Drosophila. Approximately two-thirds of disease genes are conserved in Drosophila, and gene-specific fly models exhibit behavioral changes that are related to symptoms of epilepsy. Models are based on behavior readouts, seizure-like attacks and paralysis following stimulation, and neuronal, cell-biological readouts that are in the majority based on changes in nerve cell activity or morphology. In this review, we focus on behavioral phenotypes. Importantly, Drosophila modeling is independent of, and complementary to, other approaches that are computational and based on systems analysis. The large number of known epilepsy-associated gene variants indicates a need for efficient research strategies. We will discuss the status quo of epilepsy disease modelling in Drosophila and describe promising steps towards the development of new drugs to reduce seizure rates and alleviate other epileptic symptoms.One in three epilepsy cases is drug resistant, and seizures often begin in infancy, when they are life-threatening and when therapeutic options are highly limited. An important tool for prioritizing and validating genes associated with epileptic conditions, which is suitable for large-scale screening, is disease modeling in  Approximately 50% of cases can be traced to single genetic mutations4. The identification of these genes by worldwide consortia is an important step toward the development of new therapeutic approaches because modeling the disease conditions requires a powerful genetic approach. Given the rapid progress in stem cell technology, patient-derived models are one attractive avenue of investigation5. However, the heterogeneity of human genomes poses challenges, as differences in genetic background can alter the phenotype produced by a given mutation. These challenges can be partially overcome in the Drosophila model through crossings to achieve isogenized lines, as well as the standardization of transgenic insertions to reach a stable genetic background9. However, genetic drift remains a concern when transgenic lines are kept over many generations and can lead to differing results from different labs that ostensibly use the same stocks.Epilepsy has a strong hereditary component; over the past decade, more than 100 causative epilepsy genes have been discovered by worldwide epilepsy screening consortia10. Even increased dosage or combination therapy has not produced a significant reduction in the fraction of intractable cases. In addition, epilepsy patients can develop further comorbidities despite seizure suppression, including intellectual disability (ID) and autism spectrum disorder12.Anti-seizure drugs (ASDs) aim to control epileptic symptoms and improve quality of life in epilepsy patients. Despite very good results for two-thirds of those affected, seizures remain refractory, or drug-resistant, in one-third of patients15. Readouts of larval motor coordination have also been employed in seizure-associated studies16. An intriguing alternative to screens searching for genetic modifiers is the Janelia Fly Olympiad project. In this project, 2,205 specific anatomical regions were acutely silenced using tsshi or overexcited using TrpA1 in order to track behavior changes automatically utilizing computer vision algorithms18. This technical approach registered a broad range of behavior patterns, including locomotion, coordination, and climbing that together reflect epilepsy-associated paradigms19. Righting behavior and side-steps, for instance, have been previously employed to quantify epilepsy-associated phenotypes21. The combination of increasingly efficient behavior readouts and genetic screening libraries enabling the regulation of nearly all genes with tissue-specific promotors holds great promise for future screens, in which the advantages and limitations of the chosen readout  and its interaction with ogre/Innexin1, but also stress-sensitive B (sesB)39. Despite substantial divergence in these genes between human and Drosophila, their role in human seizure progression and in blocking seizures means that the insights from the Drosophila model nevertheless hold translational value46.A functionally related group of gap junction genes has been identified owing to the characteristic induced shaking, beginning with uncoordinated and 42 more are linked to further epilepsy-relevant readouts in the FlyBase Human Disease Model Report  of the genes that can mutate to produce mechanically induced phenotypes co-occur with those that are temperature dependent. This is consistent with findings in the paralytic (para) mutant bss1para, which demonstrated that, in spite of effects on global brain activity underlying responses to mechanical or thermal stress, the effects are linked to distinct brain regions21. While a strong focus in behavioral experiments has been placed on activity bursts resembling seizures, other approaches attempt to capture paroxysmal epileptic dystonia or paralysis48.Electroconvulsive stimulus. Detection of such non-seizuring events extends the range of epilepsy modeling in Drosophila and has benefited most from electrophysiological readouts30. Hyperexcitation in the central nervous system was induced through short electroconvulsive electric pulses lasting 0.1 to 0.5 milliseconds up to 3 second pulses51. Electric stimulation at larval stages allows investigation of adult-lethal seizure alleles in Drosophila such as slamdance (sda), as it induces characteristic tremors in mutants that are visible in the high-contrast mouth hooks that can be seen in the otherwise translucent body51. The paradigms detailed in this and the previous two sections elicit heightened brain activity from where the giant fiber system plays an important role conveying stereotypic, seizure-like motor responses to the motor system, with the interesting exception of flight muscle49. This commonality suggests a degree of convergence in generating epilepsy-associated behavior patterns.Genetic stimulus. Mutants in the para locus, such as the bsspara hypermorph allele as well as knockdown alleles (as discussed in ‘Tissue-specific genetic screening’ below), have been thoroughly investigated for seizure-like behavior52. The alpha subunit of voltage-gated sodium channels Para mediates neuronal action potentials. The human orthologs SCN8A, SCN2A, and SCN1A are strongly associated with pathogenic epilepsy phenotypes and carry several of the most frequent alleles associated with epilepsy55. Drosophila carrying the bsspara mutation are sensitized to induced seizure-like attacks, notably even through optogenetic stimuli56. bsspara mutants have also received attention for the identification of genetic interactors that suppress seizuring, among them gilgamesh (gish), which encodes a casein kinase 1 that is highly conserved in humans57. Genetic seizure modifier genes and downstream interacting genes are efficiently identified in Drosophila. An alternative case of predisposition for seizure-like behavior is the metabolic epilepsy model for PNPO deficiency (OMIM #610090). Induction through a sugar-only diet in mutants of the fly gene sugarlethal (sgll), the orthologue of PNPO, elicited seizures without additional acute stimuli58.tsshibire (tsshi) the most prominent example. shi encodes a dynamin and one of its human orthologs, DNM1, is linked to an early infantile epileptic encephalopathy (OMIM #616346). These alleles produce proteins that lose function at conditions warmer than room temperature. Dynamin’s functional relevance for epilepsy modeling was demonstrated by suppressing the seizure-like phenotypes of the well-known bang-sensitive genes para, easily shocked (eas), and sda through heat activation of the tsshi allele59. A further genetic rescue is the suppression of seizures in the TBC1D24 epilepsy and DOORS Syndrome (OMIM #220500) ortholog skywalker (sky), which was mediated by the shi effector Synaptojanin (Synj)34.Some bang-sensitive alleles are temperature sensitive, with Circadian rhythm. An environmental stimulus is required to induce seizure-like behavior in Drosophila carrying genetic mutations, with rare exceptions such as the prickle mutant splpk or the zydeco mutant 1zyd when coupled with one out of several repo>dStim knockdown lines61. One potential way to observe behavioral changes without triggering an acute response is to monitor sleep or activity patterns, and mutants that have seizure-like phenotypes often also have abnormal sleep. For example, shorter or more fragmented sleep periods were observed in the uncoordinated mutant Shaker (Sh)62, which is homologous to the epilepsy gene KCNA2 (OMIM #616366), and for sky34. Several other well-studied bang sensitivity alleles are also sleep defective, including those affecting the epilepsy gene orthologs h channelI (Ih), para, and shi, as well as two direct interactors of Sh potassium channels, quiver (qvr), and Shaker cognate b (Shab)64. This link between sleep abnormalities and seizures requires further investigation in humans and in animal models to better understand its applicability in clinical epilepsy research.67. Despite this progress, most underlying mechanisms leading to epileptic diseases remain to be elucidated4. Systematic classification of causative genes is currently based on phenotypic patient descriptions. Genetic trends in the age of onset were determined through phenotypic distinction of 12 different groups, while at the same time a major overlap of the genotype–phenotype spectrum became apparent48. Mutations in genes coding for ion channel subunits underlie a high number of patient cases among hereditary epilepsies68. An example are causative variants in SCN1A which are the most frequent cause of Dravet syndrome: more than 80% of patients carry variants of this ion channel subunit and are in general highly resistant to treatment69.The identification of over 100 rare genetic epilepsies suggests a broad range of disease mechanisms, such as synaptic transmission defects, channelopathies, and transcriptional, metabolic, and transport defectsin vivo model can reveal brain-specific genetic interactors involved in epilepsy. Screens for suppressors of seizure-inducing alleles can reveal novel drug targets and point to related functional pathways. Previous large screens have identified genetic suppressors of seizure-like phenotypes. Alleles disrupting neuronal communication were also identified in non-ion channel genes, e.g. eas mutant seizure-like behavior or sky/TBC1D24 and synj/SYNJ1. In fact, the link in Drosophila between synj and epilepsy was found before human patient variants in SYNJ1-causing untreatable epilepsy had been identified72. Beyond screening for phenotypical resemblance to seizures, suppressor screens, for instance in the genetic background of bsspara mutant animals, determined genetic interactors of epilepsy-associated genes57. Suppressors have been identified in both unbiased and selective genetic screens, e.g. for sky/TBC1D2473.To identify potential new treatments for Dravet syndrome and further drug-resistant conditions effectively, a high-throughput 75.A large inhibitory RNA (RNAi)-based screen for behavioral phenotypes resulting from gene inactivation in glial cells has identified a number of bang-sensitive mutants, which are orthologs of human epilepsy-associated genes with roles in glial and, interestingly, glia–neuron interactionsDrosophila community has developed 192 genetically inbred, or isogenized, lines known as the Drosophila melanogaster Genetic Reference Panel (DGRP), which enable the connection of epilepsy-associated phenotypes with multiple endogenous genetic loci, potentially leading to the discovery of new genetic modifiers76.In addition to mutant and knockdown models, the Drosophila protein-coding genes77. These genetic libraries by the Vienna Drosophila Resource Center and the Transgenic RNAi Project at Harvard Medical School have the advantage of tissue-specific knockdown, whereas knockout lines, such as the MiMIC insertions which introduce insertion cassettes, often constitutively abrogate gene expression79. The RNAi-based screens are as versatile as the promotor lines combined with transcript knockdown, yet their effect strength has been found to vary and should be assessed independently from the phenotype80. Another 10,000 fly lines enabling knockdown in different neuronal and glial subsets have been generated in the Fly Light project81. Similar resources that take advantage of the CRISPR–Cas9 gene targeting system are under development  knockdowns, while highly useful, are variable in the degree to which they inactivate the target gene. Libraries of fly stocks that express Cas9 and UAS-driven short-guide RNA elements will enable tissue-specific complete knockouts of any targeted gene84. Akin to genetic interactor screens, the interplay of seizure phenotypes with pharmacological inhibitors has been investigated, based on both known ASD as well as on novel substances. Picrotoxin-induced seizuring or genetic models such as eas flies served as screening models85. Testing known ASDs discovered responsiveness to pharmacological inhibitors such as phenytoin and nifedipine, but also to gabapentin, compounds that are effective against severe seizure pathologies85. Just as in human epilepsies, screens found no single drug compound which could effectively suppress the many different underlying genetic changes in Drosophila11.The development of epilepsy treatments in animal models focuses on seizure-like readouts that can be suppressed through genetic interaction or application of pharmacological compounds87. ID and autism spectrum disorders are examples of strongly co-occurring pathologies which are beginning to be integrated into current epilepsy and neurodevelopmental disease models in small animal models or in drug trials90.One potential limitation of focusing on seizure-like behavior readouts in the fly model is the omission of the wider phenotypic spectrum in epileptic encephalopathies, regarding both the method of treatment and its timing11. ASD alternatives are surgical intervention, neurostimulation, ketogenic diet, and lifestyle adjustment. Recent clinical studies suggested cannabidiol-based treatment as a therapeutic intervention in drug-resistant therapies and successful seizure suppressors in a Drosophila model92. Despite the enormous amount of research and the increasing number of available ASDs, there still remain epileptic patients who cannot be treated11.Patients with drug-resistant epilepsy have a restricted quality of life and higher mortality, and the drug resistance may have cognitive and psychosocial consequences4. The large number of underlying genes combined with the small patient groups affected by individual variants require inexpensive and rapid methodologies to test anti-seizure measures. As discussed above, while stem cell-based methods potentially reveal precision medicine solutions, at the current moment Drosophila genetic screening offers rapid and economical insight into the genetic function and interactome of conserved genetic variants.The number of epilepsy-associated genes was expanded drastically when a whole-exome study of more than 17,000 individuals detected vast numbers of ultra-rare variantsmtd/OXR1 and sky/TBC1D24 variants cause epilepsies that were found to be linked to reactive oxygen species levels and modified by anti-oxidants at the level of neurons as well as the entire brain, based on humanized Drosophila transgenic models in which the cDNA of patient variant OXR1 or TBC1D24 was expressed in the brain48. Humanization of the most frequent genetic cause underlying Dravet syndrome, point mutations in SCN1A, have enabled detailed studies of the effect conveyed by many of the mutations on protein function93. By understanding the mechanistic and functional connections between epilepsy-associated genes, a step from many unconnected single disorders to functional networks becomes more tangible. A third way to assess in which wider gene networks these single factors are functionally required is based on systems biology, specifically on brain transcriptomics95. Computational approaches have the potential to connect mouse and human in vitro data with Drosophila epilepsy in vivo models to obtain integrated conserved functional networks. This approach will hold validity across a range of disorders affecting the central nervous system8.In a complementary direction, unbiased study of molecular pathways and genetic interactions connects known genes with each other as well as with new genes or unexpected causative variants in epilepsy. For example, 96. Based on epilepsy genes identified by the International League Against Epilepsy (ILAE), previous computational approaches have been validated in cellular and mouse models, leading to a proposed mechanism of valproic acid function in SCN1A patient cases and to pre-clinical confirmation of Csf1r inhibitors as novel ASD candidates95. With the advent of whole-brain single-cell transcriptome datasets, recent developments have enabled the inference of connections between the orthologs of causative monogenic epilepsy factors at unprecedented resolution and will in the future support models of epilepsy gene networks98.Epilepsy patient genetics provide unprecedented insights into common and more rare epilepsy conditions, many resistant to current treatment forms. Beyond their clinical phenotype, a shared genetic basis has been revealed that allows the investigation of converging genetic disease mechanismsGrouping disease genes based on inferred genetic interactions and confirming candidate networks in efficient animal screens could become a valuable additional tool for expanding drug classifications of pre-clinical epilepsy models, associating epilepsies on their responsiveness to treatment as a new criterion. Functional connections and ‘epilepsy gene networks’ could guide genetic and pharmacological screens and ultimately future treatment strategies.


Arabidopsis thaliana, which, due to genome duplications, has multiple paralogs of each ribosomal protein (RP) gene. We support the notion of plant evolution directing high RP paralog divergence toward functional heterogeneity, underpinned in part by a vast resource of ribosome mutants that suggest specialization extends beyond the pleiotropic effects of single structural RPs or RP paralogs. Thus, Arabidopsis is a highly suitable model to study this phenomenon. Arabidopsis enables reverse genetics approaches that could provide evidence of ribosome specialization. In this review, we critically assess evidence of plant ribosome specialization and highlight steps along ribosome biogenesis in which heterogeneity may arise, filling the knowledge gaps in plant science by providing advanced insights from the human or yeast fields. We propose a data analysis pipeline that infers the heterogeneity of ribosome complexes and deviations from canonical structural compositions linked to stress events. This analysis pipeline can be extrapolated and enhanced by combination with other high-throughput methodologies, such as proteomics. Technologies, such as kinetic mass spectrometry and ribosome profiling, will be necessary to resolve the temporal and spatial aspects of translational regulation while the functional features of ribosomal subpopulations will become clear with the combination of reverse genetics and systems biology approaches.Plants dedicate a high amount of energy and resources to the production of ribosomes. Historically, these multi-protein ribosome complexes have been considered static protein synthesis machines that are not subject to extensive regulation but only read mRNA and produce polypeptides accordingly. New and increasing evidence across various model organisms demonstrated the heterogeneous nature of ribosomes. This heterogeneity can constitute specialized ribosomes that regulate mRNA translation and control protein synthesis. A prominent example of ribosome heterogeneity is seen in the model plant,  Dictyostelium discoideum upon transition from a unicellular to a multicellular lifestyle  and a 40S small subunit (SSU). The latter decodes mRNA, and the former catalyzes the peptidyl transferase reaction that leads to the peptide bond formation of the newly synthesized proteins. The subunits are composed of rRNA and accessory ribosomal proteins (RPs). The large subunit is composed of 5S, 5.8S, and 25S rRNA, which ranges between 25S and 26S in plants but is 28S in mammals . In contogeneity  and may Ribosome biogenesis has previously been reviewed both for plants  and yeasCytosolic ribosomes comprise four mature rRNAs, i.e., 5S, 5.8S, 25S, and 18S. The process to obtain the mature RNAs starts with:RNA polymerase III mediated synthesis of 5S rRNA . Loci inArabidopsis thaliana  as depicted in tif6 and brx1-1 transcripts are differentially accumulated compared to wild type (WT) in mutant lines of the RBF REIL that are likely impaired in late cytosolic ribosome maturation and during cold acclimation  or in the case of RPs, permanently  bound to the pre-ribosomes. The RP and other RAP coding genes are transcribed and spliced in the nucleus (rp), compiled in a literature review of yeast ribosome biogenesis (2 and 27SA3 pre-rRNA processing. 2) Domains I and III are located near the polypeptide exit tunnel and the RPs binding nearby are necessary for 27SB pre-rRNA cleavage. 3) Finally, the third docking area is located near the central protuberance on the interface side of the LSU. The nearby bound RPs are necessary for 7S pre-rRNA processing and nuclear export. Whether plant-RPs conserve these sequential and spatial dependencies, remains to be tested.All RPs have specific entry points during ribosome biogenesis. Therefore, the main processing steps of ribosome biogenesis may determine when RP-specialized ribosomes can be assembled based on selection of specific RPs or paralogs instead of a non-controlled stochastic choice. Controlled assembly would mean that adjacently located RPs, if co-assembled, might be co-dependent on each other or on specific biogenesis factors. Consequently, defined ribosomal regions might be modulated by specialization mechanisms that rely on a sequential assembly line to construct functionally divergent complexes. In line with the previous idea, systematic analyses of individual ribosomal protein mutants  was conducted with rat liver ribosomes . The 79 P family , namely,P family . The othP family . AttemptP family . A more P family . The strP family .Triticum aestivum cryogenic electron microscopy (cryo-EM) map that was reported at 5.5 Å resolution  was used as reference for our visualizations (PDB ID 4v7e). Using protein BLAST comparisons, we verified the RP identity of Arabidopsis RP homologs of the protein entries linked to the macromolecular Crystallographic Information Files (mmCIF) of the wheat 80S structure model. We concluded that Arabidopsis RPs  into a bread wheat solution . Simultasolution , thus cosolution Figure 2solution . FigureSolanum lycopersicum are high  but the contribution and interplay of transcription with additional layers of control of the protein composition of ribosomes require further research.The influence of environmental and developmental cues on transcripts of plant cytosolic RP and RBF transcripts becomes increasingly evident. For Arabidopsis, a regulatory model that triggers ribosome heterogeneity was proposed based on transcriptome analyses . Such a identity  and planidentity . In Oryzstresses . The difde novo synthesis of complete ribosome complexes or synthesis of RPs followed by ribosome remodeling. In short, PTMs can create heterogeneity on a shorter time scale than possible by ribosome or RP turnover. Arabidopsis RPs undergo a great variety of covalent modifications, such as initiator methionine removal, N-terminal acetylation, N-terminal methylation, lysine N-methylation, phosphorylation and S-cyanylation  need to consider the multiple modes of ribosome heterogeneity. We think that there is reasonable doubt that ribosome heterogeneity is a mere consequence of stochastic ribosome assembly and that heterogeneity has the sole function of engineering redundancy to ensure a secure supply of the essential ribosome machinery. We support the view that evolution molded the ample structural diversity of plant ribosomes toward functionally specialized ribosomes, where PTM mechanisms act rapidly on slowly turned-over ribosome populations and For heterogeneous ribosomes to be functional, the translated proteome must be shaped by selective transcript recruiting according to external stimuli. Means of selective translation by structural changes to the ribosome at the RP level that became apparent in other organisms  remain tMany RP genes have been mutated to enable a deeper functional understanding of their gene products. These studies focus on the developmental role of single or few RP paralogs . A summarp mutants share typical features, for example, altered leaf polarity establishment, cell proliferation and shape determination  are split between nuclear and plastid encoded and can be divided in the model plant Arabidopsis into essential and nonessential components . By defiterparts . This obterparts . PRPS5 aterparts . In riceterparts .rp studies is small as compared to those analyzing genes of the cytosolic or plastidic ribosomes. Nevertheless, the already existing body of literature, reviewed elsewhere  and cold (4°C) stress with the respective control at 20°C optimized temperature. The experiment is identified as entry AT-00120 in the Genevestigator repository . We based our test-case on a compiled list of 376 Arabidopsis genes that have been annotated as members of the cytosolic ribosomal proteome. As to the procedures, the background-subtracted microarray signals of experiment AT-00120 were retrieved and imported into the R statistical programming environment. All initial matrix related transformations, object conversions and data handling were performed with the R packages stringi (version 1.4.6—https://cran.r-project.org/web/packages/stringi/index.html), reshape (https://github.com/hadley/tidyverse). Only the signals belonging to heat or cold stress subset of AT-00120 were further processed, because suboptimal temperature was reported to impact significantly RBF and RP transcripts (https://github.com/bmbolstad/preprocessCore). Afterwards the distribution of the data within treatments and genes was evaluated with density plots for treatments using the R package ggplot2  approach  and differential responses among cytosolic RP families under opposing conditions of temperature stress. Focusing on the changes that occur within RP families, we encountered instances of potential temperature-specialized RP paralogs, and, as exemplified by the inversely regulated P1/P2 P-stalk components AtRPP1D and AtRPP1A, even indications of paralog preference under heat stress  is sequestered into stress granules upon heat stress to be quickly released during stress deacclimation to resume cytosolic ribosome synthesis . This prde novo synthesis cycle. Therefore, we suggest that altered transcript abundances may rapidly translate into altered CRP stoichiometry. Studies aiming to characterize translatome-reprogramming at the onset of stress acclimation should reveal differential dynamics of RP and RP paralog synthesis and incorporation into the translating CRP.RP transcript levels of Arabidopsis roots are, however, not necessarily associated with compositional changes of the non-translating ribo-proteome . RibosomEvidence of ribosome specialization by differential paralog use alone e.g.,  or in coDespite the complexity of investigating ribosome heterogeneity and specialization, new technologies make tackling this hard problem feasible . These tde novo protein synthesis through label-assisted proteomics. Label-assisted proteomic studies monitor the kinetics of label incorporation into proteins, allowing for calculations of protein synthesis and degradation rates at biological steady states  and the University of Melbourne for funding this research via the Melbourne-Potsdam PhD Programme (MelPoPP).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.


Increasing the accessibility and affordability of hearing healthcare is a public health concern. Because low health literacy is a significant barrier to the use of existing effective healthcare services, it is critical to assess and understand health literacy deficits specific to hearing loss before implementing interventions. The purpose of this cross-sectional study was to identify differences in hearing loss health literacy among older and young adults, which is warranted because older adults are at-risk for lower levels of health literacy compared to their younger counterparts. Adults across the lifespan (n = 170) completed the Hearing Loss Health Literacy Assessment Tool, which includes self-rated ability to access/obtain, understand, and appraise hearing health information, as well as apply information to manage life with hearing loss. Results from an independent samples t-test indicated that older adults  self-reported significantly higher overall hearing health literacy than younger adults , t(168) = 4.22, p < 0.0001. Participants rated their ability to access/obtain information significantly lower than the other subscales. Age-differences in self-rated hearing health literacy exist. Findings from this study receive support from evidence indicating that the readability and suitability of the majority of patient education materials on hearing loss are not appropriate for the average U.S. adult. This investigation provides further evidence that the availability and accessibility of patient education materials on hearing loss is an important barrier that contributes to the limited use of hearing health care.


Comparative analyses have indicated that the mitochondrion of the last eukaryotic common ancestor likely possessed all the key core structures and functions that are widely conserved throughout the domain Eucarya. To date, such studies have largely focused on animals, fungi, and land plants (primarily multicellular eukaryotes); relatively few mitochondrial proteomes from protists (primarily unicellular eukaryotic microbes) have been examined. To gauge the full extent of mitochondrial structural and functional complexity and to identify potential evolutionary trends in mitochondrial proteomes, more comprehensive explorations of phylogenetically diverse mitochondrial proteomes are required. In this regard, a key group is the jakobids, a clade of protists belonging to the eukaryotic supergroup Discoba, distinguished by having the most gene-rich and most bacteria-like mitochondrial genomes discovered to date.Andalucia godoyi and used a comprehensive in silico approach to infer the nucleus-encoded portion of the mitochondrial proteome of this protist, identifying 864 candidate mitochondrial proteins. The A. godoyi mitochondrial proteome has a complexity that parallels that of other eukaryotes, while exhibiting an unusually large number of ancestral features that have been lost particularly in opisthokont  mitochondria. Notably, we find no evidence that the A. godoyi nuclear genome has or had a gene encoding a single-subunit, T3/T7 bacteriophage-like RNA polymerase, which functions as the mitochondrial transcriptase in all eukaryotes except the jakobids.In this study, we assembled the draft nuclear genome sequence for the jakobid As genome and mitochondrial proteome data have become more widely available, a strikingly punctuate phylogenetic distribution of different mitochondrial components has been revealed, emphasizing that the pathways of mitochondrial proteome evolution are likely complex and lineage-specific. Unraveling this complexity will require comprehensive comparative analyses of mitochondrial proteomes from a phylogenetically broad range of eukaryotes, especially protists. The systematic in silico approach described here offers a valuable adjunct to direct proteomic analysis , particularly in cases where the latter approach is constrained by sample limitation or other practical considerations. In the evolutionary transition from endosymbiotic bacterium to integrated cellular organelle, the mitochondrion has undergone extraordinary changes . Most ofAlphaproteobacteria, the bacterial class with which mitochondria share a specific common ancestor  have revealed that this organelle is a structural and functional amalgam of proteins having different phylogenetic origins: an evolutionary mosaic . Unexpector ..AlphaproTetrahymena thermophila, a ciliate protozoan hydrogenase and its associated maturases. However, like Stygiella, Andalucia does possess an organelle-targeted acetate:succinate CoA-transferase that might make it capable of the type of acetate-producing fermentation found in some anaerobically functioning mitochondria .All TCA cycle enzymes are present, with two distinct forms for isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase E1, and succinyl-CoA synthetase β subunit.Paracoccus denitrificans -binding domain. This protein family is highly conserved evolutionarily, throughout bacteria as well as eukaryotes, and is thought to have a role in the regulation of various iron, Fe-S, and reactive oxygen/redox reactions in cells . We retrAndalucia also encodes two distinct MAO forms, but both are specifically related to the MAO A type.Two mitochondrial outer membrane (MOM) proteins are Msp1 and monoamine oxidase (MAO), orthologs of which we identified in this study. Msp1 is an AAA+ superfamily ATPase that clears mistargeted proteins by facilitating their transfer from mitochondria to the endoplasmic reticulum . MAOs arc  and functioning in various metabolic pathways, compared to their orthologs from A. castellanii . (XLSX 19 kb)Additional file 7: Figure S3. Phylogenetic analysis of (A) RFO and (B) DUF3501 proteins. ML trees  are shown, with branch support values (rapid bootstraps) indicated when ≥51%. The root is placed arbitrarily. The RFO tree (A) is based on an alignment of 365 amino acid positions. The sequence from Ancoracysta twista is only partial, possibly explaining why it does not cluster together with sequences from other eukaryotes. Note the sequence from the gammaproteobacterium Arhodomonas aquaeolei nested among alphaproteobacterial sequences, suggesting a possible HGT event. The DUF3501 tree (B) is based on an alignment of 128 amino acid positions. (PDF 864 kb)


To describe a rare occurrence of acute vision loss and diffuse alveolar hemorrhage following a treatment of injectable gluteal cosmetic filler.A 20-year-old female underwent a cosmetic injection of unknown components for gluteal augmentation. Within hours she developed progressive shortness of breath secondary to diffuse alveolar hemorrhage. She presented to ophthalmology 6 weeks later with a history of bilateral decreased vision. Clinical examination revealed cotton wool spots and retinal hemorrhages. Fluorescein angiography demonstrated macular vascular pruning and an enlarged foveal avascular zone.The patient was observed and vision did not improve after 8 months of follow-up.These findings were attributed to a Purtscher-like retinopathy secondary to systemic inflammation induced by the filler and/or direct microembolization of the injected material or fat. To the best of the authors’ knowledge, this is the first documented case of diffuse alveolar hemorrhage and ischemic bilateral vision loss in a patient undergoing gluteal augmentation with dermal filler. Minimally invasive cosmetic procedures are being performed with increasing frequency as a means to circumvent surgery. Soft tissue filler injections are a frequently performed non-invasive technique, second only to botulinum toxin . Despite3 of what she described as “Venezuelan Artefill” for gluteal augmentation 6 weeks prior to presentation. The procedure was performed by an unlicensed administrator in a massage parlor. Immediately after the injection, she developed acute shortness of breath that was treated as an asthma exacerbation in the emergency room. She was admitted for persistent shortness of breath 2 days later. She developed frank hemoptysis and oxygen dependence. Computed tomography demonstrated the development of ground glass opacities of her lungs. Bronchoscopy and alveolar lavage confirmed a diagnosis of diffuse alveolar hemorrhage. Concurrently, the patient suffered a precipitous drop in her hematocrit (35% to 20%) over the course of 48 hours. She was ultimately stabilized with adequate hydration, high dose steroids, and two blood transfusions.A 20-year-old female received an injection of approximately 150 cmAfter discharge, she presented to the ophthalmology clinic with a history of acute bilateral vision loss that began the day of the cosmetic injection. The best-corrected visual acuity was 20/70 in the right eye and 20/200 in the left eye. Dilated fundoscopic examination revealed multiple cotton wool spots and intraretinal hemorrhages .Spectral-domain ocular coherence tomography  of the macula demonstrated irregularities in both the inner and outer retina without evidence of retinal edema . FluoresObservation was recommended. After 8 months of follow-up, the cotton wool spots and intraretinal hemorrhages resolved. However, the visual acuity further deteriorated to 20/200 in the right eye and 20/400 in the left eye, and the enlarged FAZ remained on fluorescein angiography . ContinuCosmetic injections by untrained practitioners in nonclinical settings have been performed for over a century. Local complications are well-documented and include granulomatous inflammatory reactions, erythema, itching, depigmentation, induration, ulceration, and tissue necrosis . Vision ®  is a third-generation PMMA product that is FDA approved for the treatment of nasolabial folds, but is often used off-label for other indications [To the authors’ knowledge, this is the first reported case of soft tissue filler injection for gluteal enhancement leading to bilateral vision loss and diffuse alveolar hemorrhage. The specific preparation of material injected in this case remains unknown. At the time of administration, the patient was told she was receiving a variation of polymethylmethacrylate (PMMA) and bovine collagen mixed with an unspecified additional ingredient and labeled as “Venezuelan Artefill” because she could not afford the pure product. Artefillications . It is iMultiple pulmonary vascular effects, including diffuse alveolar hemorrhage, have been described with subcutaneous cosmetic injections of various unconventional viscous substances including castor and corn oil, petroleum jelly, and automobile transmission fluid –8. The mThe manifestation of acute bilateral vision loss in our patient resulted from arteriolar occlusion likely due to Purtscher-like retinopathy. Purtscher-like retinopathy has multiple potential causes including fat embolism, acute pancreatitis, and renal failure. The patient demonstrated the typical clinical presentation of sudden vision loss occurring hours to days after the inciting event and presumed embolic occlusion of the pre-capillary arterioles. Ophthalmic exam revealed the characteristic cotton wool spots and retinal hemorrhages, but did not show Purtscher flecken, which is seen in 63% of patients with Purtscher’s and Purtscher-like retinopathy . PurtschAlthough the pathogenesis underlying Purtscher’s and Purtscher-like retinopathy remains unknown, multiple mechanisms proposed include microembolization of air, fat, platelets, fibrin, or leukocyte aggregates that occlude precapillaries . In addiIn summary, as the popularity of cosmetic procedures continues to grow, so will the subset of patients willing to undergo these procedures in an unregulated and precarious setting at a discounted rate. Large volume illicit injections may cause multisystem organ failure including permanent vision loss secondary to retinal vascular ischemia. This case highlights the need to raise awareness of these potentially crippling consequences to reduce the frequency of such unsafe practices.


Recently, the rapid development of biomaterials has induced great interest in the precisely targeted treatment of bone-related diseases, including bone cancers, infections, and inflammation. Realizing noninvasive therapeutic effects, as well as improving bone tissue regeneration, is essential for the success of bone‑related disease therapies. In recent years, researchers have focused on the development of stimuli-responsive strategies to treat bone-related diseases and to realize bone regeneration. Among the various external stimuli for targeted therapy, near infrared (NIR) light has attracted considerable interests due to its high tissue penetration capacity, minimal damage toward normal tissues, and easy remote control properties. The main objective of this systematic review was to reveal the current applications of NIR light-assisted phototherapy for bone-related disease treatment and bone tissue regeneration. Database collection was completed by June 1, 2020, and a total of 81 relevant studies were finally included. We outlined the various therapeutic applications of photothermal, photodynamic and photobiomodulation effects under NIR light irradiation for bone‑related disease treatment and bone regeneration, based on the retrieved literatures. In addition, the advantages and promising applications of NIR light-responsive drug delivery systems for spatiotemporal-controlled therapy were summarized. These findings have revealed that NIR light-assisted phototherapy plays an important role in bone-related disease treatment and bone tissue regeneration, with significant promise for further biomedical and clinical applications. Bone-related diseases such as cancers, infection, and trauma, are common clinical problems that lead to irreparable bone defects, which remains an intractable problem for clinicians, leading to ongoing suffering for patients, and representing a burden to healthcare systems In vivo studies in male rats also showed that an 810 nm laser could penetrate 51% through the skull and 40% through the scalp and skull combined in the prefrontal regions. In terms of 660 nm, only 5.8% of light could pass through the scalp plus skull and reach the cortical surface in vitro and in vivo. The controlled use of NIR light as an external stimulus is also highly biocompatible, with little toxicity or injurious effects on normal tissues Among several external stimuli‑responsive therapy approaches, those using light irradiation have attracted significant attention. The commonly used light for photoresponsive therapies can be generally divided into three subcategories: ultraviolet (UV) (200-400 nm), visible (Vis) 400-700 nm), and near infrared (NIR) 700-1300 nm) lights 00 nm lig00 nm, anin vitro and in vivoWith the rapid development of nanotechnology, a series of NIR light-responsive nanomaterials with specific light absorbance and conversion abilities in NIR light windows have revealed marked potential for phototherapy strategies. Many kinds of nanomaterials, such as carbon-based nanomaterials, metal nanostructures, metal sulfides or oxides, and other organic or inorganic nanostructures, have attracted interest because of their NIR light-responsive properties As a noninvasive spatiotemporal controlled treatment technology, NIR light‑assisted phototherapy has played an important role in clinical therapies of many diseases. To date, existing published reviews have mainly summarized the development of NIR light-responsive nanomaterials and their clinical applications for bioimaging and phototherapeutic effect in cancer therapies Therefore, this article systematically reviews the recent progress of NIR light‑associated phototherapies for bone-related diseases and bone tissue regeneration, intending to reach a general audience comprising not only medical scientists and clinicians, but also material scientists and engineers. Moreover, the current challenges and perspectives within the development of NIR light-assisted phototherapies for further clinical applications are proposed and discussed. A systematic understanding of NIR light-assisted phototherapies would help to achieve the desired therapeutic effects in the treatment of bone-related diseases, providing penetrating insights into precision medicine and regenerative medicine.A primary database collection was constructed for all relevant publications in English  based on three databases . The following keywords and their combination were used: (NIR OR (near infrared) OR (near infrared light) OR (near infrared laser)) AND bone AND (therapy OR treatment). The keyword search resulted in 1333 initial articles . The initial articles were screened based on the following inclusion and exclusion criteria:Inclusion criteria: 1) Articles focused on bone disease treatment, such as bone cancers, bone infections, osteoarthritis, bone metabolic diseases, bone defect repair, and bone tissue regeneration; 2) only studies using NIR light as the treatment method; and 3) only phototherapies under the NIR light irradiation within the biological window of NIR light (700-1300 nm).in vivo molecule, cell or tissue imaging); 4) articles in languages other than English; and 5) articles without full text (abstract only).Exclusion criteria included: 1) Duplicate studies; 2) books, reviews, meetings, letters, literature updates, and laboratory protocols; 3) studies using NIR light in the detection and imaging of bone-related diseases .Titles or abstracts of the initially retrieved articles were evaluated for inclusion. Each article was reviewed by two researchers independently. When the title and abstract could not be excluded, the full text of the article was evaluated. This resulted in 71 articles fulfilling the eligibility criteria. In addition, reference tracking of the included publications was completed and all the relevant articles were checked and added. Finally, a total of 81 articles were fully examined for discussion in this systematic review . The most common types of diseases were osteosarcoma (19 studies), bone metastasis of breast cancer (6 studies), implant‑related infections (8 studies), rheumatoid arthritis (3 studies), and osteoarthritis (1 study). Meanwhile, NIR light-assisted phototherapies have been used to accelerate bone tissue regeneration (13 studies). Twelve of these studies focused on NIR light‑responsive delivery systems to trigger the spatiotemporal controlled release of drugs or molecules to enhance the therapeutic effects to treat bone-related diseases.Figure 2).Moreover, 24 studies reported the photobiomodulation effects of NIR light irradiation upon cells and tissues, including 8 studies that discussed the photobiomodulation effects on stem cells, and 16 studies that focused on the effect of photobiomodulation therapy on bone healing and dental treatment . NIR light can be generated via many types of available lasers, such as Nd:YAG  or diode lasers. Most studies (45/81) delivered the NIR light irradiation via an 808 nm diode laser module with fiber coupling. By contrast, phototherapies using NIR coherent (lasers) or noncoherent infrared light  have been investigated for their photobiomodulation effects on stem cells and bone tissue regeneration. Eighteen studies reported the photobiomodulation effect of low level or high frequency NIR laser therapy, while six studies investigated the effect of NIR LED photobiomodulation on cells and tissues.The results of the literature search showed NIR light-assisted phototherapy exhibited great potential for the precise and controlled treatment of bone-related diseases and bone tissue regeneration because of its deep tissue penetration ability, high spatiotemporal resolution, and the unique biocompatibility of NIR light irradiation. Compared with other traditional therapy strategies, these phototherapies are considered as more minimally invasive, easily controlled, and precise to achieve the desired therapeutic effects and avoid damage to normal tissues. In this systematic review, these NIR light-assisted treatment strategies for bone-related disease and bone tissue regeneration are summarized, based on their mechanisms and applications.Bone cancers generally refer to malignant bone tumors, including autologous skeletal system cancers (such as osteosarcoma and chondrosarcoma) and bone metastasis of cancers from other tissues   in vivo PTT and PDT possible, offering promising options for the treatment of the primary bone cancers or problematic cancer bone metastasis from other tissues in vivo toxicity. The biosafety of these NIR light-responsive nanomaterials should be confirmed by testing normal hematology parameters to minimize the potential side effects Figure With the rapid development of nanotechnology, a series of NIR light-responsive nanomaterials have emerged and shown great advantages for efficient bone cancer phototherapy by serving as photothermal agents for PTT or as photosensitizers for PDT. et al. reported a novel kind of oxygen vacancy-rich tungsten bronze nanoparticle (NaxWO3) against breast cancer osteolytic bone metastasis, which acts via the desirable photothermal effect under NIR laser irradiation in vivo applications The efficiency of anticancer phototherapy depends on the photothermal and photodynamic performances of the employed NIR light-responsive nanomaterials. Several nanomaterials, such as carbon-based nanomaterials in vivo photothermal performance in both the NIR-I and NIR-II windows 2C), a novel kind of MXene, with an extraordinarily high efficiency of photothermal conversion for bone tumor ablation in vitro and in vivo photothermal cancer therapies in both NIR-I (36.4%) and NIR-II (45.65%) biowindows. Similarly, hybrid carbon dots (CDs)/WS2 heterojunctions have also been reported for NIR-II enhanced PTT of osteosarcoma. These CDs/WS2 heterojunctions exhibited improved cancer targeting ability after intravenous injection and efficient photothermal conversion under the 1064 nm laser irradiation. The deep tissue penetration ability in the NIR-II biowindow means that the satisfying ablation of cancer tissues can be realized at relatively low laser exposure, even when being covered by a 10 mm thick additional tissue. Although the NIR-II biowindow has attracted great attention for tumor xenografts, to date, it has rarely been used for bone cancer therapies, mainly because of a lack of suitable photosensitive agents with desirable strong absorption and high efficiency of photothermal conversion in the NIR-II biowindow Alternatively, the tissue penetration efficacy of NIR light would also influence the efficiency of anticancer phototherapies. The biological window of NIR light can be divided into NIR-I (700-1000 nm), and NIR-II (1000-1300 nm) biowindows et al. in vitro, thus playing important roles in anticancer therapies and regenerative medicine. The nanomaterials and the different radiation conditions used in current bone cancer phototherapies are summarized in Table  These nanomaterials show great promise for bone cancer therapy and bone tissue engineering.Meanwhile, nanomaterials can serve as an effective photosensitizer to generate ROS after NIR light irradiation for photodynamic therapy in bone cancer therapy. Pd‑bacteriopheophorbide has been reported as a photosensitizer that induces PDT, leading to hypoxia, necrosis, and eradication of prostate cancer bone metastasis Table Massive cancer-induced bone defects and residual cancer cells are two critical problems in the prognosis of bone cancers. It is essential to fabricate multifunctional scaffolds endowed with the ability to eradicate cancer cells and remodel bone tissue simultaneously during bone cancer therapy 2 nanoparticles-coated Ti6Al4V implant for the reconstruction of large bone defects followed by surgical resection of bone cancers 2 nanoparticle coating endowed the implant with good biocompatibility, strong NIR light absorbance, and efficient photothermal conversion ability under 808 nm laser irradiation. Similarly, magnetic iron oxide nanoparticles, such as Fe3O4 nanoparticles and M-type ferrite nanoparticles (SrFe12O19), could serve as photothermal agents in scaffolds. These scaffolds could exhibit photothermal anticancer therapeutic abilities and enhanced bone regeneration simultaneously 2 nanocrystal, a kind of semiconductor, has been reported as a new class of efficient photothermal agent. By functionalization with the CuFeSe2 nanocrystals, 3D printed BG scaffolds exhibited excellent photothermal performance under 808 nm laser irradiation 2 nanosheets, has also been introduced into BG scaffolds for anticancer treatment and bone tissue regeneration The classical metal nanomaterials have generated great interest because of their excellent photothermal conversion performance in anticancer therapies. It has been reported that bismuth-doped BG scaffolds displayed high photothermal conversion efficiency to induce hyperthermia for osteosarcoma treatment In vitro and in vivo studies of these carbon-based nanomaterial-coated scaffolds have successfully demonstrated their bifunctional properties of photothermal therapy and bone regeneration, which could pave the way for the design and fabrication of novel implants with simultaneous bone cancer killing and bone tissue-remodeling capacity.In addition, carbon-based nanomaterials, such as GO and single-/multi-walled carbon nanotubes, offer great promise for cancer photothermal therapies because of their strong NIR light absorbance, excellent photothermal performance and satisfactory cytocompatibility 3C2 MXene and Nb2C MXene, have unique structural characteristics, including a large specific surface area and adjustable physiochemical properties. They display enriched photothermal and photodynamic therapeutic effects in both the NIR-I and NIR-II biowindows, providing intriguing biomaterial platforms for the diversified treatment of bone cancers 6, a chemical compound of La and B elements, also shows efficient NIR photothermal conversion ability. The LaB6-modified TCP scaffold is an ideal option for both the ablation of bone cancers and the repair of defect regions With the rapid advance of nanotechnology, a series of new nanomaterials, including MXenes and Lanthanide (Ln) nanomaterials, have also been incorporated into bone scaffolds for cancer phototherapies recently. MXene nanosheets, such as 2D Ti1O2) from molecular oxygen after NIR light radiation and mediates the PDT. The highly active 1O2 interacts with the specific molecule in the drug delivery system, leading to a structural change that triggers drug release Controlled drug delivery systems exhibit an enhanced therapeutic efficiency as well as reduced systemic toxicity compared with conventional bone cancer chemotherapies. Photothermal (generation of heat) and photodynamic (production of ROS) mechanisms could be used to break, isomerize, or rearrange molecules and achieve drug release. After reaching the cancer tissues, the drug delivery systems can be triggered by NIR light to realize controlled release of the encapsulated therapeutics et al. Considering that the well-distributed porous structure of mesoporous silica nanoparticles (MSNs) is promising for drug loading and encapsulation, a series of NIR light-absorbing agent-doped MSNs have been designed for photochemotherapy of bone cancer. For example, Lu et al. in vitro, which enhanced the therapeutic anticancer effect. The above anticancer drug carrier systems could be administrated through intravenous injection and are mainly distributed to, and accumulated in, bone cancer tissues, displaying efficient photochemotherapeutic performance under NIR laser irradiation. The radiation parameters of NIR light-induced phototherapies, as well as the encapsulation and release efficiencies of these anticancer drug delivery systems, are summarized in Table In addition, mussel-inspired PDA nanoparticles also possess efficient photothermal conversion and intrinsic drug anchoring abilities Figure 5).In recent years, NIR light-assisted phototherapy has been recognized as a promising strategy, not only for anticancer therapy, but also for antibacterial and anti‑inflammation therapies Staphylococcus aureus at a hyperthermia of 88.8 °C for 15 min when photothermally triggered by NIR light 1O2, can damage cell membranes and cell walls of bacteria and kill the pathogens Table Massive bone defects caused by bone cancers, infections, fractures, and the increasing number of orthopedic diseases have led to a high demand for artificial implants 2/PDA-arginine-glycine-aspartic acid (RGD) decorations of bone implants, displayed synergistic photothermal and photodynamic effects for antibacterial therapy 3PO4 coating on bone implants also exhibited good photothermal effects using BS as photocatalyst 3PO4 also endows the implant with bacteriostatic properties. This NIR light-triggered inorganic hybrid semiconductor heterojunction-decoration system is biocompatible and effective to eliminate biofilms. In addition, red phosphorus has been proven to be an efficient photothermal coating on bone implants because of its good biocompatibility and more effective photothermal ability compared with BP. Its degradation products are mainly phosphate, which plays an important role in bone tissue regeneration in vitro and in vivo, without damaging the normal tissues. At the same time, the RGDC decoration on the surface of the Ti implant enhanced the adhesion, proliferation, and osteogenic differentiation of MSCs. These coating strategies on the surfaces of bone implants have provided great promise for clinical orthopedic applications.For example, mussel-inspired PDA is emerging as an excellent photothermal agent because of its good biocompatibility and efficient NIR light-responsive photothermal conversion. It has been reported that photosensitizer-loaded mesoporous PDA nanoparticles, as well as MoSet al. 2/MoS2/PDA/RGD nanorod array on Ti implants, which could kill bacteria under dual light coirradiation and promoted the osteogenic capabilities of the implants. While the photothermal conversion ability of 660 nm is relatively weak, the yield of ROS under dual light irradiation is almost the sum of individual light irradiation, which enhanced the antibacterial ability of the implants. The physical puncture of the nanorods also contributed to the antibacterial properties of the implants. In addition, a novel photo-sonotherapy strategy has also been designed by fabricating an oxygen deficient S-doped TiO2 coating on Ti implants, which exhibited significant sonodynamic and photothermal abilities Staphylococcus aureus under 15 min NIR light irradiation combined with ultrasound treatment. In addition, besides coating on the surface of bone implants, the NIR light-responsive nanomaterials could also be injected into the knee joint for the clinical treatment of periprosthetic joint infection. Liu et al.Curcuma longa, which has intrinsic antibacterial and anti-inflammatory properties. Compared with traditional therapeutic strategies, this research might offer a new alternative for periprosthetic joint infection.Hang  in vitro and in vivo, representing promising strategies to treat bone infections.In summary, the NIR light-assisted phototherapies have shown stable antibacterial photothermal and photodynamic therapeutic performancesWorldwide, the prevalence of arthritis is high, and includes over 100 types, of which the most common are rheumatoid arthritis, osteoarthritis, and inflammatory arthritis et al. L-cysteine (Cys) assisted Cu7.2S4 nanoparticles to serve as photothermal agents for PTT and photosensitizers for PDT simultaneously. This kind of nanoparticle can be injected into the disease region and has full potential for RA treatment. Meanwhile, a novel therapeutic system that combined NIR light-responsive BP nanosheets with a platelet-rich plasma (PRP)‑modified chitosan thermoresponsive hydrogel has been prepared to treat RA Table Rheumatoid arthritis (RA) is a common chronic inflammatory disease that seriously affects the quality of life of the patients, leading to long standing synovitis, degradation of cartilage and bone tissue, and increased disability. Recently, NIR light‑assisted phototherapy including PTT and PDT, has received increased attention for noninvasive and spatiotemporal controlled RA treatment et al. 2) nanosheets to control the release of dexamethasone for osteoarthritis therapy. This nanosystem could be used to treat osteoarthritis in vivo through intra-articular injection and the NIR light could control dexamethasone release remotely in the disease region, which could be modulated by adjusting the light‑radiation behavior. Similarly, cell membrane penetrating peptide‑modified mesoporous silica nanoparticles (MSNs) were reported to realize the efficient controlled release of triptolide, which is a traditional Chinese herbal medicine used to treat RA Moreover, NIR light-triggered drug delivery systems have been developed for arthritis treatment. As NIR light-triggered drug delivery systems for bone cancer chemotherapies, the photothermal and photodynamic mechanisms of NIR light could also be used to achieve drug release in arthritis treatment. Zhao Figure 6). These applications are summarized in the following sections.Recently, several strategies to accelerate bone tissue regeneration via NIR light‑assisted phototherapies have been proposed. The mild localized heat or photoelectric microenvironment triggered by NIR light irradiation can achieve noninvasive, remote, and spatiotemporally controlled cell differentiation behaviors, providing a unique strategy for bone tissue regeneration in vitro stimulation can achieve precise and orchestrated biological activities in tissue engineering. It represents an appealing strategy to use NIR light radiation as a kind of tissue penetrative stimuli to gain nonpharmacological and noninvasive control of cell differentiation behaviors. For example, Fu et al. et al. reported a kind of NIR light-absorbing carbon nitride (C3N4) sheet, which enhances cellular proliferation and differentiation through runt-related transcription factor 2 (RUNX2) activation. The photocurrent upon two photon excitation of this small sized C3N4 sheet would induce a charge transfer and increased cytosolic Ca2+ accumulation, resulting in enhanced osteogenic differentiation of stem cells and new bone formation. In addition, single‑walled graphene nanoribbons have been reported to generate photoacoustic signals under 905 nm NIR laser radiation, which could serve as an anabolic stimulus for noninvasive bone defect repair in a rodent femoral fracture site Controlling cell fate through noninvasive et al. in situ bone regeneration using porous AuPd alloy nanoparticles (pAuPds) as photothermal agents. The mild localized heat (around 40-43 °C) could greatly enhance cell proliferation and bone tissue regeneration via upregulating the expression of a series of osteogenesis-related genes and proteins. RNA sequencing analysis demonstrated that the Wnt signaling pathway was involved in in situ bone regeneration. Moreover, a new class of 2D BP nanosheets have been employed as photothermal agents for the remote control of bone regeneration By contrast, mild local heat (40-43 °C) has been reported to facilitate healing of bone defects via inducing the earlier differentiation of human MSCs (hMSCs) and promoting the maturation of osteoblasts differentiated from MSCs et al. 2+ delivery system was developed by incorporating SrCl2 and BP nanosheets into poly(lactic-co-glycolic acid) (PLGA) microspheres for bone tissue regeneration 2+ controlled by NIR light significantly improved bone tissue regeneration. These NIR light-triggered ion-release systems are suitable for bone regeneration therapies that require precise control at specific times.Although the use of NIR light-responsive drug delivery systems combining chemotherapy and photothermal therapy has exhibited versatility in bone cancer therapies, NIR light‑responsive release systems for bone tissue regeneration have not been widely used. NIR light has good tissue penetration, as well as accurate and spatiotemporal control properties. Remote control of cell fate could provide a versatile method to regulate bone regeneration therapies. Combining the use of tissue penetrative NIR light stimuli with NIR light-responsive nanomaterials is a delicate and efficient strategy for the intracellular control of stem cells. Recently, several strategies have been proposed to regulate the differentiation of stem cells based on NIR light-responsive release nanosystems. Kang et al. et al. Table Alternatively, the on-demand release of therapeutic proteins holds great promise for bone tissue regeneration. Tuncaboylu Photobiomodulation describes the photochemical reactions of cells and tissues achieved under red or NIR light irradiation with lower energy densities compared with other heat-mediated laser therapies, which induce tissue ablation or coagulation after irradiation in vitro, in which NIR light irradiation induced more in vitro cell viability of ASCs than BMSCs Stem cell therapy has attracted great interest in tissue regenerative medicine. Photobiomodulation therapy might increase the biological functions of MSCs. The primary action site of photobiomodulation under red or NIR light irradiation is the mitochondria, more specifically, cytochrome c oxidase. It is hypothesized that inhibitory nitric oxide dissociated from cytochrome c oxidase could restore electron transport and increase the mitochondrial membrane potential. By contrast, NIR light irradiation could trigger light or heat-gated ion channels and activate the change of transient receptor potential. These potential changes to mitochondria could increases the rate of respiration and ATP production, leading to a marked effect on stem cells via mitochondrial redox signaling et al. In addition, the effects of photobiomodulation therapy within different wavelengths are still being explored. Wavelengths from blue to NIR light have been demonstrated to enhance bone regeneration. Kunimatsu's group have reported that irradiation with a high frequency NIR diode laser is highly efficient to increase cell division and migration of osteoblasts via MAPK/ERK signaling and improved the bone tissue regeneration -2) or osteoblasts . The NIR light triggered anti-adhesive effect of a 4‑(hydroxymethyl)‑3-nitrobenzoic acid modified poly(ethylene glycol) coating on a substrate demonstrated these differentiation properties of MSCs, providing a new way to modulate bone regeneration in tissue engineering.Moreover, guiding the differentiation of MSCs by light is particularly attractive for bone tissue engineering and regenerative medicine. Yan's group constructed a controlled UCNPs-based substrate to guide multi-directional differentiation of MSCs by NIR light irradiation Several studies have demonstrated that NIR light irradiation therapy could be used to improve bone healing and the regeneration process. NIR light therapy at 790 nm was reported to guide bone tissue regeneration on rabbits' tibial fractures with wire osteosynthesis or internal rigid fixation et al. Furthermore, Gokmenoglu -2 was performed on the surface of the patients' cheek for 20 or 30 min every day or 60 min every week. Another study supported this conclusion, providing evidence that low level laser therapy accelerates bone remodeling and inhibits root resorption during orthodontic tooth movement in a rodent model -2) in that study was performed on the labial and palatal sides of the tooth for 15 s every other day. Moreover, Gunji et al. Tooth movement during orthodontic therapy is usually associated with bone resorption on the pressure side and bone regeneration on the tension side et al. Besides the reported capability to enhance tooth movement, NIR light-assisted phototherapy has also been applied to regulate the inflammatory and repair processes of dental trauma. LED phototherapy (940 nm) of the periodontal ligament stimulated the proliferation of fibroblasts and increased the synthesis of the extracellular matrix, accelerating periodontal tissue repair in vitro stimulation for targeted therapy because of its good tissue penetration ability, minimal invasiveness and high selectivity Table  However, the photobiomodulation effect under the direct NIR light irradiation has also shown great potential to regulate cellular behaviors and the process of bone tissue regeneration. To date, a series of in vitro and in vivo studies have demonstrated the encouraging advantages of the NIR light-assisted phototherapies for bone-related disease and bone regeneration. However, it remains challenging to translate preclinical studies into clinical applications.In summary, NIR light can serve as an effective  in vivo safety for further clinical translation in vivo performance because of their reduced toxicity, enhanced colloidal stability, and extended circulation within the body Firstly, the biosafety of NIR light-responsive nanomaterials should be considered. With the rapid development of nanotechnology, several kinds of nanomaterials, such as metal-based nanomaterials, carbon-based nanomaterials, and other related photosensitive nanostructures, have been involved in NIR light-assisted phototherapies to make them “smart”. Apart from their efficient photosensitive properties and attractive biocompatibility, there are still many challenges to overcome before clinical applications. Above all, the components of these nanomaterials should be nontoxic and biodegradable. The acute and long-term toxicity of these nanomaterials depend on their physicochemical properties, including their composition, size, shape, and surface charge. The acute and chronic pathological toxicity during the treatment period of these nanomaterials, as well as their renal or hepatobiliary elimination from the body, should be evaluated to guarantee theirSecondly, NIR light-induced hyperthermia should be well controlled according to different therapeutic needs. The main side effect of NIR light-induced phototherapies for bone-related disease treatment is that local hyperthermia followed by NIR light irradiation can not only eliminate the cancer cells, bacteria, and inflammatory cells, but also might damage the normal cells and tissues when the temperature is above the threshold. The precise regulation of photothermal effect during the NIR light irradiation treatment is essential to promote the efficacy of phototherapies and minimize the damage. Considering the different therapeutic goals in the treatment of bone-related diseases, the parameters of NIR light irradiation to generate targeted hyperthermia within a reasonable temperature range should be carefully selected Furthermore, the administration of NIR light could also be optimized. Although NIR light has deeper tissue penetration than the UV and Visual light, more effort should be made to overcome the laser penetration problem for the treatments of deep‑seated bone cancer, infection, or inflammation. The optimization of excitation conditions and the improvement of nano-bio interactions are also important. Local implantation and injection, rather than intravenous injection, could deliver effective amounts of photosensitive nanomaterials to targeted sites to obtain better therapeutic outcomes. Recently, phototherapy using an optical fiber NIR laser device was combined with surgery, in which the deep pathological lesions could be directly exposed to laser irradiation In conclusion, NIR light-assisted phototherapies have shown great potential to treat bone-related disease and bone tissue regeneration. This noninvasive, precisely targeted treatment strategy could be considered as an effective tool to promote therapeutic efficacy. With better understandings of this technology, NIR light-assisted phototherapy could serve as a promising alternative for clinical applications.


Crimean–Congo hemorrhagic fever (CCHF) is a severe human disease with mortality rates of up to 30%. The disease is widespread in Africa, Asia, the Middle East and Eastern Europe. The last few years have seen disease emergence in Spain for the first time and disease re-emergence in other regions of the world after periods of inactivity. Factors, such as climate change, movement of infected ticks, animals, and changes in human activity, are likely to broaden endemic foci. There are therefore concerns that CCHF might emerge in currently nonendemic regions. The absence of approved vaccines or therapies heightens these concerns; thus Crimean–Congo hemorrhagic fever virus (CCHFV) is listed by the World Health Organization as a priority organism. However, the current sporadic nature of CCHF cases may call for targeted vaccination of risk groups as opposed to mass vaccinations. CCHF vaccine development has accelerated in recent years, partly because of the discovery of CCHF animal models. In this review, we discuss CCHF risk groups who are most likely to benefit from vaccine development, the merits and demerits of available CCHF animal models, and the various approaches which have been explored for CCHF vaccine development. Lastly, we present concluding remarks and research areas which can be further explored to enhance the available CCHFV vaccine data. Orthonairovirus genus of the Nairoviridae family. Crimean–Congo hemorrhagic fever (CCHF) was medically recognized in 1944 in the wake of an outbreak involving military personnel stationed in the Crimean peninsula, and the medical condition was named Crimean hemorrhagic fever (CHF).1 A viral etiology and a tick-borne origin for CHF were proposed after Hyalomma marginatum tick filtrates produced the disease in human volunteers and individuals with psychiatric disorders.2 Following the Crimean peninsula outbreak, numerous epidemics of related disease conditions were described in Central Asia, Bulgaria, and the Soviet Union.1 Meanwhile, Dr. Courtois from the Belgian Congo isolated a virus from a febrile teenage boy using newborn mice in 1956, and the virus was designated Congo virus strain V3011.1 The causative agent of CHF was isolated in 1967 in newborn mice after intracerebral inoculation.4 Characterization studies of agents responsible for global tick-borne diseases, at the Yale Arbovirus Research Unit, established that the agent causing CHF was antigenically similar to Congo virus strain V3011.5 The names were subsequently combined and the virus named CCHFV.1Crimean–Congo hemorrhagic fever virus (CCHFV) is exclusively associated with a virulent disease in humans. In the absence of approved therapeutics or vaccines against the virus, treatment is predominantly supportive. CCHFV possesses a trisegmented negative-sense RNA genome and is classified within the 1 Incubation period depends on the route of transmission; 1–3 days, up to 9 days for tick transmission while after exposure to infected blood or tissues incubation period is mostly 5–6 days and reach up to 13 days.6CCHFV is sustained in an enzootic cycle encompassing ticks and several vertebrate animals with humans regarded as dead-end hosts. Sources of human infections include bite from an infected tick, close contact with blood or tissue from diseased animals, and CCHF patients. Animals do not display symptoms, but disease in humans progresses through four phases: incubation, prehemorrhagic, hemorrhagic, and convalescence.7–16 and countries yet to report CCHF but with evidence of viral circulation either from serological surveys or CCHFV isolation from ticks include Algeria, Benin, Cameroon, Equatorial Guinea, Ethiopia, Ghana, Guinea, Mali, Madagascar, Morocco, Mozambique, Niger, Somalia, Tunisia, and Zimbabwe.17–30 The presence of CCHFV in Somalia was suggested after evidence of the CCHFV in Hyalomma ticks obtained from Somali cattle and sheep exported to the United Arab Emirates.28 In the Middle East, the disease has been described in Iran, Iraq, Saudi Arabia, Oman, and the United Arab Emirates.31–35 East European countries with described CCHF disease include Albania, Bulgaria, Greece, Kosovo, Turkey, Georgia, and Russia.36–42 Portugal, Hungary, France, and Romania43–45 are at risk of CCHF based on serological evidence of viral circulation. Asian countries with described CCHF disease include Afghanistan, Kazakhstan, Pakistan, China, India, Tajikstan, and Uzibekistan.46–49CCHFV is an emerging and re-emerging virus with extensive geographical distribution, as shown in 50 Over 1660 cases were reported by Russian Pro-Med between 2005 and 2017.51 The first human case in Iran was reported in 1999, and an increase in incidence has been observed.53 A nosocomial outbreak of CCHF at a hospital in India in 2011 stands as the principal case in the country49 and from there on, sporadic cases have been reported.54 The first recorded CCHF cases in Spain were described in 2016 from an adult male (an index case) who most likely acquired infection through a tick bite and a nosocomially acquired infection by a health professional who nursed the index case.55 Even though CCHF has not been described in some countries, the presence of CCHFV in ticks and CCHFV-specific antibodies in wild and domesticated ruminants have been demonstrated.56 CCHFV endemic foci are expected to broaden in the face of climate change,57 human case movement as well as the movement of animals infested with a tick.58 However, in some regions, the observed increase in reported cases could be a product of improved awareness and diagnostic capacity. CCHFV poses a zoonotic risk with public health implications and as such CCHF is a notifiable disease. Nonetheless, with the exception of Turkey reporting more than 50 cases per year,59 CCHF cases are currently sporadic in endemic zones.CCHF cases have over the past few years been described in new regions while the disease has re-emerged in some countries after periods of inactivity. CCHF was first described in Turkey in the year 2000, and the country has the highest incidence of CCHF cases per annum with a mortality rate slightly below 5%.Hyalomma ticks, are at risk of acquiring CCHF.60 Development of an efficacious vaccine will prevent infections and possible mortality from the disease in risk groups. CCHF vaccine target population would include farmers dealing with agriculture or animal husbandry. Even though livestock does not develop CCHF, they present with transient viremia. Contact with blood or tissues from infected livestock and bites from infected ticks have been reported as transmitting CCHFV, resulting in outbreaks in farming communities.61 Besides farmers, individuals participating in religious activities such as the Muslim Eid-ul Azha who get exposed to livestock blood and tissue during animal slaughter are at an increased risk of CCHF.62 Veterinarians and abattoir workers stand a high chance of occupational exposure to tick bites or contact with viremic animal blood and thus constitute another risk group. Outdoor activities such as hiking and camping as well as some rural lifestyles where people live in close proximity to livestock pose as risk factors for tick bites and contact with infected animals.64Considering that CCHF cases are sporadic, global mass vaccinations are unlikely, thus targeting vaccinations would be ideal. People residing in endemic areas who are prone to tick bites, especially 65 often with higher case fatality rates compared with tick bite infections.66 Nosocomial outbreaks often serve as an indicator of unrecognized infections in the general population and seroepidemiological surveys carried in the wake of nosocomial outbreaks reveal prior presence of the virus in the community. Human serum CCHFV titers are high (108–1010 copies/mL) during the prehemorrhagic stage, especially in fatal cases67 and the virus has been detected in urine and saliva.68 The high CCHFV titers put medical professionals in contact with patients at risk and the nonspecific symptoms early in the course of disease exacerbates the risk. Laboratory personnel handling live virus comprise another risk group who could be a target for the CCHF vaccine. Laboratory-acquired infections arising from handling patient samples69–71 and during CCHFV research activities72 have been reported. Besides humans, livestock could be vaccinated to prevent viremia in animals and subsequent transmission to humans. Vaccinating livestock also prevents them from serving as amplifying hosts although small mammals are considered principal amplifying hosts.Nosocomial transmission constitutes a major route of CCHFV spread accounting for a large proportion of global CCHF cases,74 Experimental infection of knockout mice with CCHFV displayed some disease signs and physiological changes, which parallel findings in humans, although differences are found in rapidity of disease onset and level of lethality. The first animal models described lacked signaling either to all the three types of interferons (STAT-1−/−)75 or type 1 interferon (IFNAR−/−).76 Besides the knockout mice, a mouse model whose type 1 interferon system is temporarily suppressed IS has been described.77 The knockout models and the (IS) model are permissive to infection, succumbing to CCHFV infection within 5 days. STAT-1 knockout mice present with leukopenia, thrombocytopenia, elevated levels of serum hepatic alanine aminotransferase, and proinflammatory cytokines.75 CCHFV RNA is widespread in tissues of STAT-1−/− and IFNAR−/− mouse models.76 Additionally, a humanized mouse model was prepared by injecting NSG-SGM3 mice with CD34+ human stem cells.78 The humanized model displayed different disease patterns when inoculated with CCHF strain from Oman and Turkey. Lethal outcomes and neurological disease were only observed with the Turkish strain.78 A nonhuman primate model was described in 2018. Cynomolgus macaque infected with the Kosova Hoti CCHFV strain developed disease patterns and outcomes characteristic of CCHF human cases.79 In an independent comparative investigation in Cynomolgus macaque, animals infected with the CCHFV Afghanistan strain (Afg09-2990) and the Kosova Hoti CCHFV strain developed a clinical picture, laboratory clinical chemistry, and hematological parameters, as well as serum cytokine levels commonly seen in humans.80 However, all the 12 animals recovered, unlike the infection studies involving the Kosova Hoti strain, in which only 25% (1/4) of the animals did not meet euthanasia criteria. The nonhuman primate model has provided evidence regarding the ability of CCHFV to replicate and persist in the testes of monkeys, opening the possibility of sexual transmission.81Animal models for CCHF have recently been described. Previously, besides humans, the only other vertebrate known to be susceptible to CCHFV were newborn mice and rats. The immature immune systems of these infant rodents meant these animals could not serve as models. CCHF infections in immunocompetent animals result in transient viremia and absence of noticeable symptoms.82 Remarkably, there were no reported cases from military and laboratory personnel who were vaccinated.82 Nevertheless, the decrease in reported Bulgarian cases could have been independent of vaccine efficacy but a product of other factors. The observed reduction could have been attributed to a change in CCHFV epidemiology and ecology in the absence of deliberate intervention.83 Increased CCHFV awareness could have led to behavior change thus reduced tick exposure or a different case definition and reporting following the introduction of vaccine.83Even though there is not a globally recognized vaccine for CCHFV, there is a vaccine that has been in use in Bulgaria since 1974. The Bulgarian vaccine originated in the Union of Soviet Socialist Republics (USSR). This is an inactivated vaccine prepared from brain tissue of CCHFV infected newborn mice. Inactivation of the CCHFV particles was brought about by a combination of chloroform and heat treatment. The vaccine is administered subcutaneously multiple times in risk groups who are more than 16 years old. Between 1974 and 1996, there was a precipitous drop in cases (from 1105 to 279) reported to the Bulgarian Ministry of Health and fewer than 20 cases reported per annum after 1996.84 and the requirement for high containment facilities for propagation. Furthermore, the Bulgarian vaccine requires multiple immunizations with vaccinations every 5 years to preserve immunity. Individuals below the age of 16 do not qualify to receive the vaccine and leaves a fraction of the population without immunity to the virus. Apart from that, the efficacy of the Bulgarian vaccine is yet to be demonstrated in clinical trials.Although the mouse brain-derived vaccine has been in use in Bulgaria, the vaccine is not a viable option for widespread global use largely due to safety concerns and lack of efficacy trials. Propagating CCHFV in brain tissue of newborn mice requires biosafety level 4 (BSL-4) facilities. International approval of this vaccine is unlikely because of safety concerns surrounding the mouse neural tissue content, which has potential to cause autoimmune and allergic responsesin vitro or in vivo. In vitro proteins were produced in cell cultures, purified, and vaccinated into animal models while in vivo protein production utilized vectors, which were used to deliver genes encoding CCHFV antigens facilitating endogenous protein production. In situ antigen generation is desirable since proteins acquire post-translational modifications similar to natural infections.85Recent, CCHF vaccine development has focused on the viral glycoproteins and the nucleoprotein (NP) even though the immune correlates of protection are yet to be described. The investigated CCHFV proteins are recombinant proteins produced either 87 Besides that, the NP amino acid sequence shows the least variation88 thus, an NP-based vaccine is expected to offer protection against the diverse CCHFV strains. Recently, complete protection in knockout mice against CCHFV challenge infection has been reported after vaccination with NP-based vaccines using different expression systems.89–90 Even though the NP is an internal protein and is not expected to induce neutralizing antibodies, the NP is released from infected cells91 thus can interact with antibodies forming immune complexes capable of antiviral activity.92The role of the CCHFV NP in protection against infection and clearance of viruses is not known. However, the NP possesses features which make the antigen ideal for CCHFV vaccine development. The protein is produced in large amounts during infection and is highly immunogenic containing B and T cell epitopes.M) as well as secreted nonstructural proteins  and mucin-like domain.93–95 The secreted GP38 has been demonstrated to localize to viral and cellular membranes of cells expressing the M segment.96 The glycoproteins Gn and Gc have been largely considered as the antigen of choice for the CCHFV vaccine chiefly because they are located on the surface of virus particles and hence considered responsible for inducing neutralizing antibodies. To this end, monoclonal neutralizing antibodies against the pre-Gn and Gc glycoproteins were described.97 Recently all the neutralizing antibodies previously reported to target the pre-Gn interacted with the GP38 and none interacted with the Gn.96 mAb-13G8, a GP38 specific non-neutralizing monoclonal antibody protected IFNAR−/− mice against lethal infection, whereas Gc-specific neutralizing antibodies could not offer protection96 despite demonstrating in vitro virus neutralization.97 Passive administration of mAb-13G8 (homologous to the IbAr 10200 CCHFV strain) in IFNAR−/− mice displayed limited protection against a heterologous CCHFV Afg09-2990 strain.96 The diversity of the M segment, especially the region encoding the nonstructural proteins, has long been suspected to impact crossreactivity and ultimately, the neutralization ability against heterologous strains. One factor contributing to the diversity of the M segment is genetic reassortment. The consequence of reassortment on viral aspects, such as replication, transmission, virulence, and immunogenicity is yet to be fully investigated.The CCHFV M segment encodes a polyprotein glycoprotein precursor (GPC), which is post-translationally processed to intermediate glycoproteins (pre-Gn and pre-Gc). Further processing of the intermediary glycoproteins yields envelope glycoproteins Gn and Gc, nonstructural M protein , messenger RNA (mRNA) vaccine, and inactivated whole CCHFV particles.Drosophila Schneider 2 (S2) cells and purified. The Gn and Gc proteins formulated with the Sigma Adjuvant system separately elicited antibodies with neutralizing capacity in vitro after intraperitoneal vaccination in STAT129 mice using a prime and boost strategy  from the CCHFV IbAr 10200 strain on their surface was accompanied by a strong induction of in vitro neutralizing antibodies in an IFNAR−/− mice model, which protected 40% of the challenged mice100  system, has been developed. mice100 . Cytokin5 TCID50 of VRP) subcutaneous vaccination in IFNAR−/− mouse model.109 In contrast, a low dose (103 TCID50 of VRP) vaccination protected seven out of nine mice (110 (A virus-like replicon particle (VRP) vaccine candidate based on IbAr 10200 strain but with the GPC sequence from the Oman-1998 strain provided complete protection against lethal challenge following a single high dose  of a DNA vector encoding the mature CCHFV envelope glycoproteins (Gn and Gc) and the NP of the IbAr 10200 strain, elicited antibody and T cell immune responses, which protected IFNAR−/− mice against lethal CCHFV challenge100  or type 2 Th cell responses.pe genes . Fifty plenge100 . Curious−/− and interferon receptor antibody transiently suppressed (IS) mouse models were compared after intramuscular electroporation of a DNA expression vector encoding the entire GPC of the IbAr 10200 CCHFV strain77  compared with the IS model, with 60% , this was not statistically different. Significantly, complete protection was not achieved in both mouse models.77Vaccine immunogenicity and efficacy studies in IFNARstrain77 . Intrape−/− mice twice with a DNA vector encoding the complete NP of the CCHFV Turkey-Kelkit06 or codelivery of the DNA vectors encoding the complete NP of the CCHFV Turkey-Kelkit06 and the cluster differentiation 24 (CD24), protected animals from lethal challenge infection89 , 3′-polyA tail, 3′ and 5′ untranslated regions to enhance stability and translation of the construct. Intramuscular vaccination of IFNARction104 .102 recombinant vesicular stomatitis virus (rVSV),116 recombinant adenovirus type 5 (AdV-5),103 and recombinant bovine herpesvirus type 4 (BoHV-4).90 The MVA platform was used to deliver the GPC and the NP of the IbAr 10200 CCHFV strain. The N-termini of the GPC and NP were fused to the human tissue plasminogen activator leader sequence, whereas a V5 epitope tag was fused to the C-termini. An mH5 promoter was selected to drive gene transcription. The MVA-based vaccine constructs were administered two times intramuscularly ,scularly  and the lenge116 . The replenge103 . A primeriment90 .117 and availability of an animal model (rabbit),118 which makes it a good candidate. A BoHV-4 vector encoding the full-length NP of the CCHFV Turkey-Kelkit06 strain utilizing a prime and boost strategy provided complete protection of IFNAR−/− mice against lethal challenge infection, and partial protection was observed in the antibody passive transfer and T cell adoptive transfer experiment90  was observed with the 20 and 40 μg dose groups in IFNAR−/− mice following lethal challenge infection. The effect of the vaccine on the T cell immune response was not evaluated.The CCHFV Turkey-Kelkit06 strain was propagated in cell culture, harvested, and inactivated by formaldehyde to prepare an inactivated vaccine. The vaccine was mixed with the Imject Alum adjuvant and delivered intraperitoneally using a prime, boost, and boost strategy−/− mice . VaccineCynomolgus macaque CCHF animal models have allowed significant advancements in vaccine development.75–80 The impact of type I and/or type II interferon deficiency on CCHF vaccine-induced adaptive immune responses, however, deserves further evaluation. The disease spectrum in the Cynomolgus macaque depicts disease states seen in humans.79 Despite issues around variability in observed disease outcomes,81 the cost, and size of the animals, this immunocompetent animal model will be valuable in the development of CCHFV therapies. The addition of a humanized mouse model, which previously exhibited strain-specific virulence by producing different disease outcomes by a CCHFV Turkish and an Oman strain78 would be a valuable addition to evaluate the interplay between pathogenicity and immunogenicity.Interferon-deficient mice and the 97 thus vaccine attempts focused on the M segment. Studies have demonstrated the absence of correlation between in vitro neutralization and protection in the available mouse models.100 Future studies should probe and delineate factors responsible for the observed differences between in vitro neutralization and protective efficacy. CCHFV is genetically diverse and the concern is whether a single vaccine can protect against global CCHFV strains. So far, efficacy studies against genetic strains have only been investigated for a VRP vaccine candidate.110 Recently, NP-based vaccine candidates have also resulted in complete protection in knockout mice despite the absence of in vitro neutralizing antibodies.90 The NP has thus proved to be an important vaccine target. Since B and T cell epitopes have been mapped on the glycoproteins and NPs, efforts can be directed in developing multiepitope-based vaccines. Epitope-based vaccines would be one way to develop an effective vaccine against the diverse CCHFV strains by selecting multiple antigenic epitopes. The design of epitope-based vaccines allows B or cytotoxic T lymphocyte (CTL) epitopes to be linked together in series with helper T lymphocyte epitopes ensuring CD4 T cells and pathogen-derived molecules are appropriately primed facilitating robust humoral and CTL responses.119 One characteristic of an ideal vaccine is that it should confer long-term sterilizing immunity after single administration. Despite several vaccine strategies providing complete protection of knockout mice after viral challenge -2, IL-12, Granulocyte/macrophage colony-stimulating factor,128 and its virulence in humans is unknown. The NP from IbAr 10200 and AP92 strains did not antagonize interferon response in vitro as did the Hoti strain.129 In a study by Zivcec and colleagues,130 VLP-bearing glycoproteins from the IbAr 10200 CCHF strain displayed the reduced capacity to enter monocyte-derived macrophages. The effect in stimulating immune responses although remains to be elucidated. Considering the diversity of the CCHFV glycoproteins, it will be interesting to evaluate if differences in amino acid sequences between various global CCHFV strains does affect immunogenicity.The prototype IbAr 10200 CCHFV strain has mostly been used in vaccine studies. This prototype CCHFV strain was discovered in a tick90 BoHV-4 persists in monocytes and macrophages. Persistence in white blood cells could eliminate the need for booster doses for antigens delivered by BoHV-4. Besides that, delivering antigens by the BoHV-4 vector can enhance antigen presentation since the virus persists in monocytes and macrophages, which are antigen-presenting cells.90 Even though similar protection rates in knockout mice were obtained with vaccine constructs delivered with the BoHV-4 and AdV-5 vectors, the advantages offered by the BoHV-4 vector needs to be further explored in detail. The role played by the various expression systems in shaping CCHFV immune responses in animal models warrant investigation.The utility of a BoHV-4 viral vector in comparison to an AdV-5 and a DNA vector in NP-based vaccine development was evaluated.Since animals, particularly livestock, play an important role in CCHFV transmission cycle, the ability of vaccines to prevent viremia in livestock could reduce the rate of transmission to humans. CCHF human vaccine development has accelerated in recent years, and some vaccine studies have reported promising results in animal models. Whether these results can be translated to human clinical trials remains to be seen. Vaccine design and efficacy can be further enhanced by the delineation of correlates of CCHF protection which up to now have remained an enigma.