Such studies rely, in part, on analysis of the inorganic chemistry of preserved melanosomes and an understanding of melanosome chemical taphonomy. The extent to which the preserved chemistry of fossil melanosomes is biased by biotic and abiotic factors is, however, unknown. Here we report the discovery of hierarchical controls on the inorganic chemistry of melanosomes from fossil vertebrates from nine biotas. The chemical data are dominated by a strong biota-level signal, indicating that the primary taphonomic control is the diagenetic history of the host sediment. This extrinsic control is superimposed by a biological, tissue-level control; tissue-specific chemical variation is most likely to survive in fossils where the inorganic chemistry of preserved melanosomes is distinct from that of the host sediment. Comparative analysis of our data for fossil and modern amphibians reveals that most fossil specimens show tissue-specific melanosome chemistries that differ from those of extant analogues, strongly suggesting alteration of original melanosome chemistry. Collectively, these findings form a predictive tool for the identification of fossil deposits with well-preserved melanosomes amenable to studies of fossil colour and anatomy.Fossil melanosomes, micron-sized granules rich in melanin  Melanins have fundamental physiological roles, including coloration1, prevention of UV4 and oxidative5 damage and as a sink for metals1.Melanins are a family of chemically diverse pigments that are widespread in organisms from all kingdoms of life17. Arguments that preserved melanosomes are instead fossil bacteria19 are untenable given the abundant chemical evidence for degraded or intact melanin associated with melanosome-like microbodies in fossils23, because bacteria are not known to preserve as organic remains outside conservation media such as amber or chert, and given the lack of a taphonomic mechanism to explain such preservation. Recent developments in the study of fossil melanin and melanosomes have provided unprecedented insights into various aspects of animal biology and evolution, including the colour11, behaviour13, phylogenetic affinities15 and internal anatomy17 of ancient animals.It is well established that melanin and melanosomes can preserve within the integumentary tissues  of fossil vertebrates, often as organic remains24; this broad distribution in the fossil record provides an excellent opportunity to investigate the controls on melanin and melanosome taphonomy. Most studies have focussed on integumentary melanosomes, especially those from feathers, which can preserve as three-dimensionally preserved microbodies6 or as external moulds25. Recent experiments have shown that preservation of melanosomes as moulds can result from reaction with an oxidant, resulting in dissolution of melanosomes26. Other experimental studies using feather melanosomes have shown that elevated pressures and temperatures can result in melanosome shrinkage by ca. 10–20% of the original size28. Thermal maturation experiments have also been applied to studies of the molecular taphonomy of melanin; the observed increase in cross-linking among melanin molecular units is considered to enhance the preservation potential of melanin30. Another important taphonomic factor is the strong affinity of melanin for metals (reviewed in ref. 31). Incorporation of metals32 and other elements, e.g. S32, during fossilization can promote the preservation of melanosomes via enhanced molecular cross-linking23.Evidence of melanin and melanosomes has been reported from fossils that vary in taxonomy, age and geological setting34. Our pilot study of a limited number of fossil vertebrates17 showed that tissue-specific metal-melanosome associations are evident in some fossils. Such associations also characterise extant analogues and have been interpreted as evidence for functions of melanin in metal homeostasis17. Preservation of tissue-specific chemistries in fossils (which is difficult to explain as a wholly diagenetic phenomenon) therefore suggests that at least a component of original chemistry, and functions of melanin in metal regulation, may originate in deep time. Tissue-specific metal-melanosome associations have the potential to be a potent tool in palaeobiology for inferring fossil anatomy and the functional evolution of melanin, but only if biological and taphonomic components of the preserved chemical signature can be discriminated. Even where melanosomes from different tissues in fossil vertebrates are chemically distinct, the former typically show concentrations of metals  that are markedly higher than those in extant vertebrates35. This chemical enrichment has been attributed to an increase in the original concentrations of elements  via desiccation during fossilization10. Alternatively, elevated concentrations of elements in fossil melanosomes may reflect the incorporation of extrinsic elements (e.g. from pore fluids) during diagenesis17. These contrasting hypotheses have not been tested and thus the controls on the inorganic chemistry of fossil melanosomes are not fully resolved.The association between melanosomes and metals in extant animals is well knownHere, we test the fidelity of preservation of the inorganic chemistry of fossil melanosomes in the fossil record using 116 samples of melanosome-rich soft tissues (and associated sedimentary matrix) from 21 fossil vertebrates from nine biotas that range in age from the Early Permian to the Late Miocene .Detailed analysis of melanosome chemistry for individual fossil vertebrates reveals tissue-specific chemical signals in 14 specimens Table , S3, incEopelobates sp. from Orsberg, which shows no difference between melanosomes and the sedimentary matrix (see Supplementary Text). In some fossil specimens (see Supplementary Text), the inorganic chemistry of the skin melanosomes is more similar to that of the sediment than of other tissues; this phenomenon does not consistently apply to any particular species or vertebrate class in the dataset. Melanosomes from internal soft tissue regions are usually enriched in Zn and/or Cu , Rott, Saar-Nahe and one specimen from Monte San Giorgio show clear tissue-specific chemical differences in melanosomes. ANOVA and Tukey post hoc tests confirm that the chemical differences among all regions analysed are statistically significant for almost all elements for each fossil specimen . In contrast, fossil melanosomes from the skin and internal organs  overlap extensively in chemospace : Zn, Ca, Fe and K. Given that preserved melanosomes and sediment overlap in chemospace for most of the biotas studied , the observed broad-scale biota-level differences in melanosome chemistry almost certainly relate to broad differences in the chemistry of the host sediment among biotas. The primary controls on melanosome chemistry could therefore relate to chemical fingerprints that are generated in vivo. Studies of the relationship between diet and melanisation have produced conflicting results (discussed in ref. 36 and references therein); in particular, whether the dietary intake of macro- and micronutrients affects the tissue-specific inorganic chemistry of melanosomes is uncertain. Hence, we consider diagenetic overprinting to be the most plausible explanation for the enrichment in elements observed in some fossil vertebrates.Accurate interpretation of trace metal concentrations in fossil melanosomes requires an understanding of how melanosomes from different tissues respond to elevated concentrations of elements in pore fluids under different pH / Eh conditions, and of the original elemental concentrations In fossil vertebrates from Libros, Bohemia and Monte San Giorgio, preserved melanosomes are always separated in chemospace from their associated host sediment. These are also biotas for which melanosomes in most or all specimens exhibit tissue-specific chemical variation. Collectively, these data suggest that melanosomes that are more chemically distinct from the surrounding sediment are more likely to retain tissue-specific signals. Conversely, the clustering of the data for the other biotas suggests that melanosome chemistry converges during diagenesis, potentially due to common pathways for chemical transformation, especially metal enrichment, but this hypothesis should be tested experimentally.ca. 3 µm, versus 11–25 µm for the layer of non-integumentary melanosomes37, Supplementary Fig. Our data for melanosome-rich soft tissues from individual fossil specimens reveal broad trends in tissue chemistry relative to the host sediment: melanosomes are usually relatively enriched in Ca, Ti, Cu and Zn. The exception to this trend is the skin: in most specimens, skin melanosomes overlap in chemospace with the sediment. This may reflect the thin nature of the layer of skin melanosomes . Despite this, the chemistry of melanosome-rich soft tissues in at least some fossils is unlikely to be wholly original. For example, melanosomes in the eyespots from the Saar-Nahe amphibians  are often enriched in Ti or Mn; enrichment in these elements is not observed in melanosomes from any extant vertebrate taxon or tissue17. Similarly, fossilized melanosomes in the skin of fossil vertebrates are often enriched in Ti . This contrasts with skin melanosomes in extant vertebrates, which are usually associated with Ca and Zn17. Although it is conceivable that early-branching or basal representatives of various taxonomic groups may differ markedly in melanosome inorganic chemistry to extant analogues, the conserved nature of these chemical signals in extant taxa17 suggests that fossil members of the relevant crown groups should show a similar chemistry to extant forms. The differences in melanosome chemistry between fossil and extant vertebrates observed here are therefore likely to reflect differences in diagenetic history, in particular, exposure to pore fluids of different chemistry. Hence, fossil melanosomes with elevated concentrations of metals that are not associated with the relevant tissues in extant analogues are unlikely to preserve a real biological signal; the preserved chemistry is most plausibly interpreted as diagenetically altered, whereby concentrations of certain metals increase  during diagenesis. Hence, the enrichment of Ti and Mn in the fossil melanosomes analysed here suggests that the chemistry of the latter has been altered during diagenesis. Interpretation of other chemical signals, however, is less straightforward. The fossil amphibian melanosomes show enrichment (by up to two orders of magnitude) of other elements, all of which are known to associate with melanosomes in vivo. Taphonomic experiments could provide useful data on the extent of enrichment possible  during decay and diagenesis.Many of the fossils analysed here reveal striking differences in inorganic chemistry among melanosomes from different body regions (see Supplementary Text). These data support our findings from a more limited fossil datasetCritically, the absence of a pervasive and consistent tissue-specific chemical signal in our total dataset of fossil amphibians supports our hypothesis that extrinsic processes can modify concentrations of elements associated with fossil melanosomes, even though the latter can retain a tissue-specific signal at the level of individual specimens.10. Cu has been proposed as a biomarker for eumelanin8 and Zn, for phaeomelanin10. Highly elevated concentrations of these metals in fossil melanosomes relative to extant examples would call inferences of colour into question, providing diagenetic addition of metals cannot be excluded. Concentration of these elements in fossil melanosomes is more likely original where it is consistent with other chemical data that support a melanic origin, e.g. elevated concentrations of melanin monomers  and/or derivatives38, or suites of other diagnostic molecular fragments20. Further, taphonomic experiments are required to test whether eumelanin can incorporate Zn, and phaeomelanin Cu, during diagenesis which might further hamper interpretations of original chemistry.The evidence presented here for the diagenetic incorporation of metals  into melanosomes has implications for the use of trace metal chemistry to infer original melanin-based integumentary colour in ancient organisms33. Melanin can chelate metals from  the environment under various conditions of pH and metal availability33. This can occur without loss of molecular integrity . In Sepia melanin, Mg, Ca and Zn bind exclusively to carboxylic acid groups (COOH), Cu binds primarily to hydroxyl (OH) groups41 and Fe binds exclusively to OH or amine groups (NH)41. Biological associations between melanin and metals present in vivo can, however, be altered by changes in local pH and metal concentrations41. Further, whether the above binding relationships apply to all natural melanins, including those in vertebrates, is unknown. In our study, Ca and Fe are usually relatively enriched in the sediment (up to ca. 2500 µm/cm2 for Ca and up to ca. 3500 µm/cm2 for Fe) and rarely enriched in melanosomes from the skin and internal organs; instead, the latter tissues are usually enriched in Zn, Cu and Ti. Assuming the binding relationships above are broadly similar among invertebrate and vertebrate melanins, the elemental distributions in the fossil amphibians could reflect a decrease in ambient pH during diagenesis, promoting the chelation of Zn, Cu and Fe to melanosomes, rather than Ca41. This hypothesis is supported by experimental evidence that exposure of melanin to a concentrated metal solution can result in binding of metal ions to all functional groups available39. Unlike the sedimentary matrix, the fossil melanosomes are rarely enriched in Fe, but this is not unexpected: binding rates of Cu to melanin are higher than those for Fe39.Metal binding capacity (the ability of a molecule to bind certain elements) and affinity (the strength of the bond) are well characterized for both synthetic and natural melanins30: it contains a higher ratio of PTeCA/PTCA relative to melanin from extant vertebrates22. This has been attributed to cross-linking among DHI and DHICA units due to thermal maturation during burial22. Previous experiments29 have shown that during thermal maturation, part of the DHICA unit of eumelanin undergoes decarboxylization, yielding additional DHI. Thermal maturation therefore induces reconfiguration of the constituent monomers of melanin. The impact of this molecular reconfiguration on the metal content and binding capacity of melanin has not been explored fully. It is, however, likely that the loss of the carboxylic group (COOH) in DHICA and the generation of additional, diagenetic, DHI units would provide additional hydroxyl (OH) groups that preferentially bind Cu and Ti31; this is consistent with the elevated concentrations of these elements in the fossils.The capacity of melanin to chelate metals is controlled by its molecular structure. Melanin, especially eumelanin, comprises subunits of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Fossil melanin, however, is diagenetically altered45, resulting in a release of cations that, if pH conditions are favourable33, could potentially bind to melanin. Subsequent diagenesis may involve changes in temperature, pressure, pH and pore fluid metal concentrations. Both these extrinsic environmental variables and the intrinsic chemical properties of melanosomes  are likely to be major controls on the preserved chemistry of melanosomes in fossils.Decay typically results in pH fluctuations in and around a carcass46, the ability to characterise the chemistry of an entire fossil plus its associated sedimentary matrix is clearly fundamental to resolution of important trends in melanosome preservation both within and between specimens. Further, the approach used here provides a novel and essential mechanism for discriminating between more and less diagenetically altered fossil melanins in a rapid, non-destructive way. Future experimental studies will characterise the response of the elemental chemistry of natural melanins from different tissues to different diagenetic conditions, thus providing empirical data to aid attempts to discriminate biological from diagenetic components of the melanosome chemical signal.In conclusion, our study reveals the major controls on the chemistry of fossil melanosomes. The diagenetic history of the fossil-bearing sediments, especially the pore fluid chemistry, controls variation in melanosome chemistry at the biota-level; this is superimposed by a second, biological, control: that of tissue type, whereby individual tissues vary in melanosome chemistry. The chemistry of the depositional environment, i.e., water chemistry and/or diet  may also influence melanosome chemistry but this requires additional testing as the existing literature is inconclusive. Although discrimination of different soft tissues using melanosome chemistry is possible for fossils from some biotas, e.g., Libros, Rott and Saar-Nahe, the chemistry of fossil melanosomes cannot be considered fully original even in these fossils. Contra claims that SRS-XRF is of limited value in fossil melanin research17. The data for fossil vertebrates from the Jehol biota are described in detail in McNamara et al., in prep. SRS-XRF data for individual fossil specimens from Rott, Bohemia, Messel, Yanliao, Monte San Giorgio and Saar-Nahe are treated in detail herein.Fossil specimens are from the following institutions: Hessisches Landesmuseum Darmstadt (HLMD), Institute of Vertebrate Palaeontology and Palaeoanthropology Beijing (IVPP), Museum für Naturkunde Berlin (MFNB), National Museum of Denmark (NHMD), Natural History Museum London (NHML), Paläontologisches Institut und Museum, Universität Zürich (PIMUZ), and Capital Normal University, Beijing (CNU). Specimens include amphibians, reptiles and birds, and represent different ages and depositional environments Table . The datSamples of fossil soft tissues were dissected using sterile scalpels and placed on aluminium stubs, coated with Au/Pd and examined using a JEOL IT100 VP-SEM at an accelerating voltage of 20 kV and working distance of 8–10 mm.Neusticosaurus edwardsii, PIMUZ-T3749) and fossil-bearing slabs  were mounted at 45° to the incident X-ray beam and were spatially rastered at 50 ms / pixel dwell time with a pixel size of 25 μm x 25 μm. The entire fluorescence spectrum was collected at each data point and the intensity of fluorescence lines for selected elements  was monitored using a silicon drift Vortex detector; these elements are usually present in our samples between 0.1 and 3600 µg/cm2. Fluorescence intensities were corrected and normalized for detector deadtime and variation in I0 to allow comparison among samples. The concentrations of each element in µg/cm2 were calibrated using NIST traceable thin film elemental standards. Data processing was performed using MicroAnalysis Toolkit software47. Regions of interest for quantitative analysis were selected from soft tissues and sedimentary matrix for each fossil specimen. For each region of interest, raw pixel data was extracted and mean and standard deviation values calculated for the concentrations of each element (Supplementary Datas Synchrotron rapid scan-X-ray fluorescence data were collected at the Stanford Synchrotron Radiation Lightsource using beam line 10-2. The incident X-ray energy was set to 11 keV using a Si (111) double crystal monochromator with the storage ring containing 500 mA in top off mode at 3.0 GeV. A focused beam of 25 × 25 μm was provided by using a tungsten aperture. The incident X-ray intensity was measured with a nitrogen-filled ion chamber. Samples of fossil soft tissues  was used to assess differences in the trace element chemistry between skin and liver in extant and fossil amphibians. It was not possible to analyse these data using LDA because the latter requires a minimum of three groups to produce a chemospace, whereas only two groups  are considered here.The differences in elemental concentration among biotas were assessed using LDA in PAST. Differences in elemental concentrations among regions of interest (including melanosomes and sediment) within and among taxa were assessed using Linear Discriminant Analysis (LDA) and ANOVA in PASTSupplementary Data S1.Supplementary Data S2.Supplementary Data S3.Supplementary Data S4.Supplementary Data S5.Supplementary Data S6.Supplementary Data S7.Supplementary information, figures and data legends.


E. coli strains which is inversely correlated to (p)ppGpp basal level. Conversely, (p)ppGpp concentration is positively correlated to increased tolerance to different antibiotics such as β-lactams, vancomycin, and others. Here we review the variations in intrinsic (p)ppGpp levels and its consequences across the E. coli species.(p)ppGpp is at the core of global bacterial regulation as it controls growth, the most important aspect of life. It would therefore be expected that at least across a species the intrinsic  levels of (p)ppGpp would be reasonably constant. On the other hand, the historical contingency driven by the selective pressures on bacterial populations vary widely resulting in broad genetic polymorphism. Given that (p)ppGpp controls the expression of many genes including those involved in the bacterial response to environmental challenges, it is not surprising that the intrinsic levels of (p)ppGpp would also vary considerably. In fact, null mutations or less severe genetic polymorphisms in genes associated with (p)ppGpp synthesis and hydrolysis are common. Such variation can be observed in laboratory strains, in natural isolates as well as in evolution experiments. High (p)ppGpp levels result in low growth rate and high tolerance to environmental stresses. Other aspects such as virulence and antimicrobial resistance are also influenced by the intrinsic levels of (p)ppGpp. A case in point is the production of Shiga toxin by certain  It would thus be expected that bacterial resources would be devoted most of the time to maximizing growth rate. This may be true under some circumstances, especially, under optimal laboratory growth conditions. However, bacteria actually keep growth rate under very tight control. At the core of growth regulation is a small nucleotide that appears in two different forms—guanosine tetra- and penta-phosphate—ppGpp and pppGpp, collectively known as (p)ppGpp. The grip of (p)ppGpp on growth rate is achieved mainly through an effective inhibition of stable RNA (rRNA and tRNA) synthesis during amino acid starvation and other nutritional stresses in a process that became known as the stringent control ppGpp is synthesized by two different proteins—RelA and SpoT. These proteins evolved by duplication from a bifunctional ancestral RelA/SpoT Homolog (RSH) possessing both (p)ppGpp synthetic and hydrolytic capabilities, resulting in two proteins with overlapping functionalities ppGpp both under nutrient starvation and under normal growth conditions. The spoT1 allele contains two different mutations - a H255Y substitution in the synthetase domain (Synth) and a two-amino acid insertion between residues 82 and 83 (+QD) in the hydrolytic domain (HD), both at the NTD portion of SpoT ppGpp-synthetic activity ppGpp synthesis caused by the presence of relA1. It is therefore no wonder that both alleles often appear together in the same genome.Early in (p)ppGpp research different spontaneous alleles of  of SpoT . The two of SpoT , but itsspoT alleles, such as spoT201, spoT202 an spoT203 were isolated by selection on amino-triazole plates  considerably reduced growth rate and for that reason could be transferred only to a relA1 background, but not to a bacterium that carries a wild-type relA allele. The spoT alleles spoT202 and spoT203 consist, respectively, of T78I and R140C substitutions, both in the HD domain ppGpp level in strains bearing these alleles, the spoT201-203 mutations have probably compromised the ppGppase activity of SpoT.Later on, other spoT mutant alleles were transferred to other genetic backgrounds  and high resistance to environmental stresses (see below).It became evident that an inverse linear correlation exists between the intrinsic level of (p)ppGpp in a bacterium  and the bacterial growth rate ppGpp physiology and homeostasis were obtained by studying laboratory strains derived from the ancestral K-12 strain. To date very few attempts have been made to analyze (p)ppGpp homeostasis in natural isolates of E. coli. In two of these studies, the basal level and starvation-induced levels of (p)ppGpp were assessed in a set of strains derived from the ECOR collection  strains ppGpp a good correlation between (p)ppGpp levels and growth rate, even in a set of non-isogenic strains, would be expected. Indeed, when the intrinsic ppGpp concentrations of the ECOR isolates growing under non-limited growth conditions are plotted against their respective growth rates, an inverse correlation is observed ppGpp in exponentially growing bacteria or in response to stresses other than amino acid starvation, pppGpp was below the detection level ppGpp is inversely correlated with growth rate ppGpp fluctuation in natural isolates, ppGpp concentration was measured in 50 STEC strains growing under two different culture conditions—non-limited growth medium and phosphate starvation ppGpp directly and indirectly affects many important bacterial characteristics, such as stress responses, virulence, antibiotic resistance and persistence, biofilm formation, genome stability, and more ppGpp concentration, but instead presented the level of ppGpp relative to that of GTP+ppGpp as detailed in Cashel ppGpp obtained by the classical method are reproducible and give a reasonable estimate of (p)ppGpp status in a particular set of strains. More recent techniques for evaluating (p)ppGpp, based on Ion Chromatography-High-Resolution MS ppGpp values in isogenic and non-isogenic strains, (p)ppGpp was assessed using the classical method of formic acid extraction of n Cashel . The mosn Cashel , as the rpoS gene itself ppGpp concentration. While some strains exhibit a proportionality between the two measured entities, others display mediocre levels of RpoS but high (p)ppGpp levels ppGpp supports survival by either directly or indirectly stimulating the expression of genes involved in stress protection. The cell response to environmental stresses such as extreme pH and osmolarity, dehydration or oxidative stress is coordinated by the sigma factor RpoS , the transcription of σ70-dependent genes, i.e., the majority of bacterial genes, is considerably diminished. Hence, the stimulatory effect of (p)ppGpp on RpoS adds another layer of growth control in addition to the already discussed inhibition of stable RNA. Bacterial strains that accumulate high levels of (p)ppGpp or RpoS are less fit for growing on poor carbon sources or under nutrient limitation ppGpp and RpoS directly affect the transcription of dozens of genes and indirectly the transcription of many others ppGpp both in batch and in continuous cultures, have been conducted to date. The mutations observed in these studies are summarized in Given that (p)ppGpp is the most important source of growth rate control , suggesting that the mutation caused a reduction in (p)ppGpp intrinsic concentration that led to an increase in growth rate. The ACT domain interacts with the ribosome A site in order to activate the (p)ppGpp synthetic activity  was observed in another case of adaptive evolution of E. coli growing at 43.2° that contributed to an increased tolerance in the presence of 5% ethanol ppGpp concentration, thereby increasing growth rate. The L139S mutation occurred in the pseudo-hydrolytic domain of RelA and is therefore unlikely to affect (p)ppGpp synthesis. The other two mutations—L455N and L516P, were in the TGS and AH domains, respectively. These mutations might have affected the regulation of (p)ppGpp synthesis by RelA as both TGS and AH subunits form the elbow of the boomerang-shaped RelA that interacts with the 30S ribosome and with the deacyl-tRNA ppGpp in response to isopropanol, resulting in higher growth rates. The mutations—R77W and S472N, were, as before, in the pseudo-HD and AH domains, respectively.In another experiment, relA also appeared in 2 out of 11 populations growing in lactate minimal medium  for 144 generations acquired mutations in several genes ppGpp on growth, low concentrations of this alarmone also results in reduced levels of RpoS  ppGpp-independent fashion.The stringent response has been linked to bacterial tolerance to β-lactam antibiotics in E. coli displayed higher MIC values for trimethoprim, gentamicin and polymixin when compared to the ΔrelA or ΔrelA ΔspoT mutants ppGpp concentrations is not restricted to β-lactam antibiotics. The wild-type strain of hipA, that encodes a serine/threonine-protein kinase that belongs to a type-II toxin/anti-toxin module, (p)ppGpp production and the formation of persisters ppGpp (ΔrelA ΔspoT double mutant) the hipA7 allele did not confer any advantage regarding antibiotic persistence, suggesting that the high-persistence phenotype elicited by hipA7 is (p)ppGpp-dependent. Accordingly, overexpression of relA in the hipA7 strain increased the frequency of persisters ppGpp in the relA+ strain overexpressing hipA was as high as under amino acid starvation ppGpp. In their model hipA inactivates the glutamyl-tRNA synthetase GltX resulting in the accumulation of uncharged tRNAs which ultimately leads to the activation of RelA and (p)ppGpp synthesis.Several studies have shown a positive correlation between the expression of relA abolished the formation of ampicillin, but not of ofloxacin persistence, which required the deletion of both relA and spoT. Furthermore, by controlling the level of (p)ppGpp it has been shown that formation of ampicillin persisters required higher concentrations of (p)ppGpp than formation of ofloxacin persisters. It has also been shown that under conditions of nitrogen starvation E. coli accumulates high levels of (p)ppGpp and forms high percentages of persisters when treated with ciprofloxacin, but only in a relA+ strain ppGpp ppGpp helps propagating antibiotic resistance genes through activation of integrase in class 1 integrons.Integrons are important elements in the dissemination of antibiotic resistance genes. It has been shown that (p)ppGpp plays a role in the regulation of E. coli is very well-integrated with (p)ppGpp homeostasis Dalebroux et al. (E. coli (EHEC) to colonize the host intestine . In addition, bacteria of this pathotype harbor a 35 Kb pathogenicity island known as the Locus of Enterocyte Effacement (LEE), which carries most genes implicated in EHEC intimate adherence ppGpp concentration and EHEC virulence. EPEC (Enteropathogenic E. coli) is another diarrheogenic pathotype that carries the LEE, but unlike EHEC it does not produce Shiga toxin. EPEC strains harbor a plasmid (EAF) that encodes both the BFP fimbria associated with bacterial adherence to the intestine cells and the perABC operon whose products control the transcription of the chromosomal LEE region ppGpp on perABC expression suggests that a fine-tuned concentration of (p)ppGpp is required to maximize EPEC adherence. Even though (p)ppGpp concentrations were not assessed in different EHEC and EPEC isolates the data presented in these studies suggest that the expression of virulence genes and virulence traits are modulated by this alarmone.The expression of virulence-related genes in pathogenic x et al. . For insE. coli (STEC) is another diarrheogenic pathotype that secretes Shiga toxin, but unlike EHEC, does not harbors a LEE and, consequently, does not display intimate adherence to intestinal cells  causes recurrent infections in the urinary tract. A critical mechanism of UPEC infection is the ability to invade the bladder cells by means of Type-I fimbriae. The expression of fimbrial genes is controlled by (p)ppGpp and DksA ppGpp intrinsic levels present higher levels of virulence toward the host.The extraintestinal uropathogenic E. coli pathotype in which a populational study correlating (p)ppGpp and pathogenicity has been performed stands out as an outlier. STEC toxin production is coupled to phage induction, which is inhibited by (p)ppGpp. By inhibiting phage replication (p)ppGpp acts as a legitimate promoter of bacterial survival.Lastly, (p)ppGpp is directly associated with the pathogenicity of many bacterial species and is required for the full expression of virulence genes ppGpp is been shaped by the historical contingency of bacterial populations. There are two types of evidence that support this assertion: direct assessment of (p)ppGpp in E. coli natural isolates and the selection of relA and spoT mutant alleles in evolution experiments. These data indicate that the genes that govern (p)ppGpp synthesis and degradation are subjected to frequent microevolutionary pressures that will eventually determine the optimal concentration of (p)ppGpp in a population. Given the pleiotropic effects of (p)ppGpp in the cell, adjustments of (p)ppGpp intrinsic concentration should have broad implications on bacterial physiology ppGpp inverse correlation across the ECOR strains was significant but not perfect, which suggests that the role of this alarmone in growth is intertwined with other regulatory circuits and that bacterial physiology is always more complex than firstly assumed.The intrinsic concentration of (p)ppGpp in strains of the species ysiology . In factBS and KO drafted the manuscript. All authors contributed to the article and approved the submitted version.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.


Nanotechnology involves studying and working with matter on a nanoscale, which provides the ability to work at the atomic level and molecular level to create large structures with fundamentally new molecular organization. The field of nanotechnology presents an exciting and rapid expansion of research area that crosses the barriers among physics, chemistry, biology, life, and engineering sciences. Nanostructure materials generally called nanomaterials are the materials having at least one dimension between 1 and 100 nm. These materials exhibit novel and significantly improved physical, chemical, and biological properties, phenomena, and processes due to their nanoscale size , hydrogen production and pollutant degradation by functionalizing g-C3N4 with SnO2 , SiO2 coated ZnO nanorod arrays for UV-durable super hydrophobicity , synthesis of micro/nanoscale LiFePO4/Graphene for lithium-ion batteries , facile and efficient fabrication of bandgap tunable carbon Quantum Dots , Calcium-phosphate lipid system for potential dental application , VOC gas sensors based on molybdenum oxide , metal oxides—based semiconductors for biosensors (Serban and Enesca), ZnO nanorods for detecting reducing gases , ZnO nanomaterials: Current advancements in antibacterial mechanisms , quantum dot sensor for the detection of calcium ions , mixed electronic and ionic charge carrier localization and transport , and mesoporous silica nanoparticles for controlled release of antimicrobials for stone preventive conservation . We have also highlighted that how state of the art theoretical and experimental approaches are leading to better understanding of semiconducting materials and improved design of novel functional semiconducting materials.In this Research Topic, we present a collection of original research and review articles focussing on different aspects of functionalized inorganic semiconductor nanomaterials, including modified AgQian et al. concerns an effect of ammonium phosphate—modified Ag3PO4 on photocatalytic performance. The authors demonstrated a novel one-pot surface modification route by using ammonium phosphate solutions to improve the photocatalytic performance of Ag3PO4. It was found that ammonium phosphate played multiple promotion roles in favoring the formation of metallic Ag nanoparticles and providing the negative electrostatic field on the surface of Ag3PO4 photocatalysts, which consequently promoted the separation efficiency of photoinduced electron-hole pairs, enhanced selective adsorption of cationic dye, and increased concentration of reactive oxygen species. This work provides an alternative route to boost the photocatalytic activity of Ag3PO4.The contribution of Zada et al. investigated the functionalization of g-C3N4 with SnO2 for the photocatalytic hydrogen production and degradation of pollutants. This work is emphasized to overcome energy crises and environmental pollution. The authors synthesized g-C3N4 nanosheets and coupled them with SnO2 nanoparticles. The enhanced photoactivities were attributed to the better charge separation as the excited electrons thermodynamically transferred from g-C3N4 to SnO2 as had been confirmed from photoluminescence spectra, steady state surface photovoltage spectroscopic measurement, and formed hydroxyl radicals. It is believed that this work would provide a feasible route to synthesize photocatalysts for improved energy production and environmental purification.The work of Li et al. developed vertically aligned ZnO nanorod arrays with large area through chemical hydrothermal process. Ultra-thin SiO2 shell film was deposited on ZnO nanorod arrays through PLD, and subsequently modified by stearic acid. This SiO2/ZnO/glass structure exhibited well UV-durable super hydrophobicity and high transmittance. These properties have important applications in solar cells.The contribution of Liu S. et al. prepared LiFePO4/graphene composites by packing LiFePO4 nanoparticles in the micron graphene sheets by one-step microwave heating technique. The introduction of graphene did not affect the structure of LiFePO4 as the nanoparticles were surrounded by the graphene sheets and the micron structure guarded the stability of the material. The electrochemical analysis reveal that the LiFePO4/graphene composites have excellent high-rate performance and cycling life. The outstanding electrochemical performance, as well as the fast and efficient method, make this technology commercially viable.Jia et al. developed a facile, fast, and green method to prepare bandgap tunable CQDs solely from anthracite. The bandgap change of the as-prepared CQDs could be achieved by simply controlling the concentration of H2O2. The morphology, size and PL properties of the as-prepared CQDs indicated that the blue luminescence might be originated from the intrinsic emission, but the yellow and green luminescence might be originated from the extrinsic emission due to the new energy states created by the oxygen-containing functional groups inside the band gap of CQDs. This novel strategy for fabricating optically tunable CQDs from coal is highly promising for the high-end application of coal.Zhu et al. investigates the calcium-phosphate lipid system for potential dental application. Aljaafari research group successfully synthesized ZnO nanorods using a domestic microwave-assisted solution method and showed a smooth surface morphology and wurtzite hexagonal structure. They concluded that the fabricated ZnO NRs using the microwave method was very sensitive to CH4 and CO, where the sensitivity toward these two gases was very high compared to H2 gas. The smooth surface of nanorods could also be used as a high operating temperature sensor.The work of Jiang et al.  studied Ghosh et al. developed a simple optical aptasensor for the detection of calcium ions. The sensor had been found to have high specificity for calcium ions in comparison to other metal ions like sodium, magnesium, and potassium. The molecular apta-beacons also demonstrated successful endocytosis and FRET-based calcium ion detection in osteocyte cells when conjugated with a cell-penetrating peptide (DSS).We hope this Research Topic will attract readers, providing novel literature insights, synergistic research ideas, and enthusiasm in research and studies.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The increasing prevalence of obesity is accompanied by a rising incidence in metabolic syndrome and related pathologies such as non-alcoholic fatty liver disease. Macrophages are hypothesized to play central roles in these diseases, through their role as inflammatory mediators and as such are thought to be potential targets for future therapies. Recently, single cell technologies have revealed significant heterogeneity within the macrophage pool in both liver and adipose tissue in obesity. Thus current efforts are focused on dissecting this heterogeneity and understanding the distinct functions of the individual subsets. In this review, we discuss the current knowledge regarding macrophage heterogeneity, ontogeny and functions in the context of obese adipose tissue and fatty liver disease and attempt to align the distinct populations described to date. The transition to a more sedentary lifestyle coupled with a higher caloric intake has led to an enormous rise in incidences of obesity over the past decades. Simultaneously, this has led to a dramatic increase in the number of patients suffering from numerous obesity-linked metabolic disorders including insulin resistance and type 2 diabetes, cardiovascular disease and non-alcoholic fatty liver disease (NAFLD). In fact, since 1975, obesity levels have almost tripled worldwide and according to the World Health Organization (WHO) this accounted for ~8% of deaths in 2017 largely due to cardiovascular disease. Chronic low-grade inflammation has been suggested to be related to many of the co-morbidities of obesity including type 2 diabetes, NAFLD, steatohepatitis and cardiovascular disease . As nditions , 17, whiand BMPs , 19.Regarding their functions, Res-macs fulfill central roles in tissue homeostasis and immunity through their constant surveillance and their ability to clear foreign substances, dead cells and debris. This clearance is mediated through recognition of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) present on their cell surface. In addition to physically clearing the tissue of pathogens, dead/dying cells and other debris, Res-macs can also express multiple cytokines which contribute to the activation of other innate immune cells and the adaptive immune system. Notably, through their unique transcriptomic profiles imprinted by signals from the local niche , 15, ResWith the identification of the substantial role played by the niche in regulating Res-mac gene expression and hence functionality, one of the key questions in the macrophage field today is how does the niche change in inflammation and how does this impact Res-mac survival and function? Notably, a population of microglia with a distinct transcriptional and functional profile have recently been described in aged mice (20 months). These microglia contain lipid droplets and were hence termed lipid droplet-containing aged microglia (LDAMs) . As therSall1  and recently recruited KCs, on their way to becoming resident (CLEC4F+TIM4−) and Temp-macs (CLEC4F−TIM4−) that can either differentiate into KCs or be lost from the tissue upon return to tissue homeostasis . They are one of the largest populations of Res-macs present in the body and reside with part of their body in the liver sinusoids where they are in close contact with liver sinusoidal endothelial cells (LSECs) and part in the liver parenchyma where they are in close contact with hepatic stellate cells (HSCs) and hepatocytes . In miceeostasis , 32. In  Res-KCs , 43.in vivo  murine liver macrophages  altered their expression of a standard panel of activation associated genes including  or Egr2 . This th or Egr2 . However or Egr2  it is di+Ly6G− myeloid cells isolated from mice fed a western diet supplemented with fat, sugar and cholesterol for 16 weeks identified 3 distinct clusters of recruited macrophages (termed Mo-MFs) and a cluster of likely Res-KCs  diet for 20 weeks to induce NASH identified distinct clusters of macrophages in the NASH liver . Here onnd Gpnmb . The KC nd Gpnmb . The expnd Gpnmb , but thend Gpnmb , furthernd Gpnmb , it is nnd Gpnmb  indicatind Gpnmb . Interesnd Gpnmb .Clec4f, Timd4, Cd5l), general macrophage markers , monocyte markers (Ccr2 and Ly6c2) and the markers proposed by the studies  were assessed (Adgre1 (encoding F4/80) and Fcgr1 (encoding CD64) while still expressing the monocyte genes Ly6c2 (encoding Ly6C) and Ccr2, thus perhaps some of these populations may represent monocytes or monocytes transitioning to macrophages rather than fully differentiated macrophages (Timd4 (encoding TIM4), a marker of long-term residence of KCs in the liver  and how specific this expression is across the liver. For example, do any of the niche cells of the liver such as HSCs and LSECs which express higher levels of Igfbp7 than Res-KCs under homeostatic conditions (unpublished data) also express Igfbp7 in NAFLD/NASH and what role does this play?Another key question is if Res-KCs are not functioning as inflammatory mediators in NAFLD as these recent studies may suggest, do they play any role? One possibility is that Res-KCs may function to metabolize the ectopic lipid in NAFLD, as under homeostatic conditions Res-KCs have been shown to express a module of genes associated with lipid/cholesterol metabolism , althougtabolism . While aCd9, Trem2, and Gpnmb found across multiple models and in human fibrotic tissue as well as the location of these subsets in the tissue and their interactions with the local niche. Of note, one study in Trem2 KO mice fed a western diet found reduced pathology in the KOs compared with WT controls  macrophages were also identified in this study although their localization was not investigated  are spread throughout the adipose tissue with a significant proportion in lean mice being found associated with the vasculature and hence recently termed vasculature-associated macrophages or VAMs . VAMs arstigated . Regardistigated . Howeverstigated  moreover-tagging . However-tagging , suggest-tagging . Given trecruits . Notablyrecruits . In addiin vivo withstanding , Shglb3 and Timd4  (+ VAMs reported to be present in obese adipose tissue (+ macrophages (Mac2 and Mac3) were found to be located in the CLSs  and Mrc1 expression (encoding CD206) was lower than in other Mac populations which could suggest that these are the DP macs identified by Silva and colleagues but then restricted to the obese adipose tissue in this study   and crucially were also found in human obese adipose tissue  . The Macse mice)  and thusthe CLSs . If the e period , this cois study , 62. Oves (LAMs) . Althougpulation . Moreovee tissue .Cd9, Trem2, Gpnmb, and Spp1 .Pecam1 (encoding CD31) and Fcgr4  suggesting they are likely not the DP macs as described above (at least based on mRNA expression), and thus further comparison of these datasets is warranted. As the phagocytic macs are expanded in caloric restriction, it is also possible that this subset does not exist in the dataset from Jaitin et al., but then the suggestion that the phagocytic macs could be the DP macs should be further examined  or in all myeloid cells (Netrin-1) and as macs with similar profiles have been identified in other tissues, it will now be important to examine the role of TREM2 and the expression and function of NETRIN-1 in the distinct macrophage populations in adipose tissue and liver to determine exactly which macrophage populations are important for these differences in disease pathology.While there is still much to learn regarding the macrophage clusters in obese adipose tissue, there is clearly heterogeneity within adipose tissue macs  and a coscRNAseq . De novotabolism , 23 and tabolism , thus peent mice . Notably profile . Notablying ATMs . This coing ATMs . Conversing ATMs . This iming ATMs . While aing ATMs , as the ing ATMs  and macsogenesis , it is togenesis . While tTaken together, it is clear that there is significant heterogeneity within the macrophage pool in both obese adipose tissue and the fatty liver. This heterogeneity is made up of different subsets  but also likely include developmental intermediates and/or different activation states of the same cell subset. While single-cell technologies have helped us to understand this heterogeneity, questions regarding the specific functions of these different subsets and/or the precise definition of distinct subsets remain which will be important to understand in the coming years. Studies using full body KOs and chimeras are helping to shed light on the functions of the different populations, but further work is still required using cell-type specific KOs before we can truly assess the roles of one population over another so that we can better understand which populations we may need to target with new therapeutic approaches.AR and CS co-wrote the review. LM performed the bioinformatics analysis.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.


ABCA4-associated Stargardt disease (STGD1) relative to normal controls. Automated segmentation of retinal sublayers was performed with manual correction as needed, and thicknesses in various macular regions were compared using mixed effects models. Relative to controls , STGD1 patients  had slight thickening of the nerve fiber layer and retinal pigment epithelium-Bruch’s membrane, with thinning in other sublayers, especially the outer nuclear layer (ONL) (p < 0.0015). When comparing the rate of retinal sublayer thickness change over time , STGD1 retinas thinned faster than controls in the outer retina (ONL to photoreceptor outer segments). OCT-based retinal sublayer thickness measurements are feasible in STGD1 patients and may provide objective measures of disease progression or treatment response.Stargardt disease, the most common inherited macular dystrophy, is characterized by vision loss due to central retinal atrophy. Although clinical trials for Stargardt are currently underway, the disease is typically slowly progressive, and objective, imaging-based biomarkers are critically needed. In this retrospective, observational study, we characterize the thicknesses of individual retinal sublayers by macular optical coherence tomography (OCT) in a large cohort of patients with molecularly-confirmed,  STGD1 is caused by mutations in ABCA4  which encodes a transmembrane transporter protein responsible for preventing the toxic accumulation of bisretinoids in the retinal pigment epithelium (RPE) that can lead to photoreceptor loss7. Clinically, the disease is characterized by yellow, fleck-like subretinal deposits, predominantly in the macula, over which there may be outer retinal loss. However, there is substantial heterogeneity with regard to age of onset, degree of vision loss, rate of progression, and extent of retinal involvement10.Stargardt disease  is the most common cause of inherited macular dystrophy, with an estimated frequency of 1 in 10,00015. However, recent studies using optical coherence tomography (OCT) have suggested that photoreceptor loss may actually precede RPE loss17. Longitudinal, en face evaluations of ellipsoid zone (EZ) area have shown that rates of photoreceptor and RPE loss are either similar or higher in photoreceptors, with a greater ratio of area loss in photoreceptors than RPE19. Relatively few studies to date have quantified the degree of OCT-based retinal thinning in STGD1 or attempted to assess the rate of retinal thickness change over time21. Moreover, although paradoxical thickening of the inner nuclear layer has been described22, relatively little is known about changes within retinal layers other than the photoreceptors or RPE.Although the clinical features of STGD1 are well-described, debate remains regarding the exact sequence of tissue loss in this disease . Most imaging-based studies of progression in STGD1 have assessed the area of RPE loss, which is readily measured using en face fundus autofluorescence (FAF) imagingABCA4-associated Stargardt disease. We compare retinal thicknesses of these sublayers to an age-similar group of normal controls. We then describe regional differences in retinal thickness change based on macular zones and quantify the rate of change for various retinal sublayers over time.In this study, we evaluate retinal sublayer changes as measured by OCT in a large cohort of patients with molecularly-confirmed 2. The study adhered to the tenets of the Declaration of Helsinki and was conducted in accordance with regulations set forth by the Health Insurance Portability and Accountability Act. Institutional Review Board approval for the study was obtained from the Human Subjects Committee of the University of Iowa, and informed consent was obtained for study participation. For participants under the age of 18 years, informed consent was obtained from a parent and/or legal guardian. Demographic data including gender and age at each visit were recorded for each patient. Best-corrected Snellen visual acuity was recorded for each eye at each study date and converted to LogMAR measurements as previously described24. Data processing, image analysis, and statistical comparisons are summarized in Fig. This is a retrospective, observational study evaluating OCT volume scans from a previously-published cohort of patients with molecularly-confirmed STGD1 seen at the University of Iowa from January 2010 to June 2016Optical coherence tomography  macular volume scans from patients with STGD1 were included in the study. These scans were obtained during the course of routine clinical care and included fovea-centered macular volume scan protocols that ranged from 20 degrees × 20 degrees to 30 × 20 degrees, with 512, 768 or 1024  × 496  pixel density, and comprised of 19, 25, 31, 37, 47, or 49 B-scans. All scans taken in follow-up were acquired using TruTrack Active Eye Tracking with registration to prior scans.26.For analysis of longitudinal data, only eyes with registered volume scans separated by at least 12 months were selected. When multiple registered scans were available, only the earliest and latest scans were used. We then segmented these scans using the Iowa Reference Algorithms . These automated algorithms identify the foveal pit and segment the retina into 11 surfaces from the internal limiting membrane to the RPE with reliability similar to manual segmentation by expert human graders in normal eyesAll automatically segmented volume scans were then reviewed for accurate segmentation and foveal centration by two independent, ophthalmologist graders , and scans with quality insufficient for segmentation  were excluded. For the remainder of the volume scans deemed acceptable, graders then manually corrected errors in segmentation surfaces and fovea location using the editing functions within the Iowa Reference Algorithms software Fig. , which a27. To account for multiple measurements arising from the same patient and from the same eye, we adapted the method presented by Parker and coworkers for computing limits of agreement29. For each of the two comparisons, we first computed the difference between thickness measurements (either the uncorrected segmentation minus the grader average or Grader A minus Grader B). Next we fit the differences per patient (id) per eye and per visit to a linear mixed effects model, accounting for the correlation within patients and within eyes of a patient:We then computed retinal sublayer thicknesses per scan in three Early Treatment Diabetic Retinopathy (ETDRS) subfields  for each grader and in the uncorrected segmentations. Next, we averaged the corrected thickness values for each layer and subfield. We constructed Bland–Altman plots to assess agreement of thickness values (a) between grader average and uncorrected segmentation and (b) between both graders  for R . R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/]). We set the “control” parameter to be “lmeControl(opt = ‘optim’)”.We computed 95% confidence intervals around the mean bias, upper limit of agreement, and lower limit of agreement using a stratified bootstrap procedure. For each bootstrap  was formed by taking the measurements from the inner face of the outer segments to the inner face of RPE/Bruch’s complex, resulting in a total of nine segmented retinal sublayers for subsequent analysis. For patients with available longitudinal imaging, the rate of change in retinal sublayer thicknesses (microns/year) was calculated using the initial and most recent follow-up scan. A subanalysis of these data were performed using three combined retinal layers: (1) inner retina [nerve fiber layer (NFL) to outer plexiform layer (OPL)]; (2) outer retina ) for the R programming language .For each eye from a STGD1 patient, we estimated (a) the thickness at baseline visit and (b) the rate of change over time for each sublayer and subfield. We then statistically compared these estimates to a cohort of age-similar normal eyes also with registered, longitudinal macular volume scans for one or more eyes, collected at least 12 months apart. To perform these comparisons, we modeled the data using mixed effects modelsWe modeled thickness (in microns) as a function of years since baseline exam, group status (control or STGD1), and laterality of the eye (OD or OS). To evaluate how STGD1 status changes the rate of retinal thinning, we included an interaction term between years and group. We allowed baseline thickness and rate of change to vary by patient and by eye. Using the “lme” function from “nlme”, we specified the [main] model as `thickness ~ years + group + years:group’, specified the random effects as ‘~ 1 + years|id/eye’, and set the "control" parameter to ‘lmeControl ’. We evaluated this model for each combination of layers and subfields, for a total of 27 models .In the longitudinal analysis, the rate of change for individual sublayer thicknesses was small for each intrinsically thin sublayer . We therefore summed the thickness values for the inner retina  and outer retina  and fit an additional 6 models (2 combined layers × 3 subfields). To account for multiple comparisons after fitting 33 independent models  to the dataset, we applied Bonferroni correction to determine statistical significance . Error bars in supplemental figures represent the standard error of the mean for each group as estimated by the models.The results of this study were presented in part at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Vancouver, Canada, in April 2019 [ARVO Abstract 2193].A total of 162 Stargardt patients  were considered for the study. Of these, 127 patients had scans that could be reliably segmented; after removing eyes without longitudinal, registered scan pairs, our cohort for statistical analysis comprised 107 eyes from 63 patients . All included scans for STGD1 patients required some manual modification of segmentation surfaces. For the STGD1 group, included patients were younger at baseline  than excluded STGD1 patients , and mean visual acuity was better for included eyes (logMAR 0.7) than those we excluded (logMAR 1.1)  through outer segments (OS) Table . This waWhen evaluating the rate of thickness change in combined retinal sublayers  and the RPE, the outer retina (ONL to outer segments) of STGD1 patients thinned faster in all subfields than controls, who had relatively stable outer retina thicknesses  manual segmentation32. Our results demonstrate that OCT-based analysis of individual retinal sublayer thicknesses is feasible for the majority of Stargardt patients seen in an inherited retinal disease clinic. In contrast to segmentation used in ProgStar, where manual correction of individual sublayers per each individual B-scan was performed, we used the Iowa Reference Algorithms, which take into account 3-dimensional data from adjacent B-scans in a volume scan to segment individual surfaces26. For all subfields, the uncorrected Iowa Reference Algorithms tended to overestimate the thickness of the outer retinal sublayers as compared to grader average, especially in the center subfield where disease was most severe  Study group previously demonstrated good reproducibility of total retinal, EZ, and ONL thickness measurements on SD-OCT in a small subset (30 patients) of patients as part of a prospective natural history study33. In the inner and outer rings, the outer retinal sublayers (ONL to OS) are thin despite the RPE thickening in these regions, with the greatest difference in the ONL, consistent with prior OCT-based observations that loss of photoreceptor nuclei is observed first, followed by measurable loss of the photoreceptor inner/outer segments, and eventual RPE atrophy34.As expected, measurements of the outer retinal layers were thinner for STGD1 patients relative to controls at baseline, but the degree of thinning relative to controls varied by zone, with the greatest thinning in the center subfield, followed by the inner and outer rings. Interestingly, the RPE layer was thicker than controls in the inner and outer rings. This finding is consistent with the known pathophysiology of the disease (accumulation of toxic bisretinoids within the RPE) and confirms prior OCT-based observation that the outer retina between Bruch’s membrane and the EZ is thickened35, but the etiology of this change is unclear, whether due to neuroinflammation, gliosis, or edematous thickening. We suspect that the NFL layer thickening may be an earlier manifestation of damming of axonal flow in this layer, akin to what has been described in other heritable photoreceptor degenerations. For example, in retinitis pigmentosa, decreased photoreceptor signal is thought to result in secondary alterations of axonal transport in the inner retina and optic nerve head drusen in some patients38. Thickening of the nerve fibers on histology and OCT can be seen even in the presence of GCL thinning and in the absence of gliosis40. By contrast, the GCL was thinner in Stargardt patients versus controls, particularly in the inner ring and outer ring. We suspect that this represents a secondary pruning of the retinal ganglion cell nuclei in response to photoreceptor loss, as is seen in many models of photoreceptor disease43. Interestingly, temporal optic nerve head pallor has been described in about 10% of Stargardt patients44, and the thinning of the GCL may explain the optic nerve head pallor noted on clinical examination. Huang and colleagues previously used OCT to follow Stargardt patients (n = 45) for 4 years and demonstrated paradoxical INL thickening, which they attributed to retinal remodeling in the context of photoreceptor disease22. In our study, baseline INL thickness was thinner in all subfields for STGD1 patients versus controls. However, some individual patients in the longitudinal group were noted to have INL thickening  or thinning  in Stargardt has been described32. Studies using en face SW-AF or NIR-AF imaging have suggested that the rate of RPE loss correlates with EZ loss, particularly when the total area of abnormal autofluorescence (hypo- and hyper-autofluorescence) are considered using SW-AF; however, using NIR-AF, RPE atrophy appears to precede detectable photoreceptor loss47. When evaluating disease progression cross-sectionally using OCT, thinning of the ONL appears to the most prominent change, followed by measurable thinning of the photoreceptor inner/outer segments and RPE, as reported previously34. The overall rate of outer retina loss in our study was about 3 microns/year, and interestingly, the rate of loss was similar across all three ETDRS subfields. Unlike the outer retina, the RPE layer did not show significant thinning, likely because detection of thinning in this monolayer would require atrophic loss of the cells . Because of the packing density of photoreceptor nuclei within the ONL, incremental loss of photoreceptor cells would result in detectable retinal thinning, even without complete loss of the retinal sublayer. Thus, monitoring OCT-based retinal thickness changes within the outer retinal sublayers  may be a convenient way to detect mild changes before eventual cell loss and clinically visible atrophy. Further studies using cellular imaging  may shed further light onto the sequence of photoreceptor and RPE cell loss in STGD149.Detection of photoreceptor and RPE loss in STGD1 are method-dependent, and most of the previously-published work has used en face imaging using short-wavelength autofluorescence (SW-AF) or near-infrared autofluorescence (NIR-AF). These methods analyze disease progression by tracking area of involvement but require atrophic loss of RPE cells to measure a discrete, hypo-autofluorescent border, which can sometimes be difficult to distinguish 54. Due to the relatively slow progression of STGD1 and the variability in foveal involvement, it is difficult to follow disease progression in clinical trials using best-corrected visual acuity55, and measurements are sometimes limited by ability to fixate and by changes in fixation location56. Other tests such as Goldmann visual fields are time-consuming and subjective, while electroretinography is also technically challenging in some patients with relatively low test repeatability, limiting its ability to detect small changes over time. Microperimetry with static perimetry may provide detailed functional testing in the macula but is also time-consuming, and Stargardt patients have less well-defined correlation between structure and function than patients with age-related macular degeneration59. Thus, combining structural and functional outcomes may be more sensitive to changes than a single mode of testing alone60. As above, our data demonstrate an overall slow rate of retinal change  in patients with STGD1. However, there were numerous individual outliers relative to the group , the differences between the two groups are perhaps more convincing in this context, as retinal thicknesses typically decline with age62. We evaluated retinal thicknesses within standard ETDRS subfields, but future studies may evaluate the variations in foveal involvement and retinal thinning within different foveal zones. Due to the numerous layers analyzed in this study, we also did not include analysis of individual ETDRS subfields  within the inner or outer rings. Because this study was designed to evaluate retinal thickness changes within various retinal sublayers, we did not evaluate the degree of reflectance  of each sublayer, but this may be the focus of future studies.This study had many strengths, including the size of our study, which is the largest to date evaluating OCT-based findings in STGD1. In addition to clinical diagnosis by an inherited retinal disease expert, all included patients were molecularly-confirmed with two pathogenic variants in 63. About a third of eyes were excluded due to poor image quality or advanced disease where the retinal laminations were indistinct, so as expected, the data are biased toward patients with milder disease. Retinal thickness measurements can be affected by axial length64, but we did not have axial length measurements available on most patients to examine its impact. To evaluate retinal thickness changes over time, we chose statistical models that assume a linear change in retinal thickness over time. While these models provide a reasonable representation of retinal thickness changes for this population over a relatively short period of time , they are less ideal for evaluating the likely non-linear progression of disease over the course of an individual patient’s lifetime. Most of the patients included in this study had a slow rate of retinal thinning over time. However, a few patients were noted to be outliers, and future study may evaluate factors influencing the rate of retinal thinning. Although all patients in our study had molecular-confirmation of STGD1, the analysis of genotype and its potential impact on these results was beyond the scope of this particular study.This study was limited by its retrospective nature, with inclusion of macular volume scans acquired during routine clinical care. Because of this, there may be small differences in the data if the scans were acquired in a more standardized setting of a prospective study, and we did not evaluate the impact of scan density on our resultsTo our knowledge, this is the largest study to date evaluating OCT-based retinal sublayer thickness measurements in STGD1. Compared to controls, STGD1 eyes possess a distinct pattern of both inner and outer retinal thinning compared to age-similar normal controls that can be readily-identified and measured on OCT. Longitudinal, OCT-based assessment of the rate of retinal sublayer change is also feasible, and these measurements may provide objective ways of monitoring disease progression or treatment response for clinical trials over time. The rate of outer retinal thinning in STGD1 is generally very slow, but significant individual variations exist. These individual differences relative to the population may help identify outliers of interest for future studies using induced pluripotent stem cell-derived cell-based disease modeling and genetic analysis for possible modifier genes, which may in turn provide further insight into Stargardt disease pathogenesis and its pathophysiologic mechanisms.Supplementary Information.


Recent advances in next-generation sequencing technology have led to the production of an unprecedented volume of genomic data, thus further advancing our understanding of the role of genetic variation in clinical pharmacogenomics. In the present study, we used whole exome sequencing data from 50,726 participants, as derived from the DiscovEHR cohort, to identify pharmacogenomic variants of potential clinical relevance, according to their occurrence within the PharmGKB database. We further assessed the distribution of the identified rare and common pharmacogenomics variants amongst different GnomAD subpopulations. Overall, our findings show that the use of publicly available sequence data, such as the DiscovEHR dataset and GnomAD, provides an opportunity for a deeper understanding of genetic variation in pharmacogenes with direct implications in clinical pharmacogenomics. The DiscovEHR cohort is the result of the collaboration between Geisinger (GHS) and the Regeneron Genetics Center. It is comprised of samples of GHS patients, who consented to participate in the Geisinger MyCode Community Health initiative ,2. Protein silico analyses indicated that 1012 of the novel pharmacogene-related variants had the potential to abolish protein function . These exact genes are also identified in the gene dataset , which describes the 501 PGx variants found in the 1kG-p3 dataset from Lakiotaki et al. (2017) . To further highlight the potential clinical utility of our results, we assessed for an overlap of our findings with pharmacogenes harboring a ‘level A CPIC guideline’ annotation. We found that 11 out of 212 DMET genes from the DiscovEHR cohort lie within this annotation category [. (2017) . FinallyCYP2C19*17, CYP3A5*3, VKORC1*2). As this study is based on whole-exome sequencing data, we have an increased probability of covering and thus assessing the majority of the pharmacogenetically-relevant variants.One of the major caveats of the present study lies within the fact that several pharmacogenetically-relevant variants are usually not covered using commercially available whole-exome capturing kits , which were not investigated in the present study, since their detection requires different computational approaches.In this study, we integrated NGS data from a well-characterized cohort of 50,000 individuals (DiscovEHR) with publicly available PGx data to investigate the potential clinical utility of known and novel PGx variants. This approach led to the identification and characterisation of DMET pharmacogenomics variants not only within the DiscovEHR cohort but also within different subpopulations from the largest genomics database to date . We also showed that the use of data from a large and clinically well-characterized cohort enables the identification of variants with potential clinical pharmacogenomics relevance. Moreover, we identified a large number of novel and ultra-rare protein-damaging variants within a variety of gnomAD subpopulations, thus demonstrating that novel and rare missense and LoF PGx variants contribute to the functional variability within DMET pharmacogenes. We envisage integrating the available genotype, phenotype and clinical data related in order to directly associate protein-damaging variants with clinical variable outcomes, thus promoting the integration of pharmacogenomics into the daily clinic practice.


Rheumatoid arthritis (RA) is an autoimmune disease characterized by tumor-like hyperplasia and inflammation of the synovium, which causes synovial cell invasion into the bone and cartilage. In RA pathogenesis, various molecules in effector cells  are dysregulated by genetic and environmental factors. Synovial fibroblasts (SFs), the most abundant resident mesenchymal cells in the synovium, are the major local effectors of the destructive joint inflammation and exert their effects through the pathogenic production of molecules such as interleukin-6.To date, more than 100 RA susceptibility loci have been identified in genome-wide association studies (GWASs), and finding novel therapeutic targets utilizing genome analysis is considered a promising approach because some candidate causal genes identified by GWASs have previously been established as therapeutic targets. For further exploration of RA-responsible cells and cell type-specific therapeutic targets, integrated analysis  of the genome and intermediate traits  is crucial.This review builds on the existing knowledge regarding the epigenomic abnormalities in RASFs and discusses the recent advances in single-cell analysis, highlighting the prospects of SFs as targets for safer and more effective therapies against RA. Rheumatoid arthritis (RA) is an autoimmune disease that can severely impair mobility due to persistent synovial inflammation leading to joint destruction. In RA pathogenesis, a variety of molecules in immune cells  and mesenchymal cells are dysregulated under the influence of genetic predisposition and environmental factors. In recent years, genome-wide association studies (GWASs) have identified more than 100 RA susceptibility loci . To idenSynovial fibroblasts (SFs) are multifunctional mesenchymal cells of the joint synovium, which are localized in the synovial lining and sublining layers. In the normal synovium, SFs produce substrate proteins  and extracellular matrix (ECM)-degrading enzymes  to maintain the synovial structure. SFs also contribute to the synovial fluid composition by producing joint lubricants , and provide nourishment to the underlying articular cartilage.On the contrary, in the inflammatory synovium of RA, the lining layer thickens, and immune cells infiltrate the sublining layer. SFs proliferate, and cell-to-cell interactions or humoral factors within the synovium alter SFs into the activated phenotype Fig. . SFs invTo date, knowledge regarding the epigenomic abnormalities related to transcriptional regulation has been accumulated from studies on cultured RASFs. Notably, most conventional studies involve RASFs purified by subculture; hence, the term “culture” has been emphasized.Shroom Family Member 1 (SHROOM1) that encodes a protein involved in microtubule reconstitution during neural tissue development and cell division. Such genes as SHROOM1 may therefore constitute biomarkers for the early diagnosis of RA , LBH Regulator Of WNT Signaling Pathway [LBH], and Protein Tyrosine Phosphatase Non-Receptor Type 11 [PTPN11]), the knockdown of ELMO1, a protein-coding gene related to cell phagocytosis and movement, suppresses the migration and invasion activities of RASFs. Further, the knockdown of LBH, a protein-coding gene involved in embryonic development, increases the growth activity upstream of the LBH gene, and the enhancer activity is significantly lower in the risk allele than in the alternative allele. However, the enhancer activity is suppressed by methylation of the enhancer region in either alleles, and therefore, the RA risk SNPs and epigenomic modifications (DNA methylation) are thought to cooperatively control the enhancer activity of LBH [PTPN11, a gene encoding Src homology region 2 domain-containing phosphatase-2, is highly expressed in RASFs compared with OASFs and is known to be related to the invasion ability of SFs [PTPN11 exists is hypermethylated and is involved in enhanced cell sensitivity to glucocorticoid and PTPN11 expression [Several studies have reported the integrated analysis of the transcriptome and epigenome of cultured RASFs. Whitaker et al. identified differentially expressed genes (DEGs) and differentially methylated genes (DMGs) by comparing unstimulated RASFs and OASFs, and compared the DEGs and DMGs with candidate RA susceptibility genes obtained from different databases  in a GWAS . Among ty of LBH . PTPN11,y of SFs . The enhpression .Huntingtin Interacting Protein 1 (HIP1) [Furthermore, Ai et al. built a database consisting of transcriptomic and epigenomic information  of unstimulated RASFs and OASFs. As a result of this multilayer analysis, genes related to Huntington’s disease are reported to be activated in RASFs in addition to known pathways, and invasion of RASFs is found to be suppressed by knockdown of the gene encoding 1 (HIP1) .−THY1+ SFs localized around the blood vessels in the sublining represent a pathological subpopulation that produces inflammatory cytokines. Following this study, the Accelerating Medicines Partnership (AMP), which has been active since 2014 at the National Institutes of Health (NIH) in the USA, has conducted single-cell RNA sequencing and mass cytometry of the RA synovium and reported that CD34−THY1+HLA−DRhigh SFs form a pathological subpopulation that highly expresses IL-6 [+THY1+ SFs localize to the sublining and are involved in synovial inflammation, while the FAPα+THY1− SFs in the lining are involved in bone destruction [The recently developed single-cell RNA sequencing technology has revolutionized SF research. Mizoguchi et al. reported that single-cell RNA sequencing of RA synovium classifies SFs into at least three subpopulations . It has truction . Based otruction .CD40 whose induction by IFN-γ is significantly affected by an RA risk SNP (rs6074022). Upon chromatin remodeling in activated SFs, RA risk SNPs have been found to be enriched in clusters of enhancers  induced by synergistic proinflammatory cytokines. SEs are large clusters of enhancers collectively bound by an array of transcription factors to define cell identity, and they are hotspots for disease susceptibility [The transcriptomic and epigenomic abnormalities that characterize RASFs have been reported mainly in cultured cells; however, the position of SFs in the formation of inflammatory networks in the synovium has not been fully elucidated. One of the causes is the phenotypic differences between cultured cells and freshly isolated cells. As Wei et al. has clearly described , gene extibility . These rAs a future development, multi-layer analysis of freshly isolated SFs at the single-cell level  would be a step forward in understanding the pathophysiology of RA.Recent advances in biologics and molecular targeting drugs have led to a paradigm shift in RA treatment. However, maintaining remission with these drugs remains a challenge, and serious adverse events due to systemic immunosuppression often lead to clinical problems. An integrated analytical approach will therefore lead to the development of safer and more effective therapies targeting SFs and provide a breakthrough for next-generation clinical practice.