data, M.A.A. and M.N.A. led the writing of the manuscript with inputs from all co-authors.None declared.


Emergency medicine (EM) residency graduates have a profound impact on the quality of pediatric emergency care. Consequently, residency training programs must provide broad clinical training in both adult and pediatric emergency medicine (PEM).Designing a high-yield pediatric experience that allows EM residents to understand the progression of common illnesses and anticipate the medical and psychosocial needs of hospitalized children remains a challenge. Awareness of this need led us to develop a novel curriculum in pediatrics for EM residents – Pediatric Emergency Medicine with Follow-Up (PEMFU). The goal of this curriculum is to prepare EM residents to provide pediatric emergency care via a situated experience in pediatric medicine.Our goal is to describe the development of a novel curriculum for teaching and learning pediatric medicine in an EM residency program based on an assessment of need and structured around the conceptual framework of situated learning. We also describe the implementation of this curriculum within a single EM residency, and report early outcomes.After feedback from educators, residents, and medical directorship suggested that educational needs were unmet under the traditional pediatric ward rotation format, the ward rotation was withdrawn from our residency curriculum. In its stead, PEMFU was designed using the six-step curriculum development framework developed by Kern beginning with general and targeted needs assessments.8Through focus groups, anonymous surveys of current and former residents, and numerous discussions, we identified five key aspects of the inpatient pediatric rotation important to the development of pediatric competence within EM residency that may be unavailable in other venues.Assessed Need 1: Inpatient rotations foster interactions with patients and families, and experience with the pediatric physical examination. The new curriculum (PEMFU) would need to foster developmentally-appropriate and family-centered practice via experience caring for children and families.Assessed Need 2: EM residents must understand and anticipate the needs of hospitalized pediatric patients. PEMFU would need to allow trainees to understand the continuum of pediatric illness/injury and the evidence-based management and disposition of hospitalized children.Assessed Need 3: EM residents risk losing the collegial relationships formed by working side by side with inpatient providers and consultants. At a time when more hospitalized patients make their way through the ED, PEMFU would need to address this unintended social-professional consequence, and continue to allow EM residents to develop working relationships with other healthcare professionals.Assessed Need 4: There should be an emphasis on the critical importance of lifelong, self-directed learning to the practice of medicine. PEMFU would need to allow EM residents to reinforce the skills critical to lifelong self-directed learning in EM.situated learningAssessed Need 5: While an understanding of inpatient pediatrics remains important to the educational development of EM residents, adult learners learn better via participating in a social and professional setting that more closely mirrors their future practice. This concept of Learning objectives, mapped to the needs assessment, were developed to guide curriculum development under the aegis of a single broad goal of learning to provide excellent pediatric emergency care . A logic10PEMFU was implemented in the 2014–2015 academic year; 12 second-year resident learners rotated in the pilot season. Eight components comprise the curriculum .The bulk of the clinical experience takes place from caring for patients in the ED – the social-professional environment most meaningful to future emergency physicians.Additionally, residents round once weekly on the wards with a PEM subspecialist experienced in inpatient medicine, visiting the bedside, engaging in discussion of diagnosis and treatment as well as the psychosocial experiences of hospitalization. Residents follow up by phone with a smaller subset of discharged patients. Dedicated time has been built into residents’ schedules to accommodate these follow-up activities.As part of programmatic assessment,Clinical Assessment: Clinical faculty who supervise EM residents in the ED assess resident performance and provide formative feedback, recording this assessment in an online form. These assessments collectively contribute to the residents’ summative assessment.Direct Observation: One faculty member performs a monthly 2–3 hour direct observation session during an ED shift for each resident. Structured formative feedback is provided covering important skills of history-taking, age-appropriate pediatric physical examination, diagnostic decision-making, and a developmentally-appropriate approach to children. A self-reflection exercise is built into the feedback process, encouraging reflection for ongoing professional identity formation.Summary Assessment: A summary assessment is completed by the course director, assessing the core elements of the curriculum, including the resident’s written work product, interactions during ward rounds and teaching sessions, and group-sourced feedback from faculty, nursing, and other staff. PEMFU provides important insight into the progression of EM residents through the EM Milestones,Reactions to PEMFU have been favorable from faculty, residents, and families. Parents, in particular, appreciate that emergency providers are committed to following up with each patient, even when they are no longer specifically responsible for their care. Specific strengths cited by residents include developing close working relationships with the clinical faculty mentors, as well as increased access to pediatric colleagues, and increased individualized interactive instruction.“The PEM-FU rotation has led to improved patient care and outcomes for my ED patients.” This evaluation was performed in the course of educational quality improvement, and was exempted from review by the medical center’s institutional review board.Following Kirkpatrick’s evaluation model,Recommendations for improvement have related to specific clinical content: interpreting pediatric radiographic studies, exposure to pediatric critical care, the psychomotor aspects of interacting with children , and concerns regarding the seasonal nature of disease processes seen in the pediatric ED.A number of constraints make higher-level evaluation of educational outcomes challenging. In-training examinations, based on the American Board of Emergency Medicine Core Content, do not distinguish pediatrics from other components of EM practice; pediatric-specific knowledge measurements are difficult to ascertain. Whether this novel curriculum will have effects on important clinical outcomes in the longer term remains to be seen. Further, the literature on pediatric training within EM residency consists mainly of content recommendations; specific curricular recommendations are lacking, making comparisons between curricular approaches challenging.2Teaching the PEMFU program is effort-intensive and requires administrative support to allow time for weekly attending rounds, observation sessions, and ad hoc teaching sessions. In our model, a single PEM specialist has responsibility for supervision and planning, though this could conceivably be shared among several faculty.situated learning, in which residents learn by working clinically in the environment where their knowledge and skills will be put to use in their future professional practice (i.e. in the ED caring for ill and injured children), rather than extrapolating from a foreign learning environment. This is thought to contribute to deeper and more meaningful learning.cognitive apprenticeship; supervising faculty provide contextualized support while supervising clinical care through role modeling, coaching, and articulation techniques.20This curriculum represents an example of We know that knowledge and skills, if not used regularly, do not last. Pusic and colleagues have described “experience curves” chronicling the process of knowledge and skill accrual, followed by decay, with return to competence via interval training or experience.We have developed a novel educational method, with dedicated pediatric ED time, deliberate patient follow up, ward rounds, discussion of focused pediatric topics, and direct observation. This model could be customized to fit a variety of educational settings in pediatric or adult medicine training for emergency physicians. We believe that this novel curriculum represents one model for integrating knowledge of pediatric illness and injury – acute and longitudinal – into the emergency medicine residency education paradigm.


Eukaryotic genomes frequently exhibit interdependency between transcriptional units, as evidenced by regions of high gene density. It is well recognized that vertebrate microRNAs (miRNAs) are usually embedded in those regions. Recent work has shown that the genomic context is of utmost importance to determine miRNA expression in time and space, thus affecting their evolutionary fates over long and short terms. Consequently, understanding the inter- and intraspecific changes on miRNA genomic architecture may bring novel insights on the basic cellular processes regulated by miRNAs, as well as phenotypic evolution and disease-related mechanisms. Recent genome-wide projects have revealed an outstanding transcriptome diversity, especially of non-coding RNAs (ncRNAs), as well as a wealth of regulatory mechanisms and gene product interactions that compound the molecular basis of phenotypes . A notabThe interleaved model opens up numerous possibilities for regulatory mechanisms. For instance, products of antisense transcription, which is believed to occur in more than 30% of gene loci in humans , can regde novo origin . Although miR-490 is annotated as intergenic in chicken, predicted transcripts with an intron overlapping miR-490 are annotated. Expression analyses reveal a strongly conserved pattern among human, rhesus macaque, mouse, and chicken; indicating concomitant expression of miR-490 and CHRM2 . The host gene is a muscarinic cholinergic receptor involved in acetylcholine-mediated cardiac chronotropic (heart rate) and inotropic (strength of muscle contraction) effects . This host gene is a fucosyltransferase well-conserved throughout animals . Considering the young evolutionary age of miR-625, its expression levels and breadth are unusually high, thus being frequently altered in different types of cancer . Hence, young miRNAs (or “proto” miRNAs) hosted by old genes would gain higher expression breadth benefited by their host’s transcriptional activity, thus enabling many target interactions that, at first glance, are mostly neutral . This degeneration scenario is also most likely to happen with miRNAs emerged within young hosts, because of their general tissue-specific expression signature  retrotransposons have mediate their formation (de novo or by duplication (Nonetheless, it is possible that genomic comparisons of different individuals can give insights on the origination process of miRNAs, as previously done for other genetic elements . For insormation . miRNAs lication , but mecDrosophila and humans (In addition, comparing fixed patterns present in different species to polymorphic states observed in a group of individuals are useful tools for contrasting genomic features driven by natural selection to patterns produced by mutation bias . Notabled humans . TherefoFurthermore, as miRNAs expression and targeting has been shown to be implicated in a wide of human diseases , seed, aFrom the discussion above, it turns out that the genomic context, as an outcome of natural selection, imposes evolutionary constraints to maintain the structural and functional integrity of its genetic elements. Moreover, it can also propel the evolutionary fate of new elements that arise in a suitable environment, eventually accelerating the process of functionalization. Therefore, evolutionary models tackling the 3D chromatin organization will be of extreme value to pursue the general principles that afford those processes take place throughout genomes.GSF and MDV conceived the study. GSF, LCH, PAFG, and MDV wrote the manuscript. All authors revised and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Stem cells are critical to maintaining steady-state organ homeostasis and regenerating injured tissues. Recent intriguing reports implicate extracellular vesicles (EVs) as carriers for the distribution of morphogens and growth and differentiation factors from tissue parenchymal cells to stem cells, and conversely, stem cell–derived EVs carrying certain proteins and nucleic acids can support healing of injured tissues. We describe approaches to make use of engineered EVs as technology platforms in therapeutics and diagnostics in the context of stem cells. For some regenerative therapies, natural and engineered EVs from stem cells may be superior to single-molecule drugs, biologics, whole cells, and synthetic liposome or nanoparticle formulations because of the ease of bioengineering with multiple factors while retaining superior biocompatibility and biostability and posing fewer risks for abnormal differentiation or neoplastic transformation. Finally, we provide an overview of current challenges and future directions of EVs as potential therapeutic alternatives to cells for clinical applications. Stem cells play vital roles in maintaining cellular homeostasis and restoring it upon tissue injury. During both embryonic development and regeneration of adult tissues, stem and progenitor cells must reach specific target cells that could be either in close proximity or at a distance. Whereas embryonic development typically occurs in a controlled environment with minimal external disturbances, adult stem cells face frequently the additional challenge of operating in a harsher environment induced by trauma or infection with associated pathological and inflammatory responses. Stem cells are able to communicate with nearby and distant cells through soluble factors and direct cell-cell contact by long and thin tubular appendages such as cytonemes and cilia, as well as via detached extracellular vesicles (EVs). These vesicles possess properties of freely diffusing factors and the extensive membrane and cytoplasmic organization of cells. They range from approximately 30 nm to 3,000 nm in diameter and have a distinct biomolecular composition and, therefore, functions that depend on EV subtype, cell source, and conditions (see the sidebar titled DEFINITIONSApoptotic bodies or blebs (ABs)relatively large  cell fragments released from cells undergoing apoptosisExosomesbiological vesicles approximately 30–200 nm in diameter that originate as ILVs in MVBs (see below) and become released as EVs upon MVB fusion with the plasma membraneExtracellular vesicles (EVs)all-encompassing term for cell-derived small vesicles; includes exosomes, microvesicles, and ABsILVsintraluminal vesicles derived from invaginations of early endosomes to be contained within MVBs; referred to as exosomes upon their release outside the cellLiposomessynthetic, nanometer- to micrometer-sized lipid vesicles; frequently engineered to carry and release cargo in a targeted and controlled mannerMicrovesicles/ectosomesbiological vesicles approximately 50–300 nm in diameter that bud from the plasma membraneMVBsmultivesicular bodies derived from endosomes that invaginate inward to form ILVsNanoparticlessynthetic, nanometer-scale particles made of polymers, metals, or other materials; frequently carry and release cargo in a targeted and controlled mannerEVs were initially considered cellular debris or a way to excrete unneeded or toxic products from cells, but their ancient evolutionary origins and their conserved mechanisms of generation indicate that EVs perform essential physiological roles in cell-cell communication , 2. EVs Thus, the study of the roles of endogenous stem cell EVs, although still in its initial stages, has provided key background and rationale on using exogenous and bioengineered stem cell EVs to help treat injured or diseased tissues. Furthermore, in the context of cell therapy, exogenous cells infused intravenously (IV) are normally trapped in the lungs (the pulmonary first-pass effect) and also in other filter organs such as the liver, spleen, and kidneys , 12. TheEVs fall in the intermediate range of current biologics for tissue regeneration and immunomodulation . As EVs are loaded differentially and combinatorially with multiple types of proteins together with coding and regulatory nucleic acids, the responses they induce may be more complex and longer-lasting compared to those elicited by individual biomolecules , 10, 17.One of the most interesting and emerging functions of EVs may be in the maintenance of tissue homeostasis, as EVs released from injured tissue have been found to influence stem cells, and conversely, stem cell–derived EVs have been found to support injured tissue. This role of EVs in the cross-talk between stem cells and parenchymal cells, and how this can be exploited therapeutically, is the main subject of this review; we refer readers to several recent reviews on additional aspects of EVs , 2, theiEndocytosis, exocytosis, and EV biogenesis are functions that have evolved as ways in which cells maintain surveillance of, and in turn influence, the surrounding environment and neighboring cells, although researchers are only beginning to understand the stimuli or driving forces for EV release , 2. EVs Exosomes were first examined in detail during reticulocyte maturation to red blood cells, wherein the transferrin receptor is lost gradually by incorporation in the membranes of intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs) that are eventually released from the cell as free exosomes . MVBs orMicrovesicles, sometimes also called ectosomes, originate from outward invaginations of plasma membrane regions in a manner roughly reminiscent of the reverse of endocytosis. Microvesicles contain plasma membrane proteins as well as cytosolic proteins, nucleic acids, and other metabolites. Because microvesicles originate by plasma membrane pinching, they are exposed continuously to cytoplasmic material, unlike ILVs, which are encased within MVBs. Nevertheless, active targeting or sorting mechanisms can enrich microvesicles with nucleic acid, protein, and lipid constituents, and, akin to exosomes, the biogenesis of microvesicles could also use ESCRT to complete vesicle budding .ABs result from fragmentation of apoptotic cells and therefore are composed of plasma and organellar membranes and partially hydrolyzed nuclear and cytoplasmic material. ABs play key roles in cellular homeostasis, including induction of immunogenic tolerance in the absence of infection, which is used in animal studies and clinical trials –32. SomeAs mentioned for reticulocyte exosomes, alteration of membrane lipid and protein composition is one important function of EVs . The lipEVs contain integral membrane proteins such as tetraspanins and pentaspan proteins, peripheral membrane proteins such as lactadherin and annexins, submembrane actin and intermediate filaments, and intravesicular proteins that are either soluble or associated with the above proteins . Intereshttp://www.exocarta.org) and EVpedia (http://www.evpedia.info).EVs contain a variety of nucleic acids . CirculaIn contrast to intracellular signaling, which occurs by membrane components, soluble protein factors, and scaffolded complexes , multicoSignal transduction at the plasma membrane also initiates negative feedback loops to extinguish the signal, including by endocytosis of receptor-ligand complexes, thereby helping to distinguish persistent from spurious signals. Although endocytosis was initially thought to dampen signaling by degrading active receptor-ligand pairs, it is required for some signaling events emanating from intracellular endosomes . Thus, dThe development and growth of the embryo during development, and the actions of adult stem cells during tissue regeneration, are controlled by signals that have various concentration gradients and topologies . Soluble growth and differentiation factors, referred to frequently as morphogens, may be distributed along gradients from their source and captured on extracellular matrix or membrane proteoglycans before engaging their cognate receptors to initiate signaling. Although stem cell differentiation factors, such as Wnt and Hh, are known to be lipidated small proteins flowing along membranes from parenchymal cells or nearby niche and stromal cells to recipient stem cells , recent Drosophila embryonic development, cytonemes from wing cells orient toward source cells that produce morphogens such as Wnt, Hh, epidermal growth factor, fibroblast growth factor (FGF), and decapentaplegic; this polarized orientation and cytoneme length are typically reduced when the ligands are distributed uniformly under experimental conditions  that function in mating behavior  (Secretion of exosomes containing Hh-related peptides during cuticle development in ATPases) ; a similATPases) , 79. ThiATPases)  or to faATPases) , 81. AkiATPases) , althougApoptosis and its end products, ABs, play a critical role during embryonic development  and during adult cellular homeostasis by controlling the process of apoptosis-induced proliferation  and reguEx vivo–manufactured natural and engineered stem cell EVs administered to a patient can plausibly be used to recapitulate some or most of the therapeutic benefits of whole stem cells while potentially avoiding pitfalls associated with the latter, such as neoplastic transformation  and immuEVs are isolated based on differential ultracentrifugation , gel fil+ B cells selectively  and passive approaches [i.e., incorporation after EV secretion, such as by electroporation  or chemiectively , whereasectively . Geneticectively , 106.EZH1 and EZH2) recognizes certain lncRNAs selectively and therefore can be used to enrich lncRNAs into EVs. Other RNA types, such as P-element induced wimpy testis (piwi RNA) can be loaded in EVs by recruiting them via motifs of the interacting proteins piwi-like RNA-mediated gene silencing 1 (gene symbol PIWIL1), piwi-like RNA-mediated gene silencing 4 (PIWIL4), or piwi-like RNA-mediated gene silencing 2 (PIWIL2) (MVP) fused with CD63.Tags that bind specific types of nucleic acids are particularly relevant to engineering stem cell EVs containing RNAs targeting a particular pathway or pathways. Poly A binding protein, which binds mature mRNAs, can be used to recruit mRNAs selectively into EVs. Alternatively, a zipcode-like, approximately 25-nucleotide sequence present in the 3′-untranslated region (3′UTR) of certain mRNAs can be incorporated into the 3′UTR of the mRNA of interest and be recruited by the Z-DNA binding protein 1 (ZBP1) . Likewis(PIWIL2) . In addiLoading of EVs is not limited to biological cargo but can also include small-molecule drugs, which are typically incorporated in EVs through postisolation methods such as electroporation, transient osmotic shock, or reversible chemical covalent modification. For example, dendritic cell (DC)-derived EVs loaded with doxorubicin delivered it to breast cancer cells and inhibited tumor growth without major toxicity . SimilarMost studies of stem cell EVs have involved embryonic stem cells (ESCs), hematopoietic stem or progenitor cells, neural stem cells, endothelial progenitor cells (EPCs), and MSCs , 111–114Stem cells, such as ESCs and induced pluripotent stem cells (iPSCs), have tremendous promise, but at present they remain constrained for clinical use by safety issues that include their immunogenicity, incomplete differentiation, and neoplastic transformation , 120. InIn the subtotal nephrectomy murine model of kidney regeneration, microvesicles from MSCs, akin to intact MSCs, protected against renal injury by decreasing the levels of creatinine, uric acid, lymphocyte infiltration, and fibrosis . SimilarWe discuss briefly the effects of EVs on cancer, metastasis, and drug resistance, particularly in the context of the safety of using EVs as regenerative therapies. This is particularly relevant because tissue repair after injury involves extensive stem cell activity; a myriad of remodeling and immunoregulatory factors (including on EVs) secreted by parenchymal, niche, stromal, and stem cells; and therefore an enhanced probability that a chronic wound can develop various grades of neoplasia . ConsideThe literature is inconclusive as to whether stem cell EVs suppress or promote tumors. Part of the discrepancy may be due to the complexity of signals and pathways that are used by stem cells  to sense and home to tumors and that may overlap with mechanisms that guide them to sites of injury and inflammation –125. PerA recent, particularly illuminating study employed cre-lox techniques to provide evidence for EVs in mediating cancer metastasis . EVs froIn summary, although more data are needed, the above and related studies  implicatAs with other biological agents in clinical use, EV products will require clinical good manufacturing practice (cGMP) protocols, including cell banking and storage, scalability, stability, lot and batch tracking, pathogen screening, and other physical, chemical, and biological methods for quality control , 160 di.It is currently difficult to obtain sufficient quantities of EVs for large-scale in vivo animal studies and human clinical trials . In our One solution to the challenge of EV large-scale production would be to extract clinical-grade EVs from spent culture media (containing EVs) currently considered biotech waste . The curCIITA) or MHC genes to reduce the potential immunogenicity of EVs and make them available for a genetically diverse group of patients.In addition to the difficulty in generating sufficient quantities of EVs that possess therapeutic efficacy, there are numerous safety considerations, which include toxicity and side effects, as well as the need for pharmacokinetic studies prior to approval for clinical use. The use of animal serum–free media is also recommended for EV products, as serum contains EVs that may influence recipient cell responses . As EVs The size and complexities of EVs fall in the range between whole cells and defined single-molecule pharmaceuticals, similar to the clinically approved platelet lysate. Akin to cell-based and certain biological therapeutics, it is not possible to produce EVs under exact homogeneity from batch to batch as is done for chemically defined drugs. But this does not preclude their eventual adoption in clinical practice. Rather, just as occurs for therapy with whole cells , once thSimilar to current cellular therapies in clinical trials  that can be tested for potency and other therapeutic parameters before administration in patients, stem cell–derived EVs can undergo potency and quality control testing , 169. ThIn conclusion, although for many therapeutic applications of EVs, the specific MoA might not be completely definable because of their complex nature, EVs are arguably more amenable than cells to logistical operations such as potency assays, freeze/thawing, batch tracking, and related quality control tests. As researchers continue to develop the technologies for analyzing EVs, the quality control for their characterization is also expected to develop further.EVs are being investigated increasingly as potential biomarkers of health and pathology –175, in SELECTED COMPANIES CONDUCTING RESEARCH AND DEVELOPMENT ON EVSAethlon creates diagnostic tools to detect and quantify exosomes in blood and other fluids. Its subsidiaryExosome Sciences is testing the Hemopurifier device in clinical trials to deplete cancer exosomes from patients.Anosys Inc. (acquired by Chromos Molecular Systems Inc.) develops dendritic cell–derived EVs for new cancer therapies.Capricor Therapeutics Inc. develops cardiac-derived stem cells and their EVs to repair damaged heart tissue.Caris Life Sciences is a molecular informatics company with platforms for analysis of EVs from patients.Exosome Diagnostics develops biofluid-based diagnostics to deliver personalized precision healthcare, particularly for cancer.Exovita Biosciences develops exosome-based therapies to selectively target breast cancer cells.ReNeuron uses neural stem cell–derived exosomes to treat neurological diseases.System Biosciences (SBI) develops products for EV isolation, characterization, and labeling, including tools for next-generation nucleic acid analysis.As cell-based medications become more widespread and enter clinical practice, researchers are developing newer methods to track the fate of the administered cells (such as the use of nucleic acid barcodes), similar to what is done in toxicology and pharmacokinetics and pharmacodynamics analysis of small-molecule drugs, antibodies, and other biologics. Similar to the analysis of endogenous EVs or cell-free nucleic acids in blood and other bodily fluids, EVs released by administered exogenous stem cells could be collected from body fluids and tested as potential biomarkers. In particular, the added versatility of introducing a marker  in the injected cells allows researchers to monitor the fate of transplanted cells via their released EVs as a surrogate marker, which represents an exciting future direction. For instance, a few preclinical studies have tracked EV biodistribution and uptake using cre-lox reporter techniques , 26.The properties of EV membranes that provide them with excellent biocompatibility and greater durability , and theOverall, the properties of EVs, which derive from natural membranes and cytoplasmic material, have enabled their investigation for use as injury or disease biomarkers, potential surrogate markers for transplanted stem cells, or biosensing systems in various therapeutic and diagnostic models. Nevertheless, the utility of EVs as biomarkers of specific diseases and conditions is provisional, given the current paucity of studies in this emerging area and the difficulties and complexities of the biomarker field itself . FurtherEVs are emerging as mediators of stem cell responses during embryonic development and during adult tissue homeostasis and its restoration after injury. Parenchymal cells may emit EVs loaded with morphogens, apoptotic patterns, and immunomodulatory and other signals to activate their local stem cells or recruited bone marrow MSCs, which in turn can secrete EVs loaded with protein and nucleic acids interacting with specific pathways in recipient parenchymal cells to contribute to their healing or replacement. However, fundamental questions associated with the generation, distribution, and uptake of EVs should be addressed to fully understand the role and biology of stem cell EVs in cell-cell communication and tissue regeneration (see the sidebar titled FUNDAMENTAL OPEN QUESTIONS IN STEM CELL EV BIOLOGY AND BIOTECHNOLOGYWhat are the mechanisms and implications of EV biogenesis?To what extent does the content of EVs mirror (or differ from) their originating cells?How are stem cell EVs loaded with specific membrane or intravesicular cargo?How are EVs targeted to and taken up by specific cells?What are the roles of EV size, stability, membrane curvature, and structural presentation and multivalency of membrane receptors in their biological and therapeutic effects?What role do EVs play for morphogen distribution and signaling during development?What is the significance of endogenous stem cell EVs in tissue repair?Do EVs produced by exogenous stem cells and administered in vivo exert therapeutic effects?What are the key components of stem cell EVs responsible for therapeutic efficacy?What are the best approaches for analyzing endogenous and exogenous EVs in vivo?Can stem cell EVs bypass biological barriers, and if so, what are the mechanisms?Because EV biogenesis has evolved as an efficient type of cell-cell communication, EVs offer several advantages as therapeutics compared to cells, synthetic nanoparticles, and single molecules. As EVs are derived from cells and can be engineered to be nonimmunogenic, they can possess exceptional biocompatibility and biostability characteristics. Their small size enables EVs to avoid the pulmonary first-pass effect and to penetrate deep inside most tissues. For clinical applications, EVs offer additional advantages relative to whole cells, including lack of a nucleus to avoid neoplastic transformation, enhanced stability to freeze/thaw cycles, and greater capacity for loading with small molecules, proteins, and nucleic acids. Additionally, EVs can be engineered with specific receptors or antibodies on their surface to deliver therapeutic cargo into target cells and tissues. EVs may thus find increasing use as vehicles for gene therapy and drug delivery in medicine and as transfection reagents in biotechnology.Booming EV research has sparked the emergence of numerous companies that aim to develop EV-based therapeutics or diagnostic biomarkers (see the sidebar titled However, numerous challenges remain before stem cell EVs can be translated successfully into clinical use . In partIn conclusion, the intercellular functions of EVs honed by evolution to maintain and restore homeostasis are being tested for personalized and regenerative medicine. Bioengineered stem cell EVs are therefore anticipated to find increasing applications in their niche between molecular and cellular medicine and may play a part in regenerative therapy by maturing together with other medical advances.


Biologic-based treatment strategies for musculoskeletal diseases have gained traction over the past 20 years as alternatives to invasive, costly, and complicated surgical interventions. Spinal degenerative disc disease (DDD) is among the anatomic areas being investigated among this group, notably due to its high incidence and functional debilitation. In this review, we report the literature encompassing the use of biologic-based therapies for DDD. Articles published between January 1995 and November 2015 were reviewed, with a subset meeting the primary and secondary inclusion criteria of clinical trial results that could be sub-classified into bimolecular, cell-based, or gene therapies, as well as studies investigating the utility of allogeneic and tissue-engineered intervertebral discs. Ongoing clinical trials that have not yet published results are also mentioned to present the current state of the field. This exciting area has demonstrated positive and encouraging results across multiple strategies; thus, future bimolecular and regenerative techniques and understanding will likely lead to an increase in the number of human clinical trials assessing these therapies. The radiographic findings of degenerative disc disease (DDD) can be found in 40% of individuals younger than 30 and in more than 90% of individuals older than 50 years of age -2. WhileGiven the potential complications of these surgical interventions, attention to biologic-based therapies for DDD has gained traction. Trials of gene therapy, in addition to cellular- and acellular-based transplantations have been described in degenerative knee and metacarpophalangeal arthritis, with promising results -11. ThusClinicalTrials.gov, and Cochrane Library databases were searched for relevant studies from January 1995 to November 2015. The following keywords were queried in combination with intervertebral disc or degenerative disc disease: gene therapy,cell therapy, molecular therapy, stem cell, mesenchymal stem cell, disc cell, nucleus pulposus cell, disc chondrocyte, disc regeneration, and tissue engineering. After the initial search, 213 studies were identified. These studies’ results were reviewed, duplicates were identified, and only relevant studies were included. The primary inclusion criterion was the presence of clinical results on disc regeneration. The secondary inclusion criterion was the ability to categorize into one of the following categories: biomolecular therapy, cell-based therapy, gene therapy, and tissue engineered intervertebral disc (IVD). These categories were generated based on the literature that is available on in vivo animal studies on biologic therapy for DDD. For each study, we identified the type of study, the questionnaire-based subjective assessment on pain, and radiologic outcome measures. Following this advanced filtering, a total of 24 studies were included for discussion.The PubMed, Google Scholar, Embase, The IVD is composed of the nucleus pulposus (NP) surrounded by the annulus fibrosis (AF), sandwiched between cartilaginous endplates at the junction to the vertebral bodies located above and below the IVD. The NP is mainly composed of proteoglycans and type II collagen, which allows for the retention of water increasing the IVD’s ability to handle axial loading. The surrounding AF is more stiff and is primarily composed of type I collagen. With increasing age, the water content within the IVD decreases and results in the NP being less resilient to mechanical stressors. This progressive decrease in resiliency leads to NP fissures, which can extend into the AF. This marks the initial stages of degenerative destruction of the IVD, endplates, and associated vertebral bodies Figure .As previously stated, DDD is a multifactorial process including the progressive decline in NP hydration due to loss of proteoglycans and collagen. This decreased hydration results in loss of mechanical tension in the AF collagen fibers and results in abnormal spinal axial loading forces and segmental instability. These minor changes in stress forces on the spine can result in the development of neck or back pain and narrowing of the spinal canal over time. In early stage degeneration, the disc undergoes an imbalance of anabolic and catabolic factors that leads to extracellular matrix (ECM) degradation . SpecifiRecombinant Human Growth/Differentiation Factor-5Growth/differentiation Factor-5 (GDF-5) is a member of the transforming growth factor-b (TGF-b) superfamily and the bone morphogenetic protein (BMP) subfamily and is known to influence the growth and differentiation of various tissues, including the intervertebral disc. In vitro and in vivo experiments have shown that human recombinant GDF-5 (rhGDF-5) can stimulate gene expression and synthesis of ECM proteins such as type II collagen and aggrecan . In SeptPlatelet-rich PlasmaPlatelet-rich-plasma (PRP) is a fraction of plasma that can be produced by centrifugal separation of whole blood. A platelet contains the vast majority of biologically active molecules required for blood coagulation, such as adhesive proteins, coagulation factors, and protease inhibitors . In addiFurther evidence for PRP therapy in DDD was augmented in 2015 by a prospective, randomized controlled study assessing intradiscal PRP injections in discogenic-mediated low back pain. This study demonstrated improvements in pain and function in patients as early as eight weeks post-treatment and was sustained for up to one year . Forty-sCell-based Therapy for Disc RegenerationBiomolecular therapy likely has limited efficacy in discs with higher grades of degeneration, as the number of cells responsive to injected genes and proteins decline with progression degeneration. Cell-based therapy is the optimal treatment strategy in mid-stage degeneration because it directly addresses the decreased number of viable chondrocytes and stem cells within the diseased disc space Table .Autologous Disc ChondrocytesThe use of autologous disc cells is an alternative approach to repair damaged or chronically inflamed tissue by addressing multiple propagators of degeneration at once. From 2002 to 2006, the first study of autologous disc chondrocyte transplantation (ADCT) in a large group enrolled patients in a multicenter prospective, randomized, controlled, non-blinded study to compare the safety and efficacy of ADCT. Known as the EuroDISC trial, this study compared ADCT plus discectomy to discectomy alone to evaluate if ADCT mitigated postoperative pain. In this study, 28 participants between the ages of 18 and 60 with a body mass index below 28 and 1-level lumbar canal stenosis requiring surgical intervention were included. Patients with multiple levels of stenosis requiring operative intervention, discs with sclerosis, edema, or modic changes of grade II or III, and/or focal spondylolisthesis were excluded. Twelve patients received percutaneous ADCT 12 weeks following discectomy and 16 patients received only discectomy. The patients were followed for two years and assessed using the Oswestry low back back pain disability questionnaire (OPDQ) as the primary criterion. Secondary criteria included MRI and X-ray evaluation. They found that the cell therapy group had continual improvement in their OPDQ after the initial surgery compared to no improvement in the control group, and this results persisted at the two-year follow-up . AdditioAutologous Mesenchymal Stem CellsMesenchymal stem cells (MSCs) derived from bone marrow are one of the most well-studied cell types in regenerative medicine due to their accessibility and expandability in ex vivo conditions. Additionally, the anti-inflammatory effects of MSCs have been demonstrated in numerous animal models of injury including myocardial infarction, renal ischemia, reperfusion injury, burn wounds, and osteoarthritis -24. TherIn a similar regard, Orozco, et al. published a study of 10 patients with chronic back pain diagnosed due to DDD with autologous MSCs injected directly into the NP . These pDespite these results, the major challenge to MSCs therapy is the pain and cost associated with harvesting these cells. Since MSCs are found in adipose tissue, this poses an attractive source for harvesting and subsequent transplant due to the low-risk accessibility, and current trials evaluating their efficacy in intradiscal applications are ongoing -28.Autologous Hematopoietic Stem CellsHematopoietic stem cells are thought to be useful to treat disc degeneration due to their differentiation and proliferative capacities. However, as applies to most cells, the oxygen-poor environment poses a challenge for biologic therapies in spinal disc disease. In 2006, Haufe, et al. described a study utilizing hyperbaric oxygenation in 10 patients following percutaneous intradiscal injection of autologous hematopoietic precursor stem cells (HSCs) . UltimatAutologous Bone Marrow Aspirate/Bone Marrow Concentrated CellsHarvesting, culturing, and expanding autologous chondrocytes or MSCs is an expensive process and requires the patient to undergo at least two procedures since the harvested cells must be cultured and expanded in a lab for weeks prior to transplantation Figure .Bone marrow concentrated cells (BMCs) contain multiple stem and progenitor cells, including mesenchymal stem cells (MSCs) and can be autotransplanted at the time of surgery, precluding a second delayed intervention. Pettine, et al. evaluated the use of autologous, nonexpanded BMCs to treat moderate to severe discogenic low back pain . The stuAllogeneic Mesenchymal Precursor Cells/Mesenchymal Stem CellsAllogeneic stem cells are another method of biologic treatment for DDD being investigated that is attractive due to the low costs of harvesting and preclusion of secondary procedures. Currently, two clinical trials are underway evaluating their effects. Mesoblast, a private cellular medicine company, is conducting a double-blinded, placebo-controlled Phase 2 clinical trial of 100 patients with DDD randomized to received intradisc injection of saline, hyaluronic acid, or allogeneic mesenchymal precursor cells (MPCs) of varying concentrations in a hyaluronic acid . SimilarAllogeneic Juvenile Disc Chondrocytes7 juvenile chondrocyte cells/ml within a fibrin carrier. Ten of the 13 patients exhibited significant clinical and radiographic improvements, and interestingly eight of nine patients with the posterior annular tears had resolution of significant improvement in the degree of posterior annular tear, suggesting ongoing remodeling of the injured AF.Similar to the previous reports of autologous disc chondrocyte transplantation, groups have assessed the efficacy of allogeneic juvenile chondrocytes into degenerated discs. In a prospective cohort study, Coric, et al. demonstrated that NuQu, an injectable percutaneous fibrin-based delivery of juvenile chondrocytes, helps improve low back pain refractory to conservative measures . FifteenGene Therapy for DDDin vivo animal models has been promising, no clinical trials have been conducted using this technique in humans. Since viral carriers are often used for transfection, serious concerns involving unexpected mutations create challenges for Food and Drug Administration (FDA) approval and patient inclusion. Nonetheless, gene therapy has shown promising results in other neurologic and orthopedic diseases and thus remains an area of interest for biologic treatment strategies in DDD. Injection of biologic molecules into the disc allows for direct delivery of protein products to address the causes of degenerative disc disease at the source. However, these biological molecules and many cell-based therapies have short half-lives which preclude their longevity following injection. Gene therapy has emerged as a potential solution to this, which comprises of delivery of recombinant genes into cells. The recombinant gene can then incorporate with the DNA of the host cell allowing for long-term expression of either a proanabolic or anticatabolic protein to facilitate healing of the degenerated disc space . This stAllogeneic and Tissue Engineered IVD Transplantation for DDDin vivo and in vitro experiments using tissue engineered IVD are in their preliminary states. One promising study published by Ruan, et al. included five patients with cervical spondylosis who underwent transplantation of fresh-frozen composite disc allografts following discectomy [At present, both scectomy . The disThere have not been any human clinical trials for the implantation of tissue engineered IVD (TE-IVDs). However, there has been one human pilot study for using a biomimetic protein polymer that mimics the NP. Berlemann, et al. used NuCore® injectable nucleus , which is a protein polymer hydrogel that mimics the properties of the natural nucleus to treat post-discectomy patients . The polThe information elucidated from various studies on biologics presents an exciting new area of research for DDD. Adaptations and modifications of similar modalities used in degenerative joint arthritis may one day be applicable to spinal disc disease as an upfront therapy. Future research into the use of viral vector gene therapy, RNA interference, and micro RNAs may provide fruitful alternatives to cell-based and whole IVD-based therapies, but significant challenges need to be addressed prior to translation to human clinical trials.


Between July and August 2017, seven of 12,047 blood donations from eastern Austria, reacted positive to West Nile virus (WNV) in the cobas test (Roche). Follow-up investigations revealed Usutu virus (USUV) nucleic acid in six of these. Retrospective analyses of four blood donors diagnosed as WNV-infected in 2016 showed one USUV positive. Blood transfusion services and public health authorities in USUV-endemic areas should be aware of a possible increase of human USUV infections. In 2014, the Austrian Red Cross, Blood Service for Vienna, Lower Austria and Burgenland, initiated regular screening of all blood donated between 1 June and 30 November each year for West Nile virus (WNV) by a nucleic acid test (NAT). Positive samples have been found each year since 22]). ScrIn Austria, blood donors have to answer an extended medical questionnaire before donation. Several questions refer to visits to countries endemic for pathogens transmissible by substances of human origin including WNV. Travellers returning from WNV-endemic regions have either to be screened by WNV NAT or have to be deferred for 28 days after their return from an affected area.The goal of this communication is to raise awareness of blood transfusion services and public health authorities in countries, which have reported USUV infections: (i) of an increase of USUV detections in blood donations in eastern Austria in 2017 compared with the two previous years, and (ii) of the possibility that USUV infections may be misdiagnosed as WNV, due to tests not distinguishing between these two viruses.The latter is actually not a real problem because any flavivirus (including both WNV and USUV) infected blood donation must be discarded; nonetheless the diagnosis should be verified/falsified by virus-specific follow-up molecular tests to precisely identify the causative agent. Aetiological diagnosis might also reveal other flaviviruses such as Zika virus, dengue virus, etc. The minimum sensitivity of the Roche cobas WNV assay is approximately 13 copies/mL in a pooled sample, which corresponds to 250 copies of WNV RNA/mL for each individual blood donation . The virus loads in the USUV positive samples were low, therefore not all samples were uniformly positive in all conventional and RT-qPCRs applied, consequently only five sequences could be included in the NS5–3'UTR phylogenetic analysis . Despite770Asn and Lys2520Arg). This might suggest that host factors such as age, comorbidities, and immunosuppression, and not necessarily genetic markers of the infecting virus, influence the clinical outcome of a human USUV infection. It is also worth mentioning that none of the Austrian USUV-positive blood donors showed any clinical symptoms.The phylogenetic analyses demonstrated a close genetic relationship of the Austrian blood donor-derived USUV sequences with the recent bird-derived Austrian and Hungarian sequences , suggestAlthough USUV is a potential human pathogen, there are no specific regulations on screening blood donations for this virus. According to the general rules, only clinically healthy people are allowed to donate blood. However, as described above, the USUV-positive blood donors did not show any symptoms before and after donation. Although WNV and USUV endemic areas in Europe largely overlap , there aIn order to raise awareness and allow timely actions, we intentionally wrote this paper at this point in time when the WNV and USUV transmission seasons are still ongoing, which, depending on weather conditions, may last in central Europe until the end of October.


Many cancer cell lines have been developed, which have for a long time been available for use in basic and preclinical cancer research. However, those cell lines have the disadvantage that they do not necessarily reflect the behaviors of the original cancer cells in patients, owing to the artificial nature of their culture conditions. Therefore, cell line-derived xenograft tumor models, which are established by transplanting well-validated cancer cell lines into immunocompromised mice, have also been used for cancer research . Cell liPatient-derived xenograft (PDX) mouse models have attracted attention in recent years, with the aim of resolving such problems. PDX mouse models are established by direct engraftment of patient-derived tumor fragments into immunocompromised mice. Since PDXs have been suggested to retain morphologies, architectures and molecular signatures very close to those of the original tumors, it is probable that they have great potential for both basic and preclinical cancer research , such asThere have been several experimental protocols reported to generate PDX models, as individual research groups have their own ways to improve the success rate of PDX engraftment, although the protocols seem to share the fundamental concepts and techniques. Briefly, pieces of solid tumors or single-cell suspensions are collected from tumor tissues obtained by surgery or biopsy, and are transplanted under the skin (subcutaneous transplantation), in the same organ as the original tumors in the patients (orthotopic transplantation), or in the renal capsule in the recipient immunocompromised mouse. Subcutaneous transplantation models allow for easier cell transfer and precise monitoring of tumor formation and growth . In contet al. analyzed the dynamics of DNA copy number alterations during PDX passaging across 24 types of cancer . Indeed, the effects of cancer immunotherapy, using agents such as immune checkpoint inhibitors, might be difficult to evaluate with PDXs transplanted into these immune cell-deficient mice [PDXs seem to be a valuable model for cancer research, although it may be important to know their limitations as well. In original tumor tissues, stromal cells such as epithelial cells and fibroblasts co-exist with cancer cells, whereas in PDX models, almost all stromal cells derived from human tumors cannot proliferate continuously and are replaced by cells derived from the recipient mouse. Therefore, there are unavoidable limitations to studying tumor microenvironments using PDX models. In addition, the immune system is compromised in the mice employed for PDX models, such as nude mice (T cell-deficient), severe combined immunodeficient mice (T- and B cell-deficient) and extremely immunodeficient mice [T-, B-, and NK cell-deficient; NOG mice  [et al. well-characterized the breast cancer PDXs in this biobank, and also prepared PDX-derived tumor cells for culture, which preserved the characteristics of the original PDXs. They developed a platform of high-throughput drug screening assays with PDX-derived tumor cells, on which drug responses can be assessed and validated [Recently, a network of PDX banking of many research collaborations with potential success has been established . For exapdx.eu/) . They shalidated . Conside


Quantitative resistance has gained interest in plant breeding for pathogen control in low-input cropping systems. Although quantitative resistance frequently has only a partial effect and is difficult to select, it is considered more durable than major resistance (R) genes. With the exponential development of molecular markers over the past 20 years, resistance QTL have been more accurately detected and better integrated into breeding strategies for resistant varieties with increased potential for durability. This review summarizes current knowledge on the genetic inheritance, molecular basis, and durability of quantitative resistance. Based on this knowledge, we discuss how strategies that combine major R genes and QTL in crops can maintain the effectiveness of plant resistance to pathogens. Combining resistance QTL with complementary modes of action appears to be an interesting strategy for breeding effective and potentially durable resistance. Combining quantitative resistance with major R genes has proven to be a valuable approach for extending the effectiveness of major genes. In the plant genomics era, improved tools and methods are becoming available to better integrate quantitative resistance into breeding strategies. Nevertheless, optimal combinations of resistance loci will still have to be identified to preserve resistance effectiveness over time for durable crop protection. Plant pathogens are major limiting factors in crop production and this has led to the extensive use of chemicals to control them. Plant genetic resistance is a promising key alternative to control crop diseases and pests. However, pathogens frequently adapt to and overcome genetic resistance especially when it is determined by major genes. Thus, quantitative resistance has gained interest in recent years to address the major challenge of genetic resistance durability.Quantitative resistance can refer to an incomplete or partial level of resistance phenotype. It can also refer to a continuous distribution between resistant and susceptible phenotypes in a progeny, most often resulting from the segregation of alleles with variable effects at several loci. In contrast, qualitative resistance refers to either a complete or high level of resistance, or to a bimodal segregation of phenotypes in a progeny, classifying individuals into two distinct categories: resistant and susceptible. Accordingly, rx1, rx2, and rx3 were found to confer a high level of resistance of tomato to Xanthomonas campestris  which contribute, each with variable effect, to the phenotype of resistance to a pathogen. Quantitative resistance often confers a partial level of resistance to the plant. It does not block but only reduces pathogen multiplication, plant colonization, and/or symptom severity. However, a combination of resistance QTL can lead to total resistance in some cases, especially when QTL have strong effects . For exampestris .genetic architecture of quantitative resistance has often been associated with a small number of detected QTL, some with major effects and others with minor effects. In recent years, the development of next generation sequencing (NGS) technologies, coupled with the use of accession panels and multi-parental populations, has accelerated the discovery of a higher number of QTL controlling quantitative resistance. Markers closely linked to QTL, especially to QTL with minor effects, and marker haplotypes describing allelic diversity at QTL were identified  (Pi35) or loss-of-function susceptibility alleles . Some are expressed during a specific developmental stage  or in a specific environment , or confer resistance when overexpressed . Positional candidate genes located in resistance QTL genomic regions were also identified in recent studies, using genomics and GWA mapping approaches  . Indeed,proaches . These gproaches . Resistaproaches . In Arabance QTL .Mlo gene has been used to control powdery mildew in varieties of spring barley in Europe since 1979  (increased durability of quantitative resistance could be due to (i) the partial resistance effect exerting a low selection pressure on the pathogen population, (ii) a combination of contradictory selection pressures on pathogen evolution, (iii) a low probability of multiple pathogen mutations needed to overcome multiple QTL, (iv) a combination of different resistance-associated mechanisms, which together are more difficult to overcome, and (v) a combination of resistance mechanisms acting successively at different times in the pathogen life cycle or throughout plant development  . However potato) . The incelopment . Neverthelopment . Indeed,elopment .Different approaches have been proposed for adequately deploying major R genes and resistance QTL, with the aim of increasing the durability of crop resistance to pathogens. The sustainable management of available genetic resistance factors includes (i) the use of multi-line varieties or varietal mixtures carrying different R genes or QTL , (ii) thpyramiding major R genes have been extensively trialed  and marker-assisted recurrent selection (MARS) (Breeding schemes for d pepper . There an (MARS) . The sucn (MARS) . In seven (MARS) .The durability of QTL pyramids was rarely evaluated. Nevertheless, QTL combinations are expected to increase durability for different reasons.Venturia inaequalis strains co-inoculated in a mixture. Broad-spectrum QTL did not exert differential selection pressures between strains, whereas specific QTL decreased the frequency of some strains. Consequently, pyramiding QTL with different specificities or broad spectrum QTL may be expected to increase durability. Recently, the combination of two resistance QTL, which showed additive broad-spectrum effects in NILs, was suggested as a promising strategy for breeding durable resistance in rice against rice blast (Pyramiding resistance QTL showing a varying spectrum of action on pathogen strains may generate contradictory selection pressures on pathogen evolution. ce blast .Table 2). Resistance QTL with diversified actions on the pathogen’s life-cycle were shown for yellow rust resistance in barley NILs  underlying different putative functions, exhibited increased, broad-spectrum and stable levels of resistance in multiple environments  from Solanum vernei, which acts by masculinizing the nematode populations, was overcome after 8 years of recurrent selection in the laboratory. The speed at which the resistance was overcome depended on the genetic background into which vrnGpaV was introduced  was rapidly overcome under controlled experimental conditions (3pvr2 with three partial resistance QTL significantly increased resistance durability under the same conditions. Durability QTL controlling the frequency of plants with 3pvr2-resistance breakdown were mapped onto the pepper genome (In pepper, the major nditions . The comr genome . The evor genome . These iPhytophthora infestans in potato (Lr34 which confers resistance to multiple diseases in cereals (Xa4 which improves multiple agronomic traits by strengthening the cell wall (The development of molecular markers has led to an increase in the use of disease resistance QTL in breeding. However, minor-effect QTL are still difficult to exploit and resistance can have physiological costs for the plant . The devn potato , can be  cereals , or the ell wall . Howeverell wall .Quantitative resistance appears to be more durable than qualitative resistance and has the potential to preserve major-gene effects. However, there is still a need to adequately choose resistance QTL to create optimal combinations and limit QTL erosion. The choice of QTL could be based on their spectrum of action on different strains, their effect on pathogen life traits and their underlying molecular mechanisms . RecentlBreeding strategies for quantitative resistance still require a better understanding of the genetic, ecological and agronomic determinants of pathogen adaptation involved in QTL erosion. Further knowledge is needed on the evolutionary capacity of pathogen species, which would depend on their mutation rate, reproduction mode, dispersal ability and effective population size . Five reAll the authors listed made substantial intellectual contribution to the work, wrote the manuscript together and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Risk compensation (RC) could reduce or offset the biological prevention benefits of HIV preexposure prophylaxis (PrEP) among those at substantial risk of infection, including men who have sex with men (MSM). We investigated the potential extent and causal mechanisms through which RC could impact HIV transmission at the population and individual levels.Using a stochastic network-based mathematical model of HIV transmission dynamics among MSM in the United States, we simulated RC as a reduction in the probability of condom use after initiating PrEP, with heterogeneity by PrEP adherence profiles and partnership type in which RC occurred. Outcomes were changes to population-level HIV incidence and individual-level acquisition risk.When RC was limited to MSM highly/moderately adherent to PrEP, 100% RC (full replacement of condoms) resulted in a 2% relative decline in incidence compared to no RC, but an 8% relative increase in infection risk for MSM on PrEP. This resulted from confounding by indication: RC increased the number of MSM indicated for PrEP as a function of more condomless anal intercourse among men otherwise not indicated for PrEP; this led to an increased PrEP uptake and subsequent decline in incidence.RC is unlikely to decrease the prevention impact of PrEP, and in some cases RC may be counterintuitively beneficial at the population level. This depended on PrEP uptake scaling with behavioral indications. Due to the increased acquisition risk associated with RC, however, clinicians should continue to support PrEP as a supplement rather than replacement of condoms. Preexposure prophylaxis (PrEP) has been shown to be highly effective in preventing infection with the human immunodeficiency virus (HIV) among persons with sexual behaviors that place them at substantial risk, including men who have sex with men (MSM) . In the One public health concern has been whether PrEP will replace rather than supplement existing HIV prevention approaches such as condoms . AlthougOpen-label PrEP demonstration studies that have tracked outcomes before and after initiation of PrEP have found minimal evidence of risk compensation, as measured by both self-reported risk behaviors and non-HIV sexually transmitted infection (STI) incidence ,12,13. WIn this study, we use mathematical modeling to estimate the potential effects of condom-related risk compensation following initiation of PrEP among MSM in the US. Building on an earlier model  in whichEpiModel software platform (http://epimodel.org/), which depends on the statistical framework of temporal exponential random graph models (ERGMs) to simulate highly structured sexual networks of MSM partnerships over time. Most behavioral data to parameterize the model were derived from an HIV incidence cohort and a cross-sectional sexual network study of MSM in Atlanta [This study extends our stochastic agent-based model of HIV transmission dynamics over sexual partnership networks, originally developed to investigate the role of PrEP on HIV incidence among MSM in the United States . Our mod Atlanta ,20. This Atlanta . PartnerFor interhost and intrahost epidemiology, the HIV clinical model simulated all aspects of the natural progression of HIV after infection, with a representation of HIV viral load, HIV testing, antiretroviral therapy (ART) initiation and adherence profiles, and eventual disease-induced mortality for MSM with treatment failure. Within HIV-discordant partners, predictors of HIV transmission during anal intercourse (AI) included viral load , condom PrEP indications were modeled based on the CDC’s guidelines for clinical practice for MSM . Men werPrEP uptake was modeled, following earlier approaches , based oWe assigned PrEP medication adherence levels based on empirical data from a three-city PrEP demonstration project: 21.1% of men were categorized as non-adherent (0 pills/week), 7.0% as taking <2 pills/week (low adherence), 10.0% at 2–3 pills/week (moderate adherence), and 61.9% at 4+ pills/week (high adherence) . Use of Men discontinued PrEP also based on the CDC guidelines, which recommend reassessment of risk behavior every 12 months and potential cessation of prescriptions if no longer indicated. In our model, we replicated this by reevaluating the dynamic risk behavior of each man on PrEP at yearly intervals after PrEP initiation; if men no longer exhibited PrEP indications within the 6-month risk window, then they stopped PrEP. Men who stopped were allowed to restart PrEP if they exhibited indications at their next HIV diagnostic visit and the coverage threshold had not been exceeded.Risk compensation was modeled as the reduction in the probability of condom use at each sexual act. Condom-related risk compensation has been more common among MSM than other forms of behavioral change, such as increases in the number of partners ,15. BaseOur main sensitivity analyses varied the level of risk compensation across different PrEP adherence profiles and partnership types. We hypothesized that risk compensation, if it were to occur, would be most likely among highly adherent PrEP users and least likely among the non-adherent. Therefore, we modeled risk compensation along the same 0% to 100% gradient assuming that it occurred in increasingly wider subsets of PrEP users: high adherence only; high and moderate adherence; high, moderate, and low adherence; and high, moderate, low, and non-adherent. Secondary analyses explored compensation along the same gradient, but limited to main partnerships or known HIV-discordant partnerships; we hypothesized that these were more likely candidates than casual or unknown-status partnerships for risk compensation to occur.Because risk compensation could potentially impact both population-level epidemiology and individual-level HIV infection risk, our analyses considered both outcomes. Epidemiological outcomes tracked were the HIV incidence rate in each scenario, the percent of infections averted (PIA) compared to a scenario in which no PrEP was provided, and the number needed to treat (NNT) with one year of PrEP to prevent one new HIV infection. Individual-level outcomes included the expected number of infection events per 1000 exposures overall and by current PrEP status. To quantify use of PrEP in the full population, including men who were not indicated for PrEP, we calculated the total weeks on PrEP per 100 weeks of susceptible (HIV-uninfected) person-time. Although the coverage proportion remained constant at the 40% threshold, this person-time measure could increase if the total number of PrEP-indicated MSM increased in the population. Due to the stochastic nature of the models, we simulated each scenario 250 times, and reported means and 95% credible intervals (the middle 95% of each set of 250 observations) from across the simulations for each statistic.http://github.com/statnet/HIV-Risk-Comp/.The software code for reproducing model simulations and data analyses may be found at HIV incidence rates increased and the PIA decreased at all levels of risk compensation when it occurred among all four adherence groups, including MSM who were completely non-adherent to their prescriptions . When riIn all scenarios that varied risk compensation by medication adherence profile and partnership types, the efficiency of PrEP (as measured by the NNT) was negatively impacted . From thThis impact on the individual-level risk is shown in This decrease in total risk but increase in PrEP-stratified risk arose because of a unique form of dynamic confounding by indication for PrEP Figs  and 3. AOur model of HIV transmission dynamics suggests that condom-related risk compensation after PrEP initiation may have a minor impact on the population-level epidemiology of HIV among MSM in the US. Although potential risk compensation could simultaneously increase the individual-level acquisition risks and reduce the efficiency of PrEP, these negative outcomes would be greatest under extreme conditions of risk compensation  and uniformity across PrEP medication adherence. Counterintuitively, when risk compensation was limited to those with moderate to high adherence to PrEP, it was associated with a decline in HIV incidence, as more men became eligible for and initiated PrEP, consequently lowering the population-level incidence rates.Currently, few mathematical models of PrEP use among MSM have explored the phenomenon of risk compensation in this level of detail to disentangle the complex interaction between risk compensation after PrEP initiation and the behavioral indications for PrEP itself. PrEP models that incorporated risk compensation to date have suggested that negative countereffects on the ability of PrEP to prevent HIV would occur under relatively extreme conditions—just as our model required—that do not reflect current empirical data on behavioral change . A recenOurs may be the first study to explain the systems dynamics by which risk compensation fails to impact the HIV prevention benefits of PrEP. By modeling risk compensation as heterogeneous across PrEP medication adherence levels, we first observed a decline in HIV incidence when risk compensation was limited to those with moderate to high adherence levels. Understanding this counterintuitive phenomenon required further investigation of the individual-level HIV acquisition risks among PrEP users and non-users during CAI events; these risks declined overall after risk compensation was introduced but increased when stratified by PrEP use status, a novel form of dynamic and dependent confounding by indication. The confounder here was time on PrEP, which increased with higher levels of risk compensation, in an unexpected feedback loop: as more PrEP-using men engaged in risk compensation, non-PrEP using sex partners who had not previously been indicated for PrEP because they did not engage in CAI were now eligible. This study is subject to several limitations, related to the uncertainty in long-term PrEP medication adherence, coverage, and risk compensation patterns among MSM. In particular, we modeled risk compensation as a function of the PrEP use status of one of the members in a partnership, while in reality, condom use may be a negotiated outcome between the PrEP user and his partner. Partnership-level dyadic data on the intersection of PrEP use and sexual risk behavior among MSM couples might be influential in the model, and collecting these data as PrEP uptake increases among MSM will be essential to understand the population-level implications of PrEP. Second, our results strongly depend on the relationship between behavioral indications and PrEP uptake: as PrEP indications increased with more risk compensation, so did PrEP initiation. Longitudinal data on the complex dynamics of PrEP awareness, interest, and use in varied real world settings will be critical to targeting PrEP, as well as the continued development of models like ours. Third, the generalizability of these models is partially subject to the underlying Atlanta-based behavioral data upon which the baseline models were calibrated ,33. As sWhile early commentators suggested that risk compensation could be a significant vulnerability of emerging antiretroviral-based HIV prevention technologies ,34, evid


Individuals recently released from prison engage in risky behaviors that predispose them to contracting HIV. Women may be at increased risk in the immediate period post-release, given higher rates of poverty, food insecurity, and substance dependence and lower educational attainment compared with men.We describe gender differences in HIV risk behaviors using validated measures and assess potential mediators of this relationship using data from a cross-sectional study of 109 individuals recently released from prison.Women had higher rates of HIV drug-related risk behaviors compared with men  and HIV sex-related risk behaviors . Women also had higher mean incomes and severity of drug and alcohol use compared with men, but equally high rates of food insecurity and low levels of AIDS knowledge. In multivariate analysis, the relationship between gender and HIV drug-related and sex-related risk behaviors was attenuated by a greater monthly income , as well as severity of drug use .Women had higher rates of HIV risk behaviors compared with men post-release. Gender specific interventions may be useful in reducing risky drug-related and sex-related behaviors in the period immediately following release. Approximately eight million individuals are released from U.S correctional facilities each year and are at increased risk for HIV. Previous studies of this population describe frequent engagement in drug- and sex-related risk behaviors immediately after release  to explore the association between food insecurity and HIV risk behaviors among individuals recently released from prison within one calendar year of the interview.  and sex-related risk behaviors  in the 30 days prior to interview, as assessed by the HIV Risk-Taking Behavior Scale-PLUS (HRBS-Plus)  were women. There were no statistically significant differences between men and women released from prison in terms of age, race, educational attainment, housing, or employment  or AIDS knowledge  by gender. Women had a greater income compared with men . Despite similar rates of drug use , a higher proportion of women reported using intravenous drugs . Women were more likely to report higher severity of drug use  but not alcohol use .Women were more likely to engage in drug-related HIV risk behaviors  and sex-related HIV risk behaviors . In multivariate analyses, adjustment for age and race did not attenuate the impact of gender on drug-related , but women who engaged in transactional sex had higher composite HRBS scores compared with men who engage in transactional sex , driven by drug-related HIV risk behaviors (13.5 ± 8.9 vs. 0, p = 0.04, Figure We found that women recently released from prison reported increased engagement in drug- and sex-related risk behaviors compared with their male counterparts, in spite of similar knowledge of the risk factors for acquiring AIDS, rates of food insecurity, educational attainment and employment. Women released from prison had high severity of drug use, which increased the likelihood of engaging in high-risk behaviors. Women in our study, especially those engaging in transactional sex, reported higher rates intravenous drug use, though rates of overall drug use were not significantly different by gender. These findings suggest, like many prior studies, that strategies which acknowledge the parallel risk of illicit drug use and transactional sex among women, may also have an impact on reducing HIV transmission. (Binswanger et al. Unlike past studies, we did not find statistically significant differences in the rates of transactional sex by gender. (Binswanger et al. Our study has several limitations. The limited sample size prevented us from exploring the independent association of gender and HIV risk behaviors in a robust multivariate model. Also, our convenience sampling strategy reduces the generalizability of our study to all recently-released individuals. Our survey did not ask about other factors that impact HIV risk taking behavior, such as physical and sexual abuse as well as mental health history. Finally, our study used a well-established measure of AIDS knowledge that was designed prior to the advent of HAART, perhaps limiting its accuracy.Our findings may suggest the need for interventions targeting women focused on reducing drug addiction and providing employment opportunities that counter the financial incentives for engaging in transactional sex. Larger, longitudinal studies are needed to better understand the gender differences in HIV risk behavior engagement in this population.