Using the internet is increasingly a necessity. However, older adults may not do so due to either non-health reasons  or health-related reasons. While researchers have studied internet use among older adults, most do not discriminate whether non-use is due to health reasons or otherwise. Recent studies also reveal that older adults use the internet to keep in touch with family and friends, highlighting that limitations in internet use may be detrimental for their well-being. We therefore, examine the key correlates of health-related difficulty in internet use, and how it may affect quality of life by reducing the size of their social support networks. Data were from a national survey of older Singaporeans (n=3966) conducted in 2016-17. Multinomial logistic regression and mediation analysis were used to identify older adult subgroups more likely to experience health-related difficulty in internet use, and whether such difficulty affected older adults’ quality of life through their social support networks. Results showed that males, those of Malay ethnicity, those with less education, and those with more instrumental activity of daily living (IADL) limitations were more likely to experience health-related difficulty in internet use. Social support networks mediated the relationship between health-related difficulty in internet use and quality of life. These findings suggest that other than managing the health conditions of older adults who face health-related difficulty in internet use, offline modes of keeping them socially connected may promote their quality of life.


Notwithstanding these potential applications, PSC-derived transplanted neurons have to match the precise sub-type, positional and functional identity of the lesioned neural tissue. Thus, the requirement of highly specific and efficient differentiation protocols of PSCs in neurons with appropriate neural identity constitutes the main challenge limiting the clinical use of stem cells in the near future. In this Review, we discuss the recent advances in the derivation of telencephalic  neurons from PSCs, assessing specificity and efficiency of the differentiation protocols, with particular emphasis on the genetic and molecular characterization of PSC-derived neurons. Second, we address the remaining challenges for cellular replacement therapies in cortical brain injuries, focusing on electrophysiological properties, functional integration and therapeutic effects of the transplanted neurons.Brain injuries causing chronic sensory or motor deficit, such as stroke, are among the leading causes of disability worldwide, according to the World Health Organization; furthermore, they carry heavy social and economic burdens due to decreased quality of life and need of assistance. Given the limited effectiveness of rehabilitation, novel therapeutic strategies are required to enhance functional recovery. Since cell-based approaches have emerged as an intriguing and promising strategy to promote brain repair, many efforts have been made to study the functional integration of neurons derived from pluripotent stem cells (PSCs), or fetal neurons, after grafting into the damaged host tissue. PSCs hold great promises for their clinical applications, such as cellular replacement of damaged neural tissues with autologous neurons. They also offer the possibility to create  Stroke is usually referred to as a sudden cerebrovascular dysfunction leading to focal deficits and/or impairment of global brain functions and lasting more than 24 h . Indeed, initial studies showed that fetal dopaminergic neurons grafted in the striatum ameliorated PD symptoms, both in animal models  and in pejection .Due to the high clinical relevance of cerebral lesions, protocols to differentiate neural stem cells in telencephalic neurons are highly valuable. These protocols are frequently inspired by findings in neural development.Attempts to generate identity-specific cortical neurons require exploitation of the right embryonic developmental signals in a culture dish. The six layers of the mammalian neocortex originate from inside-out in a timely regulated manner during embryonic development and are characterized by a specific pattern of gene expression and connectivity  and migrate tangentially to the cortex and hippocampus . Severalin vitro, expressing the hippocampal markers ZBTB20, KA1 and PROX1; while a prolonged treatment results in increased expression of LMX1A and TTR, markers of choroid plexus  of interest, the next step is to evaluate if these cells can properly integrate in the host tissue, sending short and long projections to their natural targets and rebuilding damaged circuitry. Neurons suitable for transplantation must be able to integrate into the host context, produce the appropriate type of neurotransmitter and neurotransmitter receptors and develop synapses with functionally relevant targets and electrophysiological properties of mature neurons.Initial reports showed that the outgrowth from grafts of fetal neurons in adult host brain was limited and dependent on the site of injection . The firin vivo 2-photon imaging of embryonic cortical neurons transplanted 7 days after ablation of layer II-III callosal projecting neurons in the monocular visual cortex, evaluating longitudinal structural maturation, morphology, dendrite elongation and spine refinement of grafted neurons. Notably, already at 3–4 days post-transplantation, grafted neurons extended branched neurites, with identifiable growth cone and boutons, followed by the formation of apical dendrites which reached complete development within 4 weeks post-transplantation (wpt). Spine and bouton density greatly increased up to 4 wpt and then reached a plateau, while turnover rates increased at 5 wpt and subsequently stabilized after 8 wpt. Interestingly, as soon as 5 wpt, grafted cells displayed appropriate identity of pyramidal neurons and projected to the natural targets of V1 neurons, including ipsilateral visual areas, retrosplenial and entorhinal cortices and the corpus callosum. Furthermore, grafted cells received topographically correct synaptic inputs from appropriate cortical and subcortical regions, as assessed by performing monosynaptic rabies virus tracing. Overall, the authors showed that fetal neurons integrate adequately into the adult cerebral cortex, mature over time and re-establish a normal pattern of projection according to their original positional identity.More recently, As previously discussed, in the last decade several groups worked to overcome the ethical and technical challenges of using fetal tissue, developing protocols to differentiate both mouse and human embryonic stem cells into neurons with appropriate cellular identity and area specification , 2.in vitro.Among the first, Pierre Vanderhaeghen and collaborators showed that murine PSC-derived pyramidal neurons grafted into the frontal cortex of neonatal mice and analyzed after 1 month displayed specific patterns of axonal projections corresponding to visual and limbic occipital cortex, despite the frontal site of transplantation . FurtherIn subsequent studies by Vanderhaeghen and collaborators, murine and human PSC-derived neurons were transplanted in a model of visual cortex lesion in adult mice and the re-establishment of the damaged pathways was assessed from 4 to 90 days after grafting . GraftedAmong the first reports of mouse PSC used as cell therapy in animal models of stroke, in vivo projection pattern revealed that the different molecular identity acquired in vitro profoundly affect the different ability of these cells to extend axonal projections. In fact, we found that, accordingly to their molecular identity, isocortical neural precursors were not able to project once transplanted in the dentate gyrus (DG) of the hippocampus, contrary to PSC-derived hippocampal cells that properly elongated fibers to CA3 following endogenous hippocampal pathways. Instead, when grafted in their proper healthy cortical region, isocortical neurons sent projections toward the motor cortex and other cortical and subcortical targets. Interestingly, consistently with previous findings  and spontaneous post-synaptic activity in voltage or current clamp configuration . In one Action potentials of murine and human grafted neurons, transplanted both in naïve and pathological conditions, have been recorded in spontaneous conditions or in voltage clamp configuration, using a depolarizing current . Interesin utero electroporated in correspondence to the layer II-III neuron production peak during corticogenesis (E14.5). At birth, they transplanted PSC-derived neurons in the contralateral homotopic region to the electroporaton site. Starting from P21, in acute slice, they patched grafted neurons (GFP+) surrounded by TdTomato fibers arising from contralateral callosal projecting neurons. In voltage-clamp configurations, grafted neurons showed spontaneous excitatory and inhibitory post-synaptic activity (EPSC and IPSC). After light stimulation of the contralateral ChR2-electroporeted neurons, authors found post-synaptic currents in grafted neurons, although no action potentials occurred  was able to evoke spike activity in grafted neurons and found that half of recorded neurons increased their firing rate after flash presentation, with latencies and frequencies similar to naïve control neurons  grafted cells could enhance functional recovery by secreting trophic factors and molecules with a neuroprotective/neuroplastic effect ; (ii) transplanted neurons could properly rebuild damaged circuitry, reestablishing lost sensorimotor synaptic connections. Consistently, cell-based therapy efforts are either directed toward grafting cells to produce and release trophic factors on site, or toward obtaining neurons with a specific identity in order to promote actual cell-replacement. Indeed, the fact that in several reports grafted cells were not detected in transplanted animals, despite an apparent functional improvement, indicates that: (i) the beneficial effect of the grafting was not related to long-term cell integration in the host neuronal circuitry and was exerted in the initial phase before the cells degenerated and (ii) the effect might be due to secretion of beneficial molecules released from grafted cells, or from resident cells stimulated by the grafting, in early post-implantation phase .Vascular endothelial growth factor (VEGF) has been implicated in mediating cell-induced functional recovery . In factIt is generally accepted that grafted neurons could improve recovery by modulating neural plasticity. Indeed, transplanted interneurons induce plasticity in the host brain as reported by Southwell and colleagues, who demonstrated that grafting inhibitory interneurons in the adult visual cortex could re-open the critical period for ocular dominance plasticity . In lineThe final goal of neuroscientists working in cell therapy for brain diseases is to restore injured neural circuits, thus rescuing physiological impairments. Among the challenges faced by the clinical application of cell-based therapies is the choice of the cell source . Indeed, both embryonic PSCs and fetal transplantations raise ethical issues, since fetuses are hardly a reliable and scalable source of material. Thus, iPSCs represent a good choice from a translational point of view, showing low immunogenicity and posing no ethical concerns. Human Leukocyte Antigen (HLA)-matched banking of iPSCs would reduce both the time and the cost associated with the derivation of clinically compliant induced pluripotent stem cell lines (reviewed in: All authors revised, wrote, and discussed the manuscript.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.


PHIP as the top gene overexpressed in metastatic versus primary melanomas [Recent studies have assigned novel roles to pleckstrin homology domain-interacting protein (PHIP) in tumor progression , 2. PHIPelanomas , promptiPHIP silencing resulted in significantly decreased melanoma cell growth and invasion, with a concomitant suppression of the metastatic potential of melanoma cells. The survival of mice injected intravenously with melanoma cells expressing anti-PHIP shRNA was prolonged, establishing the rationale for therapeutic targeting of PHIP. PHIP copy number was elevated in a subset of melanomas using fluorescence in situ hybridization (FISH) analysis. This was somewhat surprising, given that the PHIP gene is located on 6q14, with earlier studies reporting losses of 6q in melanoma [Initial studies showed PHIP to be both a marker and mediator of melanoma distant metastasis . ImmunohPHIP copy number [PHIP copy number significantly and independently predicted survival in primary melanoma, confirming the results obtained using IHC analysis. In addition, there was a significant correlation with ulceration, a powerful histologic prognostic factor incorporated into the American Joint Committee on Cancer (AJCC) melanoma staging classification. While the prognostic importance of ulceration is well appreciated, the biological basis for its significance in melanoma progression is poorly understood. Functional studies in human melanoma cells showed that PHIP promotes glycolysis and angiogenesis [Subsequently, we assessed the prognostic impact of y number . Elevateogenesis  by regulPHIP copy number in melanoma. We developed three distinct tissue sets: cohort 1- 204 primary melanomas; cohort 2- 130 node-positive melanomas; and cohort 3- a matched set of 15 patients with primary and metastatic melanomas from patients who experienced both lymph node and distant metastasis. In cohort 1, we confirmed the independent prognostic impact of PHIP copy number in a cohort of melanoma patients derived from a separate population, and validated its significant association with ulceration.In a recent study , we undePHIP copy number. PHIP copy number was significantly increased separately in intact PTEN-expressing, in NRAS-mutant, and in BRAF-mutant coupled with intact PTEN-expressing melanomas. The enrichment of PHIP in PTEN-expressing melanomas was understandable, since both proteins operate in the PI3K pathway, suggesting that either molecular aberration is sufficient to result in pathway activation. However, PHIP enrichment in NRAS-mutant melanomas was surprising, as NRAS mutations are thought to activate both the MAPK and PI3K pathways, thereby suggesting functions for PHIP outside of the PI3K pathway. The enrichment of PHIP in BRAF-mutant, PTEN-expressing melanomas was also important, as few other oncogenic drivers have been described in this well-characterized melanoma subtype.In cohort 2, we identified the molecular subtypes of melanoma with elevated PHIP copy number elevations were present in the transition from primary melanoma to lymph node and distant metastasis. Remarkably, in the cases in which triple-matched specimens were available, there was a monotonic increase in elevated PHIP copy number, such that the mean copy number  increased from 13.5% (in primary tumors) to 21.6% (in lymph node metastases) to 55.5% (in distant metastases). Key aspects of these analyses were confirmed by analyzing The Cancer Genome Atlas (TCGA) cohort.Our studies in cohort 3 assessed whether PHIP identifies it as a biomarker on many levels, as it can be used to classify molecular melanoma subtypes, or to define disease stage or severity.Our data support the notion that PHIP is clonally selected in the transition from primary to metastatic melanoma. These copy number variations are caused by chromosome instability, which is both a hallmark of cancer and associated with increased metastatic potential . The cloPHIP is overexpressed in both triple-negative and basal-like breast cancer using TCGA cohort analysis.In a separate recent study , we analA major outstanding issue concerns whether the PHIP protein is druggable. PHIP does not have enzymatic activity that can be readily targeted. The PHIP protein does contain two bromodomain motifs, one of which was recently shown to be targetable . HoweverNRAS-mutant melanoma and driver-negative tumors). In summary, PHIP is a highly versatile protein with diverse cellular functions, supporting its enrichment in distinct cancer subtypes, and representing a novel target for therapy. Additional research will be required to fulfill the promise of PHIP both as a biomarker and as a target for cancer therapy.Taken together, these studies have identified an exciting role for PHIP in the progression of melanoma and other cancers, and as a potential therapeutic target. In the biomarker realm, PHIP is one of few markers whose prognostic role has been demonstrated at both the DNA and protein levels. The enrichment of PHIP in melanoma metastases is quite unprecedented, and suggests the important survival advantage it confers to melanoma cells, resulting in the progressive copy number elevations observed in melanoma progression. Separately, the enrichment of PHIP in various molecular subtypes identifies it as a rational target for therapy of tumors in which therapeutic targeting has been difficult to achieve (e.g.,


In vivo, these signaling activities contribute to host defense against pathogens, mediate the development of neuroinflammation, influence wound repair and may facilitate cancer maintenance and progression. Here, we review the roles of FXII in innate immunity as they relate to non-sterile and sterile immune responses.Factor XII (FXII) is the zymogen of serine protease, factor XIIa (FXIIa). FXIIa enzymatic activities have been extensively studied and FXIIa inhibition is emerging as a promising target to treat or prevent thrombosis without creating a hemostatic defect. FXII and plasma prekallikrein reciprocally activate each other and result in liberation of bradykinin. Due to its unique structure among coagulation factors, FXII exerts mitogenic activity in endothelial and smooth muscle cells, indicating that zymogen FXII has activities independent of its protease function. A growing body of evidence has revealed that both FXII and FXIIa upregulate neutrophil functions, contribute to macrophage polarization and induce T-cell differentiation.  In vivo, FXIIa initiates coagulation via the fibrin-forming “intrinsic” pathway and promotes inflammation via the bradykinin (BK)-producing kallikrein kinin system comprising, high molecular weight kininogen (HK) and plasma prekallikrein (PK). Together, FXII, PK, and HK are termed plasma contact activation system. Furthermore, FXIIa may modulate components of the complement and fibrinolytic systems however to date, these activities have only been demonstrated in vitro. There has been renewed interest in FXII due to the recognition that several substances  support FXII auto-activation in vivo (F12−/−) animals are protected from thrombosis without impaired hemostasis (Factor XII (FXII) is the zymogen of serine protease, factor XIIa (FXIIa). FXII is converted to its active enzyme (FXIIa) by plasma kallikrein (PKa) or by its unique ability to auto-activate following binding to artificial or biologic surfaces . In vivo in vivo –7 and prmostasis –11. Simiin vivo F−/− animaAlthough the enzymatic activities of FXII have been thoroughly studied and the FXIIa protease domain structure is known , few zymDespite its role in pathologic thrombosis, FXII deficiency is rare in humans indicating that it may contribute to homeostatic functions. Here, we review the role of FXII in non-sterile and sterile inflammation. We focus on its differential contribution to host immune responses to infectious pathogens and contrast these to newly appreciated FXII functions in chronic, sterile inflammation.First, FXII directly binds to the surface of bacteria, fungi, viruses as well as on neutrophils and neutrophil extracellular traps (NETs) where it autoactivates  from patients with hereditary angioedema type III (HAEIII), where O-linked glycosylation at position 309 is lost and inactivation by C1 esterase inhibitor during activation by plasmin is reduced , FXII exposure of peripheral blood monocytes resulted in enhanced interleukin 1 (IL-1) activity and LPS was also essential for FXII- and FXIIa-triggered expression of IL-6 and IL-23 by splenic dendritic cells  and HK (which binds to domains II and III), compete for binding and their interaction with uPAR is mutually exclusive  zymogen FXII is able to directly influence innate immune functions, and (ii) FXII and FXIIa exert distinct and separate cellular effects.Here, we focus on how FXII- and FXIIa-mediated responses in innate and adaptive immune cells are integrated in the activation, regulation and effector mechanisms that lead to diverse pathologies and extend beyond their role in contact activation.Wound healing is an intricate process that consists of multiple phases, each of which is indispensable for proper tissue repair. Prompt initiation and resolution of each wound healing phase namely, hemostasis, inflammation, proliferation, and tissue remodeling, is critical for promoting repair and avoiding scar formation. While neutrophils play a central role in the inflammatory phase, recent evidence suggests that their prompt removal is equally important for orderly wound progression through subsequent phases of healing . Animal F12−/−) mice and this is a bone marrow-endowed function , vascular endothelial damage, and a hypercoagulable state. This conventional paradigm dramatically shifted with the observation in recent years that neutrophils significantly contribute to thrombosis, termed thromboinflammation , 79, 80.in vitro . Intravein vitro .While multiple models have demonstrated the critical role of both FXII and neutrophils in thrombosis, some exceptions seem to exist. FXII and neutrophils were recently shown to be dispensable for vascular occlusion in the large veins of the head following combined “knockdown” of natural anticoagulants, protein C and antithrombin . HoweverF12−/− mice are less prone to developing CNS inflammation and FXII worsened features of experimental autoimmune encephalomyelitis (EAE). Pharmacologic blockade of FXIIa was protective against EAE, independently of Factor XI and kallikrein-kinin generation  play a key role in the development of inflammation and tissue damage during CNS autoimmunity –85. Inteneration . Mechanineration .in vivo. Far beyond its fundamental property to autoactivate, FXII engages in complex receptor interactions that cooperatively influence immune cell behavior and contribute to physiologic and pathophysiologic responses. The details of the signaling pathways downstream of FXII are still incomplete. The challenge now is to elucidate the subtle molecular details of this versatile orchestrator and its impact on in vivo processes that determine health and disease.In summary, new perspectives have emerged that call for a reappraisal of the role of FXII TR and ES wrote and edited all versions of the manuscript.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.


Since the discovery of RNA interference (RNAi), scientists have made significant progress towards the development of this unique technology for crop protection. The RNAi mechanism works at the mRNA level by exploiting a sequence-dependent mode of action with high target specificity due to the design of complementary dsRNA molecules, allowing growers to target pests more precisely compared to conventional agrochemicals. The delivery of RNAi through transgenic plants is now a reality with some products currently in the market. Conversely, it is also expected that more RNA-based products reach the market as non-transformative alternatives. For instance, topically applied dsRNA/siRNA (SIGS – Spray Induced Gene Silencing) has attracted attention due to its feasibility and low cost compared to transgenic plants. Once on the leaf surface, dsRNAs can move directly to target pest cells  or can be taken up indirectly by plant cells to then be transferred into the pest cells. Water-soluble formulations containing pesticidal dsRNA provide alternatives, especially in some cases where plant transformation is not possible or takes years and cost millions to be developed . The ever-growing understanding of the RNAi mechanism and its limitations has allowed scientists to develop non-transgenic approaches such as trunk injection, soaking, and irrigation. While the technology has been considered promising for pest management, some issues such as RNAi efficiency, dsRNA degradation, environmental risk assessments, and resistance evolution still need to be addressed. Here, our main goal is to review some possible strategies for non-transgenic delivery systems, addressing important issues related to the use of this technology. From the earliest days of agriculture, mankind cultivated the land to feed their descendants, allowing for an increase in population growth over the years. Now, thousands of years later, modern agriculture is facing one of its biggest challenges: How are we going to produce food in a profitable, efficient, and sustainable way to feed about 10 billion people by 2050? Agricultural productivity has been facing several issues that limit crop production below its maximum potential, namely damage by insects, diseases, and competition with weeds. For instance, insects are responsible for 20 to 40% of yield loss . Moreovehttps://www.pesticideresistance.org/search.php).In an attempt to reduce the damage caused by pests, growers rely heavily on synthetic chemicals, which have been developed and applied since the 1930s. Pesticides allowed growers to increase production, improve product quality, and yield better profits. In 2012, growers around the world spent nearly $56 billion on pesticides, amounting to nearly 6 billion pounds of chemicals used in 2011 and 2012 . The high amount of chemicals used every year is leading to an increase in pesticide resistance, with a significant increase in resistance cases in insects  . RNAi, aDiabrotica virgifera virgifera is considered a milestone in the use of RNAi technology in agriculture  is a promising way to induce gene silencing in a specific pest  the delivery of dsRNA molecules or (2) the direct delivery of small RNAs (sRNAs). Currently, there are two major classes of sRNAs acting on the RNAi pathway: microRNAs (miRNAs) and small-interfering RNAs (siRNAs). MiRNAs are endogenously derived and involved in the regulation of gene expression, while siRNAs can be of exogenous origin from viruses or artificial supply , or of ei.e., via transgenic plant, foliar spray, or trunk injection), they need to enter the cell of a target organism to trigger gene silencing. This process can occur through (a) direct or (b) indirect uptake  (Although a number of RNAi pathways use dsRNAs to generate sRNAs (d siRNA) , in insed siRNA) . Once ind siRNA) . The siRd siRNA) . After ud siRNA) .Caenorhabditis elegans, the RNA-dependent RNA polymerase (RdRp) enzyme synthesizes secondary siRNAs by targeting single-stranded RNA molecules (ssRNA) and synthesizing a second strand, consequently generating dsRNA molecules and producing a systemic spread of the RNAi signaling  or by non-transformative strategies through a spray-induced gene silencing (SIGS) process (RNAi in crop protection can be achieved by plant-incorporated protectants (PIPs) through plant transformation ( process Table 2. process .Bt (Bacillus thuringiensis) toxin with RNAi for insect control was released for cultivation in Canada and a year later in the USA (Plum pox virus (PPV), took 20 years to reach the market  to control insects (8%) and diseases (27%) or to improve specific plant traits (65%), with an increase in approved events over the last years Figure 2 the USA . In genee market . Also, te market . Therefoe market .Studies are being carried out prospecting non-transformative approaches to control insects, diseases, nematodes, and weeds, and it is expected that RNAi-based products will reach the market in the form of sprayable products for foliar application, trunk injection, root dipping, or seed treatment as direct control agents . The RNAThe development of resistance is an important point regarding the use of non-transformative delivery strategies. Although dsRNAs longer than 200 nucleotides result in many siRNAs post-cleavage, maximizing the RNAi response and reducing resistance issues , in transgenic plants there is a continuous supply of dsRNA, which increases the selection pressure and favors resistance development in the population. The development of RNAi resistance may be related to a reduction in cellular uptake , mutatioNon-transformative delivery methods can be developed for use on several crops, targeting pests in different regions. Although GM event approval is more complicated, RNA-based non-transformative products will also undergo regulation procedures, although they will probably be less complicated and time-consuming than for GM plants. Also, an important aspect related to the legislation of non-transformative products is that RNA-based biopesticides will probably need to be approved in only the producing country, unlike GM plants, which needs approval in both import and export countries.Based on the advances made in the last decades regarding the use of RNAi in crop protection, it is believed that this technology will soon reach growers as dsRNA/siRNA-based products . The appN. benthamiana endogenous genes. However, in another experiment, researchers were unsuccessful in inducing gene silencing in plants through siRNA application, including spraying, syringe injection, or siRNAs infiltration, yet they achieved success through high-pressure spraying of siRNAs  is the first step to achieve good control results, determining the success of the technology usage. The correct choice of delivery system will expedite the entire process and save years of development and commercialization . Hence, the main non-transformative delivery methods and their applications in insect and disease management, shown in The delivery system varies according to the target organism and crop Table 1.For pests feeding/growing on stems, foliage, or fruit/seeds, foliar spraying may be an alternative for the delivery of RNA molecules. Thus, the RNA-based formulations are evaluated similarly to topical insecticides where the RNA solution is sprayed on leaves, fed to the target insects, and the effects are observed . Due to the chemical properties of RNAs, a short half-life is expected compared to chemical pesticides. Sprayable RNAs would therefore be an environmentally friendly alternative to synthetic pesticides , the phloem-feeding Asian citrus psyllid (Diaphorina citri) (Diaprepes abbreviates) on citrus leaves, showing a promising alternative to control these insects . In tomato leaves gently rubbed with dsRNA solution, the molecules were rapidly absorbed by tomato plants and were taken up by aphids (Myzus persicae), mites (Tetranychus urticae), and in fewer numbers, whiteflies   , and theariorum) . Hence, Botrytis  fungus produces small RNAs (Bc-sRNAs), which are delivered into plant cells, silencing host immunity genes (Botrytis cinerea DCL1 and DCL 2 (Bc-DCL1/2) onto the surface of fruits , vegetables (lettuce and onion), and flowers (roses), which resulted in the significant inhibition of grey mold disease development  gene in F. graminearum, resulting in the successful inhibition of fungal growth in local directly sprayed leaves as well as the distal non-sprayed leaves in barley plants  in tobacco, similarly to vaccines for animals that use dead or living (but weakened) microorganisms. These researchers used fragments of the virus’ genetic material to produce the “vaccines” (dsRNA) together with the plant’s immune system as a defense mechanism. This system opens a range of opportunities for the use of RNAi in a non-transformative approach in the control of viruses in crops.Co-inoculation of synthesized dsRNA to protect plants against a virus/viroid is effective at preventing virus infection in a range of plants through mechanical inoculation, thereby increasing the prospect for foliar dsRNA application in virus management in plants . RecentlBean common mosaic virus (BCMV) five days before exposure to viruliferous aphids . Syngenta scientists are also developing lines of biocontrol products based on RNAi to protect potato plants from the attack of the Colorado potato beetle (https://www.youtube.com/embed/BiVZbAy4NHw?ecver=1). These technologies will help growers to improve pest control in crops, resulting in increased yields and improved quality.The potential applications of SIGS for plant protection have had significant improvement due to the recent advances in nanoparticle technology. To overcome problems related to dsRNA stability, a double-layered hydroxide (LDH) nanoparticle was developed and combined with dsRNA molecules to yield “BioClay” . The clas aphids . The resThe use of trunk injection to deliver dsRNA to control insects has been tested and showed great progress, especially in perennial plants such as citrus. Developed citrus plants  and grapevines were treated with 2 g of dsRNA in 15 L of water solution applied by root drench and injection into the trunk, and dsRNA was taken up into whole plant systems over three months . In citrM. domestica and V. vinifera plants were restricted to the xylem vessels and apoplast, being efficiently translocated  and siRNAs delivered through petiole absorption or trunk injection to slocated . Due to carboxylesterase (Ces) and CYP18A1 genes from the brown planthopper (BPH), Nilaparvata lugens, significantly knocked down these genes, resulting in high mortality when BPH nymphs were fed on treated plants  and Vg (Vitellogenin) genes resulted in a significant reduction in gene expression, indicating that RNAi can be efficiently employed through vegetable delivery in plant-sap-feeding insects  could effectively control psyllids and leafhoppers for up to 57 days . They wed plants . This st insects . The del insects , and theMany microbes such as viruses, bacteria, yeasts, and fungi can be engineered to generate a vector for RNAi induction through the continuous production of dsRNA into the host, and this is being considered as a promising dsRNA delivery method for insect and disease management .CYP genes in Manduca sexta were targeted through the engineering of Tobacco Rattle Virus (TRV) for dsRNA delivery in Nicotiana attenuata  and observed lower fecundity and a pronounced death of crawlers after feeding on recombinant TMV-infected plants  plants with recombinant TMV-expressing RNAi effectors also resulted in a decrease in Bactericera cockerelli progeny production after feeding (Citrus tristeza virus (CTV), a common virus of citrus, with D. citri truncated abnormal wing disc (awd) RNA sequence triggering awd gene silencing after D. citri nymphs fed on infected plants, causing wing malformation and mortality in adult insects (Potato virus X (PVX) engineered with Bursicon and V-ATPase gene sequences significantly reduced the population of the cotton mealybug (Phenacoccus solenopsis) after insects fed on Nicotiana tabacum plants inoculated with the recombinant PVX (Flock house virus (FHV) to induce gene suppression through RNAi in S2 cells derived from D. melanogaster embryos and insects at the adult stage. The recombinant FHV carrying the target gene sequences caused significantly higher mortality (60–73% and 100%) than the wild type virus (24 and 71%) in both S2 cells and adult insects, respectively (Virus-induced gene silencing (VIGS) is a naturally occurring and very effective defense system that is consistent with the normal dynamics of host–pathogen interactions and which is widely harnessed as a powerful tool for the study of gene function in plants . VIGS isttenuata . Also, pe phloem . To demod plants . Similar feeding . In anot insects . The Potnant PVX . Furthernant PVX . For insectively .Helicoverpa armigera) larvae exposed to an artificial diet coated with engineered bacteria for five days showed high mortality and inhibition in the expression levels of target genes, causing drastic reductions in body weight, body length, and pupation rate , a serious insect pest of the North American forests, bacterial expression of dsRNA resulted in target-gene knockdown and a subsequent reduction in body mass and egg masses (Mythimna separate), a study showed that oral delivery of bacterially expressed dsRNA led to RNAi effects, with knockdown of target genes, reduction of body weight, and increased mortality  was observed due to the resulting knockdown of five target genes tested, which caused significant mortality and reduced body weight gain in treated beetles  commonly utilized in insect and disease management studies are costly synthetic molecules or are produced through time-consuming, laborious procedures. To overcome the shortages of these methods, the potential of delivering dsRNA expressed in bacteria has been investigated, providing an alternative method for large-scale target gene screening  strain has been used to produce large amounts of dsRNA since it lacks the enzyme that degrades dsRNAs (E. coli HT115 containing dsRNA targeting the Sugarcane mosaic virus (SCMV) coat protein gene were used in maize plants as a preventive spray and they inhibited the SCMV infection (Pepper mild mottle virus (PMMoV), PPV, and TMV to protect plants against these pathogens. The application of crude bacterial preparation via spray onto tobacco plant surfaces provided protection against infection from these viruses  from Israeli acute paralysis virus (IAPV) infection (Bombus terrestris), which upon being fed on IAPV virus-specific dsRNAs, showed decreased mortality , a dsRNA product designed to protect honeybees , enzymatically synthesized, which can be performed in vitro  , and, after all, is still naked dsRNA that under field conditions presents a shorter half-life. Thus, dsRNA formulation is a promising alternative to increase stability and boost the efficiency of gene silencing in recalcitrant species in Lepidoptera and Hemiptera, allowing plants to be protected for longer.DsRNA production costs have been dropping significantly over the last years, from ∼ $12,500 USD per gram in 2008 to less than $60 USD per gram in 2018 , with anin vitro  or in vi dsRNAs) . AlthougS. exigua , where liposome encapsulation was not efficient to protect the dsRNA, leading to inefficient RNAi in this species  clay nanosheet, provided high dsRNA stability under field conditions, increasing the residual period of dsRNA on plants and protecting them from virus infection for up to 30 days compared to naked dsRNA . Guanyla. exigua  and Spodugiperda , and theugiperda  and Blatugiperda . In Euched dsRNA . However species .E. coli bacteria, allowing for the mass production of encapsulated ready-to-spray dsRNA . This technology is based on bacteria engineered with a plasmid to produce naturally occurring proteins such as capsids, which are then co-transformed with another plasmid coding for the target dsRNA or siRNA together with a sequence called the “packing site”. The double-transformed E. coli are then purified, resulting in self-assemble particles that have encapsulated the desired RNAs. These particles protect the RNAs and enhance resistance to adverse environmental conditions, and, once sprayed, they are expected to be taken up by the insect rapidly  developed a system where APSE RNA Containers (ARCs) are produced by  rapidly . The devT. vaporariorum due to the low dsRNA uptake by the insects , Tribolium castaneum (Coleoptera), A. pisum (Hemiptera), and M. sexta (Lepidoptera), resulting in target gene silencing with no effects over non-target species  from the National Council for Scientific and Technological Development (CNPq) in Brazil. ES is a recipient of a scholarship from the Coordination for the Improvement of Higher Education Personnel (CAPES) in Brazil. The Foundation Research-Flanders (FWOVlaanderen) in Belgium, the EUCLID project (No. 633999) and the COST (European Cooperation in Science and Technology) under grant agreement No. CA15223 also supported this work.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.


The term metastasis is widely used to describe the endpoint of the process by which tumour cells spread from the primary location to an anatomically distant site. Achieving successful dissemination is dependent not only on the molecular alterations of the cancer cells themselves, but also on the microenvironment through which they encounter. Here, we reviewed the molecular alterations of metastatic gastric cancer (GC) as it reflects a large proportion of GC patients currently seen in clinic. We hope that further exploration and understanding of the multistep metastatic cascade will yield novel therapeutic targets that will lead to better patient outcomes. Gastric cancer (GC) is the fourth most common cancer and second leading cause of cancer-related deaths worldwide . Over 70Metastasis is the main cause of cancer mortality >90%) and a critical step that hampers the development of anti-cancer therapy due to its systemic nature and resistance to existing therapeutic drugs , 7. Meta0% and a GC most commonly metastasises to the liver, peritoneum, lung, bone and lymph nodes  either tLocal invasion occurs when tumour cells no longer obey the delineation of the basement membrane (BM), and the invasive front infiltrates the neighbouring tumour-associated stroma and surrounding normal tissues. Three major players facilitate this process: epithelial–mesenchymal transition (EMT), matrix metalloproteinases (MMPs) and the stromal environment, within which alterations and interactions amongst various molecular processes determine the tumour cells’ invasive propensity .EMT describes the dissociation of tightly knitted epithelial cells and subsequent transdifferentiation into motile and invasive mesenchymal cells . In the In GC, a number of signalling pathways have been found to regulate EMT, with the PI3K/AKT, MEK/ERK and WNT/β-Catenin pathways taking leading roles  and erythropoietin-producing hepatocellular A2 (EphA2) which belongs to hydroxylase superfamily and protein-tyrosine kinase family, respectively, have both been suggested to induce EMT via the Wnt/β-catenin signalling pathway and further stimulate GC development and metastasis , 38. AnoThe BM is an important regulator of cellular behaviour in addition to its passive role in supporting surrounding tissues . In cancHelicobacter pylori infection can stimulate the upregulation of MMP-1, which could further enhance the potential of GC metastasis [MMP-1 is an interstitial collagenase that plays a role in the degradation of type I collagen (a major ECM component of stomach mucosa) –52. One tastasis .MMP-2 and MMP-9 belong to the family of type IV collagenases or gelatinases. Both have been reported to contribute to vessel invasion and lymph node metastasis in intra-mucosal GC by degrading type IV collagen, which enabled infiltration of lymph capillaries , 53. CerMMP-7 (matrilysin) is another MMP that is highly expressed in GC , 58. It MMP-11, also known as Stromelysin-3, is distinct from other MMPs as it can only weakly degrade the main components of ECM. Additionally, it needs to be proteolytically cleaved and activated intracellularly by Furin-like serine proteinases prior to its relocalisation to the cell membrane , 66. MMPMMP-14 belongs to one of the six membrane-anchored MMPs, unlike the majority which are secreted proteins . MMP-14 While most of the studies thus far focus on cancer cells-derived MMPs, emerging evidence indicate that MMPs (including MMP-2 and MMP-9) can also be secreted by the surrounding stromal cells, such as endothelial cells, fibroblasts, myofibroblasts and inflammatory cells . These fWhen cancer cells reach the surrounding stroma following EMT and BM penetration, the next step involves overcoming the barriers to allow further infiltration. Recent studies revealed that tumours function as a complex multicellular organ composed of both cancer cells and tumour stroma with significant interactive cross-talks . It is tStromal cells can heighten the aggressiveness and invasiveness of cancer cells through different molecular signalling pathways. For instance, interactions between neutrophils and MSCs via an IL-6–STAT3 axis lead to neutrophil activation and MSCs differentiation into CAFs, which provide a pro-inflammatory habitat. These stromal cells in turn collaboratively induce angiogenesis and invasiveness of GC cells to stimulate metastatic dissemination . FurtherAngiogenesis represents a tumour response to the hypoxic and nutrient-deficient environment driven by uncontrolled cellular proliferation and consequent explosive enlargement of tumour bulk , 81. ThiRecent studies have shown that the growth of lymphatic vasculature, also known as lymphangiogenesis, either around the tumour or in the sentinel lymph nodes, is associated with increased incidence of lymphatic metastasis , 86. In Collectively, these findings provide evidence that interactions between cancer cells and the tumour-associated stromal microenvironment could establish a potential positive-feedback loop, which provides substantial contributions to GC progression and metastasis. Accordingly, it is reasonable to hypothesise that tumour malignancy may be suppressed or even reversed by normalising the stromal environment.During the path of local invasion, cancer cells may encounter blood vessels or lymphatics to facilitate movement towards distant pre-metastatic niches. Alternatively, they may reach and penetrate beyond the serosa to initiate intraperitoneal seeding or direct invasion into neighbouring organs. Here we focus on intravasation which describes the process in which cancer cells gain access into the tumour-associated vasculatures located in the gastric submucosa , 43, 93.Intravasation can be accelerated by molecular alterations that improve the potency of cancer cells in transendothelial invasion. Accumulating evidence has shown the positive correlation among vascular invasion, intratumoral angiogenesis and distant metastasis , 95. ForApart from its role as passive channels for tumour cell dissemination, emerging evidence also illustrated that lymphatic vessels actively stimulate recruitment of tumour cells to lymph nodes, immune regulation and cancer cell survival , 89. TheIn addition to distant metastasis, ~10–20% of GC patients were found to harbour peritoneal metastasis that have likely arisen from exfoliated cancer cells through penetration of the gastric serosa , 98, 99.Following successful intravasation into the circulation, the disseminating cancer cells, now termed circulating tumour cells (CTCs), must survive the precarious microenvironment en route to new sites of dissemination. The exposure to blood introduces stressors such as haemodynamic shear forces and recognition by the innate immune system. Furthermore, CTCs must also gain the ability to survive in the absence of substratum –102.The concept of 'anoikis' represents a form of programmed cell death triggered by loss of ECM attachment in epithelial cells . AnoikisAuxiliary pro-metastatic signals exist during intravascular transition in the circulation to aid cancer metastasis. Emerging evidence has shown that the interaction between platelets and cancer cells, more specifically the formation of emboli, are constructive in priming CTCs for intravascular survival , 109–111Despite the theoretical possibility that CTCs can be deposited at any metastatic niche within or surrounding both circulation systems, clinical observations have shown that certain cancer types have a higher probability of giving rise to metastasis in certain target organ(s) because of exosome-initiated pre-metastatic niches formation. For example, GC tends to form distant metastasis in the liver, peritoneum, lung, bone and lymph nodes . Two hypExtravasation represents the exiting of circulating cancer cells out of the vessel lumen to establish new sites of metastasis. There are two recognised forms of extravasation dependent on vessel diameter. Firstly, CTCs with adhesive molecules on the surface can attach to and penetrate the endothelium of the vessel walls irrespective of vessel size . AlternaNaturally, factors that promote vasculature permeability are associated with increased extravasation. Calponin h1, an actin-binding protein which is mainly expressed in smooth muscle cells, plays a role in stabilising the actin filament system. Calponin h1 deficiency can induce the fragility of blood vessels and peritoneum, leading to the increased incidence of extravasation and tumour metastasis . AccumulFurthermore, studies focusing on targeting extravasation has led to the discovery of a double anti-angiogenic decoy receptor, double anti-angiogenic protein (DAAP), which simultaneously targets VEGF-A and angiopoietins to block tumour-associated angiogenesis and vascular leakage . Hence tExosomes are membranous nanoparticles 40–50 nm in diameter and they can be released by both tumour cells and surrounding stromal cells, which will interact reciprocally to modulate immune responses, remodel tumour microenvironments and facilitate cancer metastases –125. TheGC-derived exosomes can modulate immune responses. For example, GC-derived exosomes can stimulate macrophages to generate a pro-inflammatory microenvironment via activation of nuclear factor κB (NFκB) signalling pathway, resulting in increased cell proliferation and migration . SimilarIn terms of tumour microenvironment remodelling and cancer metastasis, mounting evidence indicates that GC-derived exosomes can initiate or accelerate pre-metastatic formation , 129. FoNevertheless, researchers are utilising the unique features of exosomes for drug delivery. Exosomes are loaded with drugs or siRNA to target the tumour regions , 134. ExDespite intensive efforts, limitations still exist in the study of exosomes as they are mainly restricted to in vitro co-culture or in vivo injection using labelled-exosomes, which is markedly different from their physiological location and concentration . These cGiven the divergent microenvironment of the metastasised sites from that of the stomach, successfully extravasated cells need to adapt to the foreign microenvironments in order to survive and colonise. Currently, there are two universally acknowledged mechanisms by which cancer cells adapt to their new microenvironment: (1) cell autonomous programmes and (2) non-autonomous programmes .For cell autonomous programmes, disseminating tumour cells (DTCs) acquire molecular alterations to increase their colonising ability. Colonising abilities are normally evaluated by detecting pulmonary metastases after intravenous (IV) injection in immune-deficient mice. For example, ectopic expression of RUNX3 repressed lung colonisation of GC cells in nude mice . SimilarHowever, clinical observations showed that relapses are often detected long after removal of the primary tumour, spanning from months to years even when there was no previous evidence of metastasis . This imLatency represents a state in which metastatic cancer cells undergo proliferative quiescent in order to escape from immune clearance, attack from the new microenvironment and the surrounding growth inhibitory signals. They remain latent until certain, currently unknown factors re-activate their proliferative potency. At present, our knowledge of the underlying mechanisms of latent metastasis is limited due to the lack of mouse models that faithfully recapitulates the metastatic process and microenvironment.Over the past decades, research progress on GC metastasis-related molecular alterations has provided valuable knowledge for deciphering this complex biological phenomenon. Although by no means comprehensive, we have rapidly gained an appreciation for the importance of stromal cells and the microenvironment. Nevertheless, due to the complexity and systemic nature of metastasis, a number of fundamental questions concerning the mechanisms of GC metastasis remain unanswered.The major hurdle in the study of tumour metastasis is the lack of a mouse model with a competent immune system that can perfectly mimic the entire metastatic cascade. Therefore, this bottleneck imposes restrictions on in-depth study of the latter stages in the GC metastatic cascade. Attempts to establish a better metastatic mouse model have recently achieved intriguing progress, such as the implementing genome-wide or high-throughput screening approaches into immune-competent mice for identification of novel regulators of metastases , 146. InMetastatic cancer cells that have successfully intravasated into the circulation system can survive and extravasate efficiently (>80%) . This phMechanistically, apart from the widely reported function of MMPs in the degradation of ECM, recent studies have revealed alternative roles of MMPs in metastasis, such as regulation of growth signals, apoptosis, tumour vasculature, inflammation and non-proteolytic functions . MoreoveWe hope that our understanding of the evolution of cancer metastasis continues to excel at this impressive pace, and that some of these findings will be translated into clinical use, especially in light of the current proportion of patients with metastatic GC.Neither the submitted paper nor any similar paper, in whole or in part, other than an abstract or preliminary communication, has been submitted to or published in any other primary scientific journal. All the authors are aware of and agree to the content of the paper, copyright assignment and authorship responsibility.


Observer-driven pattern recognition is the standard for interpretation of medical images. To achieve global parity in interpretation, semi-quantitative scoring systems have been developed based on observer assessments; these are widely used in scoring coronary artery disease, the arthritides and neurological conditions and for indicating the likelihood of malignancy. However, in an era of machine learning and artificial intelligence, it is increasingly desirable that we extract quantitative biomarkers from medical images that inform on disease detection, characterisation, monitoring and assessment of response to treatment. Quantitation has the potential to provide objective decision-support tools in the management pathway of patients. Despite this, the quantitative potential of imaging remains under-exploited because of variability of the measurement, lack of harmonised systems for data acquisition and analysis, and crucially, a paucity of evidence on how such quantitation potentially affects clinical decision-making and patient outcome. This article reviews the current evidence for the use of semi-quantitative and quantitative biomarkers in clinical settings at various stages of the disease pathway including diagnosis, staging and prognosis, as well as predicting and detecting treatment response. It critically appraises current practice and sets out recommendations for using imaging objectively to drive patient management decisions. Biomarkers derived from medical images inform on disease detection, characterisation and treatment response.Quantitative imaging biomarkers have potential to provide objective decision-support tools in the management pathway of patients.Measurement variability needs to be understood and systems for data acquisition and analysis harmonised before using quantitative imaging measurements to drive clinical decisions.Interpretation of medical images relies on visual assessment. Accumulated and learnt knowledge of anatomical and physiological variations determines recognition of appearances that are within “normal limits” and allows a pathological change in appearances outside these limits to be identified. Observer-driven pattern recognition dominates the way that imaging data are used in routine clinical practice Fig. . A semi-In some clinical scenarios, automated quantitation may be more objective and accurate than manual assessment; thresholds can be applied above or below which a disease state is recognised and subsequent changes interpreted as clinically relevant . Unlike The need for absolute quantitation (versus semi-quantitative assessment) in decision-making should be clearly established. Absolute quantitation is demanding and resource intensive because hardware and software differences across centres and instrumentation and their evolution impact the quality of quantified data. Rigorous on-going QA and QC are essential to support the validity and clinically acceptable repeatability of the measurement, and efforts are on-going within RSNA and the ESR and other academic societies. Critically also, definitive thresholds to confidently separate normal from pathological tissues based on absolute quantitative metrics often do not have wide applicability or acceptance.18FDG PET/CT studies use the Deauville scale (liver and mediastinum uptake as reference) as the standard for response assessment in lymphoma [Semi-quantitative readouts of scores based on an observer-recognition process are widely used because visual interpretation often has proven adequate and is linked to outcome. For example, MRI scoring systems for grading hypoxic-ischaemic injury in neonates using a combination of T1-weighted (T1W) imaging, T2-weighted (T2W) imaging and diffusion-weighted imaging (DWI) have shown that higher post-natal grades were associated with poorer neuro-developmental outcome . In cervlymphoma . Semi-qulymphoma , thyroidlymphoma  and bladlymphoma  tumours.lymphoma  and gradlymphoma . Manual lymphoma  and predlymphoma . These plymphoma .The simplest quantitative measure used routinely is size. Size is linked to outcome in both non-malignant and malignant disease . VentricQuantitative imaging biomarkers that characterise tissue features  can provide information that characterises a disease state and reflects histopathology. Multiple quantitative features can be incorporated into algorithms for recognising disease and its change over time . This involves an informatics style approach with data built from atlases derived from validated cases. Curation of anatomical databases annotated according to disease presence, phenotype and grade can then be used with the clinical data to build predictive models that act as decision-support tools. This has been proposed for brain data  but requQuantitation in ultrasound imaging has derived parameters related to cardiac output (left ventricular ejection fraction), tissue stiffness (elastography) and vascular perfusion (contrast-enhanced ultrasound) where parameters are related to blood flow. Ultrasound elastography is an emerging field; it has been shown to differentiate liver fibrosis , benign CT biomarkers are dependent on a single biophysical parameter, differential absorption of X-rays due to differences in tissue density, either on unenhanced scans or following administration of iodine-based contrast agent, which increases X-ray absorption in highly perfused tissues. Other developments have utilised tissue density as a parameter in multicentre trials for quantification of emphysema (COPDGene and SPIROMICS) –57 and iMRI is more versatile than US and CT because it can be manipulated to derive a number of parameters based on multiple intrinsic properties of tissue  and how these are altered in the presence of other macromolecules  and externally administered contrast agents (Gadolinium chelates). Perfusion metrics have also been derived with arterial spin labelling, which does not require externally administered agents . The app18FDG PET/CT studies is mainly performed by standardised uptake values (SUVs), although other metrics such as metabolic active tumour volume (MATV) and total lesion glycolysis are being introduced in studies and the clinic [18FDG activity), physical  and physiological factors (FDG kinetics and patient biology/physiology) [Quantitation of e clinic , 95. Thesiology) . To mitisiology) , 97 and siology) . Newer tRadiomics describes the extraction and analysis of quantitative features from radiological images. The assumption is that radiomic features reflect pathophysiological processes expressed by other “omics”, such as genomics, transcriptomics, metabolomics and proteomics . HundredUnlike biopsies, radiomic analyses, although not tissue specific, capture heterogeneity across the entire volume , potentiAutomated quantitative assessments rather than scoring systems are easier to incorporate into artificial intelligence systems. For this, threshold values need to be established and a probability function of the likelihood of disease vs. no disease derived from the absolute quantitation (e.g. bone density measurements) . AlternaFor assessing treatment response Table , the keyTo become clinically useful, biomarkers must be rigorously evaluated for their technical performance, reproducibility, biological and clinical validity, and cost-effectiveness . Table 4Technical validation establishes whether a biomarker can be derived reliably in different institutions (comparability) and on widely available platforms. Provision must be made if specialist hardware or software is required, or if a key tracer or contrast agent is not licensed for clinical use. Reproducibility, a mandatory requirement, is very rarely demonstrated in practice  because Once a biomarker is shown to have acceptable technical, biological and clinical validation, a decision must be made to qualify the biomarker for a specific purpose or use. Increasingly, the role of imaging in the context of other non-imaging biomarkers needs to be considered as part of a multiparametric healthcare assessment. For example, circulating biomarkers such as circulating tumour DNA are often more specific at detecting disease but do not localise or stage tumours. The integration of imaging biomarkers with tissue and liquid biomarkers is likely to replace many traditional and more simplistic approaches to decision-support systems that are used currently.The cost-effectiveness of a biomarker is increasingly important in financially restricted healthcare systems where value-based care is increasingly considered . However


Our understanding of the molecular basis of aging has greatly increased over the past few decades. In this review, we provide an overview of the key signaling pathways associated with aging, and whose modulation has been shown to extend lifespan in a range of model organisms. We also describe how these pathways converge onto autophagy, a catabolic process that functions to recycle dysfunctional cellular material and maintains energy homeostasis. Finally, we consider various approaches of therapeutically modulating these longevity pathways, highlighting exercise as a potent geroprotector. In the past two decades, the molecular signatures of aging have been started to be uncovered. A remarkable conservation of these cell signaling pathways has been shown across various invertebrate and vertebrate species Kenyon, . AutophaCaenorhabditis elegans revealed a general decline in activity in various tissues, including the intestine and neurons -1 Receptor in this organism] live significantly longer than their wild-type counterparts. They demonstrated that RNAi-mediated knockdown of the autophagy gene bec-1 significantly reduced the lifespan of the daf-2 mutants, clearly identifying autophagy as a process that is required for the increased longevity of this mutant , AMP-activating protein kinase (AMPK), and Sirtuin pathways Kenyon, . In thisC. elegans worms with a loss-of-function mutation in their daf-2 gene experienced a >2-fold extension in lifespan compared to wild-type. Inhibition of this pathway in vertebrate models has also been shown to extend lifespan, but to a lesser degree and in a more inconsistent manner. Female 129/SvPas mice heterozygous for the IGF-1 receptor null allele (Igf1r+/−) have been shown to live significantly longer (33%) than wild-type females, while male mutant mice demonstrated no such lifespan enhancing benefits  increase in lifespan compared to wild-type controls  experienced significant extensions in lifespan  pathway was the first pathway to be shown to affect aging  and IGF-1 levels  and a supply of amino acids  . Although a potent promoter of autophagy, Rapamycin treatment is associated with various negative side-effects, including immunosuppression and the potential induction of insulin resistance via off-target inhibition of mTORC2  is another intervention that has also been shown to promote longevity and beneficially modulate the previously described longevity pathways Kenyon, . AlthougAn important future research direction is a thorough assessment of the relative contribution of each of the pathways discussed in this review to autophagy regulation in individual organs and tissue systems. Significant effort has been made to show that organ-specific blockade of autophagy has deleterious effects, including skeletal muscle (Carnio et al., In summary, autophagy has convincingly been shown to play a pivotal role in healthspan and lifespan extension. In this review, we have discussed various cell signaling pathways whose modulation has been shown to have beneficial effects on longevity, and how autophagy is a necessary mediator of these effects. We have also presented current pharmaceutical therapies, exercise and dietary restriction as effective ways to modulate many of these pathways to increase or preserve autophagic activity, thereby acting as a potent geroprotector.JW, AB, and DL wrote the manuscript.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.


Lysosomal diseases (LDs), also known as lysosomal storage diseases (LSDs), are aheterogeneous group of conditions caused by defects in lysosomal function. LDsmay result from deficiency of lysosomal hydrolases, membrane-associatedtransporters or other non-enzymatic proteins. Interest in the LD field isgrowing each year, as more conditions are, or will soon be treatable. In thisarticle, we review the diagnosis of LDs, from clinical suspicion and screeningtests to the identification of enzyme or protein deficiencies and moleculargenetic diagnosis. We also cover the treatment approaches that are currentlyavailable or in development, including hematopoietic stem cell transplantation,enzyme replacement therapy, small molecules, and gene therapy. Lysosomes are membrane-bound organelles, which contain, among other components,hydrolytic enzymes that operate in an acidic environment . Lysosome.g., alpha-galactosidase inFabry disease). Alternatively, LDs may be caused by deficiencies in lysosomalmembrane-associated transporters or other non-enzymatic proteins .According to the WORLDSymposia® official list of lysosomal diseases, 66 clinicalconditions related to 53 distinct genes are recognized as LDs , which partiallyexplains the involvement of different organs. Issues with the targeting of enzymesto lysosomes, defective membrane proteins, and abnormal excretion of substrates mayalso cause lysosome enlargement and functional impairment. Thus, the wide range ofsymptoms in LD may be explained by the activation of several deleterious processes,such as the release of acid hydrolases into the cytoplasm causing cellular damage,the dysregulation of apoptosis or the abnormal accumulation of lipids causingdefective transport of substrates into and out of the lysosomes.LD symptomatology depends on the stored substrate and organs affected by thisaccumulation. Usually, substrate accumulation occurs in the organs where they aresynthesized  can be carried out either before or simultaneously with theenzyme activity measurement. A biomarker is generally an analyte that indicatesthe presence and/or extent of a biological process, which is in itself usuallydirectly linked to the clinical manifestations and outcome of a particulardisease , allow theidentification of biomarkers but with low sensitivity and specificity. Forinstance, thin-layer chromatography of oligosaccharides is generally used butshould soon be replaced by quantitative methods . For quaFor Fabry disease (FD), the analysis of globotriaosylsphingosine (lyso-Gb3) ispreferable to globotriaosylceramide (Gb-3), because there is not a clearcorrelation between Gb3 levels and the clinical manifestation or severity of thedisease. DBS provides a convenient, sensitive, and reproducible source tomeasure lyso-Gb3 levels for diagnosis, initial phenotypic assignment, andtherapeutic monitoring in patients with FD  is chitotriosidase (ChT). ChTactivity has been shown to correlate well with various clinical parameters andhas been used to monitor and adjust the treatment, despite being not specificfor GD. It should be mentioned that there are a significant number ofindividuals in the general population (~1:20) with low chitotriosidase activitydue to a common polymorphism. One alternative option for these cases is thepulmonary and activation-regulated chemokine (CCL18/PARC)  has been suggested as a biomarker for thepresence and progression of Krabbe disease (KD). PSY can be analyzed in blood,DBS, and cerebrospinal fluid. The psychosine concentration in patients with theinfantile form of KD is at least four-fold higher than in asymptomatic newbornswith low galactosylcerebrosidase activity, and nearly one order of magnitudegreater than in healthy newborns (For mucopolysaccharidoses (MPS), the investigation can start with urinaryscreening tests. Glycosaminoglycans (GAGs) are the most common and widely usedbiomarkers for MPS and several qualitative and quantitative methods have beenused to-date . Sensitivity and specificityof the dye-spectrometric and TLC methods are not sufficient to detect all typesof MPS, especially MPS III and MPS IV -hydroxycholesterol, which is reduced in the plasmaand cerebrospinal fluid (CSF)  and Niemann-Pick C (NP-C)], but is not a reliablebiomarker due to the overlap between the levels observed in patients and healthycontrols. Lysosphingomyelin (lyso-SPM) levels in DBS seem to be a goodalternative to sphyngomyelin. However, in patients with ASMD deficiency,lyso-SPM concentration does not correlate with the amount of residual enzymeactivity in DBS or with patient age. The analysis of chitotriosidase or filipinstaining of free cholesterol in fibroblasts lacks sensitivity and specificityfor NP-C detection (Tetrasaccharide glucose (Glc4) is the most well-known biomarker for Pompe disease(PD) but is not specific, and its use for diagnostic purposes may be limited, chorionic villi, amniotic fluid, and cultured amniocytes, among others.Most enzymatic assays for LD research rely on spectrofluorometry, which usesenzyme-specific substrates with a fluorogenic radical to generate a fluorophoreproduct that will absorb energy at a specific wavelength and then emit it atanother longer wavelength to determine the quantity of product produced.Spectrophotometry is also a widely used technique based on chromophores thatexcite themselves and emit colors depending on the energy released by the changefrom the basal to the excited state .Precise molecular diagnosis (MD) is of great importance for LDs, not only toconfirm the enzymatic diagnosis but also to ascertain a definitive diagnosis incomplex situations.e.g.,multiple sulfatase deficiency and mucolipidoses II and III,), and forconfirmation of the diagnosis in X-linked conditions where the specific enzymedeficiency is not informative , due to the overlap of theenzyme activities with the normal controls range. In addition, MD is essentialfor confirmation of the diagnosis in cases where the functional defect does notinvolve an enzyme deficiency  . Moreover, MD isuseful for those showing multiple enzyme deficiencies (disease) .MD can be made using DNA or RNA and utilizes a range of different molecularapproaches, such as Sanger sequencing, restriction fragment length polymorphism(RFLP) analysis, amplification-refractory mutation system (ARMS), multiplexligation-dependent probe amplification (MLPA), real-time PCR, andhigh-resolution melting. Currently, massive parallel sequencing technology, alsoknown as next-generation sequencing (NGS), allows for the sequencing of largegenomic regions in a short time period at relatively low cost, replacing thetraditional analysis of individual genes and exon-by-exon sequencing. NGSapplications include the sequencing of PCR-amplified genomic regions,whole-exome sequencing (WES), and whole-genome sequencing (WGS).DNA sequencing is the primary clinical technique to identify mutations in LDs,but sequencing often does not detect intragenic or whole-genedeletions/duplications. Therefore, comparative genomic hybridization (CGH) usingoligonucleotide arrays has been implemented in cytogenetic and moleculardiagnostic laboratories as a robust, rapid, and sensitive assay for detectingtargeted gene deletions , although it has been consideredas a complementary approach in some cases. The use of biomarkers is also notmoving forward as a first-tier option for NBS of LDs, either because theanalysis time per sample is too long for high-throughput NBS, or due to the highfalse-positive rates (3–5% in some reports). However, these methods are expectedto be extremely valuable for second-tier analyses (in positive cases identifiedby enzyme activity assay), especially when the same DBS can be used, which wouldavoid patient recall and parental anxiety. As an example, for metachromaticleukodystrophy, the most promising approach for NBS is the analysis ofsulfatides in DBS by MS/MS, since arylsulfatase A cannot be measured in DBS andits pseudodeficiency is quite common (Hematopoietic stem cell transplantation (HSCT) is a known treatment for LDs dueto the remarkable properties of self-renewing enzyme-producing cells to secretethe deficient enzyme and colonize enzyme-deficient tissues, allowing constantintercellular enzyme exchange. In this cross-correction process, secretedenzymes can be taken up from the reticuloendothelial system by deficient cellsvia the mannose-6-phosphate (M6P) or mannose receptors and transported tolysosomes where substrates can be properly degraded  is the fact that donorcells can cross the blood-brain barrier (BBB) and thus improve neurologicalimpairment. However, CNS repopulation is a very slow process, which usuallytakes several months to occur, thus justifying the need for early HSCT toimprove CNS impairment . FinallyLysosomes are cytoplasmic organelles that contain a variety of hydrolases. Agenetic deficiency in the enzymatic activity of one of these hydrolases willlead to the accumulation of the material meant for lysosomal degradation .ERT was first successfully administered to humans with LDs over 25 years ago andwas the first therapy that directly addressed the underlying mechanism causing agenetic disease. ERT is based on the intravenous infusion of a recombinantenzyme , which is taken up into the cell throughmembrane receptors  and replaces thecatalytic action of the missing or non-functional lysosomal enzyme .GD was the first LD for which the recombinant human β-glucocerebrosidase enzymewas developed and approved by the FDA in 1991 . ERT hasERT represents a major advancement in the treatment of genetic disorders.However, the development and implementation of large-scale ERTs has unmaskedseveral challenges in the treatment of LDs . RecombiN-acetylglucosaminidase to the CNS , which acts in patients withamenable mutations by stabilizing the enzyme alpha-galactosidase andfacilitating lysosomal trafficking. It has been shown to decrease the leftventricular mass index and reduce the incidence of renal, cardiac orcerebrovascular events  are emerging as the vector of choice forex vivo approach). A notable accomplishment, thisapproach was tested by injecting modified cells carrying the Arylsulfatase A(ARSA) gene to prevent disease manifestations in ninepatients with infantile metachromatic leukodystrophy. ARSAexpression levels were restored, and eight patients (89%) had no diseasesymptoms  and transplanting these cells to thepatient (Based on the promising results from these initial trials, new studies andapproaches are currently being tested. These studies include new technologies inpreclinical and clinical stages, such as genome editing (Although they account for less than 1% of hereditary diseases, LDs have gainedsignificance exceeding this proportion by concentrating a large number of successfulexamples of treatments for genetic conditions. Hematopoietic stem celltransplantation, enzyme replacement therapy, substrate reduction therapy, andpharmacological chaperones, are some of the approved treatment modalities that arebenefiting thousands of LD patients around the world. The treatment of CNSmanifestations is still a major challenge, but the administration of ERT to thebrain via Trojan horses or IT/ICV, as well as gene therapy/gene editing strategies,should change this picture in the near future. Patients are usually identified afterclinical suspicion, in most cases, through the identification of specific enzymedeficiencies associated with the majority of these conditions. There are a growingnumber of sensitive and specific biomarkers being reported that could help to screenfor these conditions, support the diagnosis, and provide useful information fortreatment monitoring. The development of high-throughput methods, especially basedon the use of DBS, is making newborn screening feasible for several LDs. Thecombination of early diagnosis with effective therapies is bringing practicalalternatives and hope for patients and families affected by LDs.


In recent years, the development of metamaterials and metasurfaces has drawn great attention, enabling many important practical applications. Focusing and lensing components are of extreme importance because of their significant potential practical applications in biological imaging, display, and nanolithography fabrication. Metafocusing devices using ultrathin structures  with superlensing performance are key building blocks for developing integrated optical components with ultrasmall dimensions. In this article, we review the metamaterial superlensing devices working in transmission mode from the perfect lens to two-dimensional metasurfaces and present their working principles. Then we summarize important practical applications of metasurfaces, such as plasmonic lithography, holography, and imaging. Different typical designs and their focusing performance are also discussed in detail. Therefore, it is of great scientific and application significance to break through the diffraction limit and achieve super-resolution focusing and imaging effects. Some information of the subwavelength structure (the object) is hidden in the evanescent wave. The intensity decays rapidly with increasing distance from the object and the attenuation speed is proportional to the spatial frequency. Recently, more metamaterial-enabled technologies have been reported, including computing metastructures [In recent years, surface plasmons and related devices ,21,22,23ructures , near-zeructures , curviliructures , acoustiructures , and so ructures ,85. Although Veselago first proposed a negative index medium in 1968 , a new cDue to the diffraction limit, the imaging resolution of conventional optical lenses can only reach half of the wavelength of the incident light. By recovering and enhancing the high-frequency evanescent wave carrying detailed information of the object, perfect lenses based on metamaterials with negative refractive indices are expected to break through this optical diffraction limit and achieve super-resolution imaging. Due to the loss of light waves propagating in metal, how to enhance the high-frequency evanescent wave signal more efficiently and convert it into a propagating wave to participate in imaging to better achieve far-field super-resolution imaging and how to further increase the near field ultra-high resolution focused spot depth and reduce the far field focused spot size are the focus of further research on surface plasmon-enabled lenses. Pendry first proposed the perfect lens theoretically using negative index media , which cAs shown in After achieving the near-field superlensing effect, how to further project the near-field image into the far-field becomes critical. A hyperlens  can realize far-field super-resolution imaging with low losses, which has shown notable potential for real-time biomolecular imaging and nanolithography. Although the silver film can dramatically improve the imaging quality of nanostructures and achieve super-resolution imaging, its working distance is very small and can only be limited to the near field region since the evanescent wave rapidly decays away from the surface of the silver film superlens. Liu et al. further proposed and experimentally verified a hyperlens design that can achieve far-field super-resolution imaging effect . The neaThe main limitation for current superlens and hyperlens designs is that most of them need very complex designs and some of them have very limited functional wavelength range. Simple designs with dynamic engineering functionalities are desirable. To simplify lens designs with acceptable cost and test their working principles, different analytical models have been used to verify the superlensing mechanism, such as finite difference time domain (FDTD) method and finite element method (FEM). However, these commercial software packages have inevitable limitations and disadvantages such as inherent mismatch due to frequency dispersive and time discretization . TherefoIn this section, we review recent interesting focusing devices triggered by metasurfaces which have been well studied and the most important advantage of these metasurface-enabled devices over bulky optical components is that they are highly convenient for integration with electronics. Furthermore, these thin metasurface designs can generate geometric phase changes and enable more interesting artificial properties that natural materials do not possess. The basic working mechanism is that gradient metasurfaces can introduce space-variant phase (Pancharatnam–Berry phase) changes and, therefore, they can provide many new features ,108,109 V-shaped nanoantennae metasurfaces were first reported by Yu and coworkers . After tThe measured intensity distributions b also inFurthermore, a reconfigurable metasurface lens has been experimentally demonstrated with stretchable polydimethylsiloxane (PDMS) substrates . The refTo further investigate the near field properties of metasurfaces, Spektor and coworkers  employedx) nanoposts which can function as truncated multimode waveguides. Moreover, wavefront shaping using a disorder-engineered metalens can enable access to a wide optical space and stable optical focusing function with high-quality.Another important application of a metalens is for biological imaging thanks to their unique capability of wavefront shaping. Most recently, Jang and co-workers experimentally demonstrated a disorder-engineered metalens which can enable the individual input–output responses . Note thTo conclude, we have reviewed the development of superlensing and metafocusing devices and their potential applications. Due to the recent development of metasurfaces, as well as the exploration of new optical functions, dynamic plasmonic lensing devices and more practical achievements have been developed, paving the way for new plasmonic imaging and focusing technologies. The current developing trends for actively tunable metalens seem to be towards higher modulating speed and large-scale manufacturing or even massive production. However, researchers still need to investigate the feasibility to achieve higher resolution and better optical performance since the main limitations of the metadevices are still challenging, which may need great efforts and investigations, such as developing adaptive metalenses, which can manipulate focal length, astigmatism, and shift simultaneously . In addi


Biological neural networks produce information backgrounds of multi-scale spontaneous activity that become more complex in brain states displaying higher capacities for cognition, for instance, attentive awake versus asleep or anesthetized states. Here, we review brain state-dependent mechanisms spanning ion channel currents  to the dynamics of brain-wide, distributed, transient functional assemblies . Not unlike how microscopic interactions between molecules underlie structures formed in macroscopic states of matter, using statistical physics, the dynamics of microscopic neural phenomena can be linked to macroscopic brain dynamics through mesoscopic scales. Beyond spontaneous dynamics, it is observed that stimuli evoke collapses of complexity, most remarkable over high dimensional, asynchronous, irregular background dynamics during consciousness. In contrast, complexity may not be further collapsed beyond synchrony and regularity characteristic of unconscious spontaneous activity. We propose that increased dimensionality of spontaneous dynamics during conscious states supports responsiveness, enhancing neural networks' emergent capacity to robustly encode information over multiple scales. Brain activity transitions between healthy states, including stages of sleep, restful and aroused waking, as well as pathological states such as epilepsy, coma, and unresponsive wakefulness syndrome. From such a diversity of brain states, phenomenological categories encompassing similar spatio-temporal activity patterns can roughly, but usefully, be defined: unconscious  and conscious  brain states. At the macroscopic, global scale, unconscious brain states are dominated by high voltage, low frequency oscillatory brain activity related to the microscopic alternation of synchronous neuronal spiking and near silence  to macroscopic brain activity , describing both spontaneous and evoked dynamics. Toward linking interpretations of studies between scales, mesoscopic data (micrometers to millimeters—populations of thousands to tens of thousands of neurons) have been useful to inform models of neuronal assemblies. The perspective concludes by discussing a hypothesis best tested with a multi-scale understanding of brain function: the global complexity of neural activity increases in conscious brain states so as to enhance responsiveness to stimuli. We suggest responsiveness may depend on the capacity of neural networks to transiently collapse the dimensionality of collective dynamics—in particular neural assemblies sensitive to stimulus features—into evoked low-dimensional trajectories supporting neural codes .Both spontaneous and evoked  neural sPatterns of neocortical regions activated in resting state networks have been successfully retrieved using eigenmodes of the structural connectivity matrix, i.e., the possible oscillatory patterns at frequencies allowed by white matter tract lengths  of single cells slowly oscillates between hyperpolarized and depolarized potentials associated with alternating periods of silence  and AI-like firing   channels, which leads to depolarization of cells and suppression of spike-frequency adaptation. At low neuromodulatory concentrations, during unconscious states, K+ leak channels are constitutively open and activity-dependent K+ channels open when neurons spike, allowing K+ ions to exit cells thus hyperpolarizing the membrane. Accumulating self-inhibition in the form of spike-frequency adaptation during Up periods results in the transition to Down states. Conversely, spike-frequency adaptation wears off during Down states, allowing noise fluctuations  are observed. Cash et al. have elegantly shown that K-complexes (graphoelements characteristic of sleep stage 2) are complementarily observed both at microscopic and macroscopic scales . Similarly, enhanced dimensionality associated with conscious states could subserve neural information through the collapse of complexity in neural assemblies tuned to encode particular representations.All authors listed have made a substantial, direct and intellectual contribution to the work, 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.