The environment of space provides many challenges to the human physiology and therefore to extended habitation and exploration. Research to define and countermeasures to address these challenges have been explored for well over 40 years yet success in alleviating these challenges has been inadequate, due in part to the lack of investigative tools (subject number and technology) to monitor and diagnose conditions in real time and by the overall lack of a relevant data base to understand the central causative agent(s) responsible for physiological adaptation and homeostasis in space ,2,3,4,5.REDOX equilibrium is cyclic biofeedback related biochemistry. OSaD control/REDOX homeostasis encompasses many aspects of the human physiological response to the space environment and if mitigated properly may normalize a portion of the adverse phenomena seen in space flight combinatorial events and suggest relevant countermeasures for those situations, thus increasing the level of safety and occupational health for each crew member during long duration flight and facilitate improved performance during exploration class missions. The generation of ROS/RNS in the human physiology is a normal part of human systems biology ,8. OxidaSpace travel and persistent habitation of reduced gravity or non-terrestrial environments poses many problems which are in some respects similar to the systems biology of terrestrial living associated with numerous disease states and the progression of aging  6,7]. H,7. H6,7]This Special Issue of IJMS presents some recent supporting data and possible countermeasure approaches. Most of the conditions exemplified in 2O2) as it via Haber-Weiss reaction interacts with ferric and other ions reduced by superoxide radicals [Mammalian, specifically human systems biology depends on a complicated and somewhat fragile series of dynamic checks and balances to constantly provide homeostasis. An essential part of this regulatory process is the production and scavenging of excess ROS and RNS to maintain an appropriate balance of free radicals (FRs) in the physiology. Some of the FR species particularly important for space physiology are nitric oxide (NO) as it has profound cardiovascular and sub-cellar metabolic impacts, hydrogen peroxide  are limited normally to two 6-month tours ,4,5. Thi56Fe ion radiation [56Fe ions (200 cGy) were responsible for increases in marrow cells production of mineralized nodules ex vivo regardless of radiation type or dose; and 56Fe (200 cGy) inhibited osteoblastogenesis by more than 90% (5 weeks and 1 year post-IR). After 5 weeks, irradiation (protons or 56Fe) resulted in minimal changes in gene expression levels during osteoblastogenesis, however a high dose 56Fe (200 cGy) resulted in bone loss, increased Catalase and Gadd45 gene expression, and the radiation damage seemed to be mitigated by the additions of superoxide dismutase (SOD) [Alwood et al. observed the effects of radiation on the process of osteoblastogenesis by studying the skeletal structure of C57BL6J mice exposed to either low LET (low energy transfer) protons (50 cGy) or high LET adiation . They suse (SOD) .In a simulated microgravity culture system (RWV) Shanmugarajan et al. investigated the combined effects of gamma radiation and microgravity by monitoring the maturation of a hematopoietic cell line RAW 264.7 into mature osteoclasts. In short, results of the investigation demonstrated radiation alone at 100 cGy may stimulate osteoclast cell fusion determined via giant multinucleated cells GMCs presence and the expression of the signature genes tartrate resistant acid phosphatase (Trap) and dendritic cell-specific transmembrane protein (Dcstamp). Notably, in 1G controls osteoclast cell fusion decreased in doses above 0.5 Gy. By comparison to radiation exposure, simulated microgravity resulted in increased levels of cell fusion, and the effects of radiation and microgravity are additive. Remarkably, simulated microgravity culture effects on osteoclast stimulatory transmembrane protein (Ocstamp) and Dcstamp expression was substantially higher than the radiation effect, inferring that microgravity may increase the synthesis of adhesion molecules more than radiation  and thusN-acetyl cysteine, show promise in the arrest of oxidative stress in bone as well. Though these are reports from Earth-based studies, still they suggest important avenues to be pursued in the space flight arena to combat OSaD during prolonged flight [In light of the excellent work by Alwood et al. and Shanmugarajan et al. ,11 it wod flight . In their review, Tahimic and Globus intertwine another important facet of space flight and the complex environment that consists of changes in the general physiology including bone, muscle and the vascular system; all subject to systemic OSaD effects. Microgravity forces selective environmental pressures, causing a cephalad fluid shift, profound reductions in mechanical loading of bone and muscle, and a reduced immune competency concomitant with inflammation, coupled with galactic cosmic radiation (GCR) and high energy particles (HZE). Here the authors review spaceflight-induced perturbations in calcium homeostasis and specific physiological reductions in bone mass (osteopenia), which pose protracted risks for tissue repair and skeletal health. Space analogue rodent models for these types of studies such as hind-limb unloading (HLU) result in not only bone but also vascular anomalies reminiscent of aging like vascular density (rarefication), and vasodilation responses. These responses tied to excess ROS/RNS, (NO) and inflammation are implicated both in diseases of aging, like osteoporosis and atherosclerosis, and pursuant to insults such as radiation exposure as seen in spaceflight. ROS can directly facilitate bone osteoclastogenesis, leading to resorption and bone loss during aging and estrogen deficiency may be partly attributed to OSaD mechanisms, and thus portends to link the complex phenomena of bone and muscle loss, vascular deconditioning, and immune functions together in an intricate syndrome . To address the cardiovascular system as related to ROS, Takahasi et al. reviewed the effects of OSaD on the cardiovascular system in the presence of reduced gravity environments the central constituents being microgravity and radiation. Prior research has shown that 3–4-week HLU resulted in increased superoxide anion levels in the carotid and adjacent arteries of rats. In this investigation, eNOS expression was upregulated in the carotid artery. The effect of microgravity on the cardiovascular system seems to be different depending on the region. This is not necessarily surprising due to the graded simulation of HLU. Longer HLU studies presented increased superoxide levels, elevation of pro-oxidative enzymes NOX2 and NOX4 and reduction of important anti-oxidative enzymes like Mn-SOD and GPx-1, an effect not seen in mesenteric arteries. Once again, the inflight studies demonstrate synergistic ROS effects by the combination of radiation and microgravity. In other HLU-7-day studies on mouse brains, low dose radiation (LDR) and HLU evidenced lipid oxidation in the brain cortex, but in neither of the independent conditions alone. In 9-month studies of ROS effects, lipid peroxidation and expression of NOX2 were seen in both LDR and HLU yet the combinatorial effect was still greater . Thus, it would appear that the physiology may be resistant to short term single event upset and thereby conceal larger problems in evidence when model studies incorporate multiple stressors as seen in actual microgravity.We see a myriad of factors impinge on the human physiology in space. Microgravity and increased radiation are environmental catalysts for very complex systemic responses driven to abate a cascade of negative events realized singularly in space. For example, at the metabolic and immune levels the fluid cephalic shift disrupts the entire constituency of the blood and vascular compartment driving loss of body fluids and necessitating the cannibalizing of excess blood cells now too numerous in the reduced plasma volume, consequently launching inflammatory responses and metabolic destabilization ,2,4,5.Tahimic and Globus  note theAnselm and colleagues followed up on the excellent work of Blaber and team by assessing metabolic pathways and conditions of mouse liver physiology after 30 days of flight on the Russian Bion-M1 spacecraft. After 30 days in space one cohort of the mice were allowed a 7-day re-adaptation while the remainder were not. Analyses of the liver proteomic profiling included shotgun mass spectrometry and label free quantification. The analyses yielded 1086 known proteins and 12,206 unique peptides and statistical testing by ANOVA revealed 218 up-regulated and 224 down-regulated proteins in the post-flight compared to the other groups . Amino aInvestigators Burns and Manda address the vast complexities of the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) metabolism as inferential of OSaD in their review through an in depth description of the Warburg effect (WE), originally described to yield insights into aberrant cancer metabolism which include features of increased cellular glycolysis and remarkable changes to mitochondrial function and mtDNA repair now known to be associated with other human disease states . Of note2 during spacesuit operations and be exposed to mild hyperoxia about 50% of the mission length overall [As we turn to countermeasures we see that in many of the previous manuscripts and reviews cited potential mitigating agents are either physiologically endemic, naturally occurring, or pharmacologic ,15,16,17 overall ,4. The prospects for robust OSaD countermeasures to combat elevated radiation and microgravity effects are essential for planning long duration space missions. Velalopoulou et al. detail in their publication the prospects for the use of LGM2605, a synthetic derivative of the flaxseed lignan secoisolariciresinol diglucoside (SDG), which has been proven to reduce OSaD-related biomarkers in the presence of radiation . LGM2605In conclusion, the 10 significant and elegant manuscripts published in this Special Issue illustrate the intricacies, relevance, and impacts of unstable ROS generation and persistent OSaD in the space flight environment, which are, in many ways, emblematic of the phenomena of aging. Recent data released by NASA on the human Twins Study further illustrates the changes to and potential damage sustained by the human physiology while inhabiting an unrelentingly hostile environment, especially considering the results of analyses of inflammatory cytokines, RNA and DNA analyses and microbiome research conducted by Drs. Snyder, Mason, and Turek, respectively . We woul


C). It is, however, anticipated that knowledge of its function will shed light on its contribution to neurodegenerative diseases and suggest ways to interfere with the cellular toxicity central to them. Consequently, efforts to elucidate its function have been all but exhaustive. Building on earlier work that uncovered the evolutionary descent of the prion founder gene from an ancestral ZIP zinc transporter, we recently investigated a possible role of PrPC in a morphogenetic program referred to as epithelial-to-mesenchymal transition (EMT). By capitalizing on PrPC knockout cell clones in a mammalian cell model of EMT and using a comparative proteomics discovery strategy, neural cell adhesion molecule-1 emerged as a protein whose upregulation during EMT was perturbed in PrPC knockout cells. Follow-up work led us to observe that PrPC regulates the polysialylation of the neural cell adhesion molecule NCAM1 in cells undergoing morphogenetic reprogramming. In addition to governing cellular migration, polysialylation modulates several other cellular plasticity programs PrPC has been phenotypically linked to. These include neurogenesis in the subventricular zone, controlled mossy fiber sprouting and trimming in the hippocampal formation, hematopoietic stem cell renewal, myelin repair and maintenance, integrity of the circadian rhythm, and glutamatergic signaling. This review revisits this body of literature and attempts to present it in light of this novel contextual framework. When approached in this manner, a coherent model of PrPC acting as a regulator of polysialylation during specific cell and tissue morphogenesis events comes into focus.Much confusion surrounds the physiological function of the cellular prion protein (PrP C), all three approaches have been pursued with moderate successes summarized in comprehensive earlier reviews (Arguably the most successful strategies for elucidating the function of a protein are (a) to extrapolate it from the known function of its binding partners, (b) to interpret the phenotypic consequences of deleting the gene coding for it, or (c) to derive it by studying the function of homologous proteins and place its role in the context of evolution. For the cellular prion protein . According to this model, PrPC acts upstream of a signaling loop, which modulates the expression of the polysialyltransferase (polyST) ST8SIA2 , thereby determining levels of polysialylation of NCAM1 , during the execution of specific cell plasticity programs  undergoes profound physicochemical changes that give rise to rogue conformers known as PrP Scrapie (PrPSc), a term derived from Scrapie disease in sheep, the first known prion disease. The human prion gene is coded on the short arm of Chromosome 20  superfamily. The human genome codes for two NCAM paralogs, known as NCAM1 (commonly referred to as NCAM or N-CAM) and NCAM2 . The human 24 exons . ExpressNCAM-120 . Whereasmophilic  and hetemophilic  interactST8SIA2 and ST8SIA4 genes map to Chromosome 15, Band q26 and Chromosome 5, Band q21, respectively  and ST8SIA4  was initially thought to be restricted to NCAM1 and the mechanism of its attachment unknown . SubsequGLG1; Although NCAM1 represents by far the most prominent polySia protein acceptor, polySia modifications in mammals are also found on a small number of other proteins, namely, the α-subunit of the voltage-gated sodium channel , CD36 Y, neuropiC-related phenotypes can be grouped, examples for each of the aforementioned three types of phenotypes can also be identified in the literature surrounding PrPC.How does polySia contribute to cellular function? The polySia modification of NCAM1 alters its hydrodynamic radius and confers negative charge. These properties are critical for phenotypes that have been linked to polySia-NCAM1, which can be broadly grouped into those that (a) facilitate cellular morphogenesis programs ranging from proliferation and migration to neuritogenesis and fasciculation by affecting protein interactions that mediate cell-cell or cell-substrate contacts; (b) refine cellular responses to guidance cues, or (c) modulate inputs of brain circuitry or activity of ion channels . Althougin vitro polysialylation of NRP2 and SynCAM1  but were most impacted by the depletion of PrPC  of NCAM1 or (b) of one or both polySTs, as well as (c) digestion of polySia by endosialidases. Because NCAM1 is the predominant polySia acceptor in the brain (accounting for > 95% of this modification), phenotypes related to polysialylation in neuronal paradigms are most likely NCAM1 related. The situation is more complicated in certain hematopoietic stem cell (HSC) lineages, where polySia might be attached to one of the other polySia acceptor proteins (see earlier).C in certain stroke paradigms  acquiring mesoderm-like properties . More spC deficiency phenotype reported to date has been the observation of a gastrulation arrest in zebrafish embryos made to express reduced levels of PrP-1, one of two PrPC orthologs in this organism, through a morpholino-based knockdown approach , neurogenesis gives rise to glia or neuroblasts, which upon further differentiation migrate rostrally to the olfactory bulb OB; , before Ncam1-deficient mice  was provided by the observation that the OB was reduced in size by 30% in ent mice . Studiesent mice . In thesent mice . A more ent mice .St8sia2 knockout mice, indicating that even minor reductions of polysialylation are sufficient to cause migration deficits of SVZ-derived neuroblasts  main bundle and a smaller infrapyramidal bundle (IPB) that innervates dendrites on basal CA3 pyramidal cells and normally crosses the pyramidal cell layer to connect to the SPB within the CA3 field and in proximity to the hilus. As early as 1997, it was reported that PrP the IPB . More sp the IPB . Signifiout mice  and St8sent mice  as well ent mice  and miceent mice  but not ent mice . Notablyent mice  is highlout mice . The impout mice .Figure The mossy fiber phenotype has mostly been discussed as a deficit of lamination and fasciculation caused by the modulation of cell surface interactions. Loss of polySia could either increase fasciculation by increasing interactions between axons or reduce fasciculation by increasing interactions with the environment. Alternatively, the mossy fiber phenotype could be explained by alterations of the retraction and pruning of neuritic processes and axons, a major developmental mechanism known to contribute to neuronal maturation. It has long been known that mossy fibers first send out, then retract filamentous extensions to nearby cells during their transit through the hilar region of the dentate gyrus . This coC and ST8SIA2-mediated polysialylation. Interestingly, NRP2 is a target for polysialylation but polySia-NRP2 is exclusively produced by ST8SIA4  and 21 (CCL21) are known to be essential in this context. Significantly, polySia on DC cells facilitates this migration in a manner that depends on the presence of a basic motif present at the C-terminus of CCL21 and, presumably, involves its interaction with the polySia negative charge cluster . This chC to influence polysialylation levels extends to ST8SIA4 and non-NCAM1 substrates in certain cellular contexts. If so, it will be attractive to specifically investigate if PrPC influences in immune cells the expression of ST8SIA4, thereby modulating microglia or macrophage activation or CCL21-dependend migration of DCs. Although this question has not yet been directly addressed, it was reported that transgenic overexpression of CCL21 from an insulin promoter causes chronic inflammatory disease in the pancreas that was characterized by PrPSc accumulation in prion-infected mice in this peripheral organ that in wild-type mice is spared by PrPSc infection . In humans, FFI can be caused by a single amino acid change within the prion protein sequence that replaces an aspartate with an asparagine residue at Position 178 on a background of the common methionine or methionine polymorphism at PrP in mice . While t in mice . The ins in mice . A more nt brain  and alsont brain , with hint brain . Investint brain , 2001. Tnt brain . Althougnt brain .C, one of the first prominent phenotypic changes observed in Prnp−/− mice was an altered circadian rhythm . Both PrPC and polySia-NCAM1 have been shown to impact NMDAR signaling, possibly, by modulating local copper concentrations (see later). In support of this model, copper has been shown to cause a delay in circadian neural activity rhythms  denotes a complex postsynaptic response to extracellular glutamate exposure, widely considered the cellular correlate to memory and learning. Central to LTP are the functional interplay of two types of glutamate receptors, AMPA receptors (AMPARs) and NMDARs. Briefly, when glutamate binds to AMPARs, their ion channels open causing Nasynapses . Indicat polySia . The int polySia  and acti polySia .Ncam1-deficient mice by inhibiting GluN2B or by scavenging free glutamate with GTP  family of metal ion transporters . The evoebrafish . ImportaC and ZIP transporters surfaced when it was established that two of these transporters (ZIP6 and ZIP10) can be crosslinked to PrPC in Neuro-2a neuroblastoma cells exposed to low levels of formaldehyde  genome, a PrPC-like ZIP6-related pseudogene could be identified, which is, like the Prnp gene, devoid of introns within its protein coding sequence and also lacks the sequence elements coding for the transmembrane domain observed in intact ZIP transporter genes . Consistent with this assertion, the polySTs responsible for its polysialylation may have evolved around 500 million years ago  stable cell-cell adhesion complexes need to be replaced with dynamic cell-substrate interactions; (b) instead of providing structural integrity, the cytoskeleton need to convey cell movement; and (c) the cell needs to have systems in place that alert it to guidance cues and inform it on when to stop moving and re-establish connections to neighboring cells. Thus, although a defect in the control of NCAM1 polysialylation may turn out to be the single most profound change observed in PrPaffected . In partC-NCAM1 heteromer in cellular plasticity programs did not evolve abruptly, NCAM orthologs have been linked to these programs even in cells and species whose genomes do not code for polySTs. For example, fasciclin2 (Fas2), a fruit fly (Drosophila melanogaster) NCAM ortholog, is required for sensory axon guidance . Thus, consistent with their coinciding evolutionary emergence, NCAM1 and ZIP transporters harboring PrPC-like ectodomains may have evolved to collaborate during the execution of cell plasticity programs. A need for these programs materialized during metazoan development to promote a motile lifestyle. Later in evolution, with the emergence of complex body plans, elements of these programs could be adapted for developmental programs that required the migration of defined cell populations. Components required for NCAM1 polysialylation only emerged much later. These may have served the purpose to further enhance the ability of preexisting plasticity programs to cope with both an unprecedented complexity of cell surface receptor interactions and an increasing complexity of body plans. In particular, the migration of specialized cell populations in different parts of the developing body would be expected to require a more specialized toolkit to choreograph this movement in time and space, possibly requiring further differentiation of players involved. Perhaps adapting to this need, the subbranch of the ZIP transporter family comprising PrPC-like ectodomains expanded and diversified in the vertebrate lineage  molecular interactions of PrP, (b) phenotypes of PrP deficiency models


PrimPol is a human deoxyribonucleic acid (DNA) polymerase that also possesses primase activity and is involved in DNA damage tolerance, the prevention of genome instability and mitochondrial DNA maintenance. In this review, we focus on recent advances in biochemical and crystallographic studies of PrimPol, as well as in identification of new protein-protein interaction partners. Furthermore, we discuss the possible functions of PrimPol in both the nucleus and the mitochondria. Tw. Tw42]. −/−PrimPol cells. Moreover, no further decrease in replication rate was observed when PolDIP2 was depleted in −/−PrimPol cells. These data suggest that PrimPol and PolDIP2 work epistatically in the same pathway to promote DNA replication in the presence of UV damage [Recently it was also shown that the DNA polymerase activity of human PrimPol is stimulated by interaction of its AEP domain with PolDIP2  50]. Po. Po50]. Importantly, PolDIP2 also interacts with PCNA, the regulatory p50 subunit of replicative Pol δ and several translesion DNA polymerases ,70,71. Ithe interaction between PolDIP2 and PrimPol is mediated by the N-terminal region of PolDIP2 that also contains the mitochondrial targeting signal that is likely cleaved off upon mitochondrial entry [PolDIP2 has been found both in the nucleus and mitochondria. While Klaile et al. showed that endogenous human and rat PolDIP2 localized primarily in the cytoplasmic and nuclear fractions, with lesser amounts entering the mitochondria , Cheng Xal entry . The trual entry . TherefoFinally, to date, no proteins that act to stimulate the primase activity of PrimPol have been reported excepting for RPA ,68. Ther−/−PRIMPOL knockout mice are viable but −/−PRIMPOL deficient cells show replication stress, genetic instability and defects in mitochondrial replication [lication . It is llication .PRIMPOL-deficient tumors in breast cancer patients diagnosed with invasive lobular and ductal carcinoma, and the −/−PRIMPOL tumors were found to exhibit a mutation load that was nearly twice as high as in PRIMPOL-proficient tumors.Nevertheless, the established roles of PrimPol in DNA damage tolerance and mitochondrial DNA replication suggest that PrimPol mutations could lead to some inherited diseases including cancer predisposition and mitochondriopathies. To date, however, little is known about the influence of PrimPol defects on human health. Recent analysis of gene expression data from The Cancer Genome Atlas (TCGA) demonstrated anti-mutagenic activity of PrimPol in genome maintenance and suggested a possible protective role of PrimPol in human breast cancer . The stuPRIMPOL mutation, a naturally occurring minor PrimPol variant, has been reported to potentially associate with human disease [Y89D variant has dramatically lower processivity than wild-type PrimPol, which in turn causes a significant slowing of replication fork progression and increased UV-sensitivity in vivo [Y89D variant and the human ocular disease high myopia. This mutation was found in a single family and four additional sporadic patients with high myopia. Within the family, the mutation is inherited in an incomplete autosomal dominant manner, and when heterozygous, a lighter form of the disease results [Y89D variant was found among individuals with high myopia, other forms of genetic eye diseases and normal controls [Only one missense  disease . This mi in vivo . Zhao et in vivo  hypothes results . Neverthcontrols .Many translesion DNA polymerases promote the resistance of cancer cells to chemotherapy ,78. As Pde novo priming of DNA synthesis downstream of the lesion. Since its discovery in 2013, extensive biochemical and structural studies have provided insight into the biochemical properties and functions of PrimPol and the mechanisms of its DNA polymerase and primase activities. Growing evidence suggests that the main biological function of human PrimPol during replication of chromosomal DNA is a repriming of stalled replication downstream of DNA damage or naturally occurring obstacles. However, the mechanisms that regulate the repriming by PrimPol in cells are yet to be understood. The functions of PrimPol in mitochondria and the association of PrimPol defects with human diseases are also largely unexplored and constitute important directions for future research.Many human translesion DNA polymerases have been found and characterized in the last 15 years, and PrimPol is the most recent DNA polymerase to be identified. PrimPol is unique due to its dual activities as a translesion DNA polymerase and a primase. These activities provide extreme flexibility of DNA damage bypass as PrimPol can contribute to bypass through classical TLS, by skipping the lesion and/or by


HIV infection is associated with an increased risk of cardiovascular disease (CVD). Chronic low-grade immune activation is likely one of the driving mechanisms. This systematic review provides an overview of the evidence addressing the relation between immune markers and surrogate markers of CVD (except CIMT) in HIV infection.A systematic search was performed in PubMed, Embase and Cochrane Library identifying all articles from 1996 to April 2015. It addressed the relation between immune markers and surrogate markers of CVD (except Carotid Intima-media Thickness) in HIV-positive adults. Two authors, using predefined criteria, independently conducted the selection of articles, critical appraisal and extraction of the data. Analysis focused on immune markers that were assessed most frequently. The review was conducted according to the PRISMA guideline and performed as part of an overarching review registered with PROSPERO (CRD42014010516).Twenty-nine articles were selected, describing 34 immune markers and nine different CVD surrogate outcomes: coronary calcium score (13 times) and flow-mediated dilation (10 times) were used most frequently. Twenty-seven studies had a cross-sectional design. CRP, IL-6 and sVCAM-1 were assessed most frequently. None of the immune markers were clearly associated with any of the surrogate CVD outcomes. No effect estimate could be calculated due to marked heterogeneity in study populations, immune markers, outcomes and statistical approaches.This review could not identify a clear association between any of the immune markers and surrogate CVD outcomes. This may reflect a true lack of association, or may be explained by heterogeneity across studies and lack of follow-up data. Future research should focus on longitudinal studies measuring a select set of immune markers and surrogate CVD outcomes awaiting the primary outcome of clinical cardiovascular events. Highly active antiretroviral therapy (HAART) has markedly increased life expectancy among persons infected with human immunodeficiency virus (HIV). However, it has become clear that patients infected with HIV have an increased risk for developing cardiovascular disease (CVD). MultipleTo provide insight in the burden of cardiovascular disease in HIV infected individuals while awaiting results from longitudinal studies, a multitude of surrogate markers for CVD have been used. A recent summary of the current body of evidence regarding potential associations between markers of immune activation and carotid intima media thickness (CIMT) could not draw a clear conclusion due to the heterogeneity in data. This sysCRD42014010516). Results addressing the relation of markers of immune activation, CVD and CIMT were published in January 2016.[http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42014010516)This systematic review was conducted according to the guidelines provided by PRISMA S1 Chec and it iary 2016. http://wth 2015 using terms and synonyms for HIV, immune markers and surrogate markers for CVD other than CIMT, covering all evidence from 1996 onward. Terms were limited to title and abstract  using a set format.Given heterogeneity of studies, a descriptive analysis, grouped by outcome and most frequently assessed immune markers, was conducted. When possible, percentages of common baseline characteristics were calculated.Surrogate outcomes of CVD were divided in two groups: outcomes using imaging techniques and outcomes assessing arterial stiffness. Imaging techniques were subdivided in two main categories: 1) coronary angiography and coronary artery calcium score, and 2) all other imaging techniques, namely 18FDG PET scan, SPECT and MRI scan of aortic and carotid arteries. Arterial stiffness outcomes were FMD, PWV, PWA, ABI and carotid artery stiffness. Differences in outcome protocols were not taken into account in this review.Outcome was reported as a positive association, an inverse association, no association or no data. To combine information from various types of studies reporting often more than one risk estimate per outcome, data were reduced by choosing only one effect estimate per immune marker per surrogate CVD outcome per article. To select the effect estimate, the following hierarchical order was used: 1) outcome of multivariable analysis. If not reported, 2) outcome of univariable analysis. If there was no significant outcome in multi-or univariable analysis, the outcome was scored as 3) ‘No association’. If no data were reported, although the methods section specified that the relation between immune marker and outcome has been studied, the outcome was reported as 4) ‘no data’. In this review C-reactive protein (CRP) refers to both the regular CRP measurement and to the high-sensitive CRP assays.The updated search identified 169 articles, 64 articles were screened using full text, and 29 articles were finally selected . Article2, (standard deviation (SD) 1.9kg/m2) and nearly 40% of all participants were current smokers. Duration of HIV infection ranged between 24 weeks and 16 years. Ten studies[A total of 3,559 HIV positive patients were included, whereof the majority were male  ; mean agn studies–20 inclun studies,12,21CAC score was used as endpoint in 13 studies,,17,22–30All studies were critically appraised on eight items; clarity of inclusion criteria; moment of inclusion; standardization of measurement of determinant and outcome; missing data at baseline or follow-up; missing data on potential eligible participants; and blinding and adjustment for confounders . A risk Six Smarkers were evaluated three or more times in relation to coronary calcium score or coronary angiography . CRP wasCRP was assessed in four studies using 18FDG PET scan,31 or SPSix markers were evaluated three times or more in relation to arterial stiffness . CRP wasLongenecker et al. used a cA large number of immune markers have been investigated for predictive value in relation to surrogate markers of cardiovascular disease in HIV positive patients. CRP, IL-6 and sVCAM-1 were addressed most frequently; neither CRP, IL-6, sVCAM-1 nor any of the other markers showed a clear relation with any of the surrogate CVD markers.These results complement the findings of a recent review assessing immune marker choice in relation to CVD or CIMT. CRP, IL-6 and s-VCAM-1 were also the most frequently assessed markers in relation to CIMT, and the lack of a clear relation between any of these immune markers and surrogate CVD outcomes that is currently found is in line with the findings in relation to CIMT.Although there is ample evidence that levels of CRP,38 and IAlthough the prognostic role of s-VCAM-1 levels in the prediction of CVD in not as clear, an association with signs of endothelial damage  or inflammation  was to be expected as s-VCAM-1 is expressed on the endothelial surface in case of endothelial inflammation. However,The two possible explanations why this review did not show an association between any of the markers and surrogate CVD outcomes could be that either there is no association, or associations are not yet clear due to methodological and qualitative constraints in the available evidence.The first explanation, no association, could be due to the fact that, despite surrogate outcomes and overt CVD being clearly related,–53 and CThe second explanation, lack of an association due to methodological and qualitative constraints, could be due to a marked heterogeneity in choice of both immune markers, surrogate outcomes and ways of analysis . In addition, studies were not primarily designed or powered to detect an association between immune markers and outcome and study designs were mainly cross-sectional (27 out of 29 studies). Follow-up time of the two prospective studies was too short to detect significant vascular alterations and all studies included a relatively young population (average age 45 years), in which atherosclerotic vascular changes and–burden are expected to be low.Finally, possible associations might be masked by heterogeneity in patient populations, duration of HIV diagnosis, use and duration of antiretroviral therapy (ART), viral suppression rates and CD4 counts, as they differ substantially in, and between, studies. All these factors have been previously reported to influence both the inflammatory response and the risk of cardiovascular morbidity and mortality.–55 MoreoThis systematic review synthesizes the available information on immune-markers in relation to surrogate markers of CVD (except CIMT) in HIV-infected patients from 1996 up to April 2015. The main focus of this review was to provide an overview of which immune markers were assessed, how frequently these markers were assessed and which surrogate CVD outcomes were chosen, as well as to summarize the current evidence on the relation between immune markers and outcomes.The most frequently assessed immune markers (CRP and IL-6) have been shown to be related to CVD morbidity and mortality in both the general population and in the HIV-infected population, and surrogate CVD outcomes have been shown to be related to CVD in the general population. This is the first time that the relation between immune markers and surrogate CVD outcomes is addressed in a systematic way in the context of HIV infection. The major strength of this review is that it contributes to a clear, global understanding of existing knowledge regarding immune markers and surrogate CVD outcomes.Limitations to be considered include that only one outcome per marker per article was considered which might have resulted in an overly optimistic impression of associations as negative outcomes are underreported; that positive outcomes include univariable associations which might in part be confounded; and that outcomes were grouped in categories  which might have resulted in specific associations (to specific parts of an outcome) being overlooked.This review provides an overview of the current literature regarding the association between immune markers and surrogate markers of cardiovascular disease other than CIMT in HIV-positive individuals. Most frequently assessed immune markers were CRP, IL-6 and s-VCAM-1; most frequently assessed surrogate markers for CVD were CAC and FMD.No relation between any of the immune markers and any of the surrogate outcomes could be detected. This may be due to the cross-sectional design, heterogeneity in patient populations, the variety in immune marker choice and surrogate CVD outcomes. The search for the association of immune markers in relation to surrogate CVD outcomes in a cross-sectional study design should be reconsidered.Future research should focus on longitudinal studies measuring immune markers and surrogate CVD outcomes awaiting the primary outcome of clinical cardiovascular events.S1 Checklist(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableND: no data.(DOCX)Click here for additional data file.


Substantial evidence have also demonstrated the potential of aspirin for the prevention and treatment of cancer. Multiple clinical studies have demonstrated a link between long-term aspirin use and a reduction in the incidence and mortality of several cancer types, including colorectal, stomach, esophageal, breast, lung, prostate and liver cancers.3 In this News and View, we have highlighted the emergence of aspirin as a chemoprevention agent and its role as an adjuvant therapy in cancer, focusing mainly on hepatocellular carcinoma (HCC)  that has been established to treat pain, fever and inflammation. Evidence demonstrated that whenever a cell is injured, prostaglandin is released and NSAIDs such as aspirin can help prevent and relieve these symptoms of injury by blocking the action of cyclooxygenase (COX), the enzyme responsible for the synthesis of prostaglandins. Aspirin acts by acetylating platelet COX-1 and as a consequence, also irreversibly inhibiting platelet function.ma (HCC) .4 HBV transgenic mice treated with dual aspirin–clopidogrel therapy showed reduction in overall liver damage, inflammation and fibrosis. The dual therapy also reduced disease progression and prolonged overall survival.4 By analyzing data from the Taiwan National Health Insurance Research Database, the use of aspirin or clopidogrel was also significantly associated with better overall survival and disease-free survival for patients with hepatitis B virus (HBV)-related HCC following liver resection surgery.5 To explore the protective effects of antiplatelet therapy against HCC, Lee et al. conducted a retrospective analysis of the risk of HCC in 1674 patients with CHB and whose HBV DNA levels were suppressed by antivirals to <2000 IU/ml. Risk was compared between patients received antiplatelet treatment  and patients who were not treated. The primary and secondary outcomes were development of HCC and bleeding events, respectively. During the study period, the antiplatelet-treated group showed a significantly lower risk of HCC compared to the untreated, regardless of the antiplatelet agent. While treatment with aspirin alone was not associated with a higher bleeding risk, however, antiplatelet therapy containing clopidogrel may increase the overall risk of bleeding.3 A nationwide cohort study from Taiwan has also concurred that NSAIDs or aspirin use associated with a reduced risk of HCC recurrence.6 Moreover, an independent study from The National Institutes of Health (NIH) of the United States has also shown that aspirin use was associated with a decreased risk of developing HCC and death from chronic liver disease (CLD), whereas nonaspirin NSAID use was only associated with reduced risk of CLD death.7Because of the dismal prognosis of HCC, chemoprevention offers an appealing strategy. In 2012, it was demonstrated in the chronic hepatitis B (CHB) mouse model that dual antiplatelet therapy with aspirin and clopidogrel could prevent HCC and improve survival.8 Randomized controlled trials also showed that aspirin reduced the risk of metastasis of adenocarcinomas, especially in patients with metastatic colorectal cancer.9Besides evidence emerging to support aspirin has potential value of chemoprevention, the antitumor effects of aspirin have also been examined. The association between platelets and cancer progression is well recognized and multiple mechanisms have been proposed to explain the complex interactions between platelets and tumor cells. Aspirin is an antiplatelet drug and its anticancer activity has been extensively investigated using cancer cell lines, animal models as well as clinical trials.10 Moreover, significant survival improvement was also reported when aspirin was taken at the time of embolization for HCC.11 Also, there is a current prospective randomized controlled trial registered in China to investigate the effect of sorafenib combined with aspirin in preventing patient risk for postoperative surgical recurrence of HCC (NCT02748304). The primary outcome of the trial is to measure the 5-year overall survival and the secondary outcome is to measure the 5-year disease-free survival and treatment-related bleedings. Unfortunately, the design of most of these trials suffer from the lack of biomarker-targeted selection of subpopulations of HCC patients.Sorafenib is currently the only FDA-approved molecular inhibitor for the systemic therapy of advanced HCC. However, its high cost, a marginal benefit increase and often severe side effects are the major clinical challenges associated with the treatment of HCC with sorafenib. Being an example of inflammation-related cancer and the chronic inflammatory state in response to liver damage and viral infection appears to be necessary for its initiation and development, the syngergistic therapeutic effect with dual treatment of sorafenib and aspirin has therefore been explored. Aspirin in combination with TACE has been reported to improve overall survival in treating patients with unresectable HCC.12 Previous studies have suggested that the observed pro-metastasis effect of sorafenib resulted from the downregulation of the expression of oxidoreductase HTATIP2, a tumor suppressor in HCC and stromal cell-derived factor 1-α (SDF1-α) expression in tumor microenvironment of HCC. Aspirin suppresses COX2 and SDF1-α expression leading to the increase expression of HTATIP2 and therefore counteracts the pro-invasion and pro-metastasis effects of sorafenib in HCC.14 However, we observed that the expression of HTATIP2 was not significantly modulated when expression data were analyzed in the TCGA-LIHC public data set (http://cancergenome.nih.gov/) and in a data set established in our laboratory for HBV-related HCC (access at Array Express with the codes E-MEXP-84 and E-TABM-292).15 Therefore, the synergistic antitumor effect observed for aspirin and sorafenib cannot be attributed solely to the upregulation of HTATIP2. In our recent publication,12 we demonstrated that the synergism observed with combining sorafenib and aspirin was mediated by the repression and activation of specific apoptosis-related genes. We have demonstrated that by combining low-dose sorafenib and aspirin, the synergistic antitumor effects observed are related to the simultaneously silencing of long-chain-fatty-acid CoA ligase 4 (ACSL4) and the induction of growth arrest and DNA damage inducible beta (GADD45B) expression. Furthermore, clinical evidence has independently corroborated that survival of HCC patients expressing ACSL4highGADD45Blow was significantly poorer compared to patients with ACSL4lowGADD45Bhigh expression, thus demonstrating the potential clinical value of combining aspirin and sorafenib to treat HCC patients expressing ACSL4highGADD45Blow, offering a clear strategy of evidence-driven precision medicine in HCC. In conclusion, aspirin has emerged as a promising chemopreventive and chemotherapeutic agent for HCC.As appropriate patient selection has been implicated to contribute significantly for achieving clinically meaningful results in cancer drug discovery and development, our recent publication addresses the mechanistic aspects of the synergistic effects of aspirin and sorafenib in the combination therapy for HCC.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The ischemic penumbra assessment is essential for the subsequent therapy and prediction of evolution in patients with acute ischemic infraction. Although controversial as a perfect equivalence to penumbra, perfusion-weighted imaging (PWI)-diffusion-weighted imaging (DWI) mismatch may predict the response to thrombolysis. Due to the reliance of PWI on contrast agents, noninvasive alternatives remain an unmet need.We reported a 65-year-old man complained of paroxysmal hemiplegia of his right limbs and anepia for 2 days, whereas the symptoms lasted for about 12 hours when he admitted to the hospital.We diagnosed it as acute ischemic stroke caused by the left middle cerebral artery stenosis.Susceptibility-weighted imaging (SWI), multimodal magnetic resonance imaging (MRI) work-up which includes conventional MRI sequences , DWI, PWI.His DWI-SWI mismatch was comparable to that of DWI-PWI at admission, suggesting that DWI-SWI could predict ischemic penumbra in patient with acute infarction. He refused the digital subtraction angiography examination or stenting, and he was treated with aspirin, atorvastain, and supportive treatment. The patient received a reexamination of the conventional MRI and SWI 11 days later. Expansion of the infarction in the affected MCA territory resulted from the penumbra indicated by the mismatch between DWI-SWI.SWI can be used as a noninvasive alternative to evaluate the ischemic penumbra. Besides, SWI can provide perfusion information comparable to PWI and SWI is sufficient to identify occlusive arteries. Assessment of ischemic penumbra is essential for initiating thrombolytic therapy and for predicting evolution or deterioration in patients with acute ischemic infraction. Although controversial as a perfect equivalence to penumbra, perfusion-weighted imaging (PWI)-diffusion-weighted imaging (DWI) mismatch is considered to predict the response to thrombolysis. Due to the reliance on contrast agents in PWI, noninvasive alternatives remain an unmet need. SWI is capable of providing additional clinical information complementary to conventional magnetic resonance imaging (MRI) sequences. SWI has been extensively used in the evaluation of various neurological disorders, including traumatic brain injury, neoplasms, intracranial calcification, vascular malformations cerebral venous thrombosis, spontaneous hemorrhagic transformation or postthrombolytic hemorrhagic transformation, intraarterial clot, microbleeds or iron deposition associated with neurodegenerative disorders, etc.,2 In addition, due to its ability to mirror the pathophysiology of the acute ischemic stroke, SWI has recently attracted attention in the identification of penumbra, as well as in the prediction of the evolution or the deterioration of acute ischemic infarction.–4In the hypoperfused brain tissue, for example, the increased oxygen extraction fraction via the metabolic compensation mechanism results in the elevation of the ratio of deoxyhemoglobin to oxyhemoglobin of the venous system of the brain, which contributes to the magnetic susceptibility difference between veins and the surrounding ischemic brain tissue. have found that the increased susceptibility due to the elevated deoxyhemoglobin-to-oxyhemoglobin ratio led to the visualization of prominent vein over the affected cerebral hemisphere on SWI. Interestingly, the SWI-DWI and the mean transit time (MTT)-DWI mismatches were found to be associated with deterioration of acute ischemic stroke. In this regard, SWI might illustrate misery perfusion with a compensatory increase of the oxygen extraction, and SWI is potentially a noninvasive alternative to PWI in predicting penumbra. We herein report a case in which SWI was used to evaluate penumbra.Kesavadas et al2Written informed consent was gained from the patient for the publication of the case report and accompanying images. The ethical approval of this study was waived by the ethics committee of The First Hospital of Jilin University because it was a case report.3A 65-year-old man complained of paroxysmal hemiplegia of his right limbs and anepia for 2 days, whereas the symptoms lasted for about 12 hours when he admitted to the Department of Neurology in the First Hospital of Jilin University. MRI was performed with a Siemens 3T Trio Tim scanner  by using a standard head coil. The following MRI sequences were obtained in the axial plane with the following parameters: spin echo (SE) T1WI ; fast spin echo (FSE) T2WI ; FSE inversion recovery (IR) FLAIR: ; DWI ; MRA ; PWI ; and SWI . All these sequences were done with a slice thickness of 6 mm, unless otherwise specified, a gap of 1.2 mm, field of view (FOV) of 256 mm × 256 mm, incentive number of 1. No obvious abnormalities were revealed on T1WI  for the support.


Although the precise origins of kidney macrophages remain controversial, recent studies have revealed that embryonic macrophage progenitors initially migrate from the yolk sac, and later from the fetal liver, into the developing kidney. Once in the kidney, tissue-specific transcriptional regulators specify macrophage progenitors into dedicated kidney macrophages. Studies suggest that kidney macrophages facilitate many processes during renal organogenesis, such as branching morphogenesis and the clearance of cellular debris; however, little is known about how the origins and specification of kidney macrophages dictate their function. Here, we review significant new findings about the origins, specification, and developmental functions of kidney macrophages.The adult kidney hosts tissue-resident macrophages that can cause, prevent, and/or repair renal damage. Most of these macrophages derive from embryonic progenitors that colonize the kidney during its development and proliferate  Macrophages are specialized  phagocytic cells that facilitate wide-ranging processes in diverse species. As well as their roles in host immunity and inflammation, macrophages are important in processes such as limb regeneration in salamanders , before the embryonic circulation is established  are generated by hemogenic endothelium, primarily in the dorsal aorta in the aorta-gonad-mesonephros region  and (2) the engraftment of adult circulating macrophage progenitors  fetal-generated macrophages that self-renew  fetal-geFrom the start of kidney development to the end of life, kidney-resident macrophages derive from all the progenitor waves described above: early yolk sac EMP-derived macrophages  kidney begins to develop at ~E10.5, when the ureteric bud (the precursor of the collecting duct and ureter) emerges from the caudal end of the Wolffian/nephric duct in response to glial cell-line-derived neurotrophic factor . At this stage, no monocytes (CD45+CD11bhiF4/80loLy6C+ cells) are present within the kidney , the percentage of yolk sac-derived macrophages in the kidney was found to decrease exponentially from E13.5 to postnatal week 6  to show that yolk sac-derived macrophages make a minimal contribution to the pool of kidney macrophages by postnatal week 10. As yolk sac-derived macrophages are highly proliferative , and do not exhibit high rates of apoptosis , it has been speculated that this rapid decrease in the proportion of yolk sac-derived macrophages must be explained by their dilution by the marked later arrival of fetal monocytes  can be classed as HSC-derived, whereas those that do not go through a Flt3+ stage (Flt3Cre/GFP−) can be said to be HSC-independent. Epelman et al.  and availability .A recent hypothesis proposed to explain the differences in macrophage origins between tissues is the niche competition model, as proposed by Guilliams and Scott . In thisaccessible to macrophage precursors via the systemic circulation throughout most of embryonic development , the receptor for chemokine Cx3c motif ligand 1 (Cx3cl1), is important for macrophage migration and colonization of the early embryo . Conversely, kidneys of Cx3cr1−/− embryos have greatly diminished macrophage numbers at E14.5 and in adulthood . In Cx3cr1-deficient mice, this fungus was cleared from all organs tested apart from the kidney. Consequently, Cx3cr1−/− mice uniformly succumbed to infection due to uncontrolled fungal proliferation in the kidney and renal failure , kidney macrophages exhibit increased expression of the transcriptional regulators aryl hydrocarbon receptor (Ahr), nuclear factor of activated T cells 1 and 2 (Nfatc1 and Nfatc2), and interferon regulatory factor 9 (Irf9)  accelerates growth and ureteric bud branching in cultured kidney explants , are providing insights. In D. melanogaster, the Malpighian tubules perform the function of the kidney and hemocytes are analogous to macrophages. Hemocytes deposit type IV collagen around the developing renal tubules, which sensitizes tubular cells to the BMP ligand, Decapentaplegic , it is difficult to relate macrophage functions in other developing mammalian organs with the creation of new nephrons. Here, some invertebrate species, such as the fruit fly . Macrophages facilitate endothelial anastomosis in development (Fantin et al., Moreover, macrophages are sensitive to low oxygen levels, and can promote angiogenesis in response to hypoxia (Cattin et al., + macrophages are reportedly pro-angiogenic and pro-lymphangiogenic (Cho et al., + lymphatic endothelia are closely associated with Lyve-1+ macrophages in the developing kidney, and they speculated that the latter facilitate lymphatic development during renal organogenesis. This idea is supported by evidence that macrophages can express VEGF-C to stimulate lymphangiogenesis (Kerjaschki, A subset of macrophages in the developing kidney express lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1; Lee et al., Although it seems likely that kidney macrophages will facilitate the processes described above, more studies are undoubtedly required before we fully appreciate the functional requirements of macrophages during renal development Figure .in situ throughout development and adulthood. Kidney macrophages are endowed with a unique genetic programme that allows them to promote normal renal organogenesis and to maintain the health and function of the adult organ. It will be extremely challenging to fully characterize the factors involved in kidney macrophage specification, and to understand how this dictates the functions of a given macrophage. Nevertheless, this is a challenge that should be faced, as it may expose new therapeutic opportunities to prevent and treat a range of developmental and pathological conditions.In many organs, including the kidney, tissue-resident macrophages are predominantly derived from embryonic macrophages. In the kidney, these embryonic macrophages are mainly generated from fetal monocytes, which are specified and self-renew To better understand kidney macrophage specification, future studies should utilize single-cell transcriptomic technologies to spatiotemporally classify the phenotypes of distinct macrophage populations in the developing kidney. By identifying clusters of macrophages with distinct gene expression patterns, and determining where they localize in the kidney, it may be possible to link environment-specific signals to the phenotypes and functions of a macrophage.As macrophages can promote kidney growth, they could potentially be used therapeutically to assist renal development in babies at risk of preterm birth. Increasing macrophage recruitment to the developing kidney through treatment with chemokines such as Csf-1 and/or Cx3cr1 could conceivably promote kidney growth and nephron endowment. However, macrophages can also promote abnormalities in kidney development, such as cyst formation (Karihaloo et al., The inflammatory properties of kidney macrophages and recruited circulating monocytes are implicated in the initiation and progression of injury and scarring in the adult kidney as well as kidney regeneration and healing (reviewed in Rogers et al., DM conceptualized the review, wrote the manuscript, and prepared the figures. JH reviewed and 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.


A master promoter of cell growth, mammalian target of rapamycin (mTOR) is upregulated in a large percentage of cancer cells. Still, targeting mTOR using rapamycin has a limited outcome in patients. Our recent results highlight the additional role of mTOR as a tumor suppressor, explaining these modest results in the clinic. The serine/threonine kinase mammalian target of rapamycin (mTOR) is a central regulator of mammalian cell growth. mTOR forms 2 complexes, termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). While both complexes are stimulated by growth factors, only mTORC1 can be activated by amino acids  due to their abnormal vasculature, and their general avidity to consume glutamine, tumors might constitute favorable microenvironments to induce glutamoptosis. In these conditions, the inhibition of mTORC1 during cancer therapy would prevent tumor growth, but at the same time would provide with an opportunity to cancer cells to avoid cell death. In other words, rapamycin treatment will result in a merely cytostatic effect, sustaining the survival of tumor cells. Upon treatment discontinuation, tumors will resume their growth. As a result, cancer progression will be only delayed during the period of rapamycin treatment or until the acquisition of rapamycin resistance mechanism by the cytostatic tumor cell.These tumor suppressor functions of mTORC1 highlight the complexity of the action mechanisms of central cell growth regulators, such as mTOR, and how microenvironmental cues influence their function . The assin vivo (Finally, our results open a new opportunity to explore the translational involvement of glutamoptosis for cancer therapy. In other words, can we induce glutamoptosis in tumors to specifically kill cancer cells? Microenvironments with a high concentration glutamine, or tumor types with a particular avidity for glutamine, might be particularly sensitive to glutamoptosis. Simulating glutaminolysis by artificially increasing the intracellular levels of αKG is known to induce tumor cell death In conclusion, the tumor suppressor function of mTORC1 during nutritional imbalance points at the necessity of finding alternatives to improve the outcome of mTORC1 inhibition in the clinics and questions the pertinence of the use of rapamycin as monotherapy in cancer patients.


Standardised MedDRA Queries (SMQs) have been developed since the early 2000’s and used by academia, industry, public health, and government sectors for detecting safety signals in adverse event safety databases. The purpose of the present study is to characterize how SMQs are used and the impact in safety analyses for New Drug Application (NDA) and Biologics License Application (BLA) submissions to the United States Food and Drug Administration (USFDA).We used the PharmaPendium database to capture SMQ use in Summary Basis of Approvals (SBoAs) of drugs and biologics approved by the USFDA. Characteristics of the drugs and the SMQ use were employed to evaluate the role of SMQ safety analyses in regulatory decisions and the veracity of signals they revealed.A comprehensive search of the SBoAs yielded 184 regulatory submissions approved from 2006 to 2015. Search strategies more frequently utilized restrictive searches with “narrow terms” to enhance specificity over strategies using “broad terms” to increase sensitivity, while some involved modification of search terms. A majority (59%) of 1290 searches used descriptive statistics, however inferential statistics were utilized in 35% of them. Commentary from reviewers and supervisory staff suggested that a small, yet notable percentage (18%) of 1290 searches supported regulatory decisions. The searches with regulatory impact were found in 73 submissions (40% of the submissions investigated). Most searches (75% of 227 searches) with regulatory implications described how the searches were confirmed, indicating prudence in the decision-making process.SMQs have an increasing role in the presentation and review of safety analysis for NDAs/BLAs and their regulatory reviews. This study suggests that SMQs are best used for screening process, with descriptive statistics, description of SMQ modifications, and systematic verification of cases which is crucial for drawing regulatory conclusions. The approval of a new drug is based on a balance of the substantial evidence of efficacy and its safety , 2. For SMQs are similar to a net that enables retrieval of cases of interest from MedDRA-coded databases , 7, 11. As development and use of SMQs have become increasingly important for safety analyses, a comprehensive evaluation into how SMQs are used and their impact is imperative. The objectives of this study are to characterize patterns in SMQ use and impact in the generation of regulatory decisions in the review of NDA and BLA submissions to the United States Food and Drug Administration (USFDA). Although the performance of SMQs has been discussed in previous research –18, the ® (Elsevier) to search  for USFDA Summary Basis of Approvals (SBoAs) in which use of SMQs was described. Publicly available reviews from each identified submission were extracted from the Drugs@FDA database of USFDA approved drug products [We used PharmaPendiumproducts . Documen® 2013 for information retrieved from SBoAs, which was established by the authors and verified by the corresponding author. The data included “demographic” information related to the drug and marketing application and “content” information related to the SMQ and how it was used (® version 12.1.0 (SAS Institute Inc.). Percentages have been rounded to the nearest integer. Each supplement was considered as an independent submission.A database was constructed in Microsoft EXCELwas used . DescripIn the present study, 184 submissions approved from 2006 to 2015 mentioned the use of SMQs. A total of 1350 SMQ investigations , includiThe top three therapeutic classes of therapeutics investigated with SMQs as represented by the second level of the Anatomical Therapeutic Chemical (ATC) classification system  were antThe distribution of searches initiated by the USFDA and the applicants by year of the NDA/BLA approval is displayed in Hepatic disorders (SMQ) (166 (12%)), Cardiac arrhythmias (SMQ) (100 (7%)), Ischaemic heart disease (SMQ) (62 (5%)), Central nervous system vascular disorders (SMQ) (61 (5%)), and Severe cutaneous adverse reactions (SMQ) (51 (4%)), if each sub-SMQ was incorporated into the respective first-level SMQs. These findings demonstrated the most common safety concerns, including hepatic toxicity, cardiac toxicity, central nervous system toxicity, and hypersensitivity during evaluation of marketing applications using SMQs. Moreover, the distribution of the top three most frequently searched SMQs for the top three therapeutic classes represented by the second level of the ATC classification system is shown in Drug related hepatic disorders—comprehensive search (SMQ) (41 searches (5%)), followed by Severe cutaneous adverse reactions (SMQ) (37 searches (4%)). In contrast, the most frequently searched SMQ/sub-SMQ initiated by the applicants was Anaphylactic reaction (SMQ) (24 searches (5%)), followed by Cardiac failure (SMQ) (19 searches (4%)), demonstrating the unique concerns from different perspectives. Interestingly, however, among the 55 searches described in the post-approval supplements, Embolic and thrombotic events (SMQ) (8 searches (15%)) became the most frequently-mentioned SMQs, implying different emphasis in the post-approval stage.85 first-level SMQs and 86 secondary or greater-level sub-SMQs were detected in these cases. Among the 1350 searches, the top five most frequently searched SMQs consisted of A search with narrow or broad scope terms could lead to different results emphasizing either specificity or sensitivity, while an algorithmic search utilizing the combinations of term categories is generally used in large databases to increase specificity. Although not all options are available for each SMQ, narrow searches were performed more frequently when both narrow and broad terms were available. Among 728 SMQ searches using first-level and non-hierarchical SMQs with both options, 39% of searches utilized only narrow terms, while 9% of them applied only broad terms. Fourteen percent of the searches compared the search results using both options while search options regarding the use of narrow or broad terms were not clearly specified in the rest of the searches. Although a potential 73 searches (5%) could have utilized algorithmic SMQs, only 21% of these searches reported applying the option. The relative timing of events in different term categories for algorithmic SMQs was reported in only 2 searches applying this search option.Cardiac arrhythmias (SMQ) (8 searches) and Anaphylactic reaction (SMQ) (7 searches).Five percent of total searches involved modification of terms (addition of terms: 38 searches (53%); exclusion of terms: 31 searches (43%); addition and exclusion of terms: 3 searches (4%)) which, by convention, should not be referred to as SMQ searches due to their modification , 10, 11.Drug related hepatic disorders—comprehensive search (SMQ) was most often corroborated with clinical laboratory results. In contrast, Torsade de pointes/QT prolongation (SMQ) cases were generally verified with electrocardiogram interval data. The approaches used for the top five most frequently searched SMQs are displayed in Among 1188 searches in which cases were detected for the target drug(s), verification of cases retrieved was mentioned for 470 searches (40%). “Direct verification” was performed by the investigation of AEs by e.g., studying patient narratives, checking laboratory results or vital signs. Comparison of results with other evidence, such as the report on higher MedDRA levels of AEs was an alternative approach described for 518 searches (44%) in order to help substantiate the legitimacy of safety signals. For example, the search results with MedDRA High Level Terms were often analyzed in juxtaposition to SMQ searches to assess the similarity trends in the incidence of the two datasets. The utilization of at least one approach was described in 737 (62%) searches, while application of both confirmation methods was mentioned in 251 (21%) searches. The type of data used for verification of SMQ-positive cases outside of an examination of the AE PTs depended on respective safety issues. For example, Among 1290 searches performed by either the USFDA or the applicants (excluding requests not performed), most results (763 searches (59%)) were reported using descriptive statistics. For 454 (35%) searches that included inferential statistics in their presentation of results (whether results were found or not), 39% reported a combination of the relative risk, the number of cases, and incidence, with or without a p-value; confidence intervals were reported in 98 (22%) searches. Among the 442 searches where cases were found for the target drug(s) and that used inferential statistics, direct verification of cases was mentioned for 97 (22%) searches.Severe cutaneous adverse reactions (SMQ), Anaphylactic reaction (SMQ), Angioedema (SMQ), which were often used together for the investigations of hypersensitivity were more often likely to have impact on regulatory decisions for labeling. If each sub-SMQ was not calculated separately, the SMQ Hepatic disorders was most likely to have impact on regulatory decisions for labeling (27 searches). Besides, searches with the SMQ Ischaemic heart disease were most often associated with postmarketing requirements (12 searches). Those resulting in labeling decisions most often consisted of the submissions containing antineoplastic agents (18%), based on ATC2 level classification system and those associated with postmarketing requirements most often contained the submissions of immunosuppressants (34%). Direct verification of cases retrieved and/or comparison of search results with other evidence was mentioned in 75% of the searches that seemed to have impact on regulatory actions.In addition to a summary of search results in the SBoAs, 227 searches (18% of searches performed) in 73 submissions (40% of the submissions investigated) seemed to have had a direct impact on regulatory decisions typically associated with labeling and/or postmarketing requirements . The disOur analyses suggest that SMQs are increasingly being incorporated into safety analyses from industry and the USFDA. From 2006 to 2015, the reporting of SMQ analyses has increased in frequency by the FDA and industry, alike. Requests to perform searches were frequently found in the administrative correspondence or meeting minutes with advice for or discussion of SMQ selection, recommended search options, as well as database(s) to be searched. Senior FDA reviewers, including those at the supervisory level, frequently comment on SMQ analyses found in primary reviews. While the SMQ was used, SMQ analyses and their results have clearly become an integral part of pharmaceutical safety assessment. We have comprehensively evaluated the patterns of their usage, efforts to validate their results, and their impact on the regulatory process. From these assessments, we emphasize several tenets that are found in, or derived from, the core documents describing use of SMQs  and the Use of SMQs as a tool to screen for safety signals, rather than one that produces results upon which a diagnosis may be madeUse of descriptive rather than inferential statistics to describe findings from SMQ analyses of clinical trials not designed for hypothesis testingMore regularity in the documentation of SMQ modificationSystematic verification of cases when comparisons are drawn between treatment groups or when important regulatory actions are based on the findingsSeveral properties of SMQs suggest their use is more appropriate for AE screening, than for conclusions that amount to a diagnosis or sophisticated statistical analysis. Collection of AE data, upon which the analyses are based, is a system not suited to quantitative analysis of or diagnosis based upon AEs without careful follow-up for those situations requiring further attention. Subjects make occasional visits to study sites, often after lengthy intervals of time, sometimes making the quality of information imprecise. Furthermore, the efforts needed to confirm potentially hundreds of SMQ-positive cases with trial data would be overwhelming to match the precision suggested by the level of statistical analysis. We therefore suggest that SMQs are best used in the screening process of data inspection. Descriptions of these data are most appropriately done with descriptive statistics than inferential testing, since the trials or post-marketing scenarios from which the AEs are derived are not optimally designed to collect these data. The long history of safety evaluators using “designated medical event” or “adverse events of special interest” term lists seemingly emboldens those using SMQs to adapt them on a case-by-case basis. A notable subset of searches applies a modified SMQ by term addition and/or exclusion, indicating an attempt at increasing the specificity or sensitivity. The MedDRA Maintenance and Support Services Organization in the SMQ introductory guide anticipates such alterations of SMQs .“If any modifications are made to term content or structure of an SMQ by a subscriber or user, it can no longer be called an ‘SMQ’ but it should instead be referred to as a ‘modified MedDRA query based on an SMQ’…”Acute pancreatitis (SMQ). Though a match with the AE database only requires a match on a single term, such as nausea [Reporting these results may become very confusing as investigators with different self-adapted versions of SMQs attempt to compare results. Verification or checking each case deemed “SMQ-positive” is a critical step in their use. Much like the allegorical fishing net described in our introduction, the broad web of search terms often ensnares cases based on terms that are non-specific but useful to ensure sensitivity in the setting of adequate corroboration. A clear example of this is s nausea , this isDemyelination (SMQ) has also been pointed out although only four SMQs were investigated in the study [Despite our comprehensive attempt at characterizing the regulatory use of SMQs, this study has limitations related primarily to the extent to which marketing applications are described in publicly available SBoAs. For example, because not all SMQ version numbers are available in review documents, it is difficult to determine which search options were available at the time of the submission. The percentage of directly verified cases or comparison with other evidence might be underestimated since every verification or comparison may not be mentioned in the application or review documentation. However, to the extent that information is publicly available, our research has systematically reviewed the use and influence of SMQs demonstrating their prevalence, usefulness, and factors critical for their successful application in safety analyses, which has not been comprehensively discussed before. Previous research focused more on the performance of SMQs –18. Nonehe study .In conclusion, the present study demonstrates increasing use of SMQs in reviews of marketing applications, indicating the practicality of SMQs considered by the applicants and USFDA. SMQ search strategy depended on case-by-case considerations; however, the prevalent use of certain SMQs revealed the most common safety concerns evaluated by using SMQs. These concerns included hepatic toxicity, cardiac toxicity, central nervous system toxicity, and hypersensitivity. The more frequent use of narrow searches, when both narrow and broad terms were available, implied efforts made to enhance specificity. Although direct verification of cases retrieved was not routinely performed, SMQ searches associated with regulatory decisions were usually confirmed by direct verification of cases retrieved and/or comparison of search results with other evidence. In addition, the impact on labeling and postmarketing requirements demonstrates the utility of SMQ use. The study also presents critical suggestions for successful SMQ use as mentioned above. With appropriate choice of SMQs and search options, proper statistical analysis and thorough documentation, as well as verification for cases retrieved, the SMQ is a well-recognized tool with increasing importance for the analysis of safety profiles, which could be effectively used in making regulatory decisions. Considering their increased usage, the future trends in SMQ utilization and their regulatory implications are important issues worthy of evaluation.


Lysosomal storage diseases (LSDs) describe a heterogeneous group of rare inherited metabolic disorders that result from the absence or loss of function of lysosomal hydrolases or transporters, resulting in the progressive accumulation of undigested material in lysosomes. The accumulation of substances affects the function of lysosomes and other organelles, resulting in secondary alterations such as impairment of autophagy, mitochondrial dysfunction, inflammation and apoptosis. LSDs frequently involve the central nervous system (CNS), where neuronal dysfunction or loss results in progressive neurodegeneration and premature death. Many LSDs exhibit signs of mitochondrial dysfunction, which include mitochondrial morphological changes, decreased mitochondrial membrane potential (ΔΨm), diminished ATP production and increased generation of reactive oxygen species (ROS). Furthermore, reduced autophagic flux may lead to the persistence of dysfunctional mitochondria. Gaucher disease (GD), the LSD with the highest prevalence, is caused by mutations in the GBA1 gene that results in defective and insufficient activity of the enzyme β-glucocerebrosidase (GCase). Decreased catalytic activity and/or instability of GCase leads to accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph) in the lysosomes of macrophage cells and visceral organs. Mitochondrial dysfunction has been reported to occur in numerous cellular and mouse models of GD. The aim of this manuscript is to review the current knowledge and implications of mitochondrial dysfunction in LSDs. Mitochondria are double-membrane organelles that are found in most eukaryotic cells and that execute many metabolic functions including ATP synthesis through oxidative phosphorylation (OXPHOS) . MitochoGiven their central role in cellular homeostasis, mitochondrial dysfunction has been linked to many age-related disorders including mitochondrial diseases, cancers, metabolic diseases and diabetes, inflammatory conditions, neuropathy, and neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s disease ,5. CurreLSDs are a group consisting of over 50 disorders, characterized by the progressive accumulation of undigested macromolecules in lysosomes. The massive accumulation of substances affects the function of lysosomes and impairs autophagic flux which may affect the cellular quality control of organelles such as mitochondria .Lysosomes are the primary disposal and recycling center of cells. Thus, lysosomes are involved in the degradation and recycling of extracellular material (via endocytosis) and intracellular material (via autophagy) ,12.Autophagy is an essential cellular system that consists of the degradation of cytoplasmic components within lysosomes. Autophagy pathways represent a major defense mechanism that, in addition to their role in providing cells with nutrients, also allow cell survival in response to multiple stressors.Three general types of autophagy have been described—macroautophagy, microautophagy, and chaperone-mediated autophagy—which differ in their regulation and selectivity . MacroauLysosomes are essential for autophagy, and autophagic clearance of dysfunctional mitochondria represents an important element of mitochondrial quality control . It is eOnce the mitochondrial damage becomes too extreme and causes dissipation of mitochondrial membrane potential (ΔΨm), damaged parts of mitochondria cannot fuse with the adjacent healthy mitochondria . The segIn LSDs, both lysosomal dysfunction and reduction of autophagic flux have a major impact on mitochondrial function. The dysregulation of autophagic flux may reside at the stage of autophagosome clearance, the final step in the autophagy process in which the lysosome plays a pivotal role. A significant number of damaged mitochondria and autophagosomes remained in the cytosol, suggesting inefficient and incomplete autophagic flux.Alternatively, lysosomal enzyme deficiency may translate into mitochondrial dysfunction by cellular lipid trafficking alterations ,22. In hHerein, we will review the current information regarding mitochondrial dysfunction in several LSDs, with particular interest in data derived from various cellular and animal models in Gaucher disease (GD).Pompe disease—a lysosomal glycogen storage disease caused by deficiency of acid alpha-glucosidase—is characterized by glycogen accumulation in muscle and peripheral nerves. Recently, the contribution of mitochondrial dysfunction in the pathophysiology of Pompe disease has been recognized . MultiplMSD, is a rare LSD caused by mutations in the SUMF1 gene, resulting in an accumulation of sulphatides, sulphated glycosaminoglycans, sphingolipids and steroid sulphates. Defects in both autophagy and mitophagy have been observed in the liver and brain of murine models of MSD ,28.Mitochondria in these models were found to be fragmented with reduced ΔΨm and ATP production, indicating a generalized mitochondrial dysfunction.N-acetyltransferase (HGSNAT). It has been proposed that HGSNAT deficiency and lysosomal accumulation of heparan sulfate in microglial cells followed by their activation and cytokine release result in mitochondrial dysfunction in the neurons in an animal model of the MPS IIIC -1,6-anhydro-l-idonojirimycin (NAdBT-AIJ), and more markedly by the combination of both treatments. These data suggest that targeting both mitochondria function by CoQ and protein misfolding by pharmacological chaperones can be promising therapies in neurological forms of GD. These findings support the hypothesis that secondary mitochondrial dysfunction by GlcCer accumulation aggravates the effects of the L444P/L444P mutation and therefore, the improvement of mitochondrial function may improve the activity of the mutant GCase [Mitochondrial dysfunction has also been reported in GD fibroblasts and GD mouse models. Dermal fibroblasts from three GD patients (L444P/L444P) showed reduced activity of complex I, II, III and II + III of the ETC . Mitochont GCase .On the other hand, persistent GlcCer accumulation in neurons and glial activation causes a continuous chronic inflammation that may contribute to neuronal cell death ,51. For Niemann–Pick type C (NPC) disease is an LSD caused by mutations in the NPC1 or NPC2 genes, leading to the accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes . It has Other studies have demonstrated that cholesterol content in mitochondrial membranes is significantly increased in NPC1 mouse brains and neural cells. In addition, the ΔΨm, the activity of ATP synthase, and the level of ATP are markedly decreased in NPC1 mouse brains and neurons . These rML type IV is a genetic lysosomal storage disease associated with degenerative processes in the brain, eye, and other tissues. ML type IV is linked to mutations in the ion channel, mucolipin 1 (MCOLN1)—a protein that plays a role in lysosomal/endosome function . SignifiCystinosis was the first lysosomal storage disease recognized to be due to defective lysosomal membrane transport, and it serves as a prototype for a small group of lysosomal transport disorders. The disease results in intracellular accumulation of cystine in all organs and tissues.Further studies demonstrated a significant decrease in mitochondrial ATP generation with increase ROS in cystinotic cells . ElectroThe neuronal ceroid lipofuscinoses, also known as Batten disease, are a group of LSDs that can arise from genetic mutations within 1 of 14 different genes . PatholoAlthough the biochemical defects seem to affect primarily lysosomes, the connection between this and neuronal death is not completely understood. The pathogenic mechanisms have been studied particularly in two animal models: the English setter dog and the New Zealand South Hampshire sheep (OCL6). In these models, and some of the human entities, there is evidence of mitochondrial dysfunction . This inGiven that defects in mitochondrial function and oxidative stress have been demonstrated to play a role in the pathogenesis of many LSDs, we envisioned that treatment with antioxidants and mitochondria energizers such as CoQ could exert beneficial therapeutic effects. The critical role of CoQ in mitochondrial bioenergetics and its well-known antioxidant properties represent the main rationale for its clinical use, although some of its effects may be related to a gene induction mechanism . InteresThe convergence of impaired autophagic flux and altered composition of cell membranes contributes to mitochondrial dysfunction in LSDs. The alteration of mitochondrial morphology and decreased respiratory chain activity varies in both extent and characteristics in tissues and isolated mitochondria among these diseases.We propose that lysosomal dysfunction interferes with the both the function and clearance of damaged mitochondria and that these critical pathways converge in the pathogenesis in LSDs.Finally, numerous observations in both cellular and animal models of GD suggest that mitochondrial alterations might be involved in the pathophysiology of this lysosomal disorder and may aggravate its clinical presentation.


Platanista gangetica), in which they rise dorsally as delicate, pneumatized wings anterior of the facial bones. Their position wrapping around the melon suggests their involvement in sound propagation for echolocation. To better understand the origin of crests in this lineage, we examined facial crests among fossil and living Platanistoidea, including a new taxon, Dilophodelphis fordycei, nov. gen. and sp., described herein, from the Early Miocene Astoria Formation of Oregon, USA. We measured the physical extent and thickness of platanistoid crests, categorized their relative position and used computed tomography scans to examine their internal morphology and relative bone density. Integrating these traits in a phylogenetic context, we determined that the onset of crest elaboration or enlargement and the evolution of crest pneumatization among the platanistoids were separate events, with crest enlargement beginning in the Oligocene. However, we find no evidence for pneumatization until possibly the Early Miocene, although certainly by the Middle Miocene. Such an evolutionary context, including data from the fossil record, should inform modelling efforts that seek to understand the diversity of sound generation morphology in Odontoceti.Many odontocete groups have developed enlarged facial crests, although these crests differ in topography, composition and function. The most elaborate crests occur in the South Asian river dolphin ( Dilophodelphis differs from waipatiids and squalodelphinids in having a deeply grooved lateral rostral suture between the premaxilla and the maxilla (character 3[1]); in having a small tooth diameter as a percentage of postorbital skull width (character 45[2]); in having the robust and globose/rectilinear antorbital processes of the maxilla in dorsal view (character 46[1]) and in having the ventral edge of the zygomatic process almost straight in lateral view (character 53[1]). Dilophodelphis further differs from waipatiids and allodelphinids by having an elevated antorbital region distinctly higher than the dorsal margin of the rostrum base in lateral view (character 7[1]); having a deep fossa in the frontal on the orbit roof at the level of the frontal groove (character 12[1]) and having a circle-shaped dorsal outline of the zygomatic process of the squamosal in lateral view (character 18[1]). Dilophodelphis differs from waipatiids and other platanistids in having a square-shaped pars cochlearis of the periotic (character 20[1]).Dilophodelphis also differs from the Waipatiidae in having a deep, ‘V’-shaped antorbital notch in relation to an anteriorly pointed antorbital process (character 6[1]); having a cranium distinctly shorter than wide with a ratio <0.90 (character 10[1]); wide hamular fossa of the pterygoid sinus, extending anteriorly onto the palatal surface of the rostrum (character 16[1]); transverse thickening of the anterior process of the periotic (character 23[1]) and in having a lateral lamina of the palatine (character 54[1]).Dilophodelphis differs from the Allodelphinidae in lacking an elongated rostrum (character 1[0]), having wide premaxillae at the rostrum base (character 4[1]), having the dorsal opening of the mesorostral groove anterior to the rostrum base narrower than the premaxilla (character 5[0]), having a concave lateral margin of the rostrum anterior to the maxillary flange (character 41[0]), having a deep posterolateral sulcus (character 47[0]), having a shallow premaxillary cleft (character 50[2]) and having the ventral surface of the posterior process of the periotic concave along a straight path perpendicular to its long axis (character 66[1]).Dilophodelphis belongs to the Platanistidae based on the following: medial margin of the antorbital notch made of a thin plate (character 38[1]), and dorsal surface of the vertex markedly transversely and longitudinally convex (character 39[1]). Dilophodelphis also shares the following characteristics with all the platanistids, excluding Araeodelphis: lateral rostral suture between premaxilla and maxilla deeply grooved (character 3[1]), distinct dorsal crest in the antorbital–supraorbital region (character 8[1]), antorbital process of maxilla robust and globose or rectilinear in dorsal view (character 46[1]), and the presence of a shallow premaxillary cleft (character 50[2]).Dilophodelphis differs from all other known platanistids in having an enlarged tympanosquamosal recess forming a large triangular fossa medial and anteromedial to the postglenoid process (character 55[2]). Dilophodelphis demonstrates one clear apomorphy: a deep emargination of the posterior edge of the zygomatic process by the sternomastoid fossa, in lateral view (character 52[2]). Further features that distinguish Dilophodelphis from other platanistids are discussed in the ‘Morphological comparisons’ section of the discussion. Finally, Dilophodelphis demonstrates the following possibly plesiomorphic character states : deep, ‘V’-shaped antorbital notch in relation to an anteriorly pointed antorbital process (character 6[1]); semicircular dorsal outline of the zygomatic process of the squamosal in lateral view (character 18[1]); pars cochlearis of the periotic square-shaped in ventral view (character 20[1]).3.1.2.Simocetus rayi Fordyce 2002 ; the presence of suprameatal pit of squamosal [character 57]; the presence of posterior periotic fossa of squamosal [character 59]; lateral groove or depression affecting profile of periotic as viewed dorsally [character 60(1)]; anteroposterior ridge on dorsal side [character 61(1)]; ventral surface of posterior process of periotic concave or convex along straight path perpendicular to its long axis [character 66].Our analysis supports the concept of a monophyletic Platanistoidea, which includes the living Dilophodelphis. Pneumatization first appears in the pomatodelphinines in the Early Miocene, with the appearance of Zarhachis. Therefore, the evolution of pneumatization is de-coupled from the enlargement of the supraorbital crests among the platanistoids.By mapping both the presence or absence of crest pneumatization and the relative size of the crests onto our strict consensus phylogenetic tree, we were able to visualize these traits in an evolutionary context . Our ana4.4.Dilophodelphis raises several questions about the function of supraorbital crests in platanistoids. Our only extant model for the function of supraorbital crests in platanistoids is the South Asian river dolphin (Platanista gangetica), whose prominent, concave crests are hypothesized to serve a role in echolocation because they support maxillary sinuses that lie dorsal to the melon, though experimental determination remains hard to achieve. In other odontocetes, sound produced by the phonic lips is reflected or absorbed by either the air sac system surrounding the nasal passages, or the crests themselves, producing a more anteriorly projected acoustic signal [Platanista, Pilleri [A priori we would expect that crests used to reflect sound anteriorly would have a highly concave medial surface  and that they would be denser . The density and morphology of the crests in Platanista certainly meet these requirements. However, the internal surface of the crests of Platanista are highly convoluted, which would prevent reflection of sound and instead may allow for sound to be better absorbed by the bone. Because of this configuration, we infer that if Platanista reflects sound anteriorly using its crests, it is probably doing so using the sinuses lining the crests rather than the bone itself. The acoustic mismatch between soft tissue and air is much lower than that between air and compact bone though, which logically would be a less effective method of sound reflection. More basic anatomical work on this highly endangered taxon is necessary to refine and ground truth these hypotheses.The morphology of c signal ,81. For  Pilleri  referredDilophodelphis' crests differ substantially from those of Platanista. They are robust and both laterally and dorsally expanded. Their medial surface is convex, a characteristic that would make it difficult for these structures to focus sound in any certain direction. The density of the crests of Dilophodelphis is similar to that in other platanistoids. In all species included in our analysis, the crests were denser than the zygomatic process but of similar density to the maxillae within the rostrum. The crests of Dilophodelphis are slightly denser than those of Platanista and Pomatodelphis compared with the other regions of the skull. Unfortunately, this finding may not tell us much about possible variation in crest function in these taxa . However, it is hard to imagine a scenario where loads are applied to this region of the cranium. The long, slender rostrum would be more likely to engage with an object rather than the dorsal surface of the head. Third, the crests may act as display structures. Many species of odontocetes such as male beaked whales possess elaborate display structures that allow for intraspecific or interspecific signalling. It has even been hypothesized that odontocete species may be able to sense differences in internal morphology using echolocation [Dilophodelphis. The greatly enlarged surface area of the maxilla in the supraorbital region is known to serve as an attachment point for the highly modified maxillonasolabial muscles, which are involved in sound production [Dilophodelphis seem overdesigned for this purpose, the height of the structures could provide orientations for muscle fibres necessary to control the shape of the forehead elements [Platanista contributes to facial and rostral muscle attachments. Lastly, the crests may provide space for sinuses. Mammals have evolved, or through ontogeny, developed sinus systems for a wide range of functions including weight reduction, space for chamber resonance or thermoregulation [Dilophodelphis, but think that this hypothesis is worthy of consideration given the presence and importance of the sinus system in Platanista. Future studies could assess these hypotheses using a variety of techniques. Modelling approaches, such as finite-element analysis, offer a useful mechanism to test the structural, acoustic and thermoregulatory benefits of different crest morphologies. Further work on the function of soft tissues in the acoustic system of Platanista would also greatly benefit any interpretation of crest function in fossil taxa.There are multiple possible functions for the large crests of location . Howeveroduction . Theoretelements . To the gulation . Here weLACM: Departments of Mammalogy and Vertebrate Paleontology, Natural History Museum of Los Angeles County, Los Angeles, CA, USA.USNM: Departments of Paleobiology and Vertebrate Zoology , National Museum of Natural History, Smithsonian Institution, Washington, DC, USA.YPM: Division of Vertebrate Paleontology, Yale Peabody Museum, New Haven, CT, USA.