Poecilia) that have repeatedly colonized toxic, hydrogen sulphide-rich springs. We quantified variation in body size and routine metabolism across replicated sulphidic and non-sulphidic populations in nature, modelled total organismal energy demands, and conducted a common-garden experiment to test whether population differences had a genetic basis. Sulphidic populations generally exhibited smaller body sizes and lower routine metabolic rates compared to non-sulphidic populations, which together caused significant reductions in total organismal energy demands in extremophile populations. Although both mechanisms contributed to variation in organismal energy demands, variance partitioning indicated reductions of body size overall had a greater effect than reductions of routine metabolism. Finally, population differences in routine metabolism documented in natural populations were maintained in common-garden reared individuals, indicating evolved differences. In combination with other studies, these results suggest that reductions in energy demands may represent a common theme in adaptation to physiochemical stressors. Selection for reduced energy demand may particularly affect body size, which has implications for life history evolution in extreme environments.Convergent evolution in organismal function can arise from nonconvergent changes in traits that contribute to that function. Theory predicts that low resource availability and high maintenance costs in extreme environments select for reductions in organismal energy demands, which could be attained through modifications of body size or metabolic rate. We tested for convergence in energy demands and underlying traits by investigating livebearing fish (genus  Convergent evolution, where disparate lineages exposed to similar environmental conditions independently evolve similar phenotypes, is a central theme in evolutionary diversification –3. Convevs. metabolic rate) are contributing equally to variation in energetic demands among populations. Life in extreme environments is often associated with energetic costs. Some extreme environments, such as the deep sea and caves that are subject to periodic flooding, exhibit high spatial or temporal variability in resource availability, and organisms in such environments have been documented to evolve large body size to increase metabolic efficiency and starvation resistance, allowing them to cope with periods of energy limitation . (4) Afalso see . Becausealso see , we only10(MRtot) = b*log10(mass)+a, where b was the slope and a the intercept of a regression describing the relationship between mass and metabolic rate for each population. To account for uncertainty associated with the estimation of slopes and intercepts, values for b and a were randomly chosen from within the 95% confidence interval of each parameter. The simulated values of total routine metabolic rate consequently represent estimates of the energy demand of average individuals in each population, taking into account within and among-population variation in both body mass and metabolic rate allometry. Simulated total metabolic rates were analysed using GLM with presence or absence of H2S in natural populations and drainage as fixed factors, and population (nested in the drainage by sulphide interaction) as a random factor.To test how variation in body size and RMR interact to shape organismal energy demands, we modelled total routine metabolic rates for individuals in each population based on the empirical data on size distributions and allometric metabolic rate functions . For eacvarpart command implemented in the R package vegan [In addition, we tested whether among population variation in total organismal energy demands was primarily driven by variation in body size or mass-adjusted RMR. To do so, we first calculated the average of total organismal energy demands, body mass, and mass-adjusted RMR for each population (as depicted in ge vegan . This ange vegan .10-transformed) from common-garden reared fish were combined with data from wild-caught fish from the matching sites and used as the dependent variable in a GLM. We included sex, presence or absence of H2S, drainage, and rearing environment (i.e. wild-caught vs. laboratory-reared) as independent variables. Temperature and mass (log10-transformed) were included as covariates. Three-way and four-way interactions were not significant  and thus excluded from the final model.Metabolic rate variation in wild-caught fish may merely reflect plastic responses to exposure to different environments. Hence, we conducted a common-garden experiment to test whether variation in mass-adjusted RMR between sulphidic and non-sulphidic populations has a heritable basis. Fish were collected from a subset of sites  and tranN = 2,472 individuals collected across all sites  Reduced metabolic rates may be a consequence of physiological modifications in response to variation in oxygen or energy availability among habitat types, as oxygen limitation [2S. It is possible that routine metabolic rates in presence of H2S are substantially higher due to costs associated with H2S detoxification and tissue repair. Thus, organismal energy demands in situ may not differ significantly between sulphidic and non-sulphidic populations, or they may even be higher in fish from H2S-rich environments. Nonetheless, the reduced energy demand in sulphide spring fish that we documented in absence of H2S may be adaptive, if it allows for the accommodation of costs associated with detoxification and maintenance without exceeding the maximal metabolic rate when H2S is present.Several non-mutually exclusive, proximate mechanisms could cause the documented reduced mass-adjusted RMR in sulphide spring environments. (1) Variation in RMR may simply reflect differences in activity rates or other aspects of behaviour , 64. Sinoduction . At leasrylation . Modifiemitation  as well mitation  and qualmitation  differenmitation , and genmitation . In addimitation  and dietmitation . (5) Red2S-rich habitats. This is highlighted by a positive correlation between the population-specific deviation in body size and in mass-adjusted metabolic rates from the among population average . In other words, populations with lower than average body sizes also exhibited lower than average mass-adjusted metabolic rates, indicating that modification of both traits contribute to evolutionary reductions of energy demands.While most inferences about energy demands of extremophiles have solely been drawn from analyses of metabolic rates, our study suggests that variation in body size and metabolic rates should be considered jointly. Variance partitioning indicated that variation in organismal energy demand among populations was disproportionally associated with body size. Modulation of body size upon colonization of extreme environments therefore is an important contributor to shaping organismal energy demands. Nonetheless, it is important to emphasize that evolutionary change in mass-adjusted metabolic rates was not negligible and complementary to body size reductions in populations from HOverall, our data on body size variation and oxygen consumption rates are consistent with theoretical considerations that predict reductions in energy demands of extremophiles in response to selection mediated by high maintenance costs and/or consistently low resource availability , 18. The


Exciting findings reveal that miRNAs released from exosomes modulate the expression and function of amyloid precursor proteins (APP) and tau proteins. These open up the possibility that dysfunctional exosomal miRNAs may influence AD progression. In addition, it has been confirmed that the interaction between miRNAs released by exosomes and Toll-like receptors (TLR) initiates inflammation. In exosome support-deprived neurons, exosomal miRNAs may regulate neuroplasticity to relieve neurological damage. In this review, we summarize the literature on the function of exosomal miRNAs in AD pathology, the potential of these miRNAs as diagnostic biomarkers in AD, and the use of exosomes in the delivery of miRNAs which may lead to major advances in the field of macromolecular drug delivery.Exosomes are bilipid layer-enclosed vesicles derived from endosomes and are released from neural cells. They contain a diversity of proteins, mRNAs, and microRNAs (miRNAs) that are delivered to neighboring cells and/or are transported to distant sites. miRNAs released from exosomes appear to be associated with multiple neurodegenerative conditions linking to Alzheimer's disease (AD) which is marked by hyperphosphorylated tau proteins and accumulation of A Majoritfunction . TherefoThere are many exciting findings that have emerged in the field of extracellular vesicles during the last few years. These findings that show that application of exosomal contents which causes a change in neurological damage condition provides a bright future for the diagnosis of neurodegenerative disease –6. In thβ aggregates [Just like small noncoding RNAs, miRNAs have functional effects on posttranscriptional regulation of gene expression and could serve as novel diagnostic biomarkers for AD . Profoungregates .Exosomal miRNAs and their pattern of transfer deserve more attention in order to establish their emerging roles and potential applications in AD. In this review, we summarize the existing knowledge of exosomal miRNAs and their involvement in AD, emphasizing their potential to be used as diagnostic biomarkers during the preclinical phase of AD as well as the possibility of them being used to regulate neurodegeneration.Exosomes are small-sized (between 30 and 100 nm) spherical vesicles with an endosome-derived membrane enriched with lipids such as cholesterol, ceramide, and sphingolipids. They contain cell state-specific loads of protein, mRNA, and miRNA . Some stExosomes are derived from intraluminal vesicles of late endosomal compartments named multivesicular bodies (MVBs). It is the specific contents and membrane molecules that are contained that define exosomes irrespective of the cell types they originated from. Established exosomes contain molecules that participate in MVB biogenesis like TSG101 and Alix. They could also contain proteins involved in signal transduction such as heat shock proteins HSP70, HSP90), certain cytoplasmic proteins such as tubulin and actin, Annexin and Rab family proteins, tetraspanins , and major histocompatibility complex class I (MHC-I) molecules, as well as the various transmembrane proteins [0, HSP90,The fundamental step in MVB formation involves the organization of specialized domains called tetraspanin-enriched membrane domains (TEMs). TEMs are enriched with membrane proteins that are necessary for vesicular fusion. In addition, TEMs also recruit some exosome membrane proteins which can be potential ligands for receptor-mediated internalization into the recipient cells . AnotherDuring the release of exosomes into extracellular space, any of the following could happen: (1) they may be caught by surrounding cells ; (2) they may be captured by cells within distal sites; or (3) they may enter systematic circulation and be internalized into various cells . ExosomemiRNAs are derived from long primary transcripts that are split by a complex of protein DGCR8/Pasha and RNAse III Drosha. These are shorter hairpin structures (50–120 nt) that constitute miRNA precursor (pre-miRNA) in the nucleus. The pre-miRNA is then transported into the cellular cytoplasm, converted to a short nucleotide duplex by the RNAse III enzyme Dicer, and then conveyed to miRNA-containing RNA-induced silencing complex (miRISC). In the miRISC, the single mature miRNA, which is derived from the pre-miRNA hairpin components, combines with an Argonaute protein and then migrates to specific neuronal sites , 24.Alternatively, pre-miRNA and miRNA processing machinery may be collected with RNA transport granule proteins. These molecules are subsequently exported to specific neuronal sites, where either the matured or precursor miRNAs are packaged into exosomes. It must be noted that exosome as a carrier of miRNAs is novel. A schematic representation of exosomal miRNA formation is shown in According to recent literature, the process of loading miRNAs into exosomes follows four main proposed ways even though the exact mechanisms has not been concluded on. Potential modes include the following: (1) the neutral sphingomyelinase 2- (nSMase2-) dependent pathway: the nSMase2 increases the quantum of exosomal miRNAs. When nSMase2 expression and function is inhibited specifically, the quantity of exosomal miRNAs reduces, indicating the involvement of nSMase2 in how miRNA is packaged into exosomes . (2) TheSince the CNS appears to be an abundant store of miRNA expression, it is suggested that the relevance of miRNA within the CNS is determined by neural-specific miRNA epigenetic modulative effect during brain embryogenesis . MicroarAs a carrier and transporter of miRNA, exosomes are disseminated from various cells including neurons  and astrε4 (APOE ε4) used for predicting AD [Exosomes are endosome-derived membrane vesicles that carry a variety of cellular proteins, mRNA and miRNA . Some ficting AD .γ released exosomes (IFNγ-DC-Exos) that contained miR-219 exerts functional effects including increase in myelination, reduction of oxidative stress, and improvement of remyelination [Corresponding to these findings, some specific exosomal miRNAs exhibit sufficient function in reducing memory decline and cognitive dysfunction. Application of low-level IFNlination . In addilination . Besideslination . Furtherlination . OverallCells must communicate with each other through exchange of information and substances constantly in order to maintain physiological homeostasis. It is generally accepted that several communication modes exist in CNS. Among these, the outstanding mode is through signal activation initiated by neurotransmitters in synaptic transmission between axons and their terminals. A growing number of evidence demonstrate that exosomes may be one of several distinct mechanisms to guarantee various trades of cellular information and substance transfer . The finA new exciting pathway, exosome-mediated transfer of miRNA signal from neuron to astrocyte, has been characterized: excitatory amino acid transporter 2  in perisynaptic astrocytes modulates direct miRNA-124a transfection mediated by exosome, and this leads to selective intensification of glutamate transporter1 (GLT1) protein (but not mRNA) expression in astrocytes .While neurons transfer miRNA into astrocytes through neuronal exosomes and then significantly regulate protein expression in an indirect manner , dendritInvestigation of miRNA reveals posttranscriptional modulation function as a mechanism regulated epigenetically. A considerable amount of evidence put forward by Hu et al. as well as Ismail et al. points out that miRNAs mediated by exosomes exert their effects in the recipient cell and may convert target gene expression , 41. Matβ plaques, which ultimately lead to progressive loss of neurons, are derived from processing of APP by BACE. Significantly deregulated miRNAs targeting APP like miR-193b [β generation in AD brain. Researchers have validated computational predictions and demonstrated that miRNAs recognize specific complementary sites within 3′-UTR of APP/BACE1 mRNA, and they pointed out a possible involvement of miRNA in AD [Several specific miRNAs bind to complementary sites within 3′-UTR of key genes that determine the expression of amyloid precursor protein (APP) and beta-site APP-cleaving enzyme (BACE) , 44. In miR-193b , or BACEmiR-193b , 44 inflNA in AD .β plaques in sporadic AD [Numerous findings in transgenic AD mice identify that some specific miRNA expression deviates from the general levels and that deregulation of these miRNAs can contribute to the accumulation of extracellular Aradic AD , 48. Unuradic AD . Geneticradic AD .Potentially, apart from degrading erroneous proteins, miRNAs transferred by exosomes may also alleviate protein scarcity via rectifying functional protein translation . AssociaNeuroinflammation is responsible for the “double-edged sword” effect in Alzheimer's disease. Future studies that will investigate the relationship between molecules associated with neurodegeneration and receptors that initiate inflammation will bring some understanding to the mechanism of neurodegeneration .It is conjectured that miRNAs mediated by exosomes may initiate Toll-like receptor (TLR) activation under certain circumstances , 57. SohSome findings suggest that miRNAs participate in TLR-signaling pathway at various levels, including the following: (1) targeting some components of the TLR signaling system, such as their proteins associated with signaling, regulatory molecules, and transcription factors as well as functional cytokines induced by them; (2) miRNA expression may directly be regulated by TLRs activation pathway, and (3) miRNAs may directly activate the RNA-sensing TLRs .Lehmann and his group have shown that TLR7–9 recognize specific miRNAs as ligands in the CNS. For example, miRNA let-7, which is highly expressed in microglia and neurons, interacts with TLRs to regulate factors of transcription induced by TLR. Therefore, it is inferred that let-7 binds to RNA-sensing TLR7 and consequently induces neurodegeneration through neuronal TLR7 . BesidesRapid input-restricted change in gene expression is an important aspect of neural plasticity that requires very complex mechanisms of regulating and transferring posttranscriptional mRNA. Small noncoding miRNA are positioned uniquely to support these functions by providing a nucleic acid-based specificity component for universal sequence-dependent RNA binding complexes. As a special cellular vehicle, exosomes loaded with specific miRNAs may benefit from neuroplasticity under adverse environmental conditions.Redistribution of miRNA associated with rapid depolarization in neurons was observed by Goldie and his group and provided evidence that miRNAs exert regulatory effects on the dynamics of normal synaptic function. Intriguingly, they also found that a large proportion of depolarization-associated changes attributed to the release of exosomes enriched with evolved miRNAs suggesting the essential vehicular role of exosome . Systemiγ-released exosomes (IFNγ-DC-Exos) and enriched in miRNA species reduced oxidative stress increased baseline myelination and improved remyelination following acute lysolecithin-induced demyelination [Gray matter injury is firmly associated with cognitive dysfunction. It was found that cognitive decline follows with neurodegeneration from myelin loss and that age-associated deficiency due to remyelination significantly contributed to AD progression . Researclination . Intereslination .These findings further support the hypothesis that exosomal miRNA could be used as neural plasticity regulator. The association of the abovementioned exosomal miRNAs with neurodegeneration are summarized in Current therapeutic activities for neurodegenerative disorders are restricted not only due to curative deficiency but also due to the limited understanding of their underlying mechanisms and also the difficulties posed in accurately diagnosing AD during the subclinical stages. Since pathological changes initiated years before the appearance of clinical symptoms, predictable biomarkers for the detection of AD are critical so much that preventative strategies could be applied to retard cognitive decline. In a neurodegenerative disorder research by Bellingham and his colleagues, a small RNA deep sequencing study demonstrated that neuronal exosomes contained a diverse range of RNA species which included retroviral RNA repeat regions, messenger RNA fragments, transfer RNA fragments, noncoding RNA, small nuclear RNA, small cytoplasmic RNA, and silencing RNA as well as known and novel candidate miRNA and that these circulating exosomes had a distinct miRNA signature that could be utilized for diagnosing neurodegenerative disorders .β is mediated by low-density lipoprotein protein receptor that transports it across the BBB, suggesting the possibility of other neurodegenerative biomarkers to cross this barrier. Several presumable mechanisms by which miRNAs can cross the BBB have been put forward. In substance, it is a general consensus that some pathological conditions in neurological disorders may cause perforations of BBB which consequently leads to the release of cellular components [Recent development in molecular biology has prompted the potential of biomarker research from a notion of pathogenesis and specific incident that leads to a disorder originating from biosystem deterioration. Specific profiles of exosomal miRNAs from human biological fluids such as plasma and CSF have prompted the potential application of miRNAs as diagnostic biomarkers –6. Howevmponents . In a stmponents . Inspirimponents . It has mponents . In thisAlterations that occur in a biosystem often affect substances within exosomes released by neural cells, and these enlighten the possibility of exosomal miRNAs as potential biomarkers for diverse pathological states wherein deregulated miRNAs may be used for early diagnosis. In a study, exosomal miRNAs obtained from postmortem prefrontal cortices of subjects who had been diagnosed with schizophrenia and bipolar disorders were varied significantly from matched controls . It was Analysis of biological fluids for detecting brain-associated miRNAs has been focused on CSF and blood so far. As biological fluid yielded in the choroid plexus, CSF circulates through the inner ventricular system, crosses the BBB, and is integrated into the bloodstream. On the basis of these characteristics, CSF may serve as the appropriate source containing biomarkers for AD , 5. HoweOver the last decades, new approaches to obtain large quantities of exosomes have been applied in trials including ultrafiltration, and the methods for attempting to acquire pure exosomes have been developed as utilization of monoclonal antibodies targeted membranous marker proteins of exosomes. Furthermore, exosomes have been classified according to various characteristics such as their density, morphology, size, expression of marker proteins, and intracellular origin .Studies of CSF from AD patients have applied the use of miRNA microarrays, multiplex miRNA qPCR assay, or target candidate miRNA approaches to identify differentially expressed miRNA . Recentlβ formation and this prevented deteriorations in long-term potentiation, spatial learning, and memory in the hippocampus [β formation, but the role of miR-188-3p is of critical interest, given that its recognition element in the BACE1 3′-UTR is extraordinarily conserved. Apart from the accumulation of Aβ, another neuropathological hallmark of AD, neurofibrillary tangles (NFTs) result from tauopathy which may also be regulated by miRNA as well. For example, miR-219 binds directly to the 3′-UTR of the tau mRNA and represses tau synthesis at the posttranscriptional level. Modification of the silencing mechanism of tau by miR-219 that is perturbed during neurofibrillary degeneration suggested that developing therapies for tauopathies is possible [Previous reports have demonstrated that dysfunction of miRNAs is involved in the pathogenesis of human AD and that the therapeutic potential of miRNAs could guide us towards the development of new therapeutic strategies in neurodegenerative disorder. In a study, upregulating miR-188-3p in the hippocampus suppressed BACE1 expression and Apocampus . It is upossible . In thisRecent researches have revealed that exosomes mediate horizontal delivery of distinct RNAs in intercellular communication. The utilization of exosomes for transport of certain therapeutic nucleic acids to recipient cells is considered as a potential approach for AD . InvolveThe capacity of exosomes to deliver functional miRNA pushes the hope of replacing the already known virus-based gene therapy with this . When thβ-amyloid plaque associated with AD pathogenesis [Exosomes serve as not only vehicles to deliver miRNA but also synthetic nucleic acids, for instance, short interfering RNA (siRNA) which are therapeutic compounds and have been exploited recently. Researchers obtained exosomes from self-derived dendritic cells and then decorated exosomes to express membrane protein Lamp2b and a neuron-specific RVG peptide in order to deliver cargoes specifically to recipient neural cells. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA which resulted in knockdown of BACE1 and mediated the formation of the peptide that forms ogenesis . These fBy all indications, exosomes have multiple advantages over existing miRNA delivery vehicles. Since they are derived from the patients' own cells, they would be less immunogenic than other foreign delivery vehicles. Exosomes remain relatively stable in the blood as they avoid coagulation factors and complement which are most likely due to their surface expression of CD46 and CD59 , as wellDespite these advantages, numerous issues are still being addressed before clinical implementation of this strategy. Firstly, as to whether the RVG-Lamp2b fusion-protein induces adaptive immunity after repeated administration needs to be verified. In fact, adaptive immune responses would be anticipated. Another issue not yet addressed is that the receptor coupled with RVG expressed in the surface of exosomes, alpha-7-subunit acetylcholine receptor, markedly declines in brains affected by AD . This isWith this progress in intercellular transfer researches, exosomes provide a breakthrough in the field of drug delivery, bringing an alternative source of clinical therapy.The ability of exosome-transporting miRNAs to function in neighboring or distant cells exhibits a novel insight into modification mechanisms employed in AD. Besides, our review also reveals gaps in knowledge that require further research.As novel epigenetic-related systems ensure cellular information exchange, the exhaustive mechanism that underlies uptake of exosomes containing miRNAs into neighboring or distant cells needs to be explored and this holds enormous potential for RNA interference applications. In addition, investigating the ability of exosome-mediated miRNAs to bind to TLRs in the endosomes of recipient cells is still in its infancy. Vartanian and his team hypothesized that intracellular delivery of miRNAs via exosomes brings about TLR tolerance, which is a hyporesponsive state of TLR contributing to neuroprotection . If thisAlthough previous researches have demonstrated that a panel of miRNAs within exosomes from the CSF and plasma may serve as fingerprints of early stages in AD , 4, 6, fA study that demonstrates an efficacious way of delivering functional siRNA into AD mouse brain by systemic exosome administration represents a promising approach for RNA interference applications , 10. HowThis review has summarized compelling evidence for multiple ways in which exosomal miRNAs contribute to important biological functions related to intercellular communication, neuroinflammation, and neuroplasticity. We put forward new applications of exosomal miRNAs as diagnostic biomarkers and potential gene therapy. We expect that our review will contribute to better comprehension of the roles that exosomal miRNAs play in pathological mechanisms of AD.


Zoledronic acid (ZA) is commonly used for the prevention of skeletal-related events (SREs) in patients with bony metastases. ZA is a bisphosphonate that inhibits osteoclasts, possibly through inhibition of proto-oncogenic Ras activity in osteoclasts by interfering with normal post-translational modifications of Ras. This would suggest that ZA could induce an objective tumor response in metastatic tumors in bone that harbor oncogenic Ras, as the oncogenic Ras in these tumors would require similar post-translational modifications. We recently cared for a patient with biopsy confirmed metastatic urothelial carcinoma who demonstrated a complete radiographic response to ZA. His tumor harbored an oncogenic K-Ras protein, supporting the possibility of interference with Ras activity as the mechanism for the radiographic complete response. Zoledronic acid (ZA) decreases skeletal-related events (SREs) . It may Oncogenic Ras activity is perhaps the most common oncogenic mechanism involved in human cancer, seen in about one third of human cancers . Based o2 d1) and Gemcitabine . Following treatment, residual disease was noted on the bone scan, and the patient began ZA therapy (4 mg/1 month). On follow-up scans, the patient was noted to have an objective radiographic response. Subsequent scans, as recently as June 2010, continue to show no evidence of residual bony disease.JP is a 62 year-old patient who presented in 2007 with a large primary urothelial carcinoma and apparent bony metastases. Biopsies of the primary tumor and the bony metastases confirmed urothelial carcinoma, and molecular studies demonstrated a K-ras mutation in codon 12, a known oncogenic mutation. He received 7 cycles of standard chemotherapy with CDDP (70 mg/mTo our knowledge, this is the first reported case of a patient with bony metastatic bladder cancer having a complete radiographic response to ZA. Because Ras is a potential downstream target of zoledronic acid, the presence of a K-Ras mutation in the tumor cells may offer a possible explanation for the patient’s robust response. Several independent studies have found the incidence of K-Ras mutation in bladder cancer to be a rare event . We propIn conclusion, based on our experience documented in this report, we recommend further research into the mechanism of zoledronic acid’s anti-tumor effect, especially with regard to the role of Ras. Zoledronic acid has already been proven to be an effective therapy in the prevention of skeletal-related events; therefore, many patients with bony metastatic bladder cancer, as well as other tumors, will receive this treatment in the future. A study that prospectively tests patients’ bony metastatic tumors for Ras mutations and then assesses treatment outcomes after ZA therapy could help to determine if tumors harboring oncogenic Ras are especially sensitive to zoledronic acid treatment. Confirmation of this hypothesis could lead to tailored therapies for patients with certain varieties of bony metastatic cancers.


A shift from respiration to fermentation is a common metabolic hallmark of cancer cells. As a result, glucose and glutamine become the prime fuels for driving the dysregulated growth of tumors. The simultaneous occurrence of “Press-Pulse” disturbances was considered the mechanism responsible for reduction of organic populations during prior evolutionary epochs. Press disturbances produce chronic stress, while pulse disturbances produce acute stress on populations. It was only when both disturbances coincide that population reduction occurred.This general concept can be applied to the management of cancer by creating chronic metabolic stresses on tumor cell energy metabolism (press disturbance) that are coupled to a series of acute metabolic stressors that restrict glucose and glutamine availability while also stimulating cancer-specific oxidative stress (pulse disturbances). The elevation of non-fermentable ketone bodies protect normal cells from energy stress while further enhancing energy stress in tumor cells that lack the metabolic flexibility to use ketones as an efficient energy source. Mitochondrial abnormalities and genetic mutations make tumor cells vulnerable metabolic stress.The press-pulse therapeutic strategy for cancer management is illustrated with calorie restricted ketogenic diets (KD-R) used together with drugs and procedures that create both chronic and intermittent acute stress on tumor cell energy metabolism, while protecting and enhancing the energy metabolism of normal cells.Optimization of dosing, timing, and scheduling of the press-pulse therapeutic strategy will facilitate the eradication of tumor cells with minimal patient toxicity. This therapeutic strategy can be used as a framework for the design of clinical trials for the non-toxic management of most cancers. According to the paleobiologists, Arens and West, the simultaneous occurrence of “Press-Pulse” disturbances was considered the mechanism responsible for the extinction of organic populations during prior evolutionary epochs . A “presCancer is a systemic disease involving multiple time- and space-dependent changes in the health status of cells and tissues that ultimately lead to malignant tumors . NeoplasCancer is generally considered a genetic disease where random somatic mutations underlie the origin and progression of the disease , 13–16. 2 and water for energy production. Before entering the mitochondria for complete oxidation, glucose is first split into two molecules of pyruvate through the Embden–Meyerhof–Parnas glycolytic pathway in the cytosol. As most cells are bathed in oxygen, the production of pyruvate occurs through aerobic glycolysis [Most cells of the body oxidize glucose to COycolysis . Under hycolysis . Otto Waycolysis , 36.2 = 120–160 mmHg) conditions associated with cell culture, [Although Warburg’s hypothesis on the origin of cancer has created confusion and controversy –40, his culture, , 42. Alsculture, , 43–48. culture, , 49. Theculture, , 48, 50.culture, , 51–57.The main points of Warburg’s theory are; 1) insufficient respiration is the predisposing initiator of tumorigenesis and ultimately cancer, 2) energy through glycolysis gradually compensates for insufficient energy through respiration, 3) cancer cells continue to produce lactic acid in the presence of oxygen, and 4) respiratory insufficiency eventually becomes irreversible , 58, 59.18F-deoxyglucose positron emission tomography (FDG-PET) is considered evidence for the elevated use of glucose by some tumors [Substantial evidence exists showing that many cancers avidly consume glucose and produce lactic acid –67. The e tumors . Elevatee tumors , 68. Inde tumors –74. Mamme tumors , 75–78. e tumors , 61, 79.e tumors , 43. Thee tumors , 80–85. Although the abnormal energy metabolism and mitochondrial abnormalities seen in most cancers could arise in part through oncogenic modulation of metabolism , 39, 86,2 production. Although Warburg did not address the role of TCA cycle substrate level phosphorylation in his original work [As the result of insufficient aerobic respiration, cancer cells must rely primarily on fermentation metabolism to maintain energy balance and viability. Besides substrate level phosphorylation in the cytoplasm through lactic acid fermentation, TCA cycle substrate level phosphorylation can also produce significant amounts ATP –98. In anal work , 36, an Glucose and glutamine act synergistically for driving rapid tumor cell growth. Glutamine metabolism can produce ATP from the TCA cycle under aerobic conditions. Glutamine is also a nitrogen donor for nucleotide biosynthesis and can serve as precursor for lipid synthesis under hypoxic conditions , 105. WeAmino acid fermentation can generate energy through TCA cycle substrate level phosphorylation under hypoxic conditions , 110. SuSeveral byproducts of amino acid fermentation can also accumulate in the tumor microenvironment including acetate, glutamate, alanine, succinate, and ammonia. Although acetate has been considered a potential fuel for supporting tumor cell growth , 118, acEmerging evidence indicates that macrophages, or their fusion hybridization with neoplastic stem cells, are the origin of metastatic cancer cells , 121–124Emerging evidence indicates that the function of DNA repair enzymes and the integrity of the nuclear genome are dependent to a large extent on the energy derived from normal respiration –142. PreWe recently discussed how a transition from respiration to fermentation could account for Szent-Gyorgi’s “Oncogenic Paradox”, i.e., the process by which various provocative agents  could produce cancer through a common pathological mechanism , 148. MuRick Potts, a paleoanthropologist at the Smithsonian Institution, suggested that the evolutionary success of our species has been due largely to the germline inheritance of traits that bestowed adaptive versatility –153. AdaPotts’ hypothesis is an extension of Darwin’s original theory  and can be applied to the individual cells of the organism, which exist as an integrated society of cells , 154. Thp53, K-Ras, and Raf impact negatively on mitochondrial energy efficiency thus making cells with these mutations less metabolically flexible than normal cells [K-Ras target mitochondria, thus enhancing glycolysis [The widely held notion that tumor cells have a growth advantage and are more fit than normal cells are in contrast to Darwin’s theory of evolution and also to Potts’ theory of adaptive versatility , 154. Ital cells , 156–159ycolysis . Enhanceycolysis , 162. Moycolysis . The higycolysis . Despiteycolysis . As longycolysis . This isFor example, when mice or people with tumors are placed under energy stress using dietary energy reduction (glucose restriction), many tumor cells die while normal cells survive. Indeed, the health and vitality of the normal cells improves with time under dietary energy reduction while hyper-glycolytic tumor cells undergo energetic crisis triggering apoptotic death , 167. SuIt is also possible that therapeutic energy stress could restore the microenvironment thus reversing abnormal energy metabolism and growth behavior in tumor cells not containing genetic mutations , 175. InThe greater adaptability of normal cells than tumor cells to energy stress is predicted based on the theories of Darwin and Potts . MetabolTumor cells will have difficulty surviving and growing, regardless of their complement of genomic changes, if fermentable fuels become restricted in the microenvironment. Ketone bodies and fats are non-fermentable fuels . Tumor cMark Vincent suggested how a Press-Pulse strategy could be used to target tumor cells . We haveCalorie restriction, water-only fasting, and restricted ketogenic diets reduce circulating glucose and insulin levels while elevating circulating levels of ketone bodies. Ketogenic diets (KDs) are low carbohydrate-high fat diets that are widely used to reduce refractory epileptic seizures in children , 188. Thhttps://www.charliefoundation.org).The therapeutic effects of KDs used alone or in combination with other therapies have been documented in preclinical studies for several cancer models including neuroblastoma , 200, luWe recently developed the Glucose/Ketone Index calculator (GKIC) to assess the potential therapeutic effects of various low-carbohydrate and KDs for brain cancer management . The GKIReduced glucose availability and suppression of insulin signaling will produce chronic energy stress on those tumor cells that depend primarily on glucose for growth and survival. It is important to remember that insulin drives glycolysis through stimulation of the pyruvate dehydrogenase complex , 222. ReD-β-hydroxybutyrate and acetoacetate) are produced largely in the liver from adipocyte-derived fatty acids and ketogenic dietary fat. Ketone bodies bypass glycolysis and directly enter the mitochondria for metabolism to acetyl-CoA [2, ketone body metabolism generates only NADH [D-β-hydroxybutyrate in normal cells will therefore increase the redox span between Complexes I and III, thus increasing the delta G’ of ATP hydrolysis while, at the same time, reducing ROS formation through the Complex II coenzyme Q couple [The water-soluble ketone bodies  , 234. InCalorie restriction or therapeutic fasting is anti-angiogenic, anti-inflammatory, and pro-apoptotic, and thus targets multiple cancer hallmarks , 240–243Adrienne Scheck and colleagues showed that the therapeutic efficacy of radiotherapy against the orthotopically grown GL261 mouse glioma could be greatly enhanced when combined with a commercially available ketogenic diet . Mice fePoff and colleagues demonstrated that hyperbaric oxygen therapy (HBOT) enhanced the ability of the KD to reduce tumor growth and metastasis . EvidencSome tumors use glucose as a prime fuel for growth, whereas other tumors use glutamine as a prime fuel , 262–264The VM-M3 tumor is an excellent model system for evaluating the role of glutamine as a metabolic driver of invasive and metastatic cancer. The VM-M3 tumor arose spontaneously in the brain of its syngeneic immunocompetent VM/Dk inbred mouse host . The tumThe success of the press-pulse therapeutic strategy for the metabolic management of cancer will depend on optimization of the scheduling, dosing, and timing of the various diets, drugs, and procedures used in order to achieve maximum synergistic interactions Fig. . SchedulMany of the current treatments used for cancer management are based on the view that cancer is a genetic disease. It is clear from the cancer death statistics that most current therapies are wanting in their ability to reduce the yearly death rate or to manage the disease without toxicity. Emerging evidence indicates that cancer is a mitochondrial metabolic disease that depends on availability of fermentable fuels for tumor cell growth and survival. Glucose and glutamine are the most abundant fermentable fuels present in the circulation and in the tumor microenvironment. The press-pulse therapeutic strategy is designed to target availability of glucose and glutamine thus starving tumor cells of their most important fuels and increasing their vulnerability to oxidative stress and apoptotic death. Low-carbohydrate, high fat-ketogenic diets coupled with glycolysis inhibitors will reduce metabolic flux through the glycolytic and pentose phosphate pathways needed for synthesis of ATP, lipids, glutathione, and nucleotides. DON and other similar glutamine inhibitors will deprive proliferating tumor cells of the glutamine needed for TCA cycle anaplerosis, and synthesis of glutathione, nucleotides, and proteins. Lysosomal targeting with chloroquine or similar drugs will reduce glucose and glutamine production following digestion of phagocytosed glycoconjugates and proteins. Glutamine targeting will require careful adjustments, however, as glutamine is a key metabolite needed for the immune system and for other physiological functions. Hyperbaric oxygen therapy combined with the calorie restricted ketogenic diet will kill tumor cells through apoptotic and anti-angiogenic mechanisms while also reducing inflammation in the tumor microenvironment and systemically. It is our view that the “Press-Pulse” paradigm is a compelling and parsimonious therapeutic strategy for effectively managing the vast majority of malignant cancers with minimal toxicity, as this approach will target the major energy pathways responsible for tumor cell growth and survival while enhancing the energetic efficiency of normal body cells and tissues.


We tested the hypothesis that use of artifact correction logic would improve the specificity of clinical event detection without compromising sensitivity. We showed that ventMAP could accurately detect harmful forms of OTV including excessive tidal volumes and common forms of patient-ventilator asynchrony, and that artifact correction significantly improved the specificity of event detection without decreasing sensitivity. Our multi-disciplinary approach has enabled automated analysis of high-volume streaming patient waveform data for clinical and translational research, and will advance the study and management of critically ill patients requiring mechanical ventilation.Healthcare-specific analytic software is needed to process the large volumes of streaming physiologic waveform data increasingly available from life support devices such as mechanical ventilators. Detection of clinically relevant events from these data streams will advance understanding of critical illness, enable real-time clinical decision support, and improve both clinical outcomes and patient experience. We used mechanical ventilation waveform data (VWD) as a use case to address broader issues of data access and analysis including discrimination between true events and waveform artifacts. We developed an open source data acquisition platform to acquire VWD, and a modular,  Mechanical ventilation (MV) provides life-saving therapy but if delivered improperly can cause ventilator-induced lung injury (VILI)3, and substantial patient distress if patient effort and MV support are not well-matched (known as patient-ventilator asynchrony (PVA)), that may further promote lung injury5. One of the principle mechanisms of VILI is known as volutrauma, whereby delivery of excessive tidal volumes  results in pathologic alveolar distention, cellular injury, and the development of diffuse lung injury with many of the pathologic and clinical hallmarks of the acute respiratory distress syndrome (ARDS), a common and severe form of diffuse lung injury associated with mortality of up to 50%6. Excessive distention of lung tissue may result from inappropriately prescribed ventilator settings, excessive patient effort, or from subtypes of PVA that result in incomplete exhalation in between breaths, trapping gas in the lungs and further distending tissue7. Randomized controlled trials in patients with ARDS suggest that targeting a low tidal volume ventilation (LTVV) strategy of approximately 6 ml/kg of predicted body weight and controlling PVA improve survival although studies to date have not been able to separate the effects of excessive TV from those of PVA9. Studies in ventilated patients without ARDS suggest that a LTVV strategy reduces the development of respiratory complications and hospital-acquired ARDS12. Despite over 15 years of accumulating evidence, the expected widespread translation of lung-protective MV targets into clinical practice has not occurred15.Acute respiratory failure is the most common reason for intensive care unit (ICU) admission in the U.S. and is associated with an average in-hospital mortality of approximately 30% and $54 billion in attributable yearly costs16, and most studies have been unable to collect and analyze the rich streams of ventilator waveform data (VWD) used by clinicians at the bedside to diagnose and manage pathologic patient-ventilator interactions. Manual analysis of large volumes of physiologic waveform data is limited by its labor-intensive nature, and recent data suggest that ICU clinicians perform poorly when asked to identify common forms of PVA through visual inspection of VWD, further supporting a need for standardized, automated analytic tools17.Despite its high prevalence, cost, and associated suffering, MV remains difficult to study and no well-validated, widely available analytic or clinical decision support tools exist to facilitate patient-specific, precision management of MV. Waveform data from MV- and most other life support devices- are not generally available in the electronic health record (EHR), limiting the ability to develop analytic tools. MV data from clinical studies have typically been hand-recorded only a few times per day, representing a gross under-sampling of patients who routinely take more than 20,000 breaths per day27. These studies have demonstrated an important proof of concept, namely that MV waveform data are rich in historically unrecorded information pertinent to patient-ventilator interactions, and that analysis of PVA and other forms of “off target” ventilation (OTV) may reveal associations with important clinical outcomes and processes of care. Studies to date have been limited by lack of access to ventilator data, intrusive data collection methods that may introduce observer bias29 and limit the feasibility of continuous longitudinal data collection, limited clinical validation of algorithm performance, inability to distinguish between OTV subtypes, and lack of defined analytic mechanisms to distinguish between true OTV events and waveform artifacts that may result in false positive event classification27. As these issues are not unique to MV, the development of improved MV waveform analysis software serves as a generalizable use case for the challenges facing the broader development of healthcare “big data”-specific analytics and decision support systems including barriers to data access, transmission, standardization, security, storage, and computation; incorporation of clinician-informed knowledge and heuristics into algorithms able to transform complex, high-volume raw data into actionable information while minimizing false alarms; and the development of well-engineered software solutions that allow extensibility, integration with other systems, and ultimately, provisioning of clinical decision support to the point of care32.A number of small studies have collected VWD using intrusive  or non-scalable methods of data acquisition, using a variety of analytic approaches to classifying PVA from manual annotation to power spectral analysis to the application of proprietary waveform analysis software33, continuous quality improvement, and precision medicine through clinical decision support. We assembled a multi-disciplinary team including clinicians, engineers, computer scientists, and informaticists, and developed an extensible, modular analytic engine, referred to as the ventilator multi-algorithm analytic platform (ventMAP), using rule-based logic derived from clinical bedside interpretation of MV waveforms5 to determine both inspiratory and expiratory TV, two well-recognized forms of PVA associated with hyper-inflation of the lungs, and several common types of VWD “clinical artifacts” that morphologically resemble true PVA.In this study, we aimed to develop and validate an integrated MV waveform data acquisition and analysis platform capable of unobtrusive, continuous data collection and breath-by-breath classification of OTV to support clinical outcomes research, translational patient phenotyping7. DTA occurs when a ventilator’s set inspiratory time (I-time) is shorter than a patient’s desired “neural I-time”, with ongoing patient inspiratory effort at the termination of inspiratory support resulting in the immediate triggering of a second breath without intentional exhalation 7. Unlike DTA, BSA results from either a ventilator-set or patient-triggered respiratory rate too fast to allow sufficient time for complete exhalation in between successive breaths ) and PVA. Two subtypes of PVA, referred to here as double-trigger asynchrony (DTA) and breath stacking asynchrony (BSA), cause varying degrees of incomplete exhalation in between breaths resulting in a phenomenon referred to as dynamic hyperinflationion Fig. . DTA canths Fig. . While bths Fig.  that mayWe hypothesized that the ventMAP engine would be able to measure TV with accuracy equivalent to a commercial ventilator, and that the recognition and algorithmic removal of clinical artifacts (referred to here as “artifact correction”) would significantly improve the specificity of PVA detection without compromising sensitivity. We present here the results of our derivation and validation studies.multi-algorithm analytic software platform (ventMAP) grounded in the clinical rules used in bedside MV waveform analysis to automate the quantitative analysis of OTV  to test the accuracy of ventMAP’s TVi and TVe measurement algorithms. Three separate experiments were performed using 3 different PB840s, testing ventMAP-derived TVi and TVe in a total of 1021 breaths across a range of ventilator modes, trigger mechanisms, set TV, and set inspiratory pressures using a pre-specified equivalence threshold of +/− 10%. ventMAP-derived TVi and TVe were equivalent to ventilator-derived TV across all measured conditions  across all DTA breaths in the validation cohort, with and without the use of the TV fusion algorithm.DTA and BSA both lead to dynamic hyperinflation that can be quantified by calculating the sum of two successive TVis and subtracting the intervening TVe to yield a “fused” TVi that represents the effective distending volume for the lungs Fig. . FailureIn the validation data set, mean TVi for DTAs was significantly higher when TV-fusion was employed, with a mean TVi of 293.3 ml  without TV-fusion and 562.2 ml . Clinically, prescribed TV are based on predicted body weight (PBW) derived from sex and height, with TVi of ≤6.5 ml/kg of PBW representing the standard of care for patients with severe hypoxemic respiratory failure. We used the average height of a U.S. female to normalize all DTA TVs in the validation cohort before and after TV-fusion. After identifying all potential DTA breaths and removing false positives through artifact correction, we stratified off-target breaths with and without TV-fusion as mild, moderate, or severe based on the extent to which a given breath exceeded a target of ≤6.5 ml/kg Fig. . We founmulti-algorithm analytic platform (ventMAP) to detect two main classes of off-target ventilation, namely TVV and PVA. We used customized Raspberry Pi microcomputers  operating on waveform data derived from clinical devices. Several previous studies have applied automated analytic approaches to VWD. Approaches have included the use of commercial general purpose waveform analysis software26, or including DTA and BSA in one heuristic algorithm to detect dynamic hyperinflation27. While related, DTA and BSA have important mechanistic differences that we felt merited distinct classification strategies. In this regard, DTA has historically been defined as resulting from inspiratory effort persisting beyond the end of the ventilator’s programmed inspiratory time, resulting in the triggering of another supported breath with little or no exhalation in between breaths18. In contrast, we have taken a nuanced view, defining BSA empirically as incomplete exhalation in between breaths (TVe:TVi < 90%) with a longer period (>0.3 sec) of exhalation than observed typically in DTA, representing an attempted exhalation in between successive breaths , such that ventMAP may overestimate BSA prevalence in patients with these conditions. Future research will need to model an increasingly diverse set of clinical artifacts, including the development of both morphologic and functional classification criteria such as when the detection frequency of a given artifact  exceeds physiologically plausible conditions . Finally, our study included patients and clinician-annotators from a single health system, and future studies should include data from multiple institutions including both academic and community settings to optimize generalizability.Our study also has a number of limitations. First, our manual selection of ROIs for algorithm training may have introduced selection bias. We attempted to mitigate this issue through an extensive manual survey of patient waveforms during ROI selection for the derivation data set, and through semi-random ROI selection at very low magnification for the validation data set  can automate the processing of high-volume VWD to detect common forms of OTV including TVV and PVA with high levels of sensitivity and specificity. We have also shown that common sources of clinical waveform artifacts can compromise the estimation of OTV frequency and that dedicated algorithms to detect and correct for the presence of artifacts can significantly improve the specificity of clinical event detection without compromising sensitivity. This work will help to enable future multi-center studies to delineate relationships between the cumulative dose and temporal distribution of OTV, and key patient-centered outcomes. Automated analysis of OTV will also facilitate the development of real-time clinical decision support systems to detect clinically relevant events not captured with existing methods  microcomputers running a version of the Linux operating system (Rasbian), secured out of sight on the back of Puritan Bennett model 840 (PB840) ventilators (Medtronic Corporation), and connected using a serial-USB null modem cable  attached to a serial port on the back of the PB840 screen. We developed custom scripts in the Python programming language to automatically connect to the health system enterprise network, acquire the current date and time, read the PB840’s serial port, and write ASCII-encoded pressure, flow, and time data at 50 Hz to disk. Discrete files were written every two hours to prevent data loss in the event of device failure, and to facilitate subsequent waveform visualization. Additional scripts were developed to automate hourly file back-ups to a networked study server, and an easy to use custom web-based application was developed to allow remote server-side file renaming with subject identifiers once data collection was complete, and to clear old data from RPis by study personnel with no technical background. The entire data acquisition platform and workflow was designed to be fully automated once an RPi was connected to the PB840 and plugged in.Studies of patient-ventilator interactions have been limited by difficulty in acquiring waveform data from mechanical ventilators, variously requiring proprietary software, additional instrumentation placed in-line in the ventilator circuit, and/or a direct laptop interface representing large barriers to data access in terms of human resources, cost, and potential bias introduced due to the observer effect7. In general, ventMAP processes arrays of pressure, flow, and time data from each recorded breath to derive a series of quantitative metadata such as TVi and TVe, inspiratory and expiratory times , peak inspiratory pressure, positive end-expiratory pressure (PEEP), and other derived variables , from breath start until the first data point where flow has transitioned from positive (inspiratory) to negative (expiratory) Fig. . ExperimOTV event detection algorithms were developed for DTA, BSA, in-line suctioning, ventilator disconnect, and a subset of coughs Fig. . ventMAPAnalysis of algorithm performance in both the derivation and validation cohorts was conducted with and without correction for clinical artifacts. Each algorithm’s sensitivity, specificity, and overall accuracy were estimated using logistic regression. To account for varying numbers of breaths and events across patients  to severe violation (3), respectively Fig. . To compThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Information


Basic disorders of the embodied self (BDES) encompass a cloud of related clinical constructs  that are immanently related to a profound transformation of subjectivity and with the developmental modulation of bodily awareness. They have been historically ascribed a potential role in the emergence of schizophrenia spectrum disorders. However, the clinical-phenomenological level of description has been only marginally integrated with novel insights from developmental psychopathology and neurosciences. We conducted a conceptual literature review based on clinical analysis and heuristic synthesis.Despite often occurring in prodromal/clinical at-risk states, as well as in full blown schizophrenia spectrum conditions , they are relatively neglected both in research and in routine clinical examination. Furthermore, BDES also discriminate non help-seeking genetic high risk subjects from normal controls.Within the superordinate construct of Self-disorders, BDES are a potentially relevant dimensional phenotype for the characterization of broad Schizophrenia Spectrum vulnerability. Their contextualization within a developmental and neurophysiological perspective could further amplify their value for etio-pathogenetic research.


Nicotiana benthamiana. Early RNA silencing platforms assessed for CMG resistance have been use of viral genes for co-suppression, antisense suppression or for hairpin RNAs-mediated gene silencing. Here, progress and challenges in the deployment of these approaches in the control of CMGs are discussed. Novel functional genomics approaches with potential to overcome some of the drawbacks of the current strategies are also discussed.The cassava mosaic disease (CMD), which occurs in all cassava growing regions of Africa and the Indian subcontinent, is caused by cassava mosaic geminiviruses (CMGs). CMGs are considered to be the most damaging vector-borne plant pathogens. So far, the most successful approach used to control these viruses has been the transfer of a polygenic recessive resistance locus, designated CMD1, from wild cassava to cassava cultivars. Further progress in harnessing natural resistance to contain CMGs has come from the discovery of the dominant monogenic resistance locus, CMD2, in some West African cassava cultivars. CMD2 has been combined with CMD1 through genetic crosses. Because of the limitations of the cassava breeding approach, especially with regard to time required to produce a variety and the loss of preferred agronomic attributes, efforts have been directed toward the deployment of genetic engineering approaches. Most of these approaches have been centered on RNA silencing strategies, developed mainly in the model plant  Manihot esculenta Crantz, was introduced in Africa by the Portuguese in the 16th century, and was initially grown in and around trading posts in the Gulf of Guinea in West Africa. It was subsequently introduced into East Africa from Madagascar in the later part of the 18th century  (Bemisia tabaci) or spread through infected cuttings, which are the usual mode of cassava propagation. The viral etiology of CMD was confirmed by electron microscopy of geminivirus particles (Table 1). The genome organization of CMGs is shown in Figure 1A.(CMD), which is caused by cassava mosaic geminiviruses  . CMGs ararticles . With tharticles    . The twota, TME) ,b. Severta, TME) . A clearta, TME) . The sucta, TME) .Figure 1B), all three IITA varieties recorded CMD incidences of less than 20% in high infection pressure areas in the southwestern part of the country . Since 1999, five new IITA cassava varieties containing both CMD1 and CMD2 resistances have been adopted and shown to display CMD incidences of less than 10% in the high inoculum pressure areas of southwestern Cameroon where local cultivars show 100% incidence; indeed, one of these varieties, TMS 96/0023 , displays a strong resistance to CMD.In several countries, varieties selected from the IITA germplasm have been shown to exhibit strong resistance to CMD. For example, in Uganda, where the latest CMD pandemic first emerged in the 1990s, the IITA varieties were resistant to CMD, and to a lesser extent, cassava brown streak disease (CBSD), caused by cassava brown streak viruses (CBSVs) in East Africa . Similar country . In all reversion phenomenon, which is the ability of cuttings from CMG infected mother-plants to grow into virus-free plants in the next cropping cycle. This mechanism has been attributed to incomplete systemic spread of the causal virus in infected plants  and Sweet potato chlorotic stunt virus (SPCSV) (Potato leafroll luteovirus (Subterranean clover mottle sobemovirus (Strawberry crinkle cytorhabdovirus and Strawberry mottle sadwavirus (An understudied mechanism of CMG resistance in cassava is the d plants . It was d plants . Interes cassava . It has  (SPCSV) ; potato teovirus ; subterremovirus  and stradwavirus . DespiteReversion from CMD has been shown to occur at different rates in different cassava genotypes under both field and controlled environment conditions in Cameroon .Cameroon , Uganda Cameroon , and CotCameroon . This phCameroon . This lirecovery ,b, and trecovery . A furthd plants  and do nreversion and allow the identification of markers that can be used to select high reverting cultivars as this would have a direct and immediate impact to farmers, especially given field observations by the author that reverted plants tend to be more resistant to infection by whitefly vectors.Recently, it was reported that CMD-susceptible cassava cultivars that displayed no recovery or showed a weak recovery, accumulate higher levels of viral small interfering RNAs (vsiRNAs) compared with genotypes that exhibited a strong recovery . While tTobacco mosaic tobamovirus and Alfalfa mosaic alfamovirus, respectively (Cucumber mosaic cucumovirus (CMV) satellite RNA were protected from CMV (Bean golden mosaic begomovirus (BGMV) (Nicotiana benthamiana plants expressing ACMV CP (Tomato mottle begomovirus (TMoV)-CP transgenic tobacco plants to TMoV was not correlated with CP expression (Tomato leaf curl virus (TLCV)  genes of ectively . Subsequfrom CMV . The abifrom CMV . Early efrom CMV  leading from CMV . Intrigus (BGMV)  nor Nico ACMV CP  displayepression ; similars (TLCV) . Althougormation . These PAC1, which is the only geminivirus gene that is indispensable for viral replication. Thus, N. benthamiana containing the AC1 gene from the Kenyan isolate of ACMV (ACMV-KE) under the control of Cauliflower mosaic virus (CaMV) 35S promoter. The resulting transgenic lines showed resistance to ACMV, characterized by low levels of viral DNA accumulation compared with control plants. Correspondingly, transgenic N. benthamiana plants expressing ACMV-KE AC1 with a mutation at the nucleoside triphosphate (NTP)-binding site exhibited a delay in symptom appearance and some of the plants also displayed attenuated symptoms  promoter . By the time, it had become clear that post transcriptional gene silencing (PTGS), a natural component of plant defense against viral infection mediated by siRNAs, was responsible for the resistance generated from a viral transgene  under the control of the CaMV 35S promoter. Consistent with a recent transient expression study , conferred virus immunity . This reon study , transgeoduction . TransgeN. benthamiana stably expressing Tomato golden mosaic begomovirus MP inhibited ACMV replication (Cabbage leaf curl begomovirus (CaLCV) infection  DNA displayed ACMV resistance. The DI DNA in this case was ACMV DNA B component from which large segments had been deleted, including BV1, but which retained the CR and parts of BC1 (Beet curly top virus (BCTV) or TGMV indicated that the resistance against ACMV was specific. This resistance was explained by preferential replication of the episomal DI DNA at the expense of the viral genomic components. Thus, the infecting ACMV was able to mobilize the DI DNA from the N. benthamiana genome and to replicate it episomally. This result was in agreement with previous transient expression studies where N. benthamiana plants co-inoculated with ACMV-KE and the DIs resulted in reductions in replication of both ACMV-KE DNA A and B components .Several non-viral genes have been investigated for their ability to induce host resistance to geminiviruses, including CMGs , trans-acting small interfering RNA (tasiRNA) and Clustered Regularly Interspaced Short Palindromic Repeats associated Cas9 (CRISPR-Cas9) mechanisms and their application in generating resistance to CMGs are discussed.The encouraging early results obtained from the use of hpRNA to control plant viruses have been tempered by several disadvantages associated with hpRNA-induced resistance, these include poor stability of the transgene in transformed plants, dependence on the expression levels of the antisense strand, and limited penetration of the silencing signal to the appropriate viral target due to target-sequence folding . So far, resistance to at least 12 plant viruses (including CBSV) has been generated using amiRNAs (see review in Wheat streak mosaic tritimovirus (WSMV) genome regions conferred immunity to WSMV in transgenic plants (Wheat dwarf mastrevirus (WDV) genome  genome . Even thThe encouraging results of multiplex amiRNAs offer an unprecedented opportunity in the control of CMGs, which exhibit considerable genetic variability and frequently co-infect the same plant . Therefotrans-acting siRNA (TAS) transcripts following an initial cleavage of the transcript by a RISC containing AGO1 or AGO7, depending on the specific miRNA. Two models of tasiRNA biogenesis referred to as “one-hit” and “two-hit,” respectively, have been described . In the “one-hit” model, where there is a single miRNA target site, a RISC containing AGO1-bound 22-nt miRNA, binds and cleaves the TAS transcript, under the control of AGO1 . This strategy has been used to successfully engineer resistance to plant viruses. For example, transgenic N. benthamiana harboring a TAS3 gene modified to contain tasiRNA from the AC2 and AC4 genes of Tomato leaf curl New Delhi begomovirus (ToLCNDV) exhibited strong resistance to ToLCNDV and to Tomato leaf curl Gujarat begomovirus (ToLCGV)  . Also, t viruses . Thus, tTAS genes and their promoters; this is principally because current applications use well-studied Arabidopsis TAS genes. However, we recently identified endogenous cassava TAS loci and their miRNA triggers  has been harnessed for efficient genome editing and gene regulation . In this system, Cas9 creates double-strand breaks (DSBs), leading to insertion or deletion (indel) mutations in the targeted gene  and Beet curly top curtovirus (BCTV) conferred resistance to the respective viruses in N. benthamiana plants expressing Cas9 in a virus-specific manner , was shown to specifically target and degrade BSCTV in transgenic Arabidopsis and N. benthamiana (Bean yellow dwarf mastrevirus (BeYDV) Rep-binding site, hairpin, nonanucleotide sequence, and three Rep motifs essential for rolling circle replication. This report further showed that sgRNAs targeting sequences near the BeYDV hairpin were less effective, presumably due to formation of secondary structures that impeded Cas9-sgRNA cleavage or access , expression of target genes is mostly repressed, whereas CRISPR-Cas9 knocks out the target genes. This potentially makes CRISPR-Cas9 a very efficient method to generate host resistance. However, it must be noted that an advantage of RNAi approaches over the Cas9 system is that in RNAi the guide RNAs can be extracted directly from invading nucleic acids, thus the capacity for mutational evasion by the virus is limited. In contrast, in the CRISPR-Cas9 platform, sequence determinants are encoded in the host genome, and thus targets have a greater potential for mutational evasion by evolving new virus strains.Figure 5B). Upon transcription, the tandemly arrayed tRNA-sgRNA chimera is cleaved by endogenous RNase P and RNase Z, releasing a mature sgRNAs, which direct Cas9 to multiple targets  and bentazon sensitive lethal (OsBEL) genes. Results showed that the CRISPR-Cpf1 efficiently generated specific and heritable targeted mutations in rice (phytoene desaturase (PDS) and STENOFOLIA ortholog in N. tabacum (NtSTF1) in tobacco, and Drooping leaf (DL) and Acetolactate synthase (ALS) in rice, respectively  genes. In this case, the CRISPR components were transfected to flax protoplasts to introduce double-strand breaks without integration into the genome. Because no DNA is transferred, the resulting genome-edited plants might be exempt from current genetically modified organism (GMO) regulations -based vector, which was used to deliver sgRNA targeted to the PDS gene in N. benthamiana  have been found to contain transcriptionally active endogenous geminiviral sequences that may be functionally expressed   project Together, the risks associated with transgenic cassava, a crop that has a low fertility, seed set, and germination rates are largely outweighed by the devastation caused by viruses in cassava growing regions of the world. Moreover, approaches being used in generating resistance to viruses depend on viral genomic sequences, which in some instances integrate naturally in the plant genome or occur episomally, and have been consumed by humans for centuries with no apparent ill effects.Figure 1D), new technologies will need to be deployed where breeding is unlikely to succeed. Most genetic engineering strategies depend on use of viral sequences to confer resistance and thus would be helpful in situations where there either is no natural resistance (as apparently is the case with CBSD), or the resistance is difficult to introduce through breeding.Cassava is a vital source of food and income in most tropical regions of the world and its production needs to continue to respond to food priorities of developing countries as well as to trends of a global economy. To counter cassava production constraints so as to increase yield and quality, new technologies will need to be deployed. CMD is the most important constraint to cassava production in sub-Saharan African and the Indian subcontinent. To date, introgression of CMD resistance from wild cassava has been by far the most successful CMD control strategy. Three types of CMD resistance loci, designated CMD1, CMD2, and CMD3, have been established; as a precautionary measure against the real possibility of CMD appearing on the American continent, these resistances are being introgressed into South American varieties . In spitIn the last two decades, a lot of progress has been made in identifying and understanding mechanisms of host resistance to virus infections, including particularly RNA silencing and CRISPR systems. As genome sequencing costs fall and new tools are developed to more efficiently analyze genome sequence data, new resistance factors and pathways will continue to be discovered, this would provide additional new tools for use in engineering crops for virus resistance. For example, identification of virus-resistance genes would provide opportunities for such genes to be precisely edited using non-transgenic strategies. Based on the characteristics of the recently discovered CRISPR-Cpf1 system, this approach will likely provide unprecedented new opportunities in cassava improvement through precise transient genome editing, especially given that cassava is recalcitrant to transformation.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Efficient patient-centred quality improvement requires an understanding of the system-wide areas of dissatisfaction along with evidence to identify the programs which can be strategically targeted according to specific patient characteristics and preferences. This cross-sectional study reports the proportion of chronic disease outpatients selecting 23 patient-centred improvement initiatives. Using univariate tests and multivariable logistic regressions, this multi-site study also identifies initiatives differentially selected by outpatients according to clinical and demographic characteristics. A total of 475 outpatients participated (49% response). Commonly selected initiatives included: reducing wait-times (22.3%); convenient appointment scheduling (16.0%); and receiving up-to-date treatment information (16.0%). Within univariate tests, preferences for information and service accessibility initiatives were not significantly associated with specific subgroups. However, seven initiatives were preferred according to age, gender, diagnosis status, and chronic disease type within multivariate models. For example, neurology outpatients were more likely to select assistance to manage psychological symptoms when compared to oncology outpatients (OR: 2.89). Study findings suggest that system-wide programs to enhance information provision are strategic approaches to improve experiences across patient characteristics. Furthermore, a few initiatives can be targeted to specific groups and emphasized the importance of detailed scoping analyses and tailored implementation plans when designing patient-centred quality improvement programs.  Patient-centred care (PCC) is defined as care that is respectful of and responsive to individual patient values and needs . In 2001Data from large-scale patient surveys indicate that healthcare experiences are mediated by individual demographic and clinical characteristics ,9,10. WhWhile patient-experience surveys are important to highlight the gaps in the quality of care received, there are a number of limitations in using these data to inform health service improvement. Firstly, many of these tools do not directly request patients to identify their preferences for specific quality improvement initiatives. Without a clear and actionable improvement message, patient-experience surveys may be unable to stimulate health service change ,12,13,14The touch-screen Consumer Preference Survey (Consumer-PS) was designed to enable patients to directly inform quality improvement activities by selecting specific initiatives relevant to their experiences in chronic disease outpatient settings. The survey also allows for comparisons across a range of patient characteristics and chronic diseases as it can be quickly administered to a large and diverse sample with broadly-scoped PCC quality initiatives . This isProportion of individuals selecting each quality improvement initiative; and,Initiatives that are differentially selected between individuals according to demographic and clinical characteristics.This multisite study aims to report the:This is some of the first work to provide a broadly-scoped view of initiatives across chronic disease groups in tertiary outpatient care. For health services and policy developers, this information provides a set of generic initiatives that are equally valued across a range of health service users, along with a set of patient characteristics towards which quality improvement may be efficiently targeted. Briefly, this study concluded that information-based and accessibility initiatives were commonly selected by outpatients regardless of demographic characteristics; however, a few targetable initiatives emerged, particularly for newly-diagnosed outpatients and neurology outpatients.Study design: A cross-sectional survey of outpatients was conducted according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement . Ethics Setting: Outpatients were recruited from two publically-funded tertiary hospitals located in one health district within New South Wales, Australia. This included a specialist clinic providing cardiology or neurology care and a medical oncology centre providing physician consultation and intravenous chemotherapy treatment. Participants: English-speaking adult outpatients were recruited from clinic waiting rooms or treatment areas by trained research assistants. To be eligible, participants had to have attended the clinic at least once prior to recruitment and were asked to reflect upon this experience whilst completing surveys. Assistance with touch-screen devices was provided as needed. Research assistants estimated the gender and age-range of non-consenters to ascertain possible consent bias. Measurement: Participants completed the Consumer-PS and a patient characteristic module. n = 20) and consumers (n = 27) [The Consumer-PS: The Consumer-PS includes 23 PCC quality improvement initiatives which were developed based on a structured literature review, and iterative consultation with two groups of service providers ((n = 27) . During (n = 27) . RespondA validation study reported that the average observed agreement across the 23 initiatives was 93.7% with moderate or substantial test-retest reliability ; the study also reported the high levels of respondent acceptability . For exaDemographic information: date of birth; gender; marital status (single or married/de-facto partner); highest education level achieved ; Aboriginal and or Torres Strait Islander origin; private insurance coverage; and possession of a concession card. Australian concession cards reduce healthcare costs and are restricted to pensioners, social security allowance recipients, and low-income earners.Clinical characteristics: reason for attending the clinic ; and appointment frequency within the last six months. Oncology participants completed two additional questions, relating to primary cancer site and time since receiving diagnosis, if known. Previous research with cancer patients has reported varying information and supportive care needs based on time since diagnosis and tumour type, and therefore may impact the preference for these types of initiatives . The patient characteristic module included the following: n = 23), a stringent Bonferroni significance threshold of 0.002 was used. Initiatives that reached this threshold for at least one demographic or clinical variable proceeded to the next stage;Univariate Chi Square or Fisher’s Exact tests were used to compare the proportion selecting an initiative between demographic or clinical subgroups. To reduce the number of spuriously reported associations due to the large number of initiatives tested .Participants: A total of 968 individuals were approached to participate, of which 608 individuals consented to complete touch screen surveys (62.8% consent). A total of 475 (78.1%) participants completed the survey, of which 271 (57.1%) were oncology outpatients, 135 (28.7%) were neurology outpatients, and 68 (14.4%) were cardiology outpatients. Sample demographic and clinical characteristics are available in Differences in the characteristics of those who consented versus those who declined to participate, as well as those who completed a survey versus those who did not, are available in the The proportion of individuals selecting each PCC initiative: Across the 23 initiatives, improved car parking was selected by the greatest proportion of respondents (67.2%), followed by reduced wait-times (22.3%), up-to-date information provision (16.2%), and convenient appointment scheduling (16.0%) . Comfortp-values > 0.002) across all demographic and clinical characteristics , according to demographic or clinical characteristics  likely to select receiving additional information during an appointment than those attending to discuss a diagnosed condition  and those with a high school education  were approximately three times more likely to select this initiative  to select assistance and information to manage emotional symptoms compared to medical oncology outpatients  of selecting this initiative compared to men ; attending for tests or treatments ; and who attended clinics more frequently  in the past six months  to select improved parking as compared to oncology outpatients , access to information at home (14.1%), information to manage emergencies (11.4%), and assistance and information to maintain activities of daily living (10.3%). Given the relatively higher frequency at which these initiatives were selected by study participants, health services should design programs and policies to improve information provision that can be implemented on a system-wide level. The proportion of individuals selecting information-based initiatives did not differ significantly across demographic or clinical characteristics, with the exception of two characteristics. Firstly, the odds of selecting assistance or information on financial assistance was significantly associated with age; compared to individuals between 18 and 47 years of age, those between the ages of 60 and 66 years were less likely to select this initiative. This finding highlights the need to consider the financial burden experienced by individuals who suffer income-loss as a result of illness. Secondly, relative to individuals with a diagnosed condition, individuals who were attending an appointment for an undiagnosed condition were approximately four times more likely to select additional information on treatment or condition. This finding of high information needs prior to or at the time of diagnosis aligns with closely existing studies, including systematic reviews involving cancer patients , qualitaInitiatives to improve the accessibility and accommodation of care were commonly selected: While patient concerns regarding the accommodation and amenities of care are not clinical issues, health services need to consider how such infrastructure and organizational factors may influence perceptions of care. A literature review of psycho-oncology need assessment tools suggests that health professionals may consider some patient concerns relating to these more front-line areas as outside their scope of practice ; furtherWithin this study, reduced wait-times (22.3%) and convenient appointment scheduling systems (16.0%) were commonly selected by participants and were not associated with patient characteristics. O’Brien et al.  suggesteThe accessibility of care remains an essential component to PCC and practical barriers to navigating health services can have long term patient and system implications in the form of patients delaying or limiting service use or creating additional distress. While the Consumer-PS data relating to organisational and infrastructure improvement may not directly influence healthcare professionals’ personal practice, these findings can provide the needed data to lobby for infrastructure changes with health service administrators and directors. It is also important to note that service evaluation rarely includes collecting and reviewing patient-reported data on perceived quality and accessibility of the care environment . WithoutQuality improvement is not a one-size fits all approach and preferences are influenced by demographic factors: This study sought to identify the initiatives selected by different proportions of individuals and the adjusted odds of selecting these initiatives according to demographic and clinical characteristics. For the seven initiatives significantly associated with patient characteristics, several key characteristics emerged in multivariate models including: age, gender, chronic condition, education, reason for attending, appointment frequency, and health insurance coverage. Increasing age and male gender was frequently associated with lower odds of selecting quality improvement initiatives. Within this study, compared to the youngest age group, older age groups reported decreased odds of selecting: access to information and assistance for financial, work, or insurance concerns; improved catering; information to manage emotional symptoms; and improved parking. Additionally, women were three times more likely to select being able to discuss concerns with a health professional and 2.5 times more likely to select ease of contacting the clinic. Previous studies have reported differences in patient satisfaction according to both gender and age. Results from the 2011/2012 English Cancer Patient Experience Survey, a national mail-out survey completed by 71,793 individuals, found that individuals aged 65–74 years reported a more positive experience . Concordp = 0.005). High levels of unmet emotional needs have been previously reported by individuals with neurological conditions, such as stroke and multiple sclerosis. For example, a study of long-term needs in individuals up to five years post-stroke reported that approximately 77% reported emotional problems, with the majority indicating that these needs went largely unmet [Accessing information and support for emotional concerns is particularly relevant for individuals attending for neurology services. As compared to medical oncology outpatients, neurology outpatients were significantly more likely to select emotional support . The results from the larger study will provide additional information on how the Consumer-PS, or similar surveys, can be used as a means to incorporate consumer perspectives into evaluating and improvement the delivery of PCC. In order to improve the quality of outpatient chronic disease care according to patients’ preferences and priorities, health services should focus on implementing information-based initiatives on a system-wide level. However, a few targetable initiatives emerged, such as additional emotional support for neurology outpatients. Given the number of factors associated with patient preferences’ for quality improvement, this study emphasizes the need for detailed scoping analyses to inform any quality improvement and specific concerns need to be addressed using a more tailored approach.