Delivery of bioactive factors is a very valuable strategy for articular cartilage repair. Nevertheless, the direct supply of such biomolecules is limited by several factors including rapid degradation, the need for supraphysiological doses, the occurrence of immune and inflammatory responses, and the possibility of dissemination to nontarget sites that may impair their therapeutic action and raise undesired effects. The use of controlled delivery systems has the potential of overcoming these hurdles by promoting the temporal and spatial presentation of such factors in a defined target. Hydrogels are promising materials to develop delivery systems for cartilage repair as they can be easily loaded with bioactive molecules controlling their release only where required. This review exposes the most recent technologies on the design of hydrogels as controlled delivery platforms of bioactive molecules for cartilage repair. Articular cartilage is an avascular tissue that lines the weight-bearing surface of joints, formed of organized populations of chondrocytes surrounded by their extracellular matrix (ECM)  and regulated by a metabolic balance that involves diverse signaling molecules, growth factors, and cytokines . Due to Tissue engineering and regenerative medicine approaches based on the design of biomaterials scaffolds reflecting the properties of the native cartilage may provide potent alternatives to restore a healthy and fully functional articular cartilage. Thus far, advances in biomedical devices for controlled drug delivery platforms support a new generation of products to treat such disorders where a temporal control over the pharmacokinetic profiles is required . Hence, The goal of the present work is to provide an overview of the most recent advances in the preparation of polymeric hydrogel networks as controlled delivery systems of the most relevant bioactive molecules involved in articular cartilage repair.Hydrogels are polymeric networks consisting of cross-linked hydrophilic polymers able to swell and retain a significant fraction of water within their structure but will not dissolve in water . The abiHydrogels can be classified into physical and chemical systems based on their cross-linking mechanism , 16. WhiThe use of hydrogels as controlled delivery systems of bioactive molecules in strategies of cartilage repair aims at reproducing the complex microenvironment that naturally occurs in articular cartilage in an artificial setting . ControlControlled delivery systems have been conceived to modify certain parameters of the biomolecule to be delivered including its release profile, ability to cross biological barriers, biodistribution, clearance, and stability (metabolism) . Hence, Another key parameter when designing a hydrogel-based controlled delivery system for cartilage repair is the mechanism of loading of the bioactive substance into the hydrogel network, having a strong influence on its release profile and subsequently on its therapeutic action. Incorporation of bioactive substances within hydrogel networks can be performed by physical encapsulation, physical or chemical immobilization of the biomolecules to the polymeric network, and electrostatic interaction . PhysicaAn overview of the most relevant strategies used for the fabrication of hydrogels for controlled release of bioactive substances for cartilage repair is presented in the following section .Hydrogel-based delivery systems have been used for the controlled release of bioactive factors, having a pivotal role in cartilage regeneration processes such as growth factors  and geneβ) superfamily [Growth factors are polypeptides involved in the cellular communication system, capable of transmitting signals that modulate cellular activity by either stimulating or inhibiting cellular proliferation, migration, differentiation, and/or gene expression , 51. Groerfamily  or bone erfamily , basic ferfamily , and inserfamily .Controlled delivery of growth factors from hydrogels may occur over an extended time, reducing the need for additional applications of the protein. Moreover, strictly localized release of growth factors may confine their activity to a distinct location in the proximity of the defect site reducing potential side effects . Deliverβ1 [ in vitro to target MSCs [ in vivo [β1 [Natural polymers such as alginate , 19, fibβ1  both in get MSCs  or in an in vivo , 27, sho in vivo . This stvivo [β1  or BMP-2vivo [β1  in fibrivivo [β1  and HA [vivo [β1  hydrogelβ1. Controlled release of TGF-β1 from the constructs increased cellularity with maintenance of the cell phenotype [Synthetic polymers such as PEG –40, 42, henotype .β1 and IGF-I incorporated into biocompatible hydrogels based on OPF with gelatin microparticles. When delivered to osteochondral defects in rabbits, the best histological result was observed after 3 months in vivo with IGF-I-treated defects, while these benefits were not maintained when codelivered with TGF-β1 or when TGF-β1 was delivered alone, suggesting that in this in vivo model IGF-I was superior to TGF-β1 [Most advanced strategies focused on the dual release of growth factors to achieve a synergistic effect on the enhancement of chondrogenic differentiation and on the maintenance of their phenotype. Holland et al.  investigo TGF-β1 . In the o TGF-β1 .Gene transfer via nonviral vectors (transfection) is based on the incorporation of DNA, either naked or complexed with cationic polymers or with cationic lipids (in polyplexes and lipoplexes), into the target population. Though considered as a safe method as it avoids the risk of acquiring replication competence and of insertional mutagenesis without inducing immune responses in the host, its use is limited by the low transfection efficiencies (40–50% maximum) and short-term transgene expression levels achieved –65. Whil in vivo [Local gene delivery via hydrogel scaffolds has been studied through the encapsulation of naked DNA during hydrogel formation , 67 usin in vivo , its low in vivo . Gene de in vivo , 70–75 o in vivo , 69 has  in vivo , HA [70, in vivo –75, and  in vivo , 70, 71  in vivo , 75 in aAn important limitation of the incorporation of nonviral gene transfer vectors into hydrogels is the failure of loading high DNA concentrations because of their tendency to aggregate. Incorporation of nonviral vectors into hydrogel networks often results in their aggregation as a result of the soft, loose, and charged structures of polyplexes and lipoplexes , 73. To The use of hydrogels as controlled gene delivery systems for cartilage repair is a very valuable but still developing strategy. Osteochondral and chondral units are especially promising tissues for polymeric gene delivery approaches because of the limited blood flow to the region which could cause problems in DNA polymer complex delivery and the potential for the delivered genes to induce differentiation of infiltrated MSCs . NeedhamGene transfer using viral vectors (transduction) is based on the natural cellular entry pathways of viruses from which they are derived, resulting in higher gene transfer efficiencies compared with nonviral vectors 80–90%) , 77, 78.% , 77, 7Controlled release of viral vectors from hydrogels allowing for a release pattern via a diffusion process may help overcome these issues. A summary of the main biomaterials used to produce hydrogels for controlled release of viral vectors in different tissue engineering approaches is shown in β from diluted fibrin glue hydrogels resulted in enhanced production of TGF-β and higher levels of cartilage-specific gene expression in human MSCs (hMSCs) compared with undiluted hydrogels. This fact was attributed to the more open network structure from diluted fibrin glue hydrogels compared with undiluted ones resulting in the most efficient release of rAAV TGF-β vectors [Adenoviral, lentiviral, and rAAV vectors have been encapsulated in fibrin hydrogels, taking advantage of the low immunogenicity and biodegradability of this compound and showing sustained release profiles of the vectors and expression of the transgenes of interest in different cell targets , 93–95.  vectors .Self-assembling peptides RAD16-I in a pure (RAD) form or combined with HA (RAD-HA) have been also employed to release rAAV vectors as a means of genetically modifying hMSCs . Such sy sox9 sequence leading to enhanced chondrocyte differentiation of the cells [PEO- and PPO-based “smart” or “intelligent” self-assembling, temperature-sensitive copolymers have been also utilized as efficient rAAV-mediated delivery systems due to their capacity to form polymeric micelles and to undergo sol-to-gel transition upon heating , 97. Spehe cells . Furtherhe cells . Strikinhe cells .The use of hydrogels as controlled delivery systems of bioactive molecules is a valuable strategy to achieve appropriate levels of a therapeutic factor into target places, circumventing possible limitations associated with their direct administration . Hydrogel delivery platforms can be modulated by tuning some specific parameters from the hydrogel networks as composition, cross-linking density pore size, and degradation kinetics. Hydrogel networks are promising systems for cartilage tissue engineering approaches as they exhibit many intrinsic features mimicking the ECM from cartilage showing in many studies biocompatibility with key cell populations involved in cartilage regenerative processes as chondrocytes and MSCs. in vitro might not generate similar/sufficient or adapted effects in a native environment in vivo. It will thus be fundamental to test the adaptability of the hydrogel systems in sites of tissue damage using clinically relevant, complex orthotopic animal models of cartilage defect as a means of enhancing the natural repair processes. Yet, despite remaining challenges, recent advances in the development of hydrogels as controlled release delivery systems of cartilage reparative factors are promising, new avenues of research that may clearly improve cartilage repair in patients in a close future.Although promising technological advances have been reported to produce hydrogels as delivery systems of cartilage reparative factors, large efforts are still necessary to obtain adapted hydrogel-based delivery systems that may lead to successful clinical translation in patients integrating conditions of efficacy, durability, and safety. In this sense, many relevant parameters from the hydrogel platforms such as encapsulation efficiency, interaction with and stability of the biomolecule cargo, tensile strength, resistance against dilution, gene transfer efficiency, and biocompatibility need to be accurately optimized. It will be also very important to keep in mind that a successful system


This draws on knowledge built up over several decades of remote and in situ observation, as well as recent discoveries that have shaped current understanding of early Mars. Our current understanding is that certain early martian environments fulfill several of the key requirements for microbes with NDFO metabolism. First, abundant Fe2+ has been identified on Mars and provides evidence of an accessible electron donor; evidence of anoxia suggests that abiotic Fe2+ oxidation by molecular oxygen would not have interfered and competed with microbial iron metabolism in these environments. Second, nitrate, which can be used by some iron oxidizing microorganisms as an electron acceptor, has also been confirmed in modern aeolian and ancient sediment deposits on Mars. In addition to redox substrates, reservoirs of both organic and inorganic carbon are available for biosynthesis, and geochemical evidence suggests that lacustrine systems during the hydrologically active Noachian period (4.1–3.7 Ga) match the circumneutral pH requirements of nitrate-dependent iron-oxidizing microorganisms. As well as potentially acting as a primary producer in early martian lakes and fluvial systems, the light-independent nature of NDFO suggests that such microbes could have persisted in sub-surface aquifers long after the desiccation of the surface, provided that adequate carbon and nitrates sources were prevalent. Traces of NDFO microorganisms may be preserved in the rock record by biomineralization and cellular encrustation in zones of high Fe2+ concentrations. These processes could produce morphological biosignatures, preserve distinctive Fe-isotope variation patterns, and enhance preservation of biological organic compounds. Such biosignatures could be detectable by future missions to Mars with appropriate instrumentation.This work considers the hypothetical viability of microbial nitrate-dependent Fe Mars, the red planet, has inspired the search for extraterrestrial life since the early days of the telescope, and continues to do so with perceptions of its habitability—or even inhabitation—changing with advances in exploration capabilities and knowledge of martian environments from images and data . The preFor a better understanding of the contrast between the detrimental conditions on the surface of present-day Mars and the wetter, more clement past of martian surface environments, two specific potentially habitable environments are discussed here: (1) the ancient lake bed investigated by the Curiosity rover at Gale Crater  and (2) The ancient lake bed at Gale Crater is likely to be one of many that formed within impact craters on Mars . Conglom2+ precipitates to Fe3+ precipitates in the course of the formation of the assemblage  demonstrate the diversity of potentially habitable environments (as we understand them today) on ancient Mars. In early surface environments, where the conditions were less inhospitable than the present-day, both phototrophic (solar energy-driven) and chemotrophic  primary producers may have been viable, possibly producing enough organic carbon for the subsequent development of heterotrophy and a complex web of microbial life. As the environment evolved from “warm and wet” to “cold and dry,” life would have likely become limited to the sub-surface environment , protectLaboratory-based Mars simulation experiments, using analog regolith or brine, and theoretical modeling have suggested that chemolithotrophic life could persist in the sub-surface martian environment across a wide range of pH, salinity, desiccation, and temperature .Chemolithotrophic microorganisms harvest energy from redox reactions using inorganic substrates that are available in the environment. This metabolic strategy involves the transfer of electrons donated by the inorganic substrate, through the electron transport chain for ATP production, to a final acceptor. Chemolithotrophy is pivotal for biogeochemical cycling on Earth, such as iron, nitrogen, and sulfur cycling, and for rock weathering .2+, exists as little as a few centimeters beneath the surface 2SiO4] have been detected across wide areas of the martian surface  are contained within martian crustal rocks .The iron-rich nature of Mars raises possibilities regarding the feasibility of iron biogeochemical cycling. Martian crustal geology is dominated by rocks of basaltic composition, which contain abundant FeO in quantities roughly twice those observed in comparable basalts on Earth , 2009. T surface . Indeed, surface  and largal rocks . An acti2+ directly after its dissolution from minerals such as olivine  was first proposed by A hypothetical ‘loop’ of biologically mediated martian iron cycling  to dominate , which are able to compete with abiotic oxidation at near neutral pH. On Earth, this form of metabolism is largely restricted to oxic–anoxic boundary zones, where chemical oxidation is much slower . PhylogeOn Mars, regions of higher partial pressure of oxygen in the modern sub-surface, relative to the surface, have been proposed as tolerable for microaerophiles today . King 2; howeverAnaerobic chemotrophic iron oxidation is known to occur in terrestrial anoxic waters and sediments of approximately circumneutral pH . Data frIn the absence of molecular oxygen, chemolithotrophic iron oxidizers would be limited by the availability of alternative electron acceptors, such as perchlorates and nitrates, for metabolic redox reactions .2+ oxidation when perchlorate or nitrate is provided as an electron acceptor .Nitrate is thus a more feasible electron acceptor for martian iron oxidation, having been observed as the oxidant in iron-oxidizing metabolisms of growth-phase cultures . Howeverin situ analysis of mudstone at Gale Crater by Curiosity .The geochemical evidence of nitrates on the surface of Mars comes from uriosity  and fromuriosity . It has uriosity  of the luriosity , volcaniuriosity , and mayuriosity  .Although it is not believed that nitrate deposition currently operates on the martian surface , interes2+ oxidation metabolism was identified on Earth two decades ago , but this is likely to be an under-representation of the true diversity and prevalence of these organisms  and abiotic side reactions between Fe2+ and reactive nitrogen species  a potential energy gain from coupled iron oxidation and nitrate reduction and (b) energy consumption to overcome the toxicity of Fe2+ and reactive nitrogen species  , this reotrophic  or autototrophic . At the otrophic  and may 2+-oxidizing phototrophy, in iron cycling and the production of early banded iron formations prior to the full oxygenation of the atmosphere on Earth , since it could provide a ready source of electron acceptors for NDFO organisms ; the concentration of nitrates detected at Gale Crater . Microbes that can utilize inorganic atmospheric carbon would therefore hold an advantage in the Mars environment. Although a low energy-yielding metabolism, a some species (Pseudogulbenkiania sp. strain 2002 and the hyperthermophilic archaeon Ferroglobus placidus) have been found to fix carbon autotrophically from CO2 and other inorganic sources during growth by NDFO . 5 cells g-1 sediment if present as biologically available organics in the earlier lacustrine environment, and hence could well have sustained heterotrophic NDFOs.Most NDFOs are heterotrophic and require an organic carbon source . Organic2 at levels that contributed to periodically reducing conditions  conditions, a major limiting factor for the growth of NDFO populations is the progressive encrustation of the periplasm and outer membrane by insoluble Fe3+ compounds , resulting in a decline in individual metabolic activity and cell death . Although the mechanisms remain unexplained, various extracellular Fe3+ mineral precipitates also form as by-products of NDFO metabolism, either due to the interaction of released Fe3+ ions with dissolved phosphate, sulfate, and carbonate ions, or by oxidation of extracellular Fe2+-bearing minerals ] at pH 7 (3O4) at pH 7.6 (3+ phosphates (2+/Fe3+ hydroxides)  . The nat at pH 7  or a mixt pH 7.6 . Likewisosphates , goethitosphates , or greeroxides) .2+ concentrations (50–250 μM), i.e., conditions more representative of many terrestrial NDFO sample sites . If deposited and lithified as macroscopic flocs or bands within an otherwise generally reducing sedimentary geological context, these oxidized mineral features may be visible in exposed strata and would serve as prime initial targets for further astrobiological investigation. Alternative mineralization processes such as pyritization  or silicification  could also contribute to non-specific morphological preservation of microbes in iron and sulfur-rich, predominantly basaltic, early martian environments. Microbial silicification has been observed on Earth in situ and in vivo around hot springs and under simulated conditions as well as in the fossil record  conditions, Fe minerals may promote the thermal maturation of organic matter and partly erase organic biosignatures  microorganisms oxidase Fe2+ compounds while also reducing nitrates under anaerobic, circumneutral conditions. These environments are proposed to have existed on Mars, providing the electron donors and acceptors required for NDFO metabolism. This implies that NDFO is a feasible and logical avenue for investigating hypothetical early martian life.Nitrate-dependent FeThe discovery of nitrates establishes NDFO as a viable mechanism for hypothetical, biological iron oxidation on present-day Mars. NDFO could help to close a chemotrophic ‘loop’ of biogeochemical iron cycling on Mars, by providing a potential mechanism for iron oxidation, and allowing chemotrophic iron cycling to occur in both circumneutral ancient surface waters and deep sub-surface waters throughout martian history.To test the validity of this hypothesis, further research should seek to determine the feasibility of NDFO metabolism under Mars simulation conditions and characterize any associated biomineralization processes. Should the suitability of NDFO to martian environments be supported by the outcomes of these experiments, future life detection missions could be optimized to seek the distinctive mineralized biosignatures of NDFO in the martian rock record.AP was responsible for writing the manuscript with a large amount of input and revision from KO-F, VP, and SS. JM contributed to the revision, providing experience and expertise in nitrate-dependent iron oxidation and biomineralization processes. The concept for the paper was developed in discussions between AP and KO-F with VP and SS involved from the beginning.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.


Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease that is clinically characterized by progressive muscle weakness and impaired voluntary movement due to the loss of motor neurons in the brain, brain stem and spinal cord. To date, no effective treatment is available. Ample evidence suggests that impaired RNA homeostasis and abnormal energy status are two major pathogenesis pathways in ALS. In the present review article, we focus on recent studies that report molecular insights of both pathways, and discuss the possibility that energy dysfunction might negatively regulate RNA homeostasis via the impairment of cytoplasmic-nuclear shuttling in motor neurons and subsequently contribute to the development of ALS. Amyotrophic lateral sclerosis (ALS) is a rare motor neuron disease that occurs in adults with a prevalence of ~5 per 100,000. Patients with ALS develop muscle weakness and impaired voluntary movement, largely because of degeneration of the upper and lower motor neurons , probably as a result of higher energy consumption and lower food intake  impairs mitochondrial function by disrupting the respiratory transport chain machinery  has been found to improve motor function in mSOD1-expressing or TDP-43 mutant-expressing worms  encode RNA-binding proteins that have been found to affect multiple levels of RNA processing, including mRNA transcription, RNA transport, mRNA stabilization and miRNA biogenesis  G-quadruplexes, thus suggesting the importance of the hexanucleotide repeat expansion  involved in nucleocytoplasmic transport  in a Expression of the C9ORF72 repeat expansion and the resultant DPRs is associated with abnormal mitophagy and impaired mitochondrial function (Lopez-Gonzalez et al., As noted by several recent studies and reviews, the loss of nuclear functions of many RNA-binding proteins and nuclear transport components and the gain of function and/or aggregate formation of these proteins in the cytoplasm may contribute to ALS pathogenesis (Zhang et al., Importins are key molecules in the control of nucleocytoplasmic transport because they are needed for transporting macromolecules larger than 40 kDa (such as proteins; Görlich and Kutay, Multiple lines of evidence suggest the involvement of importins in ALS. First, we have previously demonstrated that AMPK activity is higher in motor neurons of the ALS spinal cord when compared with that of non-ALS subjects (Liu et al., This alteration in the function of importin-α1 induced by AMPK phosphorylation is likely to be a common pathogenic pathway that may contribute, at least partially, to the RNA toxicity observed in ALS. In accordance with the importance of importins in ALS, up-regulation of importin α has been found to be beneficial in a fly model of ALS that exogenously expresses the C9ORF72 repeat expansion (Zhang et al., Proper RNA transport is critical for cellular RNA homeostasis. In highly polarized cells, such as motor neurons, mRNA can be transported to neurites. Motor neurons are known to have local protein synthesis in their synapses after exposure to stimuli (Yoo et al., As described above, many ALS genes encode RNA-binding proteins (RBPs, Figure C9ORF72, P-YT wrote the first draft of section Importins and AMPK in Nucleocytoplasmic Transport and RNA Transport, YC organized the manuscript and wrote the final draft.Y-JL wrote the first draft from section Abnormal Energy Homeostasis in ALS to section 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.


Cerebrovascular lesions are a frequent finding in the elderly population. However, the impact of these lesions on cognitive performance, the prevalence of vascular dementia, and the pathophysiology behind characteristic in vivo imaging findings are subject to controversy. Moreover, there are no standardised criteria for the neuropathological assessment of cerebrovascular disease or its related lesions in human post-mortem brains, and conventional histological techniques may indeed be insufficient to fully reflect the consequences of cerebrovascular disease.Here, we review and discuss both the neuropathological and in vivo imaging characteristics of cerebrovascular disease, prevalence rates of vascular dementia, and clinico-pathological correlations. We also discuss the frequent comorbidity of cerebrovascular pathology and Alzheimer’s disease pathology, as well as the difficult and controversial issue of clinically differentiating between Alzheimer’s disease, vascular dementia and mixed Alzheimer’s disease/vascular dementia. Finally, we consider additional novel approaches to complement and enhance current post-mortem assessment of cerebral human tissue.Elucidation of the pathophysiology of cerebrovascular disease, clarification of characteristic findings of in vivo imaging and knowledge about the impact of combined pathologies are needed to improve the diagnostic accuracy of clinical diagnoses. Cerebrovascular disease (CVD) is highly prevalent in brains of the elderly. However, its impact on cognition is less clear and while prevalence rates of vascular dementia (VaD) are high in clinical studies CVD is rarely found to be the neuropathological correlate of clinical dementia in post-mortem studies. In this review we highlight some of the current problems in the diagnosis of CVD and present novel approaches that may prove helpful to elucidate the impact of CVD on cognitive performance.th International Congress of Vascular Dementia by participants of the Neuropathology symposium following a discussion on current problems regarding the clinical and pathological diagnosis of VaD and CVD.This article was conceived at the 9Three diseases of cerebral blood vessels mainly contribute to vascular cognitive impairment (VCI) and/or VaD: 1) atherosclerosis (AS), 2) small vessel disease (SVD) and (3) cerebral amyloid angiopathy (CAA). AS is a degenerative vessel disorder affecting large to medium sized cerebral arteries, most commonly the basilar artery and the circle of Willis [ atherosc small veSVD encompasses three degenerative alterations of the vessel walls of smaller cerebral arteries and arterioles. The first, SVD-AS, has a similar pathogenesis to large vessel AS but affects small intracerebral and leptomeningeal arteries (200–800 μm in diameter), which develop microatheromas. The second, lipohyalinosis, affects smaller arteries and arterioles (40–300 μm in diameter) and is characterised by asymmetric fibrosis/hyalinosis associated with cholesterol-laden macrophage infiltration that can occur with or without plasma protein leakage as a result of blood–brain barrier (BBB) breakdown. The third, arteriolosclerosis, presents as concentric hyaline thickening of small arterioles (40–150 μm) that may lead to stenosis of the blood vessel [CAA is characterised by the deposition of amyloid-beta (Aβ) (predominately Aβ-40) in the vessel walls of leptomeningeal and cortical arteries, arterioles, capillaries and, rarely, veins . This re3) are frequently the result of thrombotic (AS) or thromboembolic  occlusion of the vessel lumen [3), are largely confined to the white matter and subcortical grey matter, and they are therefore primarily associated with SVD [AS, SVD and CAA can all lead to various cerebrovascular lesions (CVLs), including infarcts, haemorrhages and white matter lesions (WMLs). Ischaemic infarcts are typically observed after thrombotic or thromboembolic occlusion of large to medium arteries, often as the result of an AS plaque rupture. Haemorrhagic infarcts can occur in infarcted regions in which the remaining vessels have fragile vessel walls as a result of SVD or CAA, or they may be caused by venous obstruction; less commonly, haemorrhagic infarcts in the brain can be caused by collateral blood influx into an infarcted area . Large iel lumen . Lacunarwith SVD . Microinwith SVD , whereaswith SVD . WMLs enwith SVD –14, althCVLs can result in ‘pure’ VaD, that is, extensive vascular lesions, without widespread neurodegenerative pathology such as Alzheimer’s disease (AD) or Lewy body pathology, which explains the clinical dementia. VaD can be classified into three major forms depending on lesion distribution: multi-infarct dementia, strategic infarct dementia or subcortical vascular encephalopathy. Multi-infarct dementia is characterised by multiple lacunar infarcts and microinfarcts, as well as small and/or large infarcts in the cortex and subcortical regions. The total amount of damaged cerebral tissue results in a significant decrease in functional brain capacity, surpassing the threshold for cognitive impairment. In contrast, strategic infarct dementia is the result of a single infarct in a strategic region of the brain that results in significant cognitive deficits, for example, a single lacunar or microinfarct in the hippocampus can lead to marked memory impairment , 16. LasA large proportion of patients with dementia who have significant CVLs also exhibit more severe concomitant AD pathology , such asIn clinical population-based series, the prevalence of VaD/VCI averages 8–15.8 %  with standardised incidence rates between 0.42 and 2.68 per 1000/year, increasing with age . VaD in At present there are two fundamental issues regarding the assessment and diagnosis of VaD. First, there are no currently accepted neuropathological consensus criteria regarding the assessment of VaD, VCI, cerebrovascular pathology or related lesions . NeuropaVarious forms of cerebrovascular disorders may lead to cognitive impairment and dementia in the elderly . While pConverging evidence suggests that cerebrovascular and AD pathology exert an additive (and/or synergistic) effect on cognitive impairment. Does CVD merely reduce the cognitive threshold needed for overt clinical dementia in AD, or do both factors potentiate AD-specific pathophysiological pathways? Recent neuroimaging studies in cognitively normal elderly people aged 70–90 years suggested that vascular and amyloid pathologies are at least partly independent predictors of cognitive decline in the elderly, and that cognitive reserve seems to offset the deterioration effect of both pathologies on the cognitive trajectories .Concomitant CVLs increase the risk and severity of clinical dementia in elderly individuals meeting the neuropathological criteria for AD –55. HoweIn contrast to AD, less is known about the impact of CVD in other common neurodegenerative diseases, such as dementia with Lewy bodies (DLB) and frontotemporal lobar degeneration (FTLD). Prevalence reports of CVD in DLB are scarce, but autopsy studies reported a frequency of 20.2–34.4 % , 70, whiIn conclusion, the co-occurrence of CVD and AD in the elderly is very frequent . There iWMLs histologically encompass structural damage of the cerebral white matter as a result of white matter rarefaction . They arHPτ has been implicated as a principle instigator of degenerative axonal loss in AD. An extensive quantitative neuropathological study revealed that the burden of cortical HPτ in the temporal and parietal lobes was a predictor of WMH severity in AD , corroboWhile theoretically both cortical HPτ pathology and SVD may lead to the development of WMH, it appears that in neurodegenerative diseases such as AD, WMHs are likely to be primarily associated with cortical HPτ pathology. On the other hand, in cases without dementia and in VaD cases, SVD seems to play a role in the development of WMH, which may relate to gliovascular abnormalities and BBB damage . The claThe term cerebral microbleeds describes the radiological phenomenon of small, well-demarcated, hypointense, round or ovoid lesions detected on T2*-weighted gradient-recalled echo (T2*-GRE) and susceptibility-weighted imaging (SWI) MRI sequences . MicroblSimilar to other SVD markers, microbleeds appear to represent a potential link between stroke, brain ageing, dementia and AD , 109, buAlthough most microbleed pathological correlation studies emphasise blood leakage from nearby damaged small vessels into the brain parenchyma as a mechanism, it must not be assumed that a primary haemorrhagic process fundamentally produces all microbleeds or that the most severely affected vessels are the culprits. Alternative non-haemorrhagic mechanisms for microbleeds, particularly if no tissue damage surrounds the vessel and haemosiderin is limited to the perivascular space, include ischaemia-mediated iron store release by oligodendrocytes , phagocyIt is widely accepted that, by analogy with spontaneous intracerebral haemorrhage, the pathological processes underlying microbleeds differ according to their location in the brain, with CAA being the most notable correlate of exclusively lobar microbleeds , while ‘hypertensive arteriopathy’  is strongly associated with predominantly deep microbleeds. The majority of data to date support this hypothesis, but much of the evidence is indirect and largely based on clinical and imaging studies , 126–130With regards to CVL, novel applications of neuroimaging and biochemical methods, as well as additional investigation of neuroinflammation, have been suggested for the assessment of human post-mortem brains. Although these methods are beyond the scope of basic routine diagnostic procedures, the addition of such novel techniques may help to further elucidate the impact of CVD on cognitive performance.Post-mortem MRI provides a technique to complement research, and routine, neuropathological investigations, providing visualisation of cerebral lesions for radiological assessment or a precise location for histological examination. Direct comparison studies have found that gross MRI lesions are almost identical between human in vivo and post-mortem MRI scans , with liFrequently, post-mortem MRI is used for the detection and assessment of WMH. A recent study investigated the reliability of post-mortem MRI to assess WMH of the deep white matter: 4.7 T MRI scanning was carried out on 40 post-mortem fixed right brain hemispheres, and WMHs in the deep white matter were rated according to the Age-Related White Matter Change Scale (ARWMC)  and compCortical microinfarcts (CMI) are another common lesion found in ageing and dementia, and are considered the ‘invisible lesions’ in clinical–radiological correlation studies , visiblePost-mortem MRI has also proved a valuable tool in investigating the pathomechanisms of ischaemic stroke in the human brain. This is of major potential importance because many therapeutic interventions that have proven successful in animal stroke models have not yet been verified in human clinical trials (excluding thrombolysis and hypothermia). Developments in autoradiography of intact human brain sections have allowed for the visualisation of the ischaemic core by creating a ‘potassium map’; a method which identifies the ischaemic core by utilising the disruption of ion homeostasis and subsequent efflux of water. This method allows for the essential targeted tissue sampling of the ischaemic core to facilitate quantitative measurements of tissue components. The method for human brain sections, as described by Csiba and colleagues , is reliWhile clinical, neuroimaging and pathological assessment remain the main approaches for assessing vascular lesions and their association with cognitive impairment and other neurological disturbances, post-mortem biochemistry provides additional insights into vascular function  BiochemiAdvantages of including biochemical measurements  include the fact that they are more sensitive for the detection of hypoperfusion, they facilitate more representative sampling (e.g. up to 0.5 ml of tissue in a single homogenate compared with ~5 μl of tissue in a paraffin section) and they yield objective continuous data rather than subjective ordinal scores. Biochemical approaches were recently used to gain some understanding of the pathogenesis of cerebral hypoperfusion in VaD, AD and DLB. Measurement of the levels of myelin proteins with long half-lives but differential susceptibility to hypoperfusion confirmed a significant reduction in the perfusion of the cerebral cortex and white matter in VaD , 150. ThIn the cerebral white matter, the main abnormality associated with hypoperfusion in both VaD and AD has been demonstrated to be non-amyloid SVD . The con∼ 106-fold greater volume of tissue than a typical paraffin section) and the same homogenate can be used to measure a wide range of related molecules, allowing direct comparison between microvessel density and perfusion, vascular function, and molecules responsible for regulation of vascular growth, tone and permeability. This approach was used to assess possible causes of occipital hypoperfusion in DLB and demonstrated significant reduction in the level of VWF in the occipital cortex (a region known to be hypoperfused in DLB) but not the midfrontal cortex or thalamus [Additionally, the concentration of von Willebrand factor (VWF) in brain tissue is directly related to the density of microvessels , 155. MeThese few examples illustrate the potential of post-mortem biochemical analyses of brain tissue as a means to measure vascular function and to investigate the pathogenesis of vascular dysfunction.Aside from the hallmark pathological lesions, there is evidence to suggest a role for immunological and inflammatory mechanisms in the pathophysiology of VaD/VCI. Neuroinflammation encompasses local endothelial activation, leading to the extravasation of fluid  via a dysfunctional BBB, resulting in oedema and tissue damage in the surrounding parenchyma and eventually leading to the activation of perivascular macrophages, microglia and other glial subtypes Fig.  156–158158.Fig. Clinical studies in patients with symptomatic SVD , 160 or Elucidation of the role of neuroinflammation in the pathogenesis and pathophysiology of SVD will enable the evaluation of immunotherapies as potential therapeutic options for prevention or treatment of VCI/VaD.It becomes increasingly clear that standardised neuropathological criteria for the assessment of CVD in human post-mortem brains are needed . In ordeIn addition to the refinement of routine neuropathological scoring criteria, complementary methods such as post-mortem MRI and biochemical assessment are promising tools to investigate CVD. These should be helpful not only to better understand the pathophysiology of VCI/VaD but also to clarify the pathophysiological processes that ultimately lead to characteristic findings of in vivo imaging. The latter seems a timely need, since current assumptions regarding the ‘causes’ of WMH and cerebral microbleeds may not be accurate in all cases and, hence, negatively impact on the diagnostic accuracy of respective clinical diagnoses.


The differentiation status of tumors is used as a prognostic indicator, with tumors comprised of less differentiated cells exhibiting higher levels of aggressiveness that correlate with a poor prognosis. Although oncogenes contribute to blocking differentiation, it is not clear how they globally alter miRNA expression during differentiation to achieve this result. The pediatric sarcoma Alveolar Rhabdomyosarcoma, which is primarily characterized by the expression of the PAX3-FOXO1 oncogenic fusion protein, consists of undifferentiated muscle cells. However, it is unclear what role PAX3-FOXO1 plays in promoting the undifferentiated state. We demonstrate that expression of PAX3-FOXO1 globally alters the expression of over 80 individual miRNA during early myogenic differentiation, resulting in three primary effects: 1) inhibition of the expression of 51 miRNA essential for promoting myogenesis, 2) promoting the aberrant expression of 43 miRNA not normally expressed during myogenesis, and 3) altering the expression pattern of 39 additional miRNA. Combined, these changes are predicted to have an overall negative effect on myogenic differentiation. This is one of the first studies describing how an oncogene globally alters miRNA expression to block differentiation and has clinical implications for the development of much needed multi-faceted tumor-specific therapeutic regimens. Pathologists use the differentiation status of cancer cells as a prognostic indicator to predict the aggressiveness and stage of a given tumor. In general, a low level of differentiation, in which the tumor cells lose structural organization, grow more rapidly, and are morphologically dissimilar from the surrounding normal tissue, correlates to a higher level of aggressiveness and a poor prognosis. There are two basic mechanisms by which tumor cells can acquire an undifferentiated state. First, as shown with neuroblastoma and glioblastoma –3, the hLike many other cancers, Rhabdomyosarcoma (RMS), one of the most common soft tissue sarcomas in children, is characterized by poorly differentiated muscle cells. RMS is comprised of two main subtypes: embryonal (ERMS) and alveolar (ARMS), each defined by its unique histology, clinical presentation, therapy, and prognosis . ARMS, tIn this study we are one of the first to provide a picture of the global changes in miRNA expression that occur in the earliest stages of myogenic differentiation and to determine how the expression of PAX3-FOXO1 alters these changes, either directly or indirectly. We report that the oncogenic fusion protein mediates these changes in three distinct ways: 1) by inhibiting the expression of miRNA essential for promoting myogenesis, 2) by promoting the aberrant expression of miRNA not normally expressed during myogenesis, and 3) by altering the normal expression of miRNA. The ultimate result of these changes is predicted to be the inhibition of myogenic differentiation. Our results allow us to propose a model by which miRNAs contribute to the process of normal myogenic differentiation and to describe how the changes mediated by the oncogenic fusion protein would block normal myogenesis. Taken together, these results allow us to propose the use of miRNA-mediated differentiation therapy as a component of a multi-faceted treatment regimen for ARMS. Finally, the results presented here have implications that may be applied to other tumor models, since determining global changes in miRNA expression relative to the surrounding normal tissue may be used to identify key miRNAs that can be targeted for novel therapies for individual tumor models.To determine how miRNA expression changes during early myogenic differentiation and to examine how PAX3-FOXO1 alters these changes, we utilized mouse primary myoblasts stably transduced with the MSCV-IRES-puromycin retroviral vector (negative control), or the same retroviral vector expressing FLAG-epitope tagged PAX3-FOXO1. The transduced cells were selected with puromycin; selected cells were harvested from three independent transductions and pooled, resulting in a single mixed population for each individual construct, which removes the potential for variability that may occur from clonal effects. The observed level of PAX3-FOXO1 expression is equivalent to that of the fusion protein in ARMS tumor cell lines , 20 and In this model proliferating cells were induced to differentiate, as described in the Materials and Methods, and allowed to differentiate for 24 hours. We found that the negative control cells effectively entered the myogenic program exhibiting flattened and elongated cells that have begun to organize, align and fuse into multinucleated myotubes Figure . Taken tUsing this model, we performed miRNA deep sequencing analyses on total RNA isolated from three independent growths of stably transduced primary myoblasts. We selected time points based on the known phosphorylation status and protein expression levels of PAX3 and PAX3-FOXO1 during differentiation Figure . In the We found that in the negative control cells 90 miRNA had changes in expression over the time course of differentiation while cells expressing PAX3-FOXO1 had 82 miRNA whose expression changed. Of these miRNA, 39 of the miRNA were found to have expression changes in both the control cells and in cells expressing PAX3-FOXO1  to identify biological networks through which the targeted genes may functionally interact with one another. Finally, we performed a PubMed search to provide literature confirmation for the direct action of miRNA on target genes and/or the interaction of target genes with each other.A closer examination of the 51 miRNA whose expression changed only in the empty vector control cells revealed that they could be further categorized based on how their expression changed over the course of early differentiation . However, in contrast, the maintained increase in several of the miRNA in this category target genes required to promote the fusion or terminal differentiation of muscle cells . Taken together, and consistent with the pathological phenotype of ARMS tumor cells, alterations in the regulating miRNA would result in changes in target gene expression that would allow the initiation of myogenesis with a subsequent inhibition of myoblast fusion and terminal differentiation.A similar analysis of the miRNA whose expression changes occur only in the presence of PAX3-FOXO1 revealed that only two subclasses of changes were observed: 1) miRNA with increased expression at 30 minutes relative to proliferation with additional changes at 24 hours; and 2) miRNA with decreased expression at 30 minutes relative to proliferation with additional changes at 24 hours Table . ConsistThe miRNA in the second subcategory, in which miRNA expression decreases at 30 minutes and remains decreased at 24 hours, for the most part exhibited small changes with a majority of them having between 1.5 – 2.0 fold decrease in expression Table .Although 39 of the identified miRNA demonstrated changes in expression in both the control cells and cells expressing PAX3-FOXO1, only 6 of these miRNA exhibited similar changes in expression between the two samples (Table increase at 24 hours of expression) would result in target gene expression favorable for promoting myogenesis. In contrast, the presence of PAX3-FOXO1 promotes a large decrease in this miRNA, subsequently increasing the expression of these target genes thereby inhibiting myogenic differentiation. Similar alterations are predicted to negatively effect many biological processes essential for proper myogenesis, including organization of myofibers (miR-128-3p/Runx1 and miR-29a-3p/Dcx), myoblast fusion , expression of myogenic structural proteins , and the initiation of myogenesis .Examination of the miRNA whose expression changes in both cell types demonstrate that the alterations seen in the presence of PAX3-FOXO1 would effect target gene expression in such a way as to inhibit myogenic differentiation  translocation, an event mirrored in our 3 using the miRNeasy mini kit (Qiagen), according to the manufacturer's specifications. MicroRNA were isolated from 4μg total RNA, to generate the cDNA libraries, using the Illumina sample preparation kit according to the manufacturer's specifications. The cDNA libraries were provided a unique index identifier, allowing the clustering of several samples into a single sequencing lane, and deep sequencing analyses were performed in triplicate from three independent cell growth, RNA isolation, and cDNA library constructions.Raw fastq sequences were obtained from the Illumina Genome Analyzer II using the “Demultiplex” algorithm in the CASAVA 1.8.2 software (Illumina) that allows the identification of individual samples by “index sequences” contained within the adapters and introduced during the adapter ligation and amplification of the samples. miRNAKey, was used for data analysis at default settings. The pipeline clips the Illumina 3′ adaptor sequences, maps the clipped reads to miRBase and uses the Seq-EM algorithm to estimate the distribution of multiply mapped reads across the observed miRNAs. Sequences less than 16 bases after adaptor clipping were removed. The read counts obtained were then used for differential expression analysis between control and experimental samples using EBSeq from the R package with a False Discovery Rate (FDR) of 5%. We used the default ‘Median Normalization’ in EBSeq to make the counts comparable across samples. Resulting differentially expressed miRNA were analyzed using miRTarBase to identify experimentally validated target genes, with validation by at least two independent experimental methods. Finally, the differentially expressed miRNA and their target genes were analyzed using the Ingenuity Pathway Analysis  to identify effected pathways and biological interactions between miRNA target genes.


The Medline database was searched to identify relevant articles published until December 2016. Inclusion required that studies examined longitudinal changes in brain glutamate metabolites in patients with schizophrenia before and after initiation of first antipsychotic treatment or a switch in antipsychotic treatment. The searches identified eight eligible articles, with baseline and follow-up measures in a total of 168 patients. The majority of articles reported a numerical reduction in brain glutamate metabolites with antipsychotic treatment, and the estimated overall mean reduction of 6.5% in Glx  across brain regions. Significant reductions in glutamate metabolites in at least one brain region were reported in four of the eight studies, and none of the studies reported a significant glutamatergic increase after antipsychotic administration. Relationships between the degree of change in glutamate and the degree of improvement in symptoms have been inconsistent but may provide limited evidence that antipsychotic response may be associated with lower glutamate levels before treatment and a greater extent of glutamatergic reduction during treatment. Further longitudinal, prospective studies of glutamate and antipsychotic response are required to confirm these findings.Schizophrenia is associated with brain glutamate dysfunction, but it is currently unclear whether antipsychotic administration can reduce the extent of glutamatergic abnormality. We conducted a systematic review of proton magnetic resonance spectroscopy ( Alternatively, if current antipsychotics do not adequately address glutamatergic dysfunction, this would support the suggestion that adjunctive treatment with glutamatergic agents may have additional therapeutic benefit.Animal, postmortem, and genetic studies indicate that schizophrenia is associated with abnormalities in glutamatergic neurotransmission , but it ex vivo proton nuclear magnetic resonance (1H-NMR) following administration of clozapine and olanzapine, but not haloperidol, risperidone, or aripiprazole , usually within a specific a priori brain region of interest. Depending on the achieved resolution, 1H-MRS can provide concentration estimates for glutamate and glutamine or, at lower field strengths, the combined glutamate plus glutamine signal, which is termed Glx. The idea that antipsychotics may reduce glutamate elevation in schizophrenia is supported by a cross-sectional study that detected an elevation in Glx in non-medicated, but not in medicated schizophrenia .http://arohatgi.info/WebPlotDigitizer). Extracted data also included the reported statistical significance of the change in glutamatergic metabolite over the antipsychotic treatment period, the demographic and clinical characteristics of the sample, the antipsychotic/s prescribed and the duration of treatment, and the brain region investigated.For qualitative comparison of antipsychotic-induced change in glutamate metabolites across studies, the percentage difference in glutamatergic metabolite level over the antipsychotic treatment period was extracted from each article. This was calculated as the percentage mean difference (PMD), where PMD = [(mean glutamate metabolite level after treatment − mean glutamate metabolite level before treatment)/mean glutamate metabolite level before treatment] × 100. Where these values were not reported , 32, theThe initial search identified 51 articles, of which 40 were excluded at the title and abstract screening stage. At the full-text screening, one study was excluded as it was not available in English . Analysi1H-MRS follow-up ranged from 7 to 42 patients. Three studies recruited patients with first episode psychosis who were antipsychotic naïve or had received minimal antipsychotic exposure  = 3.06; P = 0.005]. There were no reports of significant increases in glutamate metabolites with antipsychotic treatment. Three of the eight studies did not report significant effects of antipsychotic treatment on glutamate measures in any brain area across the patient sample (Four of the eight included studies reported statistically significant glutamatergic reductions over antipsychotic treatment in at least one brain region investigated , 37, 39.t sample , 38, 40.The included studies reported mixed findings regarding associations between the degree of change in glutamate and the degree of symptomatic reduction with antipsychotic treatment Table . SignifiNone of the studies included in the review examined whether glutamate measures before antipsychotic treatment predicted the degree of subsequent symptomatic response to antipsychotic administration. However, a separate article  presentiThe main finding of this review is that the majority of studies reported a numerical reduction in glutamate metabolites following antipsychotic treatment in schizophrenia, with half of the reviewed studies finding significant reductions in at least one brain region. In contrast, no significant increases in glutamate metabolites were reported. Schizophrenia is associated with a general increase in glutamate metabolites, which varies with region and with illness stage . This reThe mean change in Glx over antipsychotic treatment was estimated from the available data as a decrease in the range of 6.5% across regions, which would require a within-subjects’ sample size of 24 patients to achieve 80% power. Only three of the eight available studies had sample sizes of 24 or more patients , 34, 37 1H-MRS include the estimation of the total concentrations of glutamate metabolites in the voxel, rather than imaging of glutamate involved in neurotransmission specifically, and inability to resolve glutamate from glutamine at lower field strengths and thereby interpretation of the combined Glx signal. Advanced methodological approaches, such as 1H-MRS at high field strengths or 13C-MRS to inspect glutamate cycling may address some of these issues in future studies.Our estimates of percentage reduction in glutamatergic metabolites following antipsychotic treatment in schizophrenia and the associated sample size calculations are limited by the availability of relatively few published studies. These studies have also investigated different brain regions, and glutamate dysfunction in schizophrenia may vary by region as well as by illness stage . In some2A antagonist activity of atypical agents (2A affinity to modulate glutamate in schizophrenia.Reductions in glutamate metabolites following antipsychotic treatment would be consistent with the cross-sectional report of elevated prefrontal cortical Glx in non-antipsychotic medicated but not antipsychotic-medicated schizophrenia . While tl agents , 41, 42.l agents –45. The The timescales over which antipsychotic effects on glutamatergic measures have been evaluated range from 4 weeks  to 7 yea1H-MRS studies, and investigated in future longitudinal studies potentially combining glutamate 1H-MRS with glutamate genetic approaches.The second aim of this article was to review the potential relationships between glutamate and the degree of antipsychotic response in longitudinal studies. Our review only identified one study prospectively examining the relationship between baseline glutamate and subsequent response, which reported higher baseline Glx/Cr across voxels in the frontal and temporal lobes and thalamus in subsequent antipsychotic non-responders than responders . Two stuAE and AB performed the literature searches and data extraction. KM and GM checked data for accuracy. All authors contributed to interpretation of the data and the final draft of the manuscript.The views expressed are those of the authors and do not necessarily represent those of the NHS, NIHR, or Department of Health.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.


Research in the last decade has shown that hematopoietic stem cells (HSCs) interact with and are modulated by a complex multicellular microenvironment in the bone marrow, which includes both the HSC progeny and multiple non-hematopoietic cell types. Intense work is gradually throwing light on the composition of the HSC niche and the molecular cues exchanged between its components, which has implications for HSC production, maintenance and expansion. In addition, it has become apparent that bidirectional interactions between leukemic cells and their niche play a previously unrecognized role in the initiation and development of hematological malignancies. Consequently, targeting of the malignant niche holds considerable promise for more specific antileukemic therapies. Here we summarize the latest insights into HSC niche biology and recent work showing multiple connections between hematological malignancy and alterations in the bone marrow microenvironment. The concept of stem cell niche, in the context of the hematopoietic system, implies that the behavior of hematopoietic stem cells (HSCs) is to a great extent modulated by their microenvironment in the bone marrow. Underlying the interest in the study of the HSC niche are two practical hypotheses: (1) precise characterization of the cell populations and factors produced by them, responsible for HSC maintenance in vivo, holds the clue for HSC expansion ex vivo; (2) knowledge of how the niche is modified in hematological diseases (such as dysplasias and leukemias) might allow selective therapeutic targeting that eliminates abnormal HSCs while restoring normal hematopoiesis. However, the complex cellular composition of the BM cavity, the multiple anatomical and functional interactions between its components—not yet fully characterized—and its apparent lack of physical compartmentalization into discrete, organized structures analogous to stem cell niches described in other tissues have complicated the cellular and molecular dissection of the HSC niche. As a consequence, significant progress in the two above mentioned directions has only begun to occur in very recent years. In this review, we discuss the most recent insights into the biology of HSCs and their niche, some of the technical difficulties that have been encountered, and recent work revealing the relationship between hematological disease and abnormalities of the HSC niche.A static, long-term HSC with fixed specific features , ultimately responsible for maintaining hematopoiesis through life.A convenient, straightforward, but simplistic model of progenitor hierarchy, in which a homogeneous population of HSC differentiates into progenitors and subsequently mature lineages along bifurcating, non-reversible, non-overlapping paths.A rigid idea of a “HSC niche”, in which—by analogy with certain non-hematopoietic tissues—the HSC is postulated to “reside” more or less statically in discrete anatomical microenvironments, the exact identity of which has remained elusive. The growing amount and complexity of information concerning the influence of many other bone marrow cells on HSC behavior has lead to postulate the existence of functionally different HSC niches, which is still not sufficiently substantiated by the experimental data available .Our idea of the HSC has traditionally been dominated by a number of concepts:In the light of the results reported over the last few years, this image is gradually evolving toward a much more dynamic model:Heterogeneity in HSC proliferation and self-renewal Analyses of the mouse and human HSC compartments have revealed variability in the cell cycle status and self-renewal capacity of individual HSCs. In the mouse, populations of “dormant” and “activated” HSCs have been identified, which are to some extent interconvertible. Dormant HSCs divide at very slow rates in homeostasis (every 145 days), exhibit the highest self-renewal and multilineage repopulation activity and, although transiently activated by bone marrow injury or by granulocyte colony-stimulating factor (G-CSF), they revert to quiescence after reestablishment of homeostasis in a non-stochastic fashion [(A)  fashion . HSCs sw fashion  and are s every 15 days, escid-IL2Rgc−/− mice, and a small fraction of HSCs is apparently Thy1- [These observations may suggest that HSC fate is initially unpredictable and occurs stochastically, but it may also in part reflect limitations in the methods currently used for the isolation and functional analysis of HSCs. Thus, despite significant improvements, the best combinations of phenotypic markers in the mouse reach about 50 % purity, measured by competitive transplant assays , 5, or ely Thy1- . TherefoHeterogeneity in multilineage differentiation capacity of HSCs Retrospective analyses of single cells or clonal transplant experiments have demonstrated different kinetics and patterns of multilineage haematopoietic reconstitution derived from individual murine HSCs. Up to 16 distinct differentiation patterns were identified, based on their relative lymphomyeloid output and kinetics [(B) kinetics –11. In tkinetics .Contribution of hematopoietic progenitor cells to hematopoiesis Traditionally, transplantation assays have served as the gold standard to assess HSC function. Under transplantation conditions, long-term reconstitution ability seems to be restricted to a small number of primitive LT-HSCs, and hematopoiesis is typically oligoclonal. However, recent studies using genetic labeling and clonal tracing of HSC have revealed a very different situation during adult steady-state hematopoiesis, in which blood cell production is highly polyclonal and predominantly maintained by “short-term HSCs” or progenitors downstream of LT-HSCs, with strong myeloid bias [(C) oid bias , 14. Moroid bias .Notwithstanding technical limitations, these data suggest that, contrasting the concept of stable and discrete HSPC populations, a more dynamic situation may exist in which there is some degree of plasticity in the proliferation and differentiation capacity of HSCs and their progeny. It is still unclear to what extent this is regulated through dynamic interactions with the microenvironment or via stochastic, cell-autonomous fluctuations in the HSC transcriptome.reg cells) have been proposed as niche components and/or critical regulators of HSC function  plus several mature hematopoietic populations  to the bone marrow is the apparent absence of compartmentalization of the latter into discrete structures sheltering the HSC. The existence of such compartments, however, has been speculated. Structures termed “hemospheres”  were shown to be enriched in CD150+ CD58− HSCs and seemed to represent sites of clonal hematopoietic cell proliferation. Hemospheres were found in sinusoidal vessels at the periphery of the BM cavity, associated with NG2+ Pdgfrb+ Nestin+ CD146+ pericytes, and required expression of VEGFR2 in the endothelium . A recenRecent evidence suggests that diverse bone marrow locations may function as niches for different hematopoietic progenitors. The evidence is stronger for lymphopoietic niches, which have been associated with IL-7-enriched bone regions , 30. AlsRecent work has highlighted the influence of different mature hematopoietic cell subsets on the regulation of the HSC niche. Among mature bone marrow cells, the greatest body of evidence implicates monocytes/macrophages as potential HSC regulators, usually indirectly through interactions with other components of the niche –36. Diff—which also differentiate from the monocyte/macrophage lineage—on HSCs through bone remodeling has remained controversial; different studies have reported that osteoclasts are either necessary, dispensable or even inhibitory for HSC maintenance and mobilization. Thus, some studies have suggested that osteoclasts promote the formation of BM niches [On the other hand, the influence of osteoclastsM niches . Also, oM niches , but theM niches . HoweverM niches . Also, tM niches .It has been recently shown that HSCs frequently localize in the vicinity of megakaryocytes, and ablation of megakaryocytes leads to cell cycle entry of quiescent HSC, suggesting that this cellular population contributes to maintaining HSC quiescence. This interaction was reported as being mediated by soluble factors such as TGF-β1, Cxcl4 and CLEC-2-mediated thrombopoietin production –44. MegaLO CXCR4HI neutrophils in the bone marrow was proposed to cause a reduction in the size and function of the HSC niche by decreasing the number of CXCL12-abundant reticular (CAR) cells and the levels of Cxcl12, thus promoting the circadian egress of HSPCs into circulation. However, circadian oscillations of Cxcl12 expression, directly induced in bone marrow stromal cells by oscillating sympathetic activity [Neutrophil clearance has been proposed as an additional regulatory mechanism of the HSC niche . Infiltractivity , might areg lymphocytes colocalize with HSCs near the endosteal surface in the bone marrow and contribute to protecting transplanted allogeneic HSCs from immune clearance, suggesting that the niche does not only nurture HSCs but also provides them with immune privilege [Trivilege .Although it has long been recognized that leukemia/lymphoma development is associated with an abnormal tissue microenvironment in the affected organs, only in recent years has experimental work begun to dissect specific roles of the HSC niche in leukemogenesis Fig. . ProgresTwo major ideas have emerged, supported by growing evidence: (1) genetic alterations of the niche, rather than the hematopoietic cell, may represent driving mutations during malignant transformation; (2) cancer cells remodel their niche into an abnormal environment that provides preferential support of malignant cells—in detriment of normal hematopoiesis—protects cancer cells from therapy-induced cell death, and/or drives disease progression. From these concepts immediately follows the idea that manipulating the leukemic niche might represent an advantageous therapeutic strategy, particularly in malignancies for which targeting the hematopoietic cells has proven inefficient.Initial studies showed that certain genetic alterations that affected broadly the BM microenvironment also promoted the development of abnormal hematopoiesis. Mice usually developed a myeloproliferative-like disease characterized by hypergranulopoiesis, extramedullary hematopoiesis, increased myeloid progenitors and eventually bone marrow failure and anemia. These phenotypes were observed upon deletion of retinoic acid receptor gamma (microenvironment dependent) , deletioDicer1 under the regulatory elements of Osterix, a gene required for osteoblastic differentiation—but not in mature osteoblasts, targeted by the regulatory elements of Osteocalcin promoter—did not only impair osteoblast differentiation, but was also found to cause a myelodysplastic syndrome characterized by dysplastic morphology, lymphoid-deficient hematopoiesis and increased HSPC proliferation and apoptosis. Key aspects of the phenotype were recapitulated by specific Osterix-cre-mediated deletion of Sbds, a Dicer-regulated gene that is mutated in some leukemias [Osterix was originally reported to label only cells committed to become osteoblasts [Subsequent studies refined this concept by modifying specific stromal cell subsets. Specific deletion of the RNA processing enzyme eukemias . Howevereoblasts , a much eoblasts –59  impaired the differentiation of hematopoietic progenitors and caused accumulation of immature myeloid cells and dysplastic neutrophils in hematopoietic tissues, resembling acute myeloid leukemia (AML). These changes were caused by expression of jagged1 in osteoblasts and consequent excessive Notch signaling in HSPCs [VEGF overexpression in rization . More drin HSPCs .Dicer1-deleted mice, rare sporadic occurrences of AML that exhibited cytogenetic alterations were reported [Ctnnb1CAosb mice, including those in an ortholog region of human chromosome 7q, which is altered in AML and myelodysplastic syndromes. Notably, transformed HSCs from Ctnnb1CAosb mice became autonomous and could transfer the AML to WT recipients [These studies demonstrated that genetic alterations in the microenvironment can trigger a pre-leukemic condition , but at the same time suggested that additional mutations are required in the hematopoietic cells to induce malignancy and/or overt leukemia. Evidence of malignant transformation was limited in most studies. In the case of reported . Most recipients .Helicobacter pylori in MALT lymphomas) in which the pathogen may provide antigenic stimulation to malignant B-cells and the tumor remits after eradication of the infection [DICER, DROSHA and SBDS was detected in MSCs—but not in leukocytes—from MDS patients compared to healthy individuals [SBDS is inactivated by mutations in the human Shwachman–Bodian–Diamond syndrome, featuring skeletal abnormalities, bone marrow failure and susceptibility to developing myelodysplastic syndrome and secondary AML [CREBPP heterozygosity in humans causes the Rubinstein–Taybi syndrome, characterized by skeletal defects and cancer predisposition, including leukemias [In spite of these experimental models providing a proof of concept, we are still lacking direct demonstration that an initial lesion in the microenvironment may play a causative role in human leukemias. However, the situation is somewhat reminiscent of that traditionally observed in extraganglionar lymphomas associated with local infectious processes  were reported to exhibit preferential homing and engraftment to the endosteal, osteoblast-rich area of the BM [Xenografted primary human AML stem cells , with decreased osteoprogenitors, endosteal-lining osteoblasts and bone mass, associated with increased CCL3 expression [SCL-tTA;TRE-BCR/ABL mice), two studies have provided evidence for the concept that leukemic cells can remodel the bone marrow environment into a self-supporting niche that impairs normal hematopoiesis. Leukemic cells were shown to stimulate excessive production of osteoblastic cells. These cells had reduced expression of HSC maintenance factors and impaired ability to support normal HSCs, while having limited impact on LSCs. Abnormal osteoblastic cells overexpressed inflammation- and myelofibrosis-related genes, contributed to bone marrow fibrosis, and their expansion was driven by TPO, CCL3 and direct contact with leukemic cells [Severe osteoblastic defects were found in a model of myeloid blast-crisis chronic myeloid leukemia  requires the loss of sympathetic regulation in the HSC niche [JAK2V617F-positive MPN patients and mice due to apoptosis induced by MPN cell-derived IL-1β. In turn, neuroglial damage compromises the survival of nestin+ cells, which are also reduced in number over the course of the disease. This loss of MSCs plays a driving role in MPN development, since selective depletion of nestin+ cells, or Cxcl12 produced by them, is sufficient to accelerate disease progression. Conversely, pharmacological compensation of neural damage blocks MPN progression (see below).Recent work from our laboratory has demonstrated that the manifestation of SC niche . SympathJAK2V617F-induced MPN, in which loss of nestin+ MSCs caused by neuroglial damage promotes disease progression [+ MSC and inhibited disease progression at both early and late stages, preventing neutrophilia, thrombocytosis, IL-1β production, bone marrow fibrosis and expansion of LSCs, while having negligible effects on normal HSCs. Similarly, treatment of MPN-affected mice with the neuroprotective molecule 4-methylcatecol, which protects sympathetic fibers and Schwann cells, abolished MPN-related neutrophilia. These results demonstrate that specific reversion of malignancy-induced changes in the bone marrow microenvironment may be sufficient to block MPN. They also prove that niche transformation represents a major driving force and a requirement for disease progression, and provide a novel, potential safe therapeutic approach where hematopoietic cell-directed therapies have previously shown limited efficacy.Growing evidence of the active participation of the HSC niche in hematological malignancies, either by providing an initiating lesion or by responding to leukemia-induced remodeling, suggests that its therapeutic targeting may have therapeutic benefit. This could be potentially achieved through inhibition of essential interactions between LSCs and supporting cells, or by reversion of niche-related changes that drive the course of the disease. We have provided direct evidence of the latter in the context of gression . Rescue However, this is very unlikely the case of more aggressive leukemias, such as AML, despite some similarities shown in a recent study suggesting that sympathetic neuropathy caused by acute myeloid leukemic cells promotes malignancy in an altered hematopoietic stem cell niche . Unlike As outlined at the beginning of this review, elucidation of the cellular and molecular interactions in the HSC niche has two major practical motivations. One is the identification of key molecules that would allow the production, maintenance and expansion of HSCs. Identification of the specific factors that contribute to maintaining stemness in vivo would facilitate the development of chemically defined culture media, avoiding the use of animal-derived culture supplements and possibly the need for time-consuming coculture protocols. The identification of specific microenvironments and factors required for the maintenance of different types of hematopoietic progenitors could make possible the selective ex vivo generation of different lineages for specific applications.Another aspect with translational potential is the identification of alterations in the bone marrow niche directly associated with, and required for, the development of each type of leukemia. It is expected that different malignancies will induce a specific set of abnormalities and will also differ in their dependence on the niche. Thus, it would not be surprising to find that aggressive, acute leukemias with accumulated mutations may become relatively cell autonomous and less sensitive to microenvironmental regulation. Most relevant will be to identify cellular processes or pathways selectively required for the maintenance of malignant progenitors—as opposed to normal HSCs—and susceptible to pharmacological targeting. The success of niche-directed therapies will likely depend on how accurately we can validate preclinical findings from mouse models in the human system, for which it will be critical to develop parallel sets of molecular markers for human HSCs and niche cells, as well as more faithful xenotransplant models.bona fide “niche cells” and indirect signals from more distant components. It would also be advantageous to clarify some of the confusion in the field resulting from the use of incompletely characterized genetic drivers and partially overlapping markers . Again,


Sir,After reviewing the published literature that documented an estimated 20% of newly diagnosed cancer cases worldwide are caused by infectious agents , and theAs expected, we found that a large number of squamous cell carcinomas contained DNA from HPV, of which 30% were high-risk HPV. Surprisingly, exogenous retroviruses of several varieties were found in 85% of squamous cell carcinomas and 47% of adenocarcinomas. Retroviruses are known oncoviruses in numerous non-human animal cancers (including adenocarcinoma of the lung) and in at least one human cancer (adult T-cell leukaemia). However, a most intriguing unexpected finding was the high incidence of DNA from hepatitis B virus (HBV) in 9/13 (69%) of squamous cell carcinomas and 12% of adenocarcinomas. HBV is a known oncovirus causing liver cancer  and may In this Letter to the Editor, the authors present a succinct and convincing discussion describing how HBV might play a role in squamous cell carcinoma of the lung, as it relates to inflammation and hit-and-run carcinogenesis. However, we emphasise caution – the detection of viral DNA  in tumour tissue does not imply causation. Nevertheless, we now appear to have a wider field of suspect microorganisms to investigate in future studies of lung cancer. Our research group is currently employing numerous techniques to substantiate our initial findings and evaluate whether a microbial signature may identify individuals at high risk for this prevalent and lethal cancer.


Rosaceae is a family with an extraordinary spectrum of fruit types, including fleshy peach, apple, and strawberry that provide unique contributions to a healthy diet for consumers, and represent an excellent model for studying fruit patterning and development. In recent years, many efforts have been made to unravel regulatory mechanism underlying the hormonal, transcriptomic, proteomic and metabolomic changes occurring during Rosaceae fruit development. More recently, several studies on fleshy (tomato) and dry (Arabidopsis) fruit model have contributed to a better understanding of epigenetic mechanisms underlying important heritable crop traits, such as ripening and stress response. In this context and summing up the results obtained so far, this review aims to collect the available information on epigenetic mechanisms that may provide an additional level in gene transcription regulation, thus influencing and driving the entire Rosaceae fruit developmental process. The whole body of information suggests that Rosaceae fruit could become also a model for studying the epigenetic basis of economically important phenotypes, allowing for their more efficient exploitation in plant breeding. Table 1) , nuts , and ornamentals  . HoweverTable 1) . For exaTable 1) . HoweverThe availability of molecular resources in different Rosaceae species has increased the genetic data collection from fruit set to pre-harvest ripening, resulting as being an important research tool . HoweverIn Rosaceae fruit crops, like in other plant families, several studies have shown that both fruit set and development (including ripening) rely on the establishment and maintenance of differential gene expression patterns  determinArabidopsis thaliana. This pathway involves the biogenesis of siRNAs (short interfering RNAs), which requires Pol IV, RDR2 and DCL3, and de novo methylation, which requires plant Pol V-dependent scaffold RNAs, AGO4-bound 24-nt siRNAs, and the de novo DNA methyltransferase DRM. Many other dedicated proteins participate in RdDM, whose precise roles have not so far been fully clarified  and non-symmetrical (CHH) contexts  and is driven by three main classes of DNA methyltransferases. The DNA methyltransferases 1 (MET1), which is an ortholog of the mammalian Dnmt1, is responsible for maintenance of CG methylation in cooperation with the VARIANT IN METHYLATION (VIM) proteins. CHG methylation is mainly maintained by a self-reinforcing loop between the plant-specific CHG DNA MTase CHROMOMETHYLASE 3 (CMT3) and the H3K9 MTase KRYPTONITE (KYP), also known as SUPPRESSOR OF VARIEGATION 3–9 HOMOLOG 4 (SUVH4) and its close homologs SUVH5 and SUVH6 , 2015. Tlarified . More relarified . An esselarified . DDM1 prlarified . CMT2 hahylation .Genome-wide studies have shown that a specific DNA methylation distribution in three contexts depends upon TE sequences occupancy in Arabidopsis and in other relatively small plant genomes . In thesMany reports reviewed mechanisms of plant DNA methylation and how they are interconnected with other epigenetic pathways, like the small RNAs responsible for guiding DRMs in CHH methylation in the RdDM process  involved in fruit patterning. The expression profile of enzymes responsible for PTMs has been determined in many fruit species, suggesting their potential role in specific moments of growth, in both early and late fruit development  leads to a co-suppression of the tomato homolog protein, resulting in various tomato phenotypes similar to those described in In the last few years many studies focused attention on the role of histone PTMs on fruit development, considering a potential role in the crosstalk between chromatin modifications and Rosa, Fragaria, and Rubus with hip, achene and berry fruit, respectively), Prunoideae (Prunus with drupe), and Maloideae  (Fragaria vesca), peach (Prunus persica), and apple  (Table 1).Because of the wide Rosaceae species phylogenetic diversity, translating into a large number of fruit types with different anatomical and physiological properties, three subfamilies are always referred to, dictated mainly by fruit structure: the Rosoideae (th pome) . To simpth pome) , for thePrunus spp. harboring drupes characterized by a lignified endocarp (stone or pit), in which seeds are enveloped, and a fleshy and juicy mesocarp surrounded by an epicarp tissue. Apple, the most important deciduous tree fruit crop grown around the world, has a pome fruit structure, defined as ‘false fruit,’ in which the floral receptacle is the fleshy edible tissue (named cortex), and where the typical organization of pericarp  characteristic of drupe is not present. Finally, the third typical Rosaceae family model is well represented by Fragaria spp. where the true fruit (achene) is localized on an accessory structure comprised of a fleshy receptacle genetic/genomic peculiar properties different from the model reference , and can be related only to Angiosperms. Fruit with fused carpels can also be classified as dry (indehiscent or non-dehiscent) or fleshy fruits, and derive only from carpels (‘true fruit’) or from accessory structures . In this articulated scenario, it is possible to collocate peach, apple, and strawberry, the three Rosaceae model plants that, on the basis of their specific fruit development pattern, result in the three different fruit types: drupe, pome, and achene, respectively , 2013. Bceptacle . It is ieference  and discFigure 1). The former is typical of pome fruits, whereas the latter of drupes. This simple representation has been improved by means of modeling approaches able to establish that for apple fruit (a pome) an expolinear equation, based on fruit diameter, is the one showing the best fit to its initial exponential growth phase followed by linear growth , the length of cell division plays a relevant role in determining the final fruit density (number of cells per volume) at harvest, as its storability and nutritional value are strongly linked to this parameter. In turn, cell division depends upon the final size of the fruit. Therefore, this stage is very short or absent in small fruits , while it lasts for 7–25 and 21–35 days in peach and apple, depending on the variety . It is wFigure 1). In this framework, we have surveyed the literature published on the transcriptional modulation of fruit development regulators, focusing our attention on TFs whose mRNA levels can be modulated at post-transcriptional level by miRNAs and by possible epigenetic mechanisms. In recent years, many miRNAs have been claimed to control fruit growth and development by controlling the interplay between hormones at signaling pathway level  have been identified as the internal factors controlling the development starting from bud endo-dormancy regulation . Regarding the role of histone modifications, in some peach cultivars an accumulation of H3K27me3 epigenetic mark associated with a repression of the gene transcription was suggested, together with decreased levels of H3K4me3 histone modification, leading to an endo-dormancy establishment/maintenance/release equilibrium  and SEPALLATA (SEP) are responsible of carpel fusion during the first steps of fruit set, confirming their fundamental attendance in floral structures definition (APETALA1 (AP1) and FRUITFULL (FUL): their functions overlap in floral meristems specification but diverge in later developmental stages during fruit growth  apple (MdMADS8/9), strawberry , peach (PpSHP) as reported by Recent discoveries indicate that a strict genetic and epigenetic control influences the entire fruit development process starting from seed formation. Indeed several pieces of evidence suggest a dynamic reprogramming of the chromatin states already in first stages of the fertilization process, with a global remodeling of the meiocyte chromatin state, by the establishment of an equilibrium between permissive-associated marks like H3K4me3 and H3K9ac and the repressive-related marks including H3K27me1, H3K27me3, and H3K9me1 . This isfinition . Their et growth . In factSEEDSTICK, STK, SHATTERPROOF, SHP, SECONDARY WALL THICKENING PROMOTING FACTOR, NTS1), between the regulation of the lignification in silique valve margin in Arabidopsis and the hardening of endocarp in peach was observed . To date, also in this case, much of the information on the genetic regulation and epigenetic control of TFs involved in later fruit growth stages are mainly elucidated in tomato. Attention is mainly focused on MADS-RIN, NON-RIPENING , and COLORLESS NON-RIPENING  considered as key TF genes for the regulation of fruit growth/ripening transition phase (PcMYB10), caused by a hypermethylation at promoter level, is correlated with the formation of green-skinned spot, suggesting a putative methylation-dependent mechanism in the regulation of anthocyanin biosynthesis during pear ripening  that is involved in fruit post-harvest related senescence (MaHIS1) and a WRKY TF was detected in response to ripening and stress responses, improving also the limited information on linker histone H1 in fruits (HMs) in sweet orange during the whole pre-harvest fruit development and post-harvest was reported following the genome sequencing of this species. In particular a genomic analysis highlighted chromosome locations, phylogenetic comparison and gene structures of HM members during biotic stress induced by blue mold infection responses  to other crops. As reported above, the Rosaceae family comprises several species with a high agronomic value but with limited knowledge about the main molecular actors involved in fruit development.To fill this gap following aspects should be considered:(1)Table 2), with an epigenetic role or regulation in fruit development, ripening and post-harvest.Although many molecular mechanisms underlying fruit development have been investigated and their genetic and, in some cases, epigenetic regulation determined, a complete view of the process needs the application of methodologies supplied in ‘omics’ era also to Rosaceae crops. Different genome-wide approaches may be considered as potential suppliers of epigenetic information regarding fruit development, with the aim of completing the genetic puzzle until now available only for some species. DNA methylome and chromatin states analyses  could be two fundamental goals enrich and improve the list of key genes, already available (see (2)DNA methylation and histone mark studies in different plant tissues and the comparison of their patterns with transcriptomes of different cell types will be pivotal to determine whether epigenetic states directly influence gene expression at specific loci involved in fruit developmental processes.(3)The knowledge of the logic and temporal sequence of the chromatin state changes that are responsible of the repression and/or activation of new gene expression programs appear essential to better understand developmental transition in fruit development in the context of chromatin.vice versa). It is well known that RNA gene silencing may be transmissible in up and downward direction determining modifications in DNA methylation levels in receiving tissues (These focus points should be faced taking into account that the majority of Rosaceae spp. are vegetatively propagated and have multiple complete sets of genetic information (polyploidy). It is well known that the majority of Rosaceae species are vegetatively propagated, bypassing the meiotic process and seed formation. Since many of the genes that control important quality traits of agronomic interest could be epigenetically regulated, it will be necessary to evaluate if the specific epigenetic marks influencing their regulation, are or are not maintained following the typical and canonical agronomic practices of plant propagation, such as clonal propagation (for cutting) or grafting. In both cases, the maintenance of peculiar epigenetic marks in the form of epialleles, controlling specific fruit quality traits, is an emerging aspect to consider in arboriculture research, together to the understanding of which potential epialleles are maintained during plant propagation and how could provide a new view in molecular selection strategies. In grafting practice, which results the most common mean to clonally propagate desirable Rosaceae tree fruit cultivars  the elucidation of ‘molecular’ interaction between scion and rootstock is a new frontier to cross, to determine whether this agronomic tool is able to transmit heritable (epi)genetic changes in the scion (and  tissues . In Sola tissues . Further tissues .ARGONAUTE1), miR172 (inhibiting APETALA2) (CNR)   mechanisms mediated by miRNAs: the first study designed to investigate miRNAs in fleshy fruits was done on tomato, where 9 conserved and 12 novel miRNAs expressed in fruit were identified . This nuPETALA2) , and miR2) (CNR) . In addihe ARF8) . In turnucidated . In P. pgulation . Interesgulation . In our of fruit . To confF. vesca (diploid) vs. Fragaria × ananassa (octoploid). Often the polyploid condition is associated to a mosaic expression profile, which involves homologous genes of different genomes. This expression variability can be driven by epigenetic gene silencing mechanisms, as reported for genus Rosa (Fragaria × ananassa), despite sometimes for them the interpretation of epigenetic regulatory mechanisms results difficult.Lastly, an additional point to consider is the presence of numerous polyploid species in Rosaceae family, even within the same genus, as well testified by nus Rosa  and can In conclusion, deepening the knowledge of the genetic and epigenetic processes regulating Rosaceae fruit development, with the aid of new ‘omics’ tools and developing innovative research approaches could represent an important priority for fruit crop improvement.SF, SV, and CB conceived and wrote the manuscript. AR helped in figures. All authors revised the MS and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A novel human protoparvovirus related to human bufavirus and preliminarily named cutavirus has been discovered. We detected cutavirus in a sample of cutaneous malignant melanoma by using viral enrichment and high-throughput sequencing. The role of cutaviruses in cutaneous cancers remains to be investigated. Protoparvovirus genus was discovered in fecal samples from children with diarrhea in Brazil and subsequently detected in samples of mycosis fungoides lesions (cutaneous T-cell lymphoma) of patients in France  analysis originally identified contigs related to human bufaviruses in de novo assembled contigs from both datasets. In light of the recently published cutavirus genomes (We subjected a clinical sample of a cutaneous malignant melanoma lesion from a patient in Denmark to enrichment of virion-associated nucleic acids and enrichment of circular DNA molecules, followed by high-throughput sequencing . BLASTn We performed phylogenetic analysis based on the NS1 and VP1 amino acid sequences . BecauseParvoviridae family, human parvovirus B19, is shown to persist in multiple tissue types, in most cases without an established correlation to disease (Cutaviruses were discovered in human fecal samples by use of metagenomics and subsequently detected in 4 of 17 samples of mycosis fungoides lesions; however, 21 skin samples, including samples from skin cancers and parapsoriasis lesions, tested negative for cutavirus (In 9 additional melanoma samples investigated in our laboratory, we did not identify contigs with similarity to those of cutavirus or bufavirus. All 10 samples were tested for cutaviral DNA by real-time PCR, but only the sample in which the cutaviral contigs were detected had positive results . We can Additional methods and details of results of testing showing cutavirus in a human cutaneous malignant melanoma biopsy sample.