In February of 2016, the World Health Organization (WHO) declared Zika virus (ZIKV) a Public Health Emergency of International Concern. This prompted a rapid response from both the private and public sector resulting in the generation of several promising vaccine candidates. In this review, we discuss published scientific efforts associated with these novel vaccines, emphasizing the immunological assays used to evaluate their immunogenicity and efficacy, and support future licensure.  Flaviviridae, genus flavivirus, which includes other human pathogens such as dengue (DEN), yellow fever (YF), Japanese encephalitis (JE), West Nile (WN) and tick-borne encephalitis (TBE) viruses. All distinguishable ZIKV strains are classified into the African and Asian/American lineages, both of which belong to a single serotype [Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) and member of the family serotype . Similarserotype . The virserotype ,4. Priorserotype . Howeverserotype . There are existing licensed vaccines for several other flaviviral diseases including DEN, YF, and JE, and protective levels of neutralizing or binding antibodies have been established for some of these viruses ,8,9,10, As with other licensed flaviviral vaccines, there is recognition that neutralizing antibodies directed against ZIKV may be used as surrogate markers for vaccine immunogenicity and protective efficacy. This has led many vaccine developers to use virus neutralization as the primary humoral immune response readout. Currently, various assay formats have been employed and a standardized protocol for neutralization testing has not been adopted. Assays include the plaque reduction neutralization test (PRNT), where endpoints are based on visual observation of viral plaques in cell monolayers after inoculation with virus incubated with test serum . Other vAlthough cell-based assays are commonly used to measure virus neutralization, alternative non-cell-based platforms, such as antibody binding tests, are concurrently being used as supplementary and/or confirmatory tests to rapidly and effectively measure the level of anti-ZIKV immunoglobulins  present in serological samples. ELISA assays using whole ZIKV purified virions or recombinant ZIKV E protein have been used for titration of vaccine-generated antibodies ,16,19,21Although virus-neutralizing antibody is the primary protection correlate for flaviviruses, measurement of cell-mediated immune responses after vaccination may provide additional useful data. For example, live-attenuated and inactivated vaccines differ in the degree of cytotoxic and helper T-cell responses that they elicit, and this may translate to differences in protective efficacy. Peripheral blood mononuclear cells were stimulated with peptide pools that encompassed ZIKV structural proteins. For vaccine responses, proportions of cytokine-positive T cells responding to the peptide pools were summed ,22.Peripheral blood mononuclear cells were stimulated with overlapping peptide pools of ZIKV structural proteins. IFNantibody ,16,17,22Both active and passive immunization have been used to assess protection from ZIKV challenge.Both adult mice and non-human primates have been used to assess vaccine immunogenicity and protection against challenge. Adult inbred mice including AG129, Balb/c, SJL, and C57BL/6 have been used for immunization and virus challenge ,20,21,22A new, lethal challenge model using C57BL/6 mice has been developed . Mice are immunized with a candidate vaccine then treated with anti-interferon monoclonal antibody prior to challenge. This model allows for vaccine responses in immunocompetent mice followed by a lethal challenge. Rhesus macaques and other non-human primates (NHP’s) have also been used for immunization and challenge studies  and to a−/− mice after challenge with ZIKV [The ability to protect both adult mice and non-human primates using anti-ZIKV immune serum globulin or purified IgG has been demonstrated in passive protection studies. Larocca et al. investigated passive protection in Balb/c mice by purifying IgG from vaccinated mice and intravenously infusing the IgG into recipient mice and challenging with ZIKV soon after. The virus-neutralizing antibody titer of the purified IgG pool could be correlated with protection from viremia after challenge with ZIKV . Abbink ith ZIKV . A similith ZIKV . Here, BAs was discussed above for virus neutralization assays, since different laboratories have their own favored assay platforms/SOPs and new assays are continually being developed, the widespread adoption of a standardized assay(s) is unlikely. Nevertheless, in order for serological test results to be compared among laboratories reference standards, especially reference antisera, are needed. To assist in this endeavor, the World Health Organization (WHO) maintains an Expert Committee on Biological Standardization as the scientific body responsible for establishing WHO reference standards. The WHO International Standard (IS) is recognized as the highest reference standard in which antibodies are assigned potencies in International Units (IU). The IS allows for comparison of assays from different laboratories, thereby helping to establish protective antibody levels after vaccination. A WHO collaborative study for testing ZIKV antibodies was organized in 2016 with 19 laboratories from six countries participating in the study. Based on neutralization and enzyme immunoassays, a ZIKV antibody IS was selected and can be requested from the National Institute of Biological Standards and Controls  for laboThe Ebola and Zika outbreaks highlighted the need for International Standards. Although establishing an IS usually requires several years, it would be advantageous to have one in place at the outset of vaccine testing and during pre-clinical development stages . The datThe development of a safe and efficacious, licensed ZIKV vaccine will require the generation and/or improvement of immunoassays that will be used to evaluate relevant immunological parameters post vaccination. The overall objective of these assays is to help elucidate the nature and types of the immune responses generated by a candidate vaccine, to include the quality and kinetics of the immune response, as well as the longevity of protection associated with the vaccine. Assays designed for this purpose should be validatable, especially if they will be used in later phase 2–3 clinical trials. Validation parameters should include the establishment of an assay cut-off or cut point (positive threshold), linearity, limit of detection (LOD), limit of quantitation (LOQ), specificity, accuracy, and precision. Durability of protective immunity is a desirable outcome for any vaccine. It has been found that long-lived plasma cells (LLPCs) are associated with life-long immunity against some viruses following infection and vaccination ,31. MiceIt will also be important to measure flavivirus cross-reactive antibodies after immunization with a ZIKV vaccine, especially in endemic areas where other flaviviruses are found. This is especially important since it has been postulated that pre-existing DENV immunity may predispose individuals to worsened ZIKV infections. Currently, the best assay for measurement of type-specific and cross-reactive antibody is the virus neutralization assay. Improvements in this assay would be beneficial to sort out homotypic (type-specific) and heterotypic (cross-reactive) antibodies responsible for protection against disease as well as pathogenicity. In one study in Brazil, ZIKV infections and disease did not correlate with DENV immune status . In anotConformational ZIKV epitopes and monoclonal antibodies have been developed that could be used for characterization of vaccine antigens and immune sera. ZIKV-neutralizing monoclonal antibodies generated from plasma cells harvested from volunteers who received a candidate Zika vaccine (ZPIV) were used for epitope mapping . Once fully characterized, these antibodies can be used in antigen binding assays  to demonstrate the presence of relevant epitopes on vaccine antigens, and in antibody competition (competitive binding) assays to determine whether the vaccine is able to elicit antibodies against these potentially important epitopes. In addition to the functional assays  aimed at measuring the ability of the ZIKV vaccine to provoke a robust immune response, characterization assays that can assess the overall protective immune response, or efficacy, of the vaccine might be useful. In this regard other antibody effector functions may be important for protection, such as complement-dependent immune cytolysis of infected cells and antibody-dependent cell-mediated cytotoxicity (ADCC). However, unlike virus-neutralizing antibodies, protection correlates (either positive or negative) for antibodies with other effector functions have yet to be established.®, is one of these. It was licensed on the basis of demonstrating in vaccinees non-inferior levels of virus-neutralizing antibody compared with antibody titers first established as protective against encephalitis for JE-Vax® (Biken) inactivated vaccine in phase 3 field trials [Regulatory views on ZIKV vaccine clinical development strategies have addressed the need for alternatives to traditional phase 3 field trials to gain licensure . Ideallyd trials .The U.S. Food and Drug Administration (US FDA) Accelerated Approval provision applies to vaccines that provide meaningful clinical benefit over existing treatments for serious or life-threatening illnesses . Even thThe US FDA has published guidance for establishing efficacy of preventive vaccines using the “Animal Rule” in “Product Development under the Animal Rule, Guidance for Industry, 2015” . The FDA4 to 106 plaque-forming units of virus, similar to a dose delivered by a mosquito. Clinical disease included mild weight loss and rash in some of the infected animals. All animals displayed decreased white blood cell counts and elevated liver transaminases. Tests for neutralizing antibodies and T-cell responses were positive. In another study, rhesus macaques infected with a clinical isolate of ZIKV developed fever, viremia, and robust excretion of ZIKV RNA in bodily fluids and the viral antigen was present in organs [The rhesus macaque has been used for both pathogenicity as well as vaccine studies and appears to meet most of the Animal Rule criteria for demonstrating vaccine efficacy. In one study, , followin organs . Congenin organs . This isVaccine studies in rhesus macaques previously cited have established that this non-human primate model may meet the FDA animal rule requirements. Human immune response data from recent phase 1–2 studies appear to follow those results found after immunization of non-human primates ,18. FurtEstablishing the efficacy of ZIKV vaccines by challenging vaccine recipients with unmodified ZIKV strains has been proposed . The demIt will be important for ZIKV vaccine developers to develop immunological assays useful for establishing vaccine efficacy. For ZIKV, a flavivirus, it appears that demonstration of virus-neutralizing or binding antibodies are important for establishing vaccine efficacy. Other flavivirus vaccines including YFV, JEV, and TBEV have established surrogate markers of neutralizing and binding antibodies as correlates of disease prevention. Animal studies support these correlates for several ZIKV vaccine candidates by direct challenge of primates as well as transfer of immune sera to mice that are then challenged with ZIKV. Human vaccine studies have also shown that immune sera from vaccinees can similarly protect mice after challenge. The rhesus macaque has been shown to develop ZIKV disease similar to humans and may be useful to establish efficacy based on parameters published by the US FDA. Validated assays will be essential for eventual license of any ZIKV vaccine candidate. Field efficacy testing may be limited due to the sporadic epidemiology of ZIKV and assay correlates will be essential for demonstration of protection against disease.


Epidemiologic studies have, variably, shown the concomitant use of statin drugs to be beneficial to cancer outcomes. Statin drugs have been FDA approved for three decades for the treatment of high cholesterol and atherosclerotic coronary artery disease and are widely used. This has engendered studies as to their influence on concomitant diseases, including cancers. In this context, statin use has been correlated, variably, with a decrease in deaths from breast cancer. However, there is no extant model for this effect, and the extent of efficacy is open to question.The overarching goal of this article is to communicate to the reader of the potential of statins to reduce breast cancer progression and mortality. This is the use as a secondary prevention measure, and not as a therapy to directly counter active cancer. First, salient aspects of statin pharmacology, as relates to cardiovascular disease, will be discussed. Second, the basic and clinical research studies that investigate statin usage in breast cancer will be presented. Additionally, statin effects in other cancer types will be included for context. Finally, proposals for future basic and clinical research studies to determine the role of statins in breast cancer management will be presented.The online version of this article (10.1186/s13058-018-1066-z) contains supplementary material, which is available to authorized users. Statins reduce blood cholesterol by inhibiting the rate-limiting step in the mevalonate pathway: HMG-CoA reductase (HMGCR). While this decreases the cholesterol carried in the blood, the mechanistic aspects also affect cell signaling, in a manner that could directly impact cell proliferation, and thereby cancer cells. Mevalonate pathway inhibition reduces the abundance of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), isoprenyl groups that critically modify small signaling G proteins involved in cell proliferation, migration, and survival pathways. The influence of the statin drugs on protein prenylation is not limited to just influencing cardiovascular pathologies but can be applied in the context of other diseases, particularly cancer in which progression depends on increased migration, survival, and ultimately proliferation.Relevant to this review, epidemiologic studies have, variably, shown the concomitant use of statin drugs to be beneficial to cancer outcomes. The aims of this article, based on literature findings both in vivo and in vitro, are to review recent studies related to statin effects on breast cancer from both a clinical and basic aspect, so as to: 1) form a consensus from the existing literature regarding the mechanisms by which statins influence cancer; and 2) establish a model of how these mechanisms influence cancer incidence, recurrence, and mortality. Existing studies suggest that statins can reduce tumor cell growth and survival, which translates to a clinical benefit of statins on reducing cancer recurrence and mortality, with limited effects on incidence of primary cancer.The first statins were isolated as potent competitive inhibitors of HMG-CoA reductase (HMGCR) by Akira Endo , motivatThe seven FDA-approved statins have similar, but not identical, pharmacokinetics  particles . StatinsIn addition to modifying lipid homeostasis, statins exert pleotropic effects on other aspects of cardiovascular pathobiology. Cell-mediated effects stem from reductions in cellular signaling from small G proteins through reducing the availability of FPP and GGPP . For exaStatin usage in the US has increased dramatically over the past several decades. Between 2003 and 2012, the percentage of adults in the US taking statins increased from 18 to 26% . The cliThis background sets the stage for the unexpected effects statins may have on cancer. As their use is widespread, there is a large overlap between people on chronic or long-term statin therapy and those with cancer. Of relevance to the present review are the basic and clinical research studies that have investigated the roles of statin drugs in cancer incidence, progression, and mortality.While few prospective clinical trials have been conducted to investigate statin therapy in cancer patients, multiple retrospective cohort studies and meta-analyses have been published. Several hypotheses motivate these studies. First, since statins lower LDL-C, this may influence carcinogenesis and tumor progression, as rapidly dividing cells require increased cholesterol for cell membrane synthesis . In addiMany studies have investigated the influence of statin therapy on the risk for developing cancer, with conflicting results. The CARE trial in 1996 published one of the first associations between statins and cancer incidence, in which pravastatin users demonstrated a statistically significant increase in breast cancer risk . SimilarThe largest studies have focused on colorectal, lung, prostate, and breast cancer. In colorectal cancer, large retrospective cohort studies demonstrated no influence of statins on disease incidence , 24. In In breast cancer, no association between statin usage and increased cancer risk has been reported in the literature . Meta-anFewer studies have investigated the influence of statins on cancer recurrence. While a consistent decrement in recurrence occurs in liver cancer , the finFurther supporting the contention that statins impact tumor progression, the majority of published data suggest that statins have a mortality benefit in most cancer types. In primary liver cancer, pravastatin and any statin usage reduces cancer-specific mortality –42. SimiIn breast cancer, the majority of studies suggest statin usage significantly reduces breast cancer-specific mortality (BCSM). A meta-analysis by Liu et al. demonstrated a 43% reduction in BCSM in women with breast cancer, which was confined to those taking lipophilic statins . A natioTwo additional studies provide convincing data for an overall benefit of statins on all site cancer-specific mortality (ASCSM). First, a meta-analysis by Zhong et al. showed a 31% and 23% reduction in ASCSM in pre- and post-diagnosis statin users, respectively . Second,Many clinical studies have demonstrated that statins can influence carcinogenesis and cancer progression. While statins do not affect the incidence of most cancers, they do exert significant benefits on recurrence and survival in many cancer types, including breast cancer. Importantly, statin recurrence and mortality benefits are most strongly seen with lipophilic statins. These clinical studies motivate further investigation of statins in basic research studies and targeted clinical trials to evaluate potential additive or synergistic effects of statins when used with standard therapies.The clinical studies discussed above have been complemented by basic research studies that focus on the mechanisms by which statins exert anti-tumor effects. Statins may affect tumor cells directly in four main ways: 1) growth suppression, 2) apoptosis induction, 3) anti-invasive and anti-metastatic effects, and 4) anti-angiogenic effects. These would be in addition to the indirect effects of lowering available cholesterol. However, the findings that most beneficial effects of statins accrue to those on lipophilic statins suggest direct effects on the tumor cells. Interestingly, the two exceptions wherein even hydrophilic statins present benefit, liver and prostate cancers, further support the direct effect hypothesis as these cancers possess the transporters that allow cellular entry of the hydrophilic statins.Previous in vitro studies have demonstrated that statins can halt cancer cell proliferation by inducing G0/G1 or G2/M arrest. Many pathways have been described that contribute to this anti-proliferative effect. First, simvastatin has been shown to induce G1 cell cycle arrest through reduction of CDK4/6 and Cyclin D1 . AdditioIn addition to suppressing growth of tumor cells, some literature reports that statins can induce apoptosis. Several pathways have been described that contribute to induction of apoptosis. First, statins have been shown to decrease the protein levels of anti-apoptotic proteins such as Bcl-2 and Bcl-xL , 64. SecHowever, other studies demonstrate no influence of statins on apoptosis. For example, simvastatin was shown to decrease proliferation of bladder cancer cells without inducing apoptosis . MoreoveStatins exert biphasic effects on angiogenesis—pro-angiogenic effects at low (nanomolar) concentrations, anti-angiogenic effects at higher (micromolar) concentrations . At low In contrast, at higher concentrations, statins negatively impact angiogenesis through multiple mechanisms. First, statins have been shown to reduce membrane localization of RhoA by blocking its geranylgeranylation, which reduced the tube-forming ability of human endothelial cells in vitro and was reversed with GGPP supplementation . This inIn the context of cancer, statins can reduce tumor angiogenesis through several different mechanisms. First, statins can directly affect the endothelial cells to reduce tumor angiogenesis. For example, simvastatin treatment was shown to decrease tumor vascularization in mice at high statin doses . Second,Many in vitro studies have suggested statins reduce the invasiveness and metastatic potential of cancer cells through multiple different mechanisms. First, statin treatment destabilizes the cytoskeletal structure of tumor cells in a RhoA/RhoC-dependent manner. Upon statin treatment, Rho delocalizes from the membrane, which causes breakdown of the actin cytoskeleton, loss of actin stress fibers and focal adhesion sites, and cell rounding , 85. MorStatins also impact tumor invasion and metastasis in ex vivo and in vivo models. Statins were shown to reduce prostate cancer migration towards human bone marrow stroma without impacting ECM adhesion . Also, lMany basic research studies have shown that statins can reduce tumor cell growth and proliferation by inducing cell cycle arrest. Additionally, some, but not all, studies suggest that statins can induce apoptosis in cancer cells. In addition to affecting tumor cell growth and apoptosis, statins can negatively impact the tumor vasculature through suppression of angiogenesis. While statins exhibit a biphasic effect on angiogenesis, their anti-angiogenic effects are observed at similar doses to those used in studies that demonstrate anti-tumor effects. While statins have been shown to reduce cancer cell migration and invasion in vitro, few studies have demonstrated a reduction in metastasis with statin treatment in vivo. These previous studies motivate the investigation of statins in the context of breast cancer metastasis, in particular dormancy and emergence, which is the main cause of morbidity and mortality in this patient population. We propose a model in which statins block the secondary EMT and outgrowth of tumor cells by preventing Ras, Rac, and RhoA prenylation Fig. .Fig. 2PrAn important consideration for repurposing statin drugs for breast cancer treatment is toxicity. Breast cancer patients already receive chemotherapeutic compounds that exhibit varying degrees of toxicities. Fortunately, statins are typically well-tolerated drugs. The most common adverse events associated with statin usage involve muscle tissues, which range in severity from myalgia to fatal rhabdomyolysis . Muscle The key concern for breast cancer patients is that the toxicity of statins leads to generalized inflammation. The initiation of inflammatory cascades can awaken dormant micrometastases to outgrow , 100. ThPrevious studies have reported mechanistic and pharmacologic insight into the use of statins as anti-tumor agents, but additional questions remain regarding their use in breast cancer. First, the efficacy of statins against breast cancer cells with different molecular subtypes is not yet well understood. The susceptibility of many breast cancer cell lines—hormone receptor-positive, HER2-positive, and triple-negative—should be investigated in order to determine whether molecular subtype plays a role in statin sensitivity. Moreover, patient-derived cells should be tested to validate the cell line data.Second, the difference in efficacy of statins against primary and metastatic tumor cells should be further investigated. Since the clinical data demonstrate statins selectively reduce breast cancer mortality and recurrence without affecting cancer incidence, this would suggest statins more selectively target metastatic cells. This may result from the evolution of tumor cells between the primary and metastatic tumor or be related to primary tumor drug penetrance. In order to better investigate the differential potency of statins against primary and metastatic tumor cells, paired biopsy samples from clinical patients should be used in in vitro, ex vivo, and in vivo models of breast cancer metastasis. This may reveal the relative importance of intrinsic differences in statin susceptibility of the primary and metastatic cells and the differences in the primary and metastatic microenvironments. Moreover, there may be a difference in statin susceptibility with the site of metastasis.Finally, it is critical to understand how statins interact with currently used breast cancer therapies, as statin use may either enhance or diminish their efficacy. For example, since statins work by inducing growth arrest and decreasing proliferation, the efficacy of chemotherapeutic agents may be compromised since they are only effective against cycling cells. If chemotherapeutic efficacy is compromised by statins, patients should stop taking their statin during chemotherapy cycles. In contrast, statins have been shown to induce oxidative stress in cancer cells, which may compromise the health of tumor cells and make them more susceptible to cytotoxic drugs. If these findings hold true, patients who don’t normally take statins should supplement their chemotherapy regimen with a lipophilic statin. Finally, since most statins are metabolized by CYP450 isozymes, they may interfere with the metabolism of chemotherapeutic drugs and possibly induce additional adverse events. Examining statins in combination with other chemotherapeutic drugs in ex vivo liver culture systems and in vivo will suggest which drug combinations may result in higher clinical efficacy or toxicity.Prospective clinical trials should investigate statin use as adjuvant therapy in breast cancer. Since the studies that demonstrate a cancer mortality benefit with statin use are quite large, prospective clinical trials would need to be conducted in a targeted patient population. For example, investigating adjuvant statin therapy in triple-negative or HER2-positive patients would be more feasible than a trial of hormone receptor-positive patients since the former two groups typically relapse earlier. Moreover, patients with nodal metastases or locally invasive primary tumors would be prime candidates for these trials, as they are at the highest risk for metastatic relapse. Patients would be randomized to a lipophilic statin (likely atorvastatin) or placebo and followed longitudinally. These trials would contain defined interim analysis periods to allow patients to crossover from placebo to statin should the statin recurrence or mortality benefits prove significant.Future basic research studies and clinical trials that build upon the results of past studies have the potential to establish statins as effective and safe agents to delay breast cancer recurrence and mortality. In the short term, studies should address whether current statin users for cardiovascular indications with a history of breast cancer should be uniformly prescribed lipophilic statins. In the long term, clinical trials should address whether statins play a role in adjuvant management of breast cancer in women who do not otherwise require a statin. It is hoped that accomplishing both of these short- and long-term goals will improve the quality of life and survival rates of women with breast cancer.Additional file 1:Chemical structures of HMG-CoA, compactin, and FDA-approved statins. The chemical structures of HMG-CoA, compactin, the first derived statin, and the seven FDA-approved statin drugs. Dates in parentheses indicate the initial approval date by the FDA, according to their website . Drug stAdditional file 2:d) or mean inhibitory concentration (Ki) of HMG-CoA and statins, respectively, for HMGCR. The statin prefixes are used instead of the whole name . Affinity and mean inhibitory concentration values are reported in nanomoles (nM) [Affinity of HMG-CoA and statins for HMGCR. The affinity (Kles (nM) –106. . Times are represented in hours (h). Log D = distribution coefficient at pH 7.4 ; chachter , McKennechachter . High-, moderate-, and low-intensity statin therapy. Statin dosages that achieve plasma reductions in LDL-C of < 30% (low-intensity), between 30 and 50% (moderate-intensity), and > 50% (high-intensity), as tested in randomized control trials (RCTs) and reported in the 2013 American College of Cardiology (ACC) and American Heart Association (AHA) 2013 guidelines . Statin


Scly gene in mice led to obesity, hyperinsulinemia, glucose intolerance, and hepatic steatosis, with SCLY being suggested as a participant in the regulation of energy metabolism in a sex-dependent manner. With the physiological role of SCLY still not fully understood, this review attempts to discuss the available literature regarding SCLY in animals and provides avenues for possible future investigation.The enzyme selenocysteine β-lyase (SCLY) was first isolated in 1982 from pig livers, followed by its identification in bacteria. SCLY works as a homodimer, utilizing pyridoxal 5’-phosphate as a cofactor, and catalyzing the specific decomposition of the amino acid selenocysteine into alanine and selenide. The enzyme is thought to deliver its selenide as a substrate for selenophosphate synthetases, which will ultimately be reutilized in selenoprotein synthesis. SCLY subcellular localization is unresolved, as it has been observed both in the cytosol and in the nucleus depending on the technical approach used. The highest SCLY expression and activity in mammals is found in the liver and kidneys. Disruption of the  The samThis review attempts to describe and discuss what is known about SCLY, focusing further on the eukaryotic enzyme. [Ser]Sec, containing the anticodon ACU. Biochemical modifications of the Sec-tRNA[Ser]Sec encompass a pseudouridine at position 55, an isopentenyladenosine at position 37, a 1-methyladenosine at position 58, and a 5-methoxycarbonylmethyluridine at position 34 that is methylated in selenium adequacy, with this methylation step regulated by the presence of a transcription factor, STAF, binding region [[Ser]Sec to the UGA recognition, promoting selenocysteine incorporation into selenoproteins. A mechanistic model for SECIS recognition of the UGA codon has been suggested Sec). Primarily, the Sec-tRNA[Ser]Sec is aminoacylated with serine by seryl-tRNA synthetase and then phosphorylated by the enzyme phosphoseryl-tRNA[Ser]Sec kinase (PTSK), forming the intermediate O-phosphoseryl-tRNA[Ser]Sec [[Ser]Sec. SecS provides a selenophosphate for the exchange [[Ser]Sec with consequent formation of thiophosphate and insertion of cysteine instead of selenocysteine into UGA sites Sec loading is occurring, which could explain the downregulation of most selenoproteins observed [Hypoxic conditions were also revealed as downregulators of the SCLY mRNA in human hepatocellular carcinoma (HepG2) cells. Interestingly, SCLY mRNA expression in these cells was upregulated by treatment with 100 nM selenite, but the downregulatory effect of hypoxia occurred regardless of selenium levels. Moreover, such a downregulatory effect was independent of hypoxia-inducible factors (HIFs) classically involved in response to hypoxic conditions, suggesting an alternative route of regulation of the selenoprotein synthesis machinery by hypoxia. Notably, gene expression of SEPHS2 was also severely downregulated by chronic hypoxia, a correlation that suggests limiting Sec-tRNAobserved . Interesobserved . Since hobserved , it coulScly mRNA expression in the liver and brain [Paradoxically, however, SCLY activity was reported as not regulated by selenium levels or the chemical form of selenium provided in the diet. Feeding rats for nine weeks with 2 ppm of either selenite, selenocysteine, or selenomethionine did not result in significant changes in activity levels of SCLY in liver, kidneys, testis, or muscle . On the nd brain , suggestScly gene expression by endocrine factors, it was reported that glucocorticoids negatively regulate Scly mRNA in mice. A study that performed RNA-sequencing analysis of the hypothalamic arcuate nuclei of mice chronically treated with corticosterone revealed a remarkable downregulation of the Scly gene in this region. Strikingly, in the first two days of glucocorticoid treatment, the mRNA for two selenoproteins, SelenoP, and iodothyronine deiodinase 2 (Dio2), were upregulated [Dio2 gene in mice was maintained upon chronic treatment with corticosterone [In terms of the regulation of egulated . The enzegulated ,76. Upreosterone , implyinA study revealed the SCLY transcript to be upregulated on the fourth day after treatment with a gonadotropin-releasing hormone (GnRH) agonist, buserelin. This upregulation occurred at the uterine endometrial tissue of Nelore cows with large preovulatory follicles and corpus luteum, which are an indication of greater receptivity after in vitro fertilization protocols . GnRH isOreochromis mossambicus) was inversely correlated with environmental salinity [Interestingly, renal SCLY expression in tilapia  had only mild effects on their neurological health, mostly when challenged with a selenium-deficient diet [−/−Scly mice also worsened their obesity when fed a high-fat diet, even when dietary selenium levels were adequate [−/−Scly mice had an attenuated phenotype, gaining weight and accumulating lipid in their fat depots but without the effects on glucose homeostasis and insulin levels. Interestingly, castration of male −/−Scly mice restored insulin levels and energy expenditure outcomes, a result that suggests a physiological role for selenocysteine decomposition in the testis in improving metabolic outcomes [Mice lacking the ent diet . This ouent diet . This obadequate , suggestoutcomes .−/−Scly mice [−/−Scly mice equally diminished levels of stress-regulated selenoproteins GPx1, selenoprotein M (SelenoM), and selenoprotein S (SelenoS) in the hypothalamus [Scly gene in Agrp neurons of the arcuate nucleus of the mouse hypothalamus led to protection against diet-induced obesity with an elevation of brown fat thermogenesis marker uncoupling protein 1 in both sexes [−/−Scly mice suggest that peripheric tissues are sorting and coordinating mechanisms to compensate for hypothalamic suppression of stress-related selenoprotein synthesis. Moreover, compensatory mechanisms are likely also regulated by sex, leading to impairment in glucose and lipid homeostasis, and inducing a metabolic syndrome-like phenotype exacerbated in the males of the Scly−/− mouse model. Overall, the results obtained in mouse models lacking the Scly gene were able to shed light on the role of this enzyme as an important player in hypothalamic synthesis of selenoproteins and the regulation of hepatic insulin-dependent glucose homeostasis, broadly connecting selenocysteine decomposition to the physiological regulation of energy metabolism.Selenium metabolism is strongly affected by sex ,88. Henc−/− mice ,86. Remathalamus ,90,91. Ith sexes . TherefoSupporting a role for SCLY in coordinating the intersection between selenium and energy metabolism, at least in the liver, is the evidence that SCLY interacts with several proteins involved in metabolism. Yeast two-hybrid screening studies using mouse Scly as protein bait revealed an array of interactors with diverse functions such as major urinary proteins (MUPs), aldehyde reductase, ATP synthase A, glutathione S-transferase, farnesyl diphosphate farnesyl transferase I, vitamin D-binding precursor, liver regeneration p-53 related protein, lysosomal pepstatin-insensitive protease, and apolipoprotein A-II, among others ,93. InteThere are currently 530 single nucleotide polymorphisms (SNPs) mapped in the human SCLY gene according to the SNP database from the National Institutes of Health of the United States of America. Among all these SNPs, only two of them have been connected with a disease condition. A genome-wide linkage and association (GWAS) analysis in Americans of Mexican origin as part of The Insulin Resistance Atherosclerosis Family Study uncovered two SCLY SNPs associated with cholesterol levels. SNP rs201606363 is in the intronic area of the SCLY gene, while SNP rs3832063 is found in the intergenic area between the gene for UBE2F and SCLY. Both ranked highest in the GWAS analysis and were determined to be associated with low-density lipoprotein and cholesterol levels . Since the first description of SCLY in 1982, several aspects of its biochemistry and biology have been elucidated. Nevertheless, significant questions remain. For instance, the subcellular localization of SCLY is still unresolved, and sorting this issue will improve our understanding of selenium metabolism and the enzyme role on it. Another unresolved issue is the particular mechanism of selenium delivery to SEPHS enzymes, as it is not currently known whether SCLY directly provides selenide to selenophosphate synthesis, or if an intermediate protein exists that receives the SCLY-produced selenide and delivers to SEPHS. Yet, as it is becoming apparent with recent studies, SCLY is possibly involved in aspects of energy metabolism, particularly in the liver. The fact that the enzyme might act as a bridge between selenium metabolism and how carbohydrate and lipid are handled in cells over different conditions is an element of the role of SCLY that needs to be more extensively sought. The impact of the actions of SCLY in human health and nutrition, particularly to matters where selenium is already known to be involved, may bring an enriched perspective on overall physiology, potentially uncovering novel molecular mechanisms dependent on SCLY.


Next-generation sequencing has shown that the majority of the human genome does not encode proteins, yet many such “non-coding” regions are actively transcribed into RNA. In particular, long non-coding RNAs (lncRNAs) have received considerable interest for their essential roles in numerous biological processes. Defined as non-protein-coding transcripts longer than 200 nucleotides, lncRNAs are frequently expressed in a tissue-specific or developmental stage-specific manner , 2. MultXist is one of the best characterized lncRNAs, and functions as an effector of dosage compensation in female cells. Xist maintains inactivation of one X chromosome by binding along its length and recruiting repressive factors to silence transcription. Immune cells, however, regulate X chromosome inactivation through diverse mechanisms that are not limited to the use of Xist RNA, as recently revealed by Syrett et al. For instance, the inactivated X in female murine plasmacytoid dendritic cells (pDCs) lacks both Xist RNA and the repressive chromatin modification H3K27me3. Interestingly, pDCs from female mice that spontaneously develop SLE-like disease show increased biallelic expression of the X-linked gene Tlr7, as compared to healthy female mice. Further delineating the mechanisms of X chromosome inactivation in immune cells will be necessary before we can develop a complete understanding of how sex and sex-linked genes contribute to autoimmune diseases.Females have a more responsive immune system than males , 7, poteRickettsia genus of Gram-negative obligate intracellular bacteria is transmitted to humans via arthropod vectors, including ticks, fleas, and lice, and can infect the vascular endothelium to cause illnesses such as Rocky Mountain spotted fever. There are increasing concerns over the prevalence of Rickettsia due to climate change and the spread of vector populations. Understanding rickettsial infection and developing novel therapeutics to mitigate its spread through the vascular endothelium will thus be crucial in the near future. Chowdhury et al. have begun to address the roles of lncRNAs in the host response to Rickettsia species using a mouse model of R. conorii infection. The authors identified two enhancer lncRNAs affecting expression of nearby genes, Id2 and Apol10b, which have not been previously studied in rickettsial infection. Furthermore, these two lncRNAs exhibit differential expression in infected macrophages compared to endothelial cells, emphasizing the cell type-specificity of lncRNAs. The resulting genome-wide analysis of lncRNA expression during rickettsial infection establishes a foundation to further study the host immune response to Rickettsia species and reveals cell type-specific signaling pathways mediated by enhancer lncRNAs.The Wang reviews in detail the known lncRNAs involved in multiple steps of the viral life cycle and infection.Some of the most interesting advancements in our understanding of lncRNAs in immunity come from the study of their roles in viral infection. While it would be intuitive to assume host lncRNAs exist to suppress viral infections, it has been shown that host lncRNAs can actually increase viral replication and pathogenesis during certain infections . This beCastellanos-Rubio and Ghosh review the disease-associated lncRNA SNPs that have been described thus far. Further inquiry and development of tools to interpret lncRNA structure and function will be necessary before the impact of lncRNA SNPs (and other genetic variants) on disease can be fully realized.GWAS have provided a more complete understanding of complex diseases. Of the more than 70,000 variant-trait associations cataloged from GWAS , a majorYang et al. review the mechanisms of circRNA biogenesis, summarize the current understanding of their function in immunity, and present approaches and limitations to studying circRNA function.While lncRNAs typically display features associated with linear messenger RNA, such as 5′ capping, alternative splicing, and polyadenylation, non-coding RNA loci can also be transcribed to form circular RNA (circRNAs). These circRNAs form closed loops without 5′ capping or polyadenylation and can contain multiple exons. Similar to lncRNAs, circRNAs are frequently expressed in a tissue-specific manner, undergo chemical modifications, and function through varied mechanisms. lncRNAs are now an established class of functional molecules with physiological relevance shown in nearly every human organ system. Their roles in immunity have been identified in immune cell differentiation, maintenance, and effector function. Given that only a fraction of lncRNAs have been functionally characterized a significant wealth of insight awaits discovery. Moreover, as personalized medicine begins to drive the use of genetic tools in the clinic, we can expect the identification of clinically-relevant lncRNA variants and the development of therapeutics that target lncRNA function.AW and GK wrote and edited the manuscript. JH-M edited 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.


The use of nanoparticles has contributed to many advances due to their important properties such as, size, shape or biocompatibility. The use of nanotechnology in medicine has great potential, especially in medical microbiology. Promising data show the possibility of shaping immune responses and fighting severe infections using synthetic materials. Different studies have suggested that the addition of synthetic nanoparticles in vaccines and immunotherapy will have a great impact on public health. On the other hand, antibiotic resistance is one of the major concerns worldwide; a recent report of the World Health Organization (WHO) states that antibiotic resistance could cause 300 million deaths by 2050. Nanomedicine offers an innovative tool for combating the high rates of resistance that we are fighting nowadays, by the development of both alternative therapeutic and prophylaxis approaches and also novel diagnosis methods. Early detection of infectious diseases is the key to a successful treatment and the new developed applications based on nanotechnology offer an increased sensibility and efficiency of the diagnosis. The aim of this review is to reveal and discuss the main advances made on the science of nanomaterials for the prevention, diagnosis and treatment of infectious diseases. Highlighting innovative approaches utilized to: (i) increasing the efficiency of vaccines; (ii) obtaining shuttle systems that require lower antibiotic concentrations; (iii) developing coating devices that inhibit microbial colonization and biofilm formation. In 1959, Richard Feynman described a process that allows one to individually manipulate atoms and molecules throughout high precision instruments. This system could be applied to design and build systems at nanoscale level, atom by atom  and its Nanotechnology refers to the area of the knowledge that designs and produces structures, devices and systems by manipulating atoms and molecules at the nanoscale level . NanoparNanobiotechnology is the area of nanotechnology focused on the biological field. Nanoparticles utilized in biology are grouped into three categories: organic, inorganic and mixed (organic/inorganic) . In receShuttle systems are commonly used for the delivery and stabilization of bioactive drugs and antimicrobial molecules, ensuring not just their specificity but also controlled release . CoatingThe use of nanomaterials as biosensors has currently a vast impact and a fast development in the usage of smart nanobiomaterials. Biosensors are accurate and offer a cost effective approach for the detection of pathogenic infectious agents in natural environment, food but also clinical specimens . NanondiThe aim of this review is to highlight and discuss the recent progress and applications of nanotechnology in the medical field (nanomedicine) focusing on the prevention, diagnosis and treatment of infectious diseases.Staphylococcus aureus, Helicobacter pylori, Shigella spp. or Escherichia coli is still not possible [Traditional vaccines have been developed using live attenuated organisms (cellular vaccines) or inactivated toxins or proteins (acellular vaccines). Recently, the development of synthetic peptide-based vaccines has shown many advantages compared with traditional vaccines, such as better safety and/or conservation. However, the peptide-based vaccines generate a weaker immune response, and the inclusion of adjuvants and/or the use of vaccine delivery systems is highly needed . Antibacpossible .Nanoparticles have several applications in nanobiomedicine, especially in the field of vaccine production where they can be applied as efficient delivery systems. Their particular nature increases cross-presentation of the peptide  and it a(i)d,l-lactic-co-glycolic acid)(PLGA) [Polymers, divided in turn into nanoparticles containing synthetic polymers, such as poly(d)(PLGA) , polyethd)(PLGA)  or polyed)(PLGA) , and natd)(PLGA) , inulin d)(PLGA)  or chitod)(PLGA) . Synthetd)(PLGA) ;(ii)Liposomes, which are biodegradable and non-toxic phospholipids. They encapsulate antigens and incorporate viral envelope glycoproteins to form virosomes. The combination of a modified cationic liposome and a cationic polymer (such as protamine)-condensed DNA is called liposome-polycation-DNA nanoparticles (LPD). They are commonly used as adjuvant delivery system in DNA vaccine studies ;(iii)Nanosized emulsions are those nanoparticles that can exist as oil-in-water or water-in-oil form. Emulsions can carry antigens inside their core to increase the efficiency of vaccine delivery or they can also be simply mixed with antigen ;(iv)Inorganic nanoparticles are non-biodegradable, they have rigid structure and controllable synthesis. Silica-based nanoparticles (SiNPs) offer the advantage of biocompatibility and have excellent properties as nanocarriers. SiNPs particles such as mesoporous silica nanoparticles (MSNs) could potentially become high-efficient, controlled-release nanocarriers in future vaccine formulations .(v)Immuno-stimulating complexes (ISCOM). They are composed of supra-molecular structures of the adjuvant Quil A and immunizing peptides, which allows selective incorporation of viral envelope proteins by hydrophobic interaction .(vi)®, RECOMBIVAX®HB against to HBV [Virus-like particles (VLP) are optimized for interaction with the immune system, avoiding the infectious components. They can induce potent immune responses, even in the absence of adjuvant . VLP bast to HBV .(vii)Self-assembling systems emerged as a consequence of an attempt to drive higher levels of protein, and consequently better immunological properties. A variety of natural proteins can be self-assembled into nanoparticles, conferring highly symmetric, stable and organized structure .Nanomaterials may have intrinsic immunomodulatory functions, acting as adjuvants or immune potentiators . AccordiThe emergence of infections together with the fast evolution of drug-resistant bacteria (superbugs), are triggering the increased ineffectiveness of actual therapies used to treat infectious diseases . ClinicaMycobacteriumtuberculosis complex (MTB) within 4 h [S. aureus [Salmonella typhimurium based on a single-stranded DNA aptamers along with silica fluorescence nanoparticles.A number of nanotechnology-based materials have been studied with the purpose of controlling and preventing infectious diseases . The phythin 4 h . Incorpothin 4 h . A combi. aureus . Wang an. aureus  have devLiposomes can recognize target toxins, and therefore they can be used for toxin detection. Liposomes labelled with fluorescent markers (such as rhodamine dyes) can be incorporated into sandwich fluoro-immunoassay on antibody-coated microtiter plates in order to detect toxins . Ahn-YooQuantum dots (QDs) are special nanocrystalline semiconductors  composedSalmonella sp. with a detection limit of 10 cell/mL. The aforementioned fluorescent nanobioprobes made on a specially designed cellulose-based swab could be applied in a large number of samples related to public health surveillance to visually detect and directly isolate pathogens in situ.In 2010, Zhang and Hu  developeSerratia marcescens with a detection range from 10 to 106 colony forming units/mL (CFU/mL) at pH 7.In 2007, Klostranec et al.  reportedDetection systems based on noble metal nanoparticles , particuMagnetic nanoparticles (MNPs) have nanoscale sizes, which mimic the size of molecules in nature, and they harbor favorable characteristics for their use in nano-biomedicine, such as imaging and therapy [ therapy . Surface3O4) or maghemite (γ-Fe2O3) nanocrystallites. IONP-biosensors have been developed for the detection of HSV-1 and adenoviruses enabling to detect five viral particles in 10 μL serum samples without previous PCR amplification steps [4 CFU/mL bacterial cells by using nano-MALDI platforms [These recognition moieties help to detect the bacteria selectively and at low concentrations ,116. Theon steps . Using Ion steps  and vancon steps  have alllatforms . Nanodialatforms . HoweverDrug resistance is of a great concern for public health. The use of high dose antibiotic treatments often generates high rates of toxicity and the development of new resistance. In addition, the costs of treatments increase while there is a major number of treatment failures and high spectrum therapies associated with an increase in the number of hospitalization days. Due to the lack of new alternatives for the treatment of infectious diseases, several classes of antimicrobial nanoparticles and nanocarriers for antibiotic delivery have been studied, as well as their effectiveness for the treatment of infectious diseases, including antibiotic resistant bacteria .Nanoparticles provide a versatile platform for the design of materials with antimicrobial properties. Their unique nanoscale as well as physical and chemical properties provide multiple attributes that facilitate the development of unique antimicrobial strategies; hence, they are emerging as weapons in our antimicrobial arsenal. These nano-antimicrobial materials can be synthesized by variety of different methods influencing subsequent antimicrobial effect . They coStaphylococcus aureus and Escherichia coli, and according to properties they have been even used to treat tuberculosis (TB) [The antimicrobial mechanism of the action of nanoparticles is not fully known. Nevertheless, the antimicrobial actions include destruction of cell membranes, blockage of enzyme pathways, alterations of microbial cell wall, and nucleic materials pathway. The applicability of nanoparticles as therapeutic agents includes a wide range of action, varying from broad spectrum antimicrobial agents, sterilization and wound healing agents, to sustained inhibitors of intracellular pathogens . The mossis (TB)  , with the implementation of tailored coating systems. These systems are based on coating the surface with nanoparticles inhibiting biofilm formation. Most studies have been focused on pathogens frequently associated with nosocomial infections such as S. aureus, P. aeruginosa, Acinetobacter baumani and K. pneumoniae [Proteus mirabilis infection and their subsequent blockage control.Nanoshuttle systems deliver antibiotics to a precise location and release them progressively in a controlled manner (shuttle systems). These systems use nanoparticles for the delivery and controlled released of several antibiotics and natural products. Nanoparticles are free to move uninhibited into cells, increasing their efficiency. Antibiotics can be released inside the microorganism, increasing the therapeutic index and reducing the overall serum concentration. As a result, the deleterious side effects decrease . An addifections . The anteumoniae   undergoing phase III clinical trials, are respectively, an inhalable liposomal tobramycin and amikacin dosage forms, claimed for the treatment of P. aeruginosa pulmonary infections in cf. [Currently, some drug delivery systems (DDS) usually named “nano-antibiotics” have been clinically-approved for human use in various infectious diseases, among them, liposomal delivery systems. Pulmaquin™ and Lipoquin™  are inhalable liposomal dosage forms of ciprofloxacin, for the treatment of serious infectious diseases encountered in cystic fibrosis (CF) or in non-CF bronchiectasis. AX-Tobra™  based on Fluidosomess in cf. .Nanobiotechnology offers multiple solutions for the prevention, diagnosis and treatment of infectious diseases. Nanoparticles can be designed to increase the activity in vaccines with low toxicity against the host. A huge number of nanoparticles can be used for the delivery and stabilization of bioactive drugs as well as antimicrobial molecules, ensuring controlled release of the drug. Due to their specificity, low dimensions, targeted delivery, controlled release properties and low cytotoxicity, nano-active systems could lead to more efficient and less invasive therapeutic outcome, contributing to the development of personalized treatment for several infectious diseases.In the superbug era, nanotechnology is offering a new approach that allows us to fight against resistant bacteria. Nanobiomedicine offers new tools to be applied in the prevention, detection and treatment of the infectious diseases, managing to decrease the co-morbidity/mortality ratios, costs and improving lifestyle quality.


One of the most coveted goals of science is to elucidate the cellular bases of complex brain functions. How does a piece of biological tissue produce accurate cognitive insights, or succumbs to a devastating neurological disease? Neurons and astrocytes are the most numerous cells in the brain, comprising >95% of the brain parenchyma, and are responsible for all aspects of rapid information processing in the mammalian brain. Monitoring of physiological changes at the individual cell level, as well as in cell populations, is essential  advanced our knowledge on signaling in submicron structures  while permitting the investigations in semi-intact tissues . In parallel, the new emerging field of optogenetics allowed the selected expression of light-activated proteins that can be used for precisely targeted photostimulation (Baker and Flannery). Other optical strategies that recently progressed include combined Ca2+ and Na+ measurements , membrane potential recordings from individual cells , or cell populations , and chemical stimulation of targeted receptors using caged compounds .In the last decades, neuroscience research has been driven by the development of novel technologies interfacing scientists with molecules, cells, neuronal tissues, or the entire brain. Certainly, optical techniques have played a major role in this progress opening the gate to unprecedented information on living neurons and astrocytes. Innovative illumination and light recording strategies  from the entire hippocampus CA1 area for extended periods of time (experiments lasting up to 12 h). Their results shed light on a rarely discussed spatial aspect of long term potentiation (LTP) induction  . Using the same experimental approach, based on extracellular labeling of brain slices with voltage-sensitive dye and state-of-the-art wide-field single-photon voltage imaging at 2,000 frames per s, Kajiwara et al. showed that the efficacy of propagation of electrical activity between two critical brain regions, from perirhinal cortex to entorhinal cortex, is strongly dependent on membrane excitability and is malleable by synaptic plasticity . Miyazaki et al. have developed a robust method for simultaneous imaging of [Ca2+]i and [Na+]i changes in neurons at high speed and high spatial resolution. Via whole-cell electrode attached to the cell body, the authors co-inject two different pairs of spectrally orthogonal Ca2+ and Na+ indicators and excite the two dyes by illuminating at two wavelengths either using LEDs or laser spots. This method allowed them to monitor synaptically evoked Ca2+ and Na+ transients in individual dendritic spines . The review article by Dong et al. describes several techniques for studying gap junctions in the central nervous system. Fluorescent compounds and dyes are at the heart of several experimental approaches designed to probe the existence, strength, or nature of gap junction coupling: injection of small tracer molecules, scrape loading/dye transfer method, and recovery after photobleaching method . The review article by Kuhn and Roome, describes experimental design for performing dendritic voltage imaging in awake behaving animals. The authors also provide a very detailed account of the physical chemistry and quantum mechanics of charge-shift voltage-sensitive dyes with a special emphasis on two dyes from the Fromherz group (ANNINE-6 and ANNINE-6+). Physical chemistry and quantum mechanics of the organic dyes explains certain practical issues of working with fluorescent dyes, such as signal-to-noise ratio, phototoxicity, shape of the emission spectrum, dye flipping from one membrane leaf to another, etc. (Kuhn and Roome).In the early days of functional optical imaging, organic dyes were used for studying membrane potential changes in excitable cells and were dubbed voltage-sensitive dyes  was carried out in head-restrained mice running on a circular treadmill. Data showed that Ca2+ signals had larger amplitude and could invade the entire dendritic tree during locomotion, while during immobility, the signal amplitude was significantly lower in both soma and dendrites, and a significant fraction of dendrites showed spatially restricted CaTs, which were not seen in the soma. The authors proposed that strong dendrite-soma coupling, associated with animal locomotion, may facilitate the spike-timing-dependent or Hebbian forms of synaptic plasticity in interneurons. In contrast, during animal quiet state, this type of activity can be reduced or even replaced by local dendritic Ca2+ signaling, which may facilitate the anti-Hebbian plasticity mechanisms . Another group working with GCaMP used the transgenic animal Thy1-GCaMP6f, expressing GCaMP6f in cortical pyramidal neurons . These authors showed that they can monitor sharp waves (SWs), carbachol-induced theta oscillations, and interictal-like spikes in brain slices. Furthermore, the authors claim that population GCaMP6f signals are fast enough for monitoring theta and beta oscillations (<25 Hz), which is an advancement for Ca2+ imaging. Quicke et al. also use a transgenic animal approach for delivery of a genetically encoded indicator to neurons. However, instead of GCaMP which is a calcium indicator, they use voltage indicator (VSFP Butterfly 1.2) to monitor membrane potential changes . Unlike the cytosolic molecule GCaMP, voltage indicators are membrane proteins . Their expression in brain tissue produces fluorescently labeled neuropil made of densely intermingled dendrites and axons, hence cellular resolution is lost. To achieve single cell resolution in voltage imaging, Quicke et al. combine recently developed sparse transgenic expression strategy . Unlike GCaMP, genetically encoded voltage indicators have poor signal to noise ratio . Finally, Storace et al. illustrate how membrane potential imaging using GEVIs  can be utilized to decode the activity of the mammalian olfactory bulb, a goal that has been elusive for decades using older experimental approaches. Here, the authors used a clever strategy to simultaneously monitor input and output of a brain region in awake animal responding to controlled odor presentations. Afferent inputs to the olfactory glomeruli were assessed by calcium imaging from axon terminals of olfactory receptor cells . Olfactory bulb outputs, on the other hand, were monitored by GEVI labeling of the mitral cells, whose axons project from the olfactory bulb into other brain regions. Simultaneous calcium and voltage imaging revealed that the output activity maps and the output signal size are much less concentration dependent than are the input maps and signals . These results contribute immensely to our current understanding of the cellular mechanism for concentration invariance of odor recognition. The Storace et al. study is an excellent example of how applications of advanced imaging technologies  will impact systems neuroscience in the future.Genetically encoded optical sensors of cell activity have become very popular in recent years, because they can be targeted to specific cell types. This is especially important in studies of the brain circuitry, because individual brain regions always include a multitude of different cell types, each type bestowed with a specific set of afferents and efferents, as well as rules for synaptic integration and patterned action potential firing. Battefeld et al. describe a versatile and open-source rapid LED switching system for one-photon imaging and photo-activation, that can be used either for quantitative ratiometric Ca2+ imaging or to combine Ca2+ imaging with optogenetic stimulation. Miyazaki et al. present an upgraded version of an imaging system, based on high speed multiplexing of light LEDs, to combine Na+ and Ca2+ imaging, a powerful approach to advance our knowledge on neuronal excitability and synaptic transmission. Ronzitti et al. review strategies for light shaping in three dimensions, including SLM-based multiplexing illumination, temporal focusing and other approaches. These rapidly developing illumination technologies open the gate to all-optical manipulation of neural circuits involving simultaneous stimulation of well-defined targets and simultaneous functional imaging of the resulting activity. Among light shaping approaches, light-sheet microscopy represents one of the most promising strategies. Using this technique, Müllenbroich et al. present an interesting improvement for imaging the transparent nervous system of zebra fish. In this application, Bessel beams are used to remove artifacts and dramatically improve sensitivity in Ca2+ imaging measurements . Finally, a novel caged glutamate receptor antagonist is presented by Palma-Cerda et al. This compound combines the fast photolysis and hydrolytic stability of this family of photoactivatable molecules with fast-equilibrating competitive action on glutamate receptors, enabling deep investigation of postsynaptic functions at the molecular level .The ability of functional imaging to address fundamental questions depends on novel cutting-edge technologies and instrumentation. 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.


Cellular parabiosis is tissue-based phenotypic suppression of cellular dysfunction by intercellular molecular traffic keeping initiated age-related diseases and conditions in long latency. Interruption of cellular parabiosis (e.g. by chronic inflammation) promotes the onset of initiated pathologies.cellular parabiosis. Such ‘cellular solidarity’ creates tissue homeostasis by buffering defects and averaging cellular functions within the tissues. Chronic inflammation is known to (i) interrupt cellular parabiosis by the activity of extracellular proteases, (ii) activate dormant pathologies and (iii) shorten disease latency, as in tumour promotion and inflammaging. Variation in cellular parabiosis and protein oxidation can account for interspecies correlations between body mass, ARD latency and longevity. Now, prevention of ARD onset by phenotypic suppression, and healing by phenotypic reversion, become conceivable.The stability of initiated latent cancers and other age-related diseases (ARD) hints to phenotypically silent genome alterations. I propose that latency in the onset of ageing and ARD is largely due to phenotypic suppression of cellular dysfunctions via molecular traffic among neighbouring cells. Intercellular trafficking ranges from the transfer of ions and metabolites (via gap junctions) to entire organelles (via tunnelling nanotubes). Any mechanism of cell-to-cell communication resulting in functional cross-complementation among the cells is called  This and the accompanying paper [The incidence of death and diverse ARD increases with about the fifth power of time, suggesting a common biological clock (with species-specific speed) and perhaps a shared common cause. A common cause would mean that it might be simpler to mitigate all ARD collectively than any particular one—reminiscent of the long-lasting simultaneous resistance of super-centenarians to all ARD.cellular parabiosis  of disease via interruption of cellular parabiosis  of advanced diseases and ageing. The ideas and proposed concepts were inspired by the literature on biology of diseases and ageing spanning over seven decades. Hence, I refer often to adequate reviews.2.In the companion paper , we arguA common quantitative relationship between oxidative protein carbonylation induced by UV and ionizing radiation and killing of bacteria and metazoan germ line cells—regardless of huge differences in cellular radiation resistance—is shown in fig. 3 of . Such coE. coli, spontaneous and UV-induced mutation rates increase with about the seventh power of protein carbonylation, while mutations emerge linearly with respect to inflicted DNA damage [Irreparable protein damage such as oxidative carbonylation is far more mutagenic than reparable DNA damage ,3 and caA damage ,8. MoreoErrors in the somatic maintenance of DNA methylation patterns, in particular, gene silencing involving hypermethylation of some specific CpG islands, are (i) age-related, (ii) more frequent than mutations, (iii) diagnostic of human biological age and (iv) predictive of remaining life ,10. ThusWhile gene silencing by DNA methylation and gene inactivation by mutation display similar phenotypes, the incidence of somatic mutations  appears Proteomes of aerobic organisms have evolved a remarkable intrinsic resistance to oxidation, yet individual proteins display great variability in their intrinsic susceptibility to oxidative damage (fig. 6 in ). The faApparently, no major vital cellular function remains unaffected by ageing . All mol3.cellular parabiosis for the compensation of functional deficiencies of individual cells in tissues by intercellular communication via molecular traffic. The result of such phenotypic cross-complementation is the homogenization, or averaging, of cellular functions across the tissue, called tissue homeostasis. Let us consider the expected impact of cellular parabiosis on ageing and diseases.The notion of parabiosis (living by proximity) derives from an experiment by Paul Bert in 1864 that revealed physiological effects of shared bloodstream between two surgically joined animals . Over a 8 times) lower incidence of cancer and other deadly diseases in whales than in mice. What is the difference between the biology of a whale, or human, cell in vivo and a similar cell in a mouse or shrew? Apparently, whale cells have evolved ways to reduce the initiation and/or prolong the latency of ageing and diseases. If we knew how, we could use that knowledge to mimic, healthwise, the biology of virtual huge humans to mitigate diseases and extend lifespan in a manner already developed during evolution. If the principles of a medicine for small animals can be found in large animals, then we should try to understand them in order to slow down ageing and mitigate ARD. Below, the description of a concept for the latency of ARD and ageing is followed by chapters integrating the current knowledge within the framework of the concept.A whale that can live 210 years has a million times more cells at risk of cancer than a mouse, yet lives cancer-free for a hundred times longer than a laboratory mouse (lifespan 2.5 years). This is referred to as Peto's paradox . On a pe3.1.Among random phenotypic mutations, most are recessive . Some malignant and senescence phenotypes appear recessive (i.e. suppressed) in cell fusion experiments (reviewed in ). ChromoPhenotypic expression of newly acquired recessive genome alterations emerges in solitary cells with the fading out of residual (non-renewing) protein activity either by protein dilution (if the cell divides) or by protein damage, aggregation or proteolysis (in quiescent and in dividing cells). However, for cells within the tissues, or confluent cell cultures, recessive and some dominant alterations can remain phenotypically silent for an indeterminate time . The larBased on published data, I propose the existence of a lasting phenotypic suppression, even phenotypic reversion, of cellular deficiencies by means of known  mechanisms for the acquisition of metabolites, as well as functional and informational macromolecules, and entire organelles, transferred and shared among neighbouring cells –25 figu. MoleculMany names, often redundant, were given to diverse extracellular structures shown, or presumed, to be the vectors of intercellular molecular traffic –25. MostFor the sake of argument, it suffices to recall that cellular contents ranging from ions and metabolites to entire mitochondria are subject to intercellular trafficking generating phenotypic suppression of the recipient cell's deficiency . TnTs din vivo is expected to functionally homogenize entire tissues and organs via dominant-positive effects on non-overlapping cellular deficiencies  is nearly 100 times greater in vivo (human tissues) than in vitro (cell cultures), suggesting that telomere maintenance in tissues—a bottleneck to cell division—might take advantage of cellular parabiosis.In a remarkable review, Harry Rubin  presenteUndesirable effects of cellular parabiosis upon health, such as increased fitness of tumour cells and cell-to-cell transfer of infectious particles, such as viruses, bacteria and prions, have been put forward in relation to TnTs ,25. IndeThe proposed cellular parabiosis  could ex3.2.Perhaps the most informative phenomenological studies on cancer are the 70-year-old initiation–promotion experiments on mouse skin by Isaac Berenblum and colleagues . They shhprt gene function, bestowing a drug resistance, was considered a ‘disease’) just to find out that precisely that experiment was published in 1981 by Newbold & Amos [Subsequently, it was shown that promotion is a cellular phenomenon, since TPA promotes malignant cell transformation initiated by radiation  and inted & Amos  with a s+ cells. Nanomolar concentration of TPA, devoid of detectable toxicity, prevents a very efficient phenotypic reversion of drug (8-azaguanine and 6-thioguanine) resistance in dense co-cultures with the drug-sensitive Hprtin vivo involves induced generation of ROS by the macrophages (to kill bacteria) and excretion of matrix metalloproteinases (MMPs) and some other serine proteases (e.g. plasminogen activators and plasmin) [Inflammation plasmin) . Presumain situ and therefore start expressing their genome-based defects  displayed by non-dividing senescent cells bearing damaged or telomere-depleted chromosomes . SenesceTumours that, like senescent cells, exert SASP-like effects including MMP excretion display poor clinical prognosis —probablyIn conclusion, inflammation appears as ‘phenotypic expressor’  or by instigating an inflammatory immune reaction. Moreover, some extracellular and intracellular misfolded and oxidized proteins form small oligomeric pore-like hydrophobic structures that kill cells when inserting into the membranes by causing fatal leakage of ions and metabolites ,51. SuchOn the other hand, abundant extracellular high molecular weight hyaluronan is required for notorious cancer resistance in the naked mole rat . hyaluro3.5.Molecular trafficking among cells is proposed to: (i) keep cells in metabolic and developmental synchrony, (ii) induce trans-differentiation of intruding heterophilic cells and (iii) keep malignant and other cellular defects in the state of phenotypic dormancy . The arrAs pointed out by John Cairns , the bas3.6.Whatever the detailed mechanism, some type of tissue-based phenotypic suppression such as cellular parabiosis is a logical necessity to account for the phenomenology of ARD ,19,21,50in vivo cellular parabiosis—of stable pathological alterations accumulated with age  displayed by culturing primary hepatocytes from old (but not young) mouse liver, can be viewed as phenotypic expression—upon the exit from iewed in ,33 and pex vivo cellular mutation rates with an increase in body size . Since 3.7.in situ during prolonged inflammation, cross-complementing cells will express their morbid phenotypes. This was documented as age-related phenotypic heterogeneity  revealed during the establishment of primary hepatocyte cultures derived from the liver of old versus young mice .Poor viability and quality of cells in primary cultures from old organs  suggest In contrast to somatic tissues, the immortality of germline cells and of malignant cells surviving passages in cell culture (without phenotypic suppression) is probably assured by constant selective amplification (overgrowth) of fittest autonomously growing cells. This is akin to the periodic selection of fastest growing sub-clones in continuous bacterial cultures. Such selection for fitness occurs with circulating primary tumour cells that undergo massive apoptotic death (anoikis) while few metastasize . Similar3.8.A basic prediction of the proposed protein-centred model for ageing and ARD is the reversibility of deleterious phenotypes upon proteome renewal . Animal heterochronic parabiosis (shared bloodstream between isogenic old and young mice) shows rapid reversion of tested ageing and ARD phenotypes . But the3.9.If the generation of ROS, the cellular management of ROS-mediated proteome damage and the efficacy of cellular parabiosis together tune the somatic life clock, all of them should be affected by the rate of oxidative metabolism, i.e. generation of ATP and ROS. ATP is the bottleneck in proteome renewal and chaperone activity, whereas ROS are the killers of proteome function . Hence, Accurate protein biosynthesis and chaperone-assisted folding are the cell's highest energy (ATP) consumers (over 75%). Therefore, selective pressure for maintaining high ATP production, along with increased defence against damage by ROS, will cease once reproduction is assured. The larger and more complex the animal, the longer the time required for its reproduction and the longer lasting must be the high-energy investment in parental proteome quality. Indeed, there is a remarkable interspecies correlation between the increase in longevity and body mass on one hand, and the decrease in the rates of translational errors  and oxidDrosophila [long-lived birds and bats with high metabolic rates reduce the damaging impact of ROS by a modified mitochondrial membrane and an increased defence against protein damage ,61; andthe very long-lived and disease-resistant naked mole rat displays increased intrinsic oxidation resistance of its proteome via an approximately 10-fold reduction in translational error rate   to ‘mechanistic studies’ of the consequences of ageing and ARD is adequate for the task. It is legitimate to dream of a better medicine based on prevention rather than therapy and on more relevant research such as to identify and act upon the root cause(s) of ARD. Currently, the research on active prevention of diseases, equivalent to vaccination, is practically neglected in favour of therapies of advanced diseases that may never succeed with the ‘war on diseases’ approach because ARD reside within our own cells. Perhaps a ‘biomimetic’ approach would be more productive since evolution has already found ways to reduce initiation and promotion of diseases in long-lived animals. In such a case, we should support free academic research providing basic knowledge necessary for prevention and healing of ARD. Instead, we have to justify fundamental research because of pretended short-term lack of utility while monumental funding continues for mission-oriented research that showed its long-term lack of utility.Validation of the concepts proposed in the two companion papers, just on common ARD, risks taking a long time. But what if they are basically correct? Obvious potential applications were mentioned throughout this and the companion paper , such as


The pivotal role of phytohormones during fruit development and ripening is considered established knowledge in plant biology. Perhaps less well-known is the growing body of evidence suggesting that organic acids play a key function in plant development and, in particular, in fruit development, maturation and ripening. Here, we critically review the connection between organic acids and the development of both climacteric and non-climacteric fruits. By analyzing the metabolic content of different fruits during their ontogenetic trajectory, we noticed that the content of organic acids in the early stages of fruit development is directly related to the supply of substrates for respiratory processes. Although different organic acid species can be found during fruit development in general, it appears that citrate and malate play major roles in this process, as they accumulate on a broad range of climacteric and non-climacteric fruits. We further highlight the functional significance of changes in organic acid profile in fruits due to either the manipulation of fruit-specific genes or the use of fruit-specific promoters. Despite the complexity behind the fluctuation in organic acid content during fruit development and ripening, we extend our understanding on the importance of organic acids on fruit metabolism and the need to further boost future research. We suggest that engineering organic acid metabolism could improve both qualitative and quantitative traits of crop fruits. True fruits are specialized plant organs found solely in angiosperms , and these unique organs are believed to have evolved to improve seed dispersal and protection . The natThe countless types of fruits present in angiosperms can be operationally organized within a few broad categories by using combinations of traits such as: (i) dehiscence or indehiscence; (ii) fleshy or dry exterior; and free (apocarpous) or fused (syncarpous) carpels . These vSolanum lycopersicum L.) mutants such as pat (pat-2 (pat-3/4 (procera mutant (pin4 (arf7 (arf8 (iaa9 (entire)  fruit set; (ii) growth; (iii) maturation; and (iv) ripening. Fruit set occurs during and after fertilization, which can be defined as the transition of a quiescent ovary to a rapidly growing young fruit and depends on the successful completion of pollination and fertilization . Additioh as pat , pat-2  . On the During fruit growth, a signal, most likely derived from seeds (sources and sinks for cytokinin and auxin), induces neighboring tissues to expand, by both cell division and expansion, with a positive correlation between seed number and fruit size . This faVitis vinifera L.) and tomatoes, which are derived from the ovary, , through apples  and pineapples (Ananas comosus L. Merril), to strawberries (Fragaria x ananassa Duch.), which are derived from the receptacle tissues or from expansion of the sepals   .Musa paradisiaca L.), papaya (Carica papaya L), and tomato (further details in Table Citrus spp.), grapes, melon (Cucumis melo L.), and strawberries (Fragaria spp.) do not show the respiratory burst and ethylene production remains at a basal level during the whole fruit development including maturation and ripening  cycle components in addition to their recognized role as energetic intermediaries in plants . Most stderstood .Arabidopsis thaliana), soybean (Glycine max) and sunflower (Helianthus annuus) can accumulate high levels of fumarate  and Arabidopsis   and malaCollectively, these data provide compelling evidence that the metabolic connections associated with TCA cycle-related organic acids are responsible, at least partially, for such changes and therefore an extensive metabolic reprogramming also occurs in fruits when changes in TCA cycle take place. It seems therefore tempting to speculate that the enzymes of the TCA cycle as potential target to further improve fruit quality. However, further demonstration of the importance of changes in organic acid levels in fruits are required to obtain a full comprehension of this process. This will most likely occur by the genetic manipulation of fruit-specific genes using fruit-specific promoters  to underActinidia deliciosa), peach, pepper, and tomato, present a highly similar metabolic pattern in which higher organic acids concentration are observed in the first stages of fruit development followed by clear reductions in their levels as maturation progresses (Prunus persica (L.) Batsch). The accumulation of organic acids is seemingly well regulated during fruit development and is differentially controlled during growth stages (P. armeniaca L. cv. Harcot), plumcot , plum , and peach (P. persica L. Batsch cv. Jinmi)  . Notably. Jinmi) .Citrus paradisi) cultivars produced in Turkey, C. reticulate) and ‘Satsuma’ (C. unshiu), sweet orange ‘Newhall’ (C. sinensis) and ‘Early Gold’ (C. sinensis), pummelo ‘HB’ (C. grandis) and grapefruit ‘Flame’ (C. paradise) was recently analyzed  fruits, it was showed that increases in sugars content are followed by considerable reductions in the content of organic acids  fruits, meaning that malate metabolism most likely regulates transitory starch metabolism and that this process is probably conserved between climacteric and non-climacteric fruits . In both lemon and lime (Citrus aurantifolia) citrate accumulation can be as high as 8% of the fruit dry weight  in fruits such as strawberries and lemons , a non-climacteric fruit, the activities of the enzymes involved in organic acid metabolism including malic enzyme (ME), ICDH, ACO, and alcohol dehydrogenase increased during the first 3 weeks of post-harvest storage. Concomitantly, increased activity of enzymes involved in sugar catabolism such as hexokinase (HXK), Susy, UDPG pyrophosphorylase and PPi-dependent phosphofructokinase was also observed , a typical climacteric fruit, have revealed that the accumulation of sugar, especially sucrose, occurs between 20 and 30 days before physiological maturation. During this stage, there is a significant increase in acid invertase (AI) activity with lower SPS and Susy activities in papaya mesocarp (Studies with papaya (mesocarp . It has mesocarp . Althougmesocarp . Particumesocarp .Prunus persica) fruits showed that malate, quinate and shikimate concentrations were high at the beginning but declined at the end of fruit development , banana, tomato, grape and apple (Mangifera indica) continue to accumulate malate throughout ripening, even at the climacteric stage  fruits  fruits . Further) fruits .VviERF045) from grape affects a range of different processes including, photosynthetic capacity, secondary metabolism, expression of key genes related to changes in epidermis and cuticle of the berry, cell expansion, as well as activation of several defense related genes , non-ripening (nor), green ripe (Gr), green-flesh (gf), colorless non-ripening (Cnr), never ripe (Nr), high pigment 1 (hp1), high pigment 2 (hp2), and dark green (dg) have been investigated in the context of ripening  underlining the importance of these metabolic pathways during fruit ripening  as demonstrated by virus-induced gene silencing (VIGS) leading to decreased content of ABA that can significantly retard the ripening , which can also contribute to changes in TCA cycle intermediates , hexoses, hexose-phosphates, and amino acids in immature green, mature green, and red ripe fruits  play an important role in increasing the contents of the organic acids such as citrate, oxaloacetate and succinate  generating SAM as a reaction product which is catalyzed by SAM decarboxylase (SAMDC) (Polyamines (PAs), another group of signaling molecules, has been extensively studied in recent years. PAs are small aliphatic amines with an important role in plant growth process including fruit ripening and . In plan (SAMDC) . SAM is  (SAMDC) .SlACS) delays ripening simultaneously improving fruit quality in tomato and increasing the PAs levels associated with down-regulation of cell wall hydrolyses. Notably, still in tomato fruit, PAs has been identified as a great contributor of fruit ripening mainly associated with the activity of both ornithine decarboxylase (ODC) and arginine decarboxylase (ADC)  , 1991. Ioduction . In grapoduction , while ioduction . Applicaoduction . Remarkaoduction . Recentlripening . PAs plaLastly, but not least important, very few studies have demonstrated the association between salicylic acid (SA) and the maintenance of fruit quality during post-harvest. Sweet cherry treated with exogenous SA revealed an effective and environmentally friendly tool to maintain fruit quality during storage associated with the maintenance of the sugar and organic acid content in the fruit as well as with enhancements of both the concentration of bioactive compounds concentration and the antioxidant activity . InteresAlthough changes in the levels of organic acids are unequivocally important during fruit ripening, it seems necessary to study post-harvest physiology in more realistic environments, which means creating links with companies involved in fruit storage and transport and breaking down the variables that affect organic acid content and other important traits. The understanding of the primary metabolism in fruit is directly connected with fruit quality and seems to be an obvious target for future improvement, however, the complicating factor in this approach is that the metabolism is very dynamic over fruit development and changes are considerable throughout the fruit growth until ripening with many signaling process. Nevertheless, emerging tools can nowadays provide the opportunity to turn this information into a mechanistic understanding of fruit quality, and ultimately to design better fruits in which studding primary metabolism alongside with modeling tools can proved novel information into a mechanistic understanding to mainly develop better fruits . AdditioGenetic engineering technologies such as CRISPR/Cas9 could be used to specifically edit the sequence  or alterFinally, to increase our understanding of the quality and how specific compounds can be changed to improve the ratio between acidity and sugar in fleshy fruits, it seems that coupling integrative approaches (omics) with systems biology is necessary. This would allow the generation of plants, or better fruits, more adapted to stress conditions. Importantly, it is a general opinion that a fruit is a reflection of the conditions to which the plant has been exposed during its development . In summConsidering the importance of amino acids profile during fruit ripening, such as glutamate, the major amino acid of ripe fruits, the usage of such tools could facilitate investigation and simultaneously increasing quality or even extending fruit shelf life, mainly with higher reparatory rate and reduced postharvest time. Teasing out the connections of organic acid metabolism with a hormones may help us understand the real contribution of each hormone on central metabolism. Moreover, rational bioengineering of plants with modified levels of organic acid would also benefit from an increased knowledge of the biochemical regulations and connections inherent to the metabolism of organic acids. The development of plants with altered organic acid composition in fruits should also take into consideration that this pathway is tightly connected with several other aspects of plant metabolism. As such, changes in organic acid metabolism within fruits may not always be beneficial, especially for plants growing under sub-optimal environmental conditions. Different lines of evidence have pointed out that changes in organic acid content might greatly improve fruit organoleptic characteristics. It is important to mention, however, that the majority of those advances have been made in model organisms as well as in some plants of agricultural relevance. To successfully transfer these advances to major food crops, which are generally more recalcitrant to genetic manipulation, still remains a great challenge. To further increase our understanding concerning how organic acids affect fruit metabolism we suggest two complementary approaches: (a) it is possible that the usage of introgression Lines (ILs), as the ones developed in tomato  could alAlthough the summary presented here provides a scaffold for understanding the connections between organic acid and hormone metabolism in fruit development, we posit that it is of pivotal importance that these emerging studies should be expanded. More fundamental knowledge is still required to identify further strategies for manipulation that would improve fruit quality and consequently fruit metabolism. It seems reasonable to anticipate that approaches such as genomics, transcriptome, proteomics and metabolomics coupled with genome editing can present itself as an important data generator that would allow the production of a mechanistic map of fruits in general and their association with phytohormones and fruit developmental changes.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.


Comparative research investigating how nonhuman animals generalize patterns ofauditory stimuli often uses sequences of human speech syllables and reportslimited generalization abilities in animals. Here, we reverse this logic,testing humans with stimulus sequences tailored to squirrel monkeys. When teststimuli are familiar (human voices), humans succeed in two types ofgeneralization. However, when the same structural rule is instantiated overunfamiliar but perceivable sounds within squirrel monkeys’ optimal hearingfrequency range, human participants master only one type of generalization.These findings have methodological implications for the design of comparativeexperiments, which should be fair towards all tested species’ proclivities andlimitations. Several comparative studies on rule learning have tested nonhuman mammals and birds usingstimuli of debatable ecological validity, such as human-spoken nonsense syllables , which included nonsense strings of spokenconsonant-vowel syllables, and the heterospecific stimuli condition(hereafter HSC), which included artificial strings made of sine wave tonestuned to squirrel monkeys’ hearing frequency range . Syllables in the CSC were divided in two easily discriminableclasses of sounds, spoken by either a male or a female speaker. Tones in the HSC alsooccupied two frequency ranges: Tones varied around a mean frequency of 2 kHz(lower tones) or around a mean frequency of 11 kHz (highertones). Notably, the heterospecific stimuli, being designed to suitsquirrel monkeys, were less familiar and perceptually conspicuous to humans than theconspecific ones. Previous studies on squirrel monkeys, marmosets, and chimpanzees,using the same . Specifically, withineach condition, participants were habituated to stimuli created as follows: In the CSC,one syllable spoken by the male speaker occurred in first and last positions of thesequence, and one to three syllables spoken by the female speaker occurred in themiddle; similarly, in the HSC, one lower tone occurred in first and last positions ofthe sequence, and one to three higher tones occurred in the middle (lower pitched stimuli (class L)and female and higher tones represented higher pitched stimuli (classH), and in each condition, habituation sequences were LHL, LHHL, and LHHHL .Stimuli were designed to assess the effect of these two conditions on humans’ ability togeneralize an ABe middle . Thus,mPrior to the experiment start, participants performed an audiometric test measuring theirability to hear tones in the frequency range of the HSC: Only successful participantswere included in the analysis of pattern rule extraction and generalization abilitiesinvestigated here .similarto or different from those heard in the habituation phase:(a) generalization of the same pattern to novel, longer sequences including more H unitsin the middle and units of similar frequencies as in the habituation  and (b) generalization over the more abstractABnA pattern, where the position of high-pitched and low-pitched stimuliwas swapped compared with the stimuli used in the habituation phase . Hence, in the structural generalization test, participantshad to classify HLnH patterns as instances of the same structural rule as theLHnL pattern heard in the habituation phase. Across all test phases,order of trials was randomized across participants. Instructions were minimized to makethis work comparable with similar experiments on nonhuman animals.The experiment started with a habituation phase that included 36 stimuli presented indifferent randomized orders across participants. This was followed by two tests  where participants had to rate new sequences of sounds as similar ordifferent to the pattern extracted in the habituation phase (p < .05). In the structural generalization test, six participantswere above chance in CSC (top-right), while only three participants achievedsignificance in HSC (bottom-right). A binomial logit GLMM provided similar results:Stimulus pattern (consistent vs. inconsistent with the habituation pattern),generalization type , and experimental condition (CSCvs. HSC) all significantly affected participants’ responses in the comparable direction but often neglect the species-specificperceptual tuning of test stimuli . Our results indicate that limitations in perceptual relevance or audibility ofstimuli may lead to failure to generalize over complex patterns. Increasing thecomparative methodological fairness of stimuli will thus enhance accuracy incross-species investigations of cognition.Click here for additional data file.Supplemental Material for Perceptual Tuning Influences Rule Generalization:Testing Humans With Monkey-Tailored Stimuli by Andrea Ravignani, Piera Filippiand W. Tecumseh Fitch in i-Perception