In most species the reproductive system ages at the same rate as somatic tissue and individuals continue reproducing until death. However, females of three species – humans, killer whales and short-finned pilot whales – have been shown to display a markedly increased rate of reproductive senescence relative to somatic ageing. In these species, a significant proportion of females live beyond their reproductive lifespan: they have a post-reproductive lifespan. Research into this puzzling life-history strategy is hindered by the difficulties of quantifying the rate of reproductive senescence in wild populations. Here we present a method for measuring the relative rate of reproductive senescence in toothed whales using published physiological data. Of the sixteen species for which data are available , we find that three have a significant post-reproductive lifespan: short-finned pilot whales, beluga whales and narwhals. Phylogenetic reconstruction suggests that female post-reproductive lifespans have evolved several times independently in toothed whales. Our study is the first evidence of a significant post-reproductive lifespan in beluga whales and narwhals which, when taken together with the evidence for post-reproductive lifespan in killer whales, doubles the number of non-human mammals known to exhibit post-reproductive lifespans in the wild. The taxonomic scarcity of this strategy suggests that it requires unusual selective pressures to evolve4. Comparative research has shown that females of only three species of mammal - humans (Homo sapien), killer whales (Orcinus orca) and short-finned pilot whales  – are known to have a statistically significant post-reproductive lifespan in the wild5. Recent work has also suggested that females of a third toothed whale species, false killer whales (Pseudorca crassidens), may also have a post-reproductive lifespan6. Here we define post-reproductive lifespans as common and prolonged female survival after the cessation of reproduction, such that a female entering the adult population can expect to live a substantial period of her life post-reproductive7. We differentiate post-reproductive lifespans from non-adaptive brief and rare survival past last reproduction resulting from the usual processes of senescence7.Why a female should cease reproducing before their expected end of life is a long-standing question in evolutionary biologyOdontoceti) it is somewhat surprising that all but one of the species known to have post-reproductive lifespans are in this group. Recent research has suggested that the demographic consequences of certain social structures are important in life-history evolution8, and the evolution of post-reproductive lifespans in particular9. Toothed whales show a remarkable diversity of social and reproductive strategies11. The diversity of social structures and reproductive strategies in the toothed whales makes them an important target group to understand the evolution of post-reproductive lifespans.Given the logistical difficulties inherent in studying a predominantly oceanic taxon such as the toothed whales . In a systematic search for age-specific corpora data in all 72 species of toothed whale we found sixteen species with age-specific corpora count data suitable for analysis: , narwhal Monodon monoceros (relative rate = 1.48), northern right-whale dolphin Lissodelphis borealis (relative rate = 1.14) and short-finned pilot whales (relative rate = 1.38). After the removal of an outlier the northern right-whale dolphin rate dropped to below one , but the other species results are qualitatively robust to the removal of outlying data.In ten of the sixteen species, a second degree polynomial best explained the relationship between corpora count and age showing that age-specific ovarian activity declined with age . Assumptions around population growth rates (see methods) mean that the Phys-PrR is reported for a static population, a shrinking population and a growing population (as: static [shrinking - growing]).Post-reproductive representation (PrR) is a population-level metric which calculates the proportion of adult female years in the population being lived by post-reproductive females. For example, in humans  approximately 40% of adult female years are being lived by post-reproductive females (PrR = 0.443)Of the ten species that show a decline in reproductive activity with age Table  in threePrR calculated from observations of births or pregnancy data is comparable to Phys-PrR in both species of pilot whale and false killer whales  for the presence of post-reproductive lifespans in any ancestral species of any toothed whales. All the other nodes show very strong support  for a lack of post-reproductive lifespans in ancestral species. Our results suggest that post-reproductive lifespans have evolved independently three times in the toothed whales: once at some point before the separation of the beluga and narwhal lineages, once in the lineage leading to short-finned pilot whales, and once in the lineage leading to killer whales Fig. .Figure 317. When taken together with the evidence for post-reproductive lifespans in killer whales from long-term individual based studies20 and non-invasive physiological studies21 our new findings effectively double the number of non-human mammals known to exhibit this unusual life-history strategy. Further, we found that post-reproductive lifespans are likely to result from at least three independent evolutionary transitions in the toothed whale lineage.Using physiological data, we have found evidence of post-reproductive lifespans in three species of toothed whale: beluga whales, narwhals and short-finned pilot whales. To our knowledge this is the first record of population-level post-reproductive lifespans in beluga whales and narwhals, and supports previous suggestions of significant post-reproductive lifespans in short-finned pilot whales22. In resident-ecotype killer whales , neither males or females disperse from their natal group: though males mate outside the group23. In ancestral humans dispersal is thought to have been female biased25. Under these demographic conditions selection will favour young females to invest resources in their own reproduction at the cost of reproduction in other group members20. Older females on the other hand can increase their inclusive fitness by aiding other group members to reproduce through grandmother and mother benefits26. Having a post-reproductive lifespan can be the optimal strategy when the inclusive fitness costs of reproductive conflict are combined with the inclusive fitness benefits of late-life helping20. Benefits to late-life helping and costs of reproductive conflict have been found in both humans and killer whales28. We predict that the two new species with a post-reproductive lifespan reported here will have demography that increases female within group relatedness as a function of age: either bisexual social philopatry with non-local mating or female dispersal with local mating9. Comparing the social structures of cetacean species with and without a significant female post-reproductive lifespan provides a unique opportunity to test the generality of demographic processes that are predicted to select for the evolution of post-reproductive lifespans29.Recent work suggests that the evolution of post-reproductive lifespans in humans and killer whales is driven by changes in local relatedness across the lifespan31. Similarly, both male and female beluga whales show high fidelity to natal summering feeding areas32, and both female and male  are regularly found in association with close kin33. Narwhal societies appear to be focussed around matrilines while migrating to summer feeding grounds35. Thus, in all three species current evidence suggests patterns of demography, that may lead to an increase in females local relatedness to their group with age, much like in resident killer whales and ancestral humans9. Further work based on individual based longitudinal studies and or population genetic studies are needed to confirm these findings.The limited information available on social structure in short-finned pilot whales, beluga and narwhal suggest that the population social structure may be based on bisexual philopatry. For example, genetic studies and observations at stranding events suggest that short-finned pilot whales live in mixed sex groups with males mating outside the group10. The males, in contrast, disperse at sexual maturity and are largely solitary, roving between female groups in search of mating opportunities10. This male-biased dispersal does not lead to the relatedness dynamics predicted to promote selection for female post-reproductive lifespans9. However, it is important to note that female relatedness to her group increasing with age does not presuppose the evolution of post-reproductive lifespans. For example, the available evidence suggests that long-finned pilot whales exhibit bisexual philopatry36 - much like short-finned pilot whales – and yet they do not have a significant post-reproductive lifespan. This highlights that it is not only demographic structures but also the balance of the costs of harming and the benefits of helping that may lead to the evolution of post-reproductive lifespans. Even within species there is considerable variation in social structure. Transient-ecotype killer whales , for example, form much smaller groups than resident-ecotype killer whales, and some males disperse from their natal group37. This would lead to very different relatedness structures in transient compared to resident-ecotype killer whale societies. It is unknown if transient-ecotype killer whales exhibit significant post-reproductive lifespans. Overall, there is a considerable amount still to be discovered about toothed whale social structure and life-history. Our results highlight the importance of the taxa for understanding the interplay between social behaviour and life history evolution.The social structures of toothed whales without a significant post-reproductive lifespan are varied (Supplementary 30). However, for many species females may stop reproduction prior to complete physiological reproductive senescence (measured via ovarian activity). For example, in rural Bangladesh women have their last child, on average, a decade before reproductive cesssation38. Detailed age-specific pregnancy data are rare for cetaceans, but do exist for short-finned pilot whales where, much like humans, the post-reproductive lifespan measured via pregnancy data is longer than the post-reproductive lifespan measured via ovarian activity  which persists in the ovary39. Corpora counts have been used to infer ovulation rate and other reproductive characteristics in a variety of Cetacean species (e.g.40). Here we use corpora counts as a measure of ovarian activity and not to estimate pregnancy rates which may differ from the corpora count42. In earlier studies corpora albicans and corpora atretica may not always have been properly distinguished39, however, as we are measuring ovarian activity, not ovulations per se this will not bias our results.We used published age-specific corpora counts to quantify the rate of reproductive senescence in female toothed whales. In Cetaceans, reproductive history can be inferred from anatomical examination of the ovaries43. Our criteria for data inclusion were: each female studied had a count of corpora and an independent estimate of age; female age structure was well represented; and that the data is presented in a clear format to be accurately obtained. Independent estimates of age were based on examination of dentine cemental layers in all species except narwhals, where the racemization of aspartic acid in the eye was used45. Recent research has shown that beluga whales deposit growth layer groups annually47. We therefore use growth layer group counts as our estimate of beluga whale age- though we note that as PrR is calculated as a proportion systematic age, estimation errors  would not affect our conclusions. Appropriate data was found for sixteen species: Atlantic white-sided dolphin Lagenorhynchus acutus48, Baird’s beaked whale Berardius bairdii49, beluga whale Delphinapterus leucas50, common bottlenose dolphin Tursiops truncatus52, false killer whale Pseudorca crassidens53, harbour porpoise Phocoena phocoena54, long-finned pilot whale Globicephala melas40, melon-headed whale Peponocephala electra55, narwhal Monodon monoceros45, Northern right-whale dolphin Lissodelphis borealis56, Pantropical spotted dolphin Stenella attenuata57, short-beaked common dolphin Delphinus delphis15, short-finned pilot whale Globicephala macrorhynchus30, sperm whale Physeter macrocephalus58, spinner dolphin Stenella longirostris59 and striped dolphin Stenella coeruleoalba57. Previous work in resident killer whales (Orcinus orca) has documented significant post-reproductive lifespans using long term individual based observations20 and the post reproductive period has been confirmed using non-invasive hormonal samples21. Currently however, to our knowledge there are no published corpora count data on killer whales of a sufficiently large sample size for a robust test of the rate of reproductive senescence and a calculation of physiological PrR (but see60). Killer whales are not, therefore, included in our analysis of ovarian activity. Data were restricted to include only data from the age of first ovulation, i.e. the age with the first non-zero corpora count. All analysis was performed in R61 with the ggplot2 package used for producing the figures62.We undertook a thorough literature search for age-specific corpora count data on all 72 recognised species of Odontocetes53, however we only use data from one population (Japan) as the second population (South Africa) may have been reproductively compromised53.It is important to note that throughout this study we refer to species, but our data is only based (with one exception) on a single population. For one species, the common bottlenose dolphin, data were available from three geographically distinct populations which we analyse independently. Data are also available from two false killer whale populations63. For some species of cetacean however, there is evidence to suggest corpora may regress, and not persist indefinitely15 and in some cases there may be multiple eggs released at a single ovulation event15. However, there is no evidence of age-related changes in either poly-ovulation or regression of corpora, which could otherwise affect our analysis of age-dependent changes in ovarian activity. Indeed, for three species  both pregnancy and corpora data are available and in both cases changes in pregnancy rate show a strikingly similar age-related pattern to changes in corpora deposition  to each of the sixteen species to investigate this declining ovarian activity , harbour porpoise (adj-R2 = 0.07) and the short-beaked common dolphin (adj-R2 = 0.10). No further analysis was performed on these species.We found a relationship between corpora count and age in thirteen of the sixteen species .For species with a decline in fecundity with age we then calculated their physiological post-reproductive representation (Phys-PrR). Post-reproductive representation is a population level measure describing the proportion of adult females years in the population that are being lived by post-reproductive females64. Bin widths were calculated in reverse: from the oldest individual. The oldest bin contains only the oldest female in the sample. The lower limit of the next bin was then selected to contain more than one whale, i.e. a greater number of females than the next oldest bin. This process continued until all females were assigned to a bin. In some cases, to fit the assumption of monotonically decreasing age cohorts the first age (youngest) bin for some species had to be smoothed to match the second youngest bin. This method will tend to underestimate late life survival, and therefore underestimate post-reproductive representation. Survival was then calculated from these age cohorts with survival assumed to be evenly spread through each age represented in a cohort. It should be noted that due to the low probability of sampling ‘rare’ ages of individuals, older whales are likely to be underrepresented in our data, further underestimating survival and the significance of the post-reproductive lifespan.The calculation of PrR is based on age-specific measures of survival and fecundity. We calculate age-specific survival from age-cohorts constructed from the original corpora data. Age-cohorts were constructed by making variable bin-widths starting at the oldest female in the study. We used these variable bin widths to construct monotonically decreasing age-cohorts, a pre-requisite for calculating survival from age-cohortsvice versa64. In the absence of detailed population growth parameters for most cetacean species, we model three population change scenarios in our calculation of Phys-PrR. Firstly, we assume a population at equilibrium, where population growth (r) = 064. Secondly we assume a population in serious decline, r = −0.1, where the total population shrinks by 10% each year. We model the largest possible population growth scenario for each species, up to r = 0.1, given the age-structure of the data64. These values are comparable to the estimated population growth rates of cetacean populations. For example, at the peak of the modern sperm whale fishery between 1945 and 1975 the best estimate of global sperm whale population decline averaged approximately 2.67% (r = −0.027) per year . In contrast, North Atlantic humpback whales (Megaptera novaeangliae) may be recovering from very severe whaling at annual growth rate of 0.073–0.08665.Calculating survival from age-cohort data assumes a stable population. If the population is not at equilibrium then calculation of survival, and therefore PrR, will be inaccurate. For example, in a growing population younger individuals will be overrepresented, underestimating late-life survival, and We used our measure of age-specific ovarian activity as a measure of fecundity. PrR is the summed life-expectancy in years after 95% of population fecundity has been completed (age M). Age M is independent of population change and therefore remains unchanged in the different growth scenarios. Because our data begin from maturity, age B  is equal to the first age present in our data. We calculated Phys-PrR for each population change scenario for all ten species with evidence of reproductive senescence.12. The reported p values are the number of these simulated populations with a higher Phys-PrR than the real Phys-PrR. Significance is reported as the result of a two-tailed test.The significance of our PrR values was calculated by simulating the life-history of individuals based on the real survival and fecundity data. We calculate the estimated Phys-PrR of 1000 populations of 1000 individuals with reproductive senescence equal to somatic senescence13 and beluga whales66) hunting pressures may have changed the demographics, with a bias to removing large (old) individuals from the population. For these species, this will lead to an underestimation of the frequency of post-reproductive females in the population, and therefore an underestimation of Phys-PrR.It should be noted that these calculations are based on a stable and representative age structure. For some species  or other suitable records of reproduction in older females . This model has a single parameter, the instantaneous rate of change between these two states . We used the ancestral state estimation function in the R package “ape”69 in order to estimate the value for this rate parameter using maximum likelihood estimation. This approach allows us to infer the likely state of post-reproductive lifespans at ancestral nodes in the phylogeny given this model of evolution. These inferences are given as proportional probabilities (range: 0 to 1) and indicate whether ancestral species are likely to have had the trait under consideration.We used a continuous-time Markov chain method70). In the absence of such published data for cetaceans we have made use of this historical physiological data, but highlight the need for, and value of, detailed individual based longitudinal demographic data in the future.All data used in this study are from published corpora counts from dissection of whale corpses. The corpses from each study come from a variety of sources (Supplementary Combined supplementary information


The first step of the adaptive immune response involves the interaction of T cells that express T-cell receptors (TCRs) with peptide-loaded major histocompatibility complexes expressed by antigen-presenting cells (APCs). Exactly how this leads to activation of the TCR and to downstream signaling is uncertain, however. Recent findings suggest that one of the key events is the exclusion of the large receptor-type tyrosine phosphatase CD45, from close contacts formed at sites of T-cell/APC interaction. If this is true, a full understanding of how close contact formation leads to signaling would require insights into the structures of, and interactions between, large membrane proteins like CD45 and other proteins forming the glycocalyx, such as CD43. Structural insights into the overall dimensions of these proteins using crystallographic methods are hard to obtain, and their conformations on the cell surface are also unknown. Several imaging-based optical microscopy techniques have however been developed for analyzing protein dimensions and orientation on model cell surfaces with nanometer precision. Here we review some of these methods with a focus on the use of hydrodynamic trapping, which relies on liquid flow from a micropipette to move and trap membrane-associated fluorescently labeled molecules. Important insights that have been obtained include (i) how protein flexibility and coverage might affect the effective heights of these molecules, (ii) the height of proteins on the membrane as a key parameter determining how they will distribute in cell-cell contacts, and (iii) how repulsive interactions between the extracellular parts of the proteins influences protein aggregation and distribution. The high specificity of the adaptive immune system is ensured by the interaction of molecular complexes on the surface of T lymphocytes called T-cell receptors (TCRs) with peptide-loaded major histocompatibility complexes (pMHCs) on antigen presenting cells (APCs). This interaction leads to activation of the TCR and further downstream signaling. However, how the TCR is triggered and initiates signaling is highly debated . DiffereThe possibility of sterically-based molecular segregation at cellular contacts poses the questions of how large proteins are organized and interact in general as well as the physicochemical mechanism driving segregation in these contacts. Various studies have been undertaken to understand this better including experiments using contacting cells , 16–18, The T-cell glycocalyx consists of mainly CD43 and the receptor-type protein tyrosine phosphatase (RPTP) CD45, which are two heavily glycosylated glycoproteins. CD43 is comprised of a mucin-like extracellular region only, whereas CD45 has a folded region as well as variable N-terminal stretches of mucin-like sequence, with mucins being unfolded and extended, serine-, threonine-, and proline-rich polypeptide segments bearing large numbers of O-linked oligosaccharides , 28. Thevia the formation of disulfide bonds; other large type II RPTPs either completely lack, or contain many fewer cysteines. Together, these observations point to the mechanical properties of the folded part of the extracellular domain of CD45 being integral to its function, as would be expected if it acts as a “lever” that excludes the attached cytoplasmic phosphatase from cell-cell contacts.The crystal structure of the folded part of CD45 has been determined and comprises a beads-on-a-string arrangement of four fibronectin-like (FN3) domains . The orgObtaining the overall dimensions of glycoproteins using crystallographic methods is often difficult due to the presence of large amounts of sugar-based side chains which tends to work against the formation of crystals. The height of the intact ectodomains of CD43 and CD45 have instead been estimated using electron microscopy. Whereas the single mucin-like extracellular region of CD43 has a length of 45 nm , the ~15Experiments have indicated that rigid membrane-anchored molecules can have markedly different surface heights due to their orientation, covering the range from being upright to lying flat on the surface depending on the conditions and their molecular properties –36. Rotavia a receptor/ligand pair, where the latter were enriched with CD45R0 (Reflection interference contrast microscopy (RICM) measures the gap size between contacting membranes by utilizing the interference pattern arising from light rays being reflected at different sample interfaces . From thh CD45R0 . From thh CD45R0 . This crh CD45R0 . The dech CD45R0 . The relh CD45R0 . Both CDThere are also other fluorescence-based methods that can be used to measure molecular height and orientation. Förster resonance energy transfer has been used extensively to map <10 nm distances and conformational changes in macromolecules . A relatHydrodynamic trapping uses the focused liquid flow from a micropipette to move and accumulate membrane-anchored molecules . It is t2 each  Schmid et al. found a relatively sharp transition between partially and fully excluded proteins, and that proteins 2 nm larger than the 10 nm gap were essentially completely excluded (2) bridging two cell surfaces and forming a 15 nm cell-cell gap (Proteins larger than the cell-cell gap generally get excluded, but the extent of exclusion can vary significantly depending on the system and the number of adhesion molecules creating the contact , 23. In cell gap  and in tcell gap . A slighcell gap  and the cell gap  gives cocell gap .−1 = 2.3 times larger than that of CD45R0. A more accurate analysis would have to take molecular interactions as well as additional membrane deformation into account, but this approximation still captures the essential behavior observed by Carbone et al. (Using a wholly non-cell based approach, Carbone et al. compared the exclusion of CD45R0 and CD45RABC in a 15 nm gap between a giant unilamellar vesicle and a supported lipid bilayer . In these et al. .in vivo could be different, especially if the membrane protein composition and density vary, as discussed above. Hence, general methods to measure the height of membrane-anchored molecules on the surface of cells are needed. Super-resolution fluorescence imaging is one technique that has shown promising developments in recent years to achieve this. However, knowing both the effective height of the protein and its crystal structure are not sufficient to predict how the protein will distribute at cell-cell contacts. The interactions between proteins and whether they can rotate also affects this. Hydrodynamic trapping can be used to measure these interactions and combined with knowledge of the protein structure reveal the conditions required for the proteins to partition into domains or aggregate on the membrane. Taking all of this into consideration, complementary techniques are needed to understand the complex behavior of these molecules and how they distribute at cell-cell contacts.We have discussed that it is not only important to know the molecular dimensions of membrane-anchored proteins to understand how they behave and distribute at cell-cell contacts but also how these proteins orient and interact on the membrane. Fluorescence-based techniques have been developed that allow the effective height of a specific membrane-anchored protein to be obtained, however, in most of the studies done to date the protein is anchored to a model membrane. These model systems have nevertheless shown that the actual height VJ, AS, SD, YL, and PJ wrote the paper. VJ, PJ, and YL made the figures.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.


Conventionally, CRM197 is isolated by fermentation of Corynebacterium diphtheriae C7 (β197) cultures, which often suffers from low yield. Recently, several recombinant approaches have been reported with robust processes and higher yields, which will improve the affordability of CRM197-based vaccines. Vaccine manufacturers require detailed analytical information to ensure that the CRM197 meets quality standards and regulatory requirements. In the present manuscript we have described detailed structural characteristics of Escherichia coli based recombinant CRM197 (rCRM197) carrier protein. The crystal structure of the E. coli based rCRM197 was found to be identical with the reported crystal structure of the C7 CRM197 produced in C. diphtheriae C7 strain (Protein Data Bank (PDB) ID: 4EA0). The crystal structure of rCRM197 was determined at 2.3 Å resolution and structure was submitted to the PDB with accession number ID 5I82. This is the first report of a crystal structure of E. coli derived recombinant CRM197 carrier protein. Furthermore, the rCRM197 was conjugated to Vi polysaccharide to generate Typhoid conjugate vaccine (Vi-rCRM197) and its immunogenicity was evaluated in Balb/C Mice. The Vi-rCRM197 conjugate vaccine was found to generate strong primary α-Vi antibody response and also showed a booster response after subsequent vaccination in mice. Overall data suggest that E. coli based recombinant CRM197 exhibits structural and immunological similarity with the C7 CRM197 and can be used as a carrier protein in conjugate vaccine development.It is established that the immunogenicity of polysaccharides is enhanced by coupling them to carrier proteins. Cross reacting material (CRM Over the past three decades, many routine childhood and adult vaccines have been developed using conjugation technology. Conjugate vaccines are developed by covalent attachment of an antigenic polysaccharide to a nontoxic carrier protein. Conjugation to a nontoxic carrier protein enhances the immunogenicity of polysaccharide antigens, enabling host defence against diseases caused by encapsulated pathogens. Conjugation transforms the T cell-independent response of polysaccharide vaccines to a T cell-dependent one. Conjugate vaccines have been demonstrated to be immunogenic and capable of inducing immunological memory and high avidity antibodies. Unlike unconjugated polysaccharides, conjugate vaccines also elicit protective responses in the immature immune system of young infants and the senescent immune system of the elderly .197) is widely used as a carrier protein in conjugate vaccines . Th. Thapo f 0.17 Å) , demonst197 and C7-CRM197, are equivalent. By superimposing the A and B chains of rCRM197 on to C7-CRM197 and its symmetry-related molecule that forms the dimer, 924 out of 998 Cα superimposed with RMSD of 0.38 Å  generated using E. coli derived rCRM197 as carrier protein was evaluated in Balb/c mice. Mice were immunized with three doses of conjugate vaccine Vi-rCRM197 and anti-Vi IgG immune response was measured by ELISA. Sera from mice immunizied with PBS was used to generate the baseline. Vi-rCRM197 immunized mice were able to elicit strong Anti-Vi IgG antibody response. Mice immunized with unconjugated Vi polysaccharide (Control) showed only basal level of Anti-Vi IgG. The Anti-Vi antibodies response also found to be bootable as seen in a steep increase in IgG titre after second dose of vaccine (Booster) (197 conjugate vaccine was found to be ~16-fold higher than teh baseline response (PBS) after booster dose wheseas un-conjugated Vi showed only ˜ 2 fold increase in the anti-Vi IgG over baseline. Noteworthy, the response of anti-Vi IgG in mice immunized with conjugate vaccine was 10-12 fold higher than the un-conjugated Vi.Immunogenicity of typhoid conjugate vaccine (Vi-rCRMBooster) . The Ant197 as carrier protein comfortably meets the WHO immunogenicity criteria for typhoid conjugate vaccine, as it induces more than four-fold Anti-Vi IgG and also exhibits a booster response in mice. The data confirm the immunological functionality of rCRM197 as carrier protein and suggests that it is able to convert T-independent response of polysaccharides in mice into T-dependent one and therefore booster antibody response is resulted. These data corroborate that rCRM197 conjugation with Vi polysaccharide in mice can be resulted in immunogenic conjugates and therefore strengthening the suitability of rCRM197 as a carrier protein for the development of conjugate vaccines.As per the recommendation of World Health Organization-Technical Report Series (WHO-TRS) on typhoid conjugate vaccine, the conjugate vaccine should induce an immune response that is at least four-fold higher than the response induced by un-conjugated Vi, and a booster response should occur after the second dose in animals  . The con197 carrier protein produced by heterologous expression in E. coli. In addition to reveal the scientific insights of rCRM197, the data presented in this work are also important in the context that regulatory guidelines on conjugate vaccines that emphasizes the need for in-depth characterization of carrier protein used in the conjugate vaccines development. Successful and broadly marketed carbohydrate conjugate vaccines are based on just a few FDA-approved carrier proteins. First-generation of carrier proteins such as DT and tetanus toxin require detoxification with formaldehyde, potentially eliminating part of the lysine residues needed for glycan attachment, thereby potentially compromising the conjugation efficacy. CRM197 is one of the most widely used and highly effective carrier protein for conjuate vaccine. Licenced conjugate vaccines such as HibTITER (Haemophilus influenzae type b associated diseases), Prevnar , and Menveo  are containing CRM197 as a carrier protein. Majority of currently marketed glycoconjugate vaccines contain CRM197 as a carrier protein and access to high quality material will support the development and production of new conjugate vaccines. The data presented herein demonstrate for the first time structural and immunological characterization of rCRM197 produced in E. coli.The present study provides the detailed structural and immunological characteristics of CRM197 has also been expressed in E. coli using pET28a expression vector [197 was expressed in Histidine-tagged form (His-CRM197) which requires a histidine removal step by enterokinase treatment. This process often poses the limitation during the scale-up of production process and inconsistencies with respect to batch to batch purity/impurity profiles of protein. The protein was purified at small scale by immobilized metal affinity chromatography (IMAC) with reasonable purity [197 expression was done in pTWIN expression vector, which releases the expressed protein from fusion tag by self-splicing with high fidelity. CRM197 protein was purified at high scale  using ion exchange chromatography and highly pure form of protein (>95%) was recovered as the final product.In a recent study, the recombinant CRMn vector . Unlike e purity . However197 proteins produced in three different expression systems . A comprehensive physicochemical analysis of the CRM197 molecules demonstrate that recombinant CRM197s expressed in E. coli are overall highly similar to those expressed in the traditional system (C. diphtheriae C7) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile and in vitro antigenicity [in vivo antigenicity/immunological characterization was lacking. As a part of further in-depth characterization, we have determined the 3D crystal structure of E. coli derived rCRM197. The rCRM197 has shown the equivalence with C7 CRM197 in terms of overall folding pattern, domain architecture and the presence of correct disulfide bonds. The absence of toxicity in CRM197 relies on a point mutation at amino acid position 52 that replaces Glycine with Glutamate (G52E) and renders protein nontoxicity. In the present study, the genetic basis of nontoxicity of E. coli based rCRM197 was also confirmed by X-ray crystallography. These data are important for scientific communities as well as industries working on conjugate vaccine development using CRM197 as carrier protein. The in vivo functionality of E. coli expressed recombinant CRM197 carrier protein has been demonstrated by conjugating it with Vi polysaccharide antigen of Salmonella Typhi thereby generating Vi-rCRM197 conjugate vaccine. The data confirm that the use of rCRM197 as a carrier protein in conjugate vaccine will be able to elicit the primary and a booster antibody response in animals. The Anti-Vi IgG elicited by Vi-rCRM197 conjugate vaccine was more than four-fold higher than the response generated by unconjugated Vi which is one of the benchmark for a protective typhoid conjugate vaccine [197 is already published [197 showed the comparable result in terms of overall fold change in Anti-Vi IgG over Vi and booster impact. The overall data presented in our study will potentially catalyse the efforts for the production of CRM197 protein in the recombinant system and its use as carrier protein in the conjugate vaccine development.Recently, we have studied the analytical comparability of five CRMgenicity . However vaccine . The comublished . The imm


Staphylococcus aureus (SA) bacteremia often requires a long treatment duration with antibiotics to prevent relapse due to the ability of SA to establish reservoirs of infection in sites such as heart and bone. These metastatic sites of infection cannot be serially sampled to monitor the clearance of SA infection. This study aimed to establish a link between persistence of circulating SA deoxyribonucleic acid (SA-DNA) and tissue reservoirs in patients with SA bacteremia.A highly sensitive quantitative polymerase chain reaction was used to measure whole blood SA-DNA and plasma-derived SA cell-free DNA (SA-cfDNA) in a set of longitudinal samples from 73 patients with confirmed SA bacteremia and correlated with clinical features.Blood SA-DNA was detected for longer than the duration of positive blood cultures. Longer duration of circulating bacterial DNA was observed in complicated SA bacteremia infections, such as endocarditis and osteoarticular infections, compared with uncomplicated bloodstream infections. In contrast, traditional blood cultures demonstrated similar time to clearance regardless of foci of infection. Plasma-derived SA-cfDNA showed concordance with blood SA-DNA levels. Baseline levels of SA-DNA were higher in patients presenting with greater clinical severity and complicated bacteremia.Prolonged levels of circulating SA-DNA in patients with complicated tissue reservoirs after clearance of blood cultures observed in this single-center study should be validated in additional cohorts to assess the potential utility for monitoring clearance of infection in patients with SA bacteremia. Staphylococcus aureus (SA) bacteremia is a major health threat and is responsible for many serious blood, tissue, and device-related infections . Se. SeP ≤ .< .0005) , which cStaphylococcus aureus cfDNA was detected in 66% of plasma samples from patients with positive BC compared with 32% of samples from patients with negative BC at the time of plasma sampling. Staphylococcus aureus cfDNA showed significant correlation to total blood SA-DNA   . The senStaphylococcus aureus DNA was detectable for a median of 6.8 days  in blood samples collected after antibiotic treatment, which was considerably longer then the median duration of detection of SA by BC of 1.3 days  (Staphylococcus aureus DNA could be detected for longer in MSSA compared with MRSA infections (15 days) . Higher 15 days) . Subject15 days) . We obse15 days) . The med15 days) . A simil15 days) . Staphylfections .Staphylococcus aureus DNA and SA-cfDNA were significantly higher in patients with PCT levels of >2 ng/mL  in patients with SA, fections , 25. StaStaphylococcus, this PCR approach must be paired with conventional microbiological diagnosis to exclude infection with other Staphylococcus strains as was done in this study. Future refinements of this assay could include multiplexing other genes to aid in increasing specificity. If the data from this pilot study is validated in larger cohorts, monitoring levels of SA-DNA during treatment may be informative as a marker of clinical resolution or relapse. Biomarkers that monitor clearance of tissue infection foci are essential to better inform the optimal duration of antibiotic treatment. This study provides preliminary evidence to support further evaluation of this rapid and sensitive SA quantitative PCR assay.Future studies in larger prospective cohorts of patients with invasive SA infections are needed to address the limitations of this small pilot study. Uniformly sampled matched BC, blood DNA, and plasma DNA samples were not available for subjects in this study, which relied on convenience sampling. Larger volumes of matched samples are needed to assess the relative abundance of SA-DNA in whole blood versus serum versus SA-cfDNA in plasma, to ascertain which sample type will be the most sensitive for monitoring response to treatment. This would help to interpret whether the small proportion of samples that were positive by BC but negative by PCR results from differences in available blood volume , differences in handling and storage of the convenience samples used in this study, or assay sensitivity. Evaluating the relationships between initial SA-DNA levels and metrics of clinical severity, such as mortality, relapse, or development of sepsis, are needed to extend the preliminary associations observed in this study. Because the current assay detects all species of Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplement-InformationClick here for additional data file.


Cancer is the second leading cause of death worldwide. Researchers have been working hard on investigating not only improved therapeutics but also on early detection methods, both critical to increasing treatment efficacy, and developing methods for disease prevention. The use of nucleic acids, or aptamers, has emerged as more specific and accurate cancer diagnostic and therapeutic tools. Aptamers are single-stranded DNA or RNA molecules that recognize specific targets based on unique three-dimensional conformations. Despite the fact aptamer development has been mainly restricted to laboratory settings, the unique attributes of these molecules suggest their high potential for clinical advances in cancer detection. Aptamers can be selected for a wide range of targets, and also linked with an extensive variety of diagnostic agents, via physical or chemical conjugation, to improve previously-established detection methods or to be used as novel biosensors for cancer diagnosis. Consequently, herein we review the principal considerations and recent updates in cancer detection and imaging through aptamer-based molecules. Cancer is a major public health problem and the second leading cause of death worldwide. The number of new cases of cancer is expected to grow rapidly as the population increases and continues to make lifestyle choices that increase cancer risk ,2. AccorThe accumulation of genetic alterations in cells together with abnormal cell division and elevated resistance to apoptosis are known factors in cancer development. These changes allow mutant cells to acquire the ability to overexpress certain proteins not expressed by normal cells . These cAptamers have recently emerged as potential molecular probes in the development of more specific and accurate cancer diagnostics and therapeutics due to their unique characteristics. Aptamers are single-stranded nucleic acid molecules (ssDNA or RNA) that recognize specific targets based on unique three-dimensional conformations . A wide Thus, we present a state-of-the-art aptamer review with a specific focus on their application to cancer. We summarize the latest advances in aptamer use for cancer diagnosis, highlighting their potential future role as promising molecules in cancer detection and imaging.12–1014 unique oligonucleotide sequences 20–80 bases in length. Protein-bound aptamers are eluted and amplified to create the library for the next round of selection. An enriched pool of potential binders generated from PCR amplification is used in the subsequent selection rounds. To increase the selection stringency, a negative selection step can be introduced. After multiple selection rounds, potential target-specific aptamers are obtained [Aptamers are single-stranded nucleic acid molecules (ssDNA or RNA) that recognize specific targets based on unique three-dimensional conformations . Aptamerobtained ,16.The resulting highly enriched pool of aptamers can be analyzed using next-generation sequencing for further characterization of individual aptamers . In factOver the past two decades, cell-SELEX technology has been used for the selection of specific aptamers with diagnostic and therapeutic purposes over a wide variety of cell surface targets, particularly for tumor cells. Multiple groups have reported the discovery of new important biomarkers in different types of cancer and other diseases through the use of cell-SELEX ,33,34,35The general protocol of cell-SELEX includes a positive selection step of incubation, elution, and amplification of binding aptamers, and a negative selection step is also necessary to remove sequences which bind to normal cells. In other words, the cell-SELEX method first starts with the incubation of the nucleic acid library with normal or control cells that express either normal surface proteins or do not express the target protein. This step is called the negative selection step. In the positive selection step, unbound aptamers are incubated with the target cancer cells. Candidate aptamers are recovered from the positive cell lines and amplified to become the library for the next round. This cycle of selection is repeated for as many rounds as necessary . ChanginDespite its enormous potential, cell-SELEX presents technical limitations such as cell conditions and the complexity of some cancer cell lines. It is important for the aptamer selection to avoid nonspecific uptake. Cell death leads to altered protein expression. Therefore, the elimination of these cells will favor the identification of specific aptamers. Second, it is important to choose the proper cell line because cell surface proteins are very complex in some cancers. Because of the complexity and heterogenicity of some cancer cells, it is critical to perform additional selection rounds against non-target cells in order to improve aptamer specificity. However, this selection process can be quite complex and may have a negative impact in terms of time and resources spent by the researcher .Currently, new selection methods based on cell-SELEX have emerged to improve the success rate of aptamer selection, such as target expressed on cell surface-SELEX (TECS-SELEX) , fluoresSeveral factors need to be taken into consideration before the selection process begins. One of these aspects is the design of the library for SELEX procedures. Researchers must decide whether they are working with DNA or RNA aptamers—a decision that depends on the purpose of the selection. DNA and RNA aptamers are functionally similar, but each has its own unique benefits. DNA aptamers are inherently more stable, cheaper, and easy to produce. RNA aptamers typically have more diverse three-dimensional conformations due to the 2′-OH group and stronger RNA-RNA interactions, which probably increase binding affinity and specificity. However, RNA aptamers need more steps between selection rounds than DNA aptamers, making the method potentially more cost- and labor-intensive. Whereas PCR is used to recover the DNA aptamer-bound sequences, RNA aptamers must first be reverse-transcribed into cDNA to enable subsequent PCR amplification and RNA in vitro transcription for the next selection round ,47.2), or O-methyl (OCH3) groups can increase the half-lives by reducing aptamer degradation. Capping the 3′end with an inverted dT residue can provide resistance to serum nucleases as well. The latest results of Kratschmer and Levy indicate that fully modified oligonucleotides  had the longest half-lives. They also demonstrated little degradation in human serum even after prolonged incubation [There are also several challenges in terms of in vivo application that remain unresolved. Aptamers are vulnerable to nuclease degradation and have short circulation times. They can be chemically modified to enhance their nuclease resistance and in vivo stability. Replacing the 2′-OH position with fluoro (F), amino , computed tomography, and other imaging techniques combined with flow cytometry, immunohistochemistry, and cancer biomarker assays in serum to reach a diagnosis in cancer patients . HoweverOne detection system used for cancer diagnosis is aptamers linked to nanoparticles (Apt-NP). The use of this chimera is a good alternative to achieve the specific extraction and detection of cancer cells in complex fluids such as blood. Due to the low molecular weight of the aptamer, this method allows cell-specific aptamers to be immobilized on the surface of nanoparticles to create an aptamer–gold nanoparticle (Apt-AuNP) system  or aptamLiu et al. developed an aptamer-conjugated gold nanoparticle strip biosensor (ANSB) for the detection of Ramos cells, even in human blood . In shorA sample containing Ramos cells and Thiol-TD05-NP migrate along the strip. The Thiol-TD05-AuNP-Ramos cell complex is captured on the test zone through the immobilized aptamer, and its accumulation produces a quantifiable red band. As a result of ANSB, approximately 4000 Ramos cells could be detected with the researcher’s eye and 800 cells with a portable strip reader. In similar studies, Medley et al. and Borghei et al. developed a colorimetric assay based on the color produced by the conjugated aptamer-nanoparticle ,77. In MA similar approach to an Apt-AuNP system was pursued using aptamers conjugated to magnetic nanoparticles (Apt-MNPs). Tan’s group developed an effective method for the detection of a minimum of 10 cancer cells in 250 μL of sample. The method consists of measuring the change of spin-spin relaxation time (ΔT2) of the surrounding media water protons. It is well-known that MNPs alone enhance the magnetic resonance signal of protons from surrounding water molecules. In this way, when aptamer-magnetic nanoparticle clusters recognize the cancer cells, they generate a magnetic field that brings a consequent decrease of the T2. Tan et al. were able to recognize human T-leukemic cells (CCRF-CEM) and colorectal cancer cell lines (DLD-1 and HCT 116) using specific aptamers for each cell line in different complex biological media, including fetal bovine serum, human plasma, and whole blood. This proof-of-concept study demonstrated the ability of the Apt-MNPs clusters to be a sensitive and specific method for cancer cells detection and diagnosis .6 cells/mL of HL-60 cells could be detected using this approach. In another study, Zhang et al. demonstrated the electrochemical detection of CCRF-CEM cells using Apt-MNPs conjugated with ferrous ferric oxide (Fe3O4). A competitive binding assay was performed in which T-acute leukemia cells competed efficiently with an AuNP-conjugated cDNA for binding specifically to the aptamer of the Fe3O4-Apt-MNP complex. The released AuNPs were treated via silver deposition in the presence of target cells. The AuNP-catalyzed silver deposition enhancement showed high sensitivity with a detection limit of 10 cells [3O4 nanoparticles (Apt-GMNPs). Ethidium bromide (EB) was intercalated into the stem of the aptamer hairpin. The presence of cancer cells caused the disruption of the hairpin structure of the aptamer, forcing the release of EB and a consequent decrease in electrochemical signal. Under optimal conditions, this technology could detect a range of leukemia cancer cells from 10 to 1 × 106 cell/mL. However, most of the ECL approaches achieve cell detection by capturing cells on the electrode surface. A novel sandwich sensing system through an aptamer-cell-aptamer architecture has been developed by several authors [63−/4−) as a redox probe. More recently, Crulhas et al. described a system to detect vascular endothelial growth factor (VEGF) and prostate-specific antigen (PSA) released by prostate cancer cells based on specific thiolated aptamers bound to a gold-covered electrode surface [A different approach was developed combining the biological recognition property of Apt-AuNP with a physical transduction technique , to achieve the detection of target cancer cells ,83. Zhan10 cells . Another10 cells . In this authors ,87,88,89 surface . This el surface .The use of aptamers for cancer diagnosis has also spread to other platforms. In 2009, Phillips and colleagues revealed the combination of aptamers with microfluidic devices to be an excellent platform for cancer diagnosis, since this device is cheap, simple, and able to detect multiple cancer cells . BrieflyQuantum dots (QDs) appear to be promising tools for cancer imaging and diagnosis because they can be easily conjugated with nucleic acids that specifically target biomolecules. QDs with distinct emission wavelengths can be ligated to different aptamers for multiple cancer detection . Kang etMolecular beacon aptamers have been used to monitor different kinds of targets, including cancer cells. A molecular beacon (MB) is a dual-labeled single stranded DNA with a fluorophore at the 5’ end and a quencher at the 3’ end. The molecule has a stem-loop structure where the fluorophore and quencher do not fluoresce when in close proximity to each other. However, if the probes hybridize to a target sequence, separating the fluorophore and quencher, they will fluoresce . Shi et Two years ago, Zhang et al. identified abnormal DNA methyltransferase activity which is closely associated with cancer, through the detection of DNA adenine methylation methyltransferase (Dam MTase) activity . Both a Fluorescent aptamer probes appear to be one of the most widely used imaging tool in aptamer research due to their low cost and high sensitivity . One of 2O3 nanoparticles (NPs) and used as MRI contrast agents. The formation of PEG-Gd2O3/aptamer-Ag NCs nanoprobes demonstrated their application as multimodal molecular imaging probes by enhancing the fluorescence emissions of each molecule in vitro [3O4 nanoparticles with a fluorescent silicon dioxide (SiO2) shell (MFS) were conjugated to TLS11a aptamers for liver cancer-cell-specific targeting and imaging [3O4-Au nanoparticle when interacted with different cancer cell lines. The authors concluded that the designed nanoprobe could both specifically bind breast cancer cells (4T1 cells) and be used as an MRI contrast agent.Magnetic resonance imaging (MRI) is an imaging technique that uses the behavior of protons in a magnetic field to construct 3D images of biological systems. In order to improve the sensitivity of this technique, small exogenous probes based on gadolinium (Gd(III)) or manganese (Mn(II)) complexes could be used as contrast agents due to their measurable influence on magnetic relaxation time (T1 and T2) . Over thin vitro . Newer cin vitro . They shin vitro . This coin vitro . In this imaging . The use imaging . The MRIDespite the vast number of aptamers mentioned in this review , only a ®) received FDA approval for age-related macular degeneration (AMD) therapy [®) [®) [®) [Regarding clinical trials for oncology treatment, several aptamers have undergone clinical evaluation for the treatment of different diseases. However, only one aptamer-based drug was approved by the Food and Drug Administration (FDA). In 2004, the anti-vascular endothelial growth factor (VEGF) aptamer, pegaptanib (Macugen therapy . This RNrapy [®) , ranibiz [®) [®) , and afl [®) [®) . Two aptamers for cancer therapy have failed to pass clinical trials: AS1411  and NOX-The number of available applications of in vitro-selected aptamers has increased significantly over the past 25 years, indicating an exciting future for aptamers as early, personalized cancer diagnostics . AptamerdK values), specificity (cross-reactivity data), secondary structure predictions and the biological, chemical, or thermal stability  of their aptamers. This would allow others to reproduce their results—a mechanism critical to the improvement of aptamer-based assays.Despite its promising future, the use of aptamers as cancer diagnostics is still mostly restricted to early stages. For several reasons, most currently-available aptamers have not gone beyond the laboratory setting. Cancer cells have complex membrane proteins and vary in composition across both tumor type and subtype, making the accurate identification of tumor cells complex. Further investigation into SELEX techniques are needed to identify high-quality aptamers with improved stability, simplified synthesis, and optimal target specificity. The use of next-generation sequencing (NGS) and bioinformatics coupled with the SELEX method appears to be the critical step needed to address these issues and push aptamers into the clinical setting . In addiIt is important to not only continue with further research and the optimization of existing aptamers, but also to focus on the development of new aptamers with new molecular targets. It is clear that the full potential of aptamer technology has not been reached. Addressing the challenges presented above is crucial to their successful future in personalized medicine. Therefore, the aptamer-based technologies discussed in this review provide an excellent alternative to traditional cancer diagnostic methods, and have the potential to soon radically change how we diagnose and treat cancer.


Solution-processed bulk heterojunction solar cells have experienced a remarkable acceleration in performances in the last two decades, reaching power conversion efficiencies above 10%. This impressive progress is the outcome of a simultaneous development of more advanced device architectures and of optimized semiconducting polymers. Several chemical approaches have been developed to fine-tune the optoelectronics and structural polymer parameters required to reach high efficiencies. Fluorination of the conjugated polymer backbone has appeared recently to be an especially promising approach for the development of efficient semiconducting polymers. As a matter of fact, most currently best-performing semiconducting polymers are using fluorine atoms in their conjugated backbone. In this review, we attempt to give an up-to-date overview of the latest results achieved on fluorinated polymers for solar cells and to highlight general polymer properties’ evolution trends related to the fluorination of their conjugated backbone. Solution-processed bulk heterojunction (BHJ) solar cells based on polymer composite active layers were first reported in 1995 ,3, thesePffBT4T-2OD polymer 2 derivative, for instance  for solar cells and to highlight general trends observed in material properties that may be helpful for future polymer solar cell (PSC) developments. The following will cover, in particular, the impact of fluorination on (i) polymer synthesis; (ii) polymer frontier orbital energy levels; (iii) active layer morphology; (iv) charge transport properties; and (v) charge generation and recombination kinetics. A final paragraph is devoted to n-type fluorinated polymers (vi).Possible physical mechanisms that may contribute to the remarkable increase in performances after backbone fluorination of different chemical systems have already been discussed in the literature. However, due to the large variety of molecular systems investigated so far and due to the complexity of the physics underlying the photovoltaic effect in organic BHJ devices, a general understanding of the impact of fluorination on the device performances is still lacking. This report therefore attempts to give an up-to-date overview of the latest results achieved on fluorinated polymers , short-circuit current density (Jsc) and fill-factor (FF), were significantly improved in regards to regiorandom BFRx copolymers  on the chemical units should have, at minimum, no detrimental impact on the reaction yield and, at most, could increase the reaction rate. Indeed, in Suzuki and Stille polycondensation, the fluorine introduction in the halogen-aryl group is known to increase the reaction rate, as the EWG introduction into the halogenated aryl is known to increase the rate of the oxidative addition step. Since this oxidative addition is considered to be the rate-limiting step of the catalytic cycle, the EWG introduction in the halogenated aryl should increase the reaction yield .…F, S…F and F…F) observed in fluorinated backbones. These factors are responsible for a significant solubility decrease of fluorinated polymers in regards to non-fluorinated analogues thiophene pro-quinoidal unit  does not increase the steric hindrance along the conjugated backbone. The lowering of the HOMO level by 0.11 eV, going from PTB5 to PTB4  induced by the backbone fluorination were attributed to differences in the active layer morphology, although the underlying physical mechanism remained unknown.In the first publications on conjugated backbone fluorination in semi-conducting polymers for PSC, the introduction of fluorine atoms was mainly reported to decrease the HOMO  and LUMO  energy levels. Indeed, fluorine is the chemical element with the highest electronegativity . Its strong electron-withdrawing nature should lower both FMO energy levels as demonstrated theoretically on the cyano electron-withdrawing group by Heeger and Brédas and experimentally by Zyung and co-workers on a poly-rivative ,51. Acco for PSC . They sh to PTB4 , leads tacceptor . ChangesPTB polymers still including a fluorine atom on the C3 position of the thienothiophene, but using side-chain carrying keto groups instead of the previously used ester groups  . They deeply investigated the structure-property relationships in this series, from the physico-chemical properties to solar cell performances. As previously observed, the fluorine substitution slightly decreases the HOMO energy level of the resulting polymers, with a difference of 0.06 eV obtained between the most fluorinated (PBF100) and the non-fluorinated polymers  calculations in order to get a deeper understanding of the experimental observations ,57,60,61Nevertheless, the experimentally observed decrease of both HOMO and LUMO levels is well reproduced by the DFT calculations. Moreover, the surface contour plots of the HOMO and LUMO levels are predicted to be rather unaffected by the fluorination , althoug…F and F…F interactions may influence the planarization  polymers  (PMMA) as a gate dielectric in top gate-bottom contact devices. The polymer exhibits an even more remarkable hole mobility of 9.0 cm2·V−1·s−1 when using a high-k P(VDF-TrFE) ferroelectric polymer as a gate dielectric. These mobility values are significantly larger than those reported previously for its non-fluorinated equivalent (3 × 10−3 cm2·V−1·s−1) ..31].PBDT[2F]T.For these materials, no difference in blend morphology was observed either by transmission electron microscopy or by photoluminescence quenching between the fluorinated and the non-fluorinated polymer. Indirect electric measurements (photocurrent measurements as a function of the effective voltage) suggest, nevertheless, that charges can be more efficiently separated and extracted in d-spacing measured by XRD on polymer films . Am. Amn-typrivative . The aut devices . Althougn-type polymers demonstrate that similar positive impacts could be expected from backbone fluorination for both components of the PSC active layer and thus pave the way towards all fluorinated polymer solar cells.These series of pioneering fluorinated Voc) increases when the HOMO of the electron-donor polymer is lowered. The introduction of the very electronegative fluorine atom into the polymer backbone also almost systematically lowers the LUMO of the polymer. If the LUMO is lowered as much as the HOMO (as is often observed), the energy band-gap and therefore the absorption wavelength range of the fluorinated polymer will remain comparable to that of its non-fluorinated analogue. An interesting auxochromic effect of the fluorine atom has sometimes been observed with a significant extinction coefficient improvement for fluorinated polymers.In this review, we presented the influence of backbone fluorination of π-conjugated polymers for photovoltaic applications. The trend observed systematically for such polymers is a lowering of their HOMO when compared to their non-fluorinated analogues. The lowering of the HOMO is of particular interest in photovoltaic applications, as the open circuit voltage , open-circuit voltage (Voc) and fill-factor (FF) values. Fluorinated electron-donor polymers are very promising candidates for this ideal polymer, as impressive photovoltaic parameters  with an overall PCE of 10.8% have been recently reported for such a polymer [On the other hand, tuning the frontier energy levels is a necessary but insufficient condition to reach high power conversion efficiency. The second generally observed trend when fluorine is introduced on the polymer backbone is the planarization of the backbone. The origin for this planarization is still under discussion, but the consequences have been clearly identified: fluorinated polymers tend to aggregate (even in solution) and the morphology they adopt in thin films when blended with PCBM is different from that obtained with their non-fluorinated analogues. We tentatively presented in this review the influence of fluorination on several macro-properties that are closely linked to each other: the morphology in thin films (pure polymer and blends), the charge carrier mobility (pure polymer and blends) and the charge carrier generation and recombination kinetics (blends). At this stage, it seems impossible to draw a complete picture of the influence of fluorination on each of these properties as some contradictory reports can be found in the literature. However, it is established that fluorinated electron-donor polymers presenting highly interesting properties for photovoltaic applications have been synthesized. The tendency of fluorinated polymers to aggregate as well as the fluorophobicity of PC polymer .Nevertheless, as exposed in the present review, complementary experimental and theoretical investigations are still needed in the field of fluorinated polymers in order to fully understand the effect of fluorine introduction into the backbone on interlinked optoelectronic and structural properties. By continuing these efforts, further increase in the photovoltaic device performances may be expected.


A cancer cell line originating from human epithelial colorectal adenocarcinoma (Caco-2 cells) serves as a high capacity model for a preclinical screening of drugs. Recent need for incorporating barrier tissue into multi-organ chips calls for inclusion of Caco-2 cells into microperfused environment.This article describes a series of systems biology insights obtained from comparing Caco-2 models cells grown as conventional 2D layer and in a microfluidic chip. When basic electrical parameters of Caco-2 monolayers were evaluated using impedance spectrometry and MTT assays, no differences were noted. On the other hand, the microarray profiling of mRNAs and miRNAs revealed that grows on a microfluidic chip leads to the change in the production of specific miRNA, which regulate a set of genes for cell adhesion molecules (CAMs), and provide for more complete differentiation of Caco-2 monolayer. Moreover, the sets of miRNAs secreted at the apical surface of Caco-2 monolayers grown in conventional 2D culture and in microfluidic device differ.When integrated into a multi-tissue platform, Caco-2 cells may aid in generating insights into complex pathophysiological processes, not possible to dissect in conventional cultures. Current methodology for evaluating novel drug candidates in preclinical models is far from being perfect due to the difference in the metabolism of laboratory animals and humans, the limitations of single component in vitro systems and the human body, and the restrictions of in silico modeling –3.The main goal of microfluidic organotypic chips is the emulation of human physiology on a miniature scale. As an in vitro approach with a great predictive power for human drug response it has a potential in the reduction of costly failures in the evaluation of drug efficacy and safety . Microfl5 liver cells and 8–6 104 endothelial cells in separate compartments and a total circulating media volume of 30 ml. Due to the use of external pumps and external media reservoirs embed in described system, tissue volumes represent only a tiny part of the overall circulating media.These are some necessary requirements for “human-on-a-chip” systems. First of all, these systems should be superior to the organotypical single organ equivalents, secondly, they should reflect systems biology of a human organism at a smaller scale by retaining functional connectivity of cultured organ and tissue representatives. A few multi-compartment microfluidic systems, which provide the simultaneous co-culture of different tissues, were described previously –12. A maWe have met the tissue-to-media volume challenge by integrating a peristaltic micropump and media reservoirs into a multi-organ-chip (MOC) of a standard microscope slide. Therefore, a minimized fluid-to-tissue ratio within the whole system was achieved, while ensuring control of a media velocity. There have been several designs produced, of which the two-tissue MOC design has proven the capability of the MOC platform to simultaneously maintain a human liver equivalent and a human skin biopsy. The selection of these two tissues has been guided by utility considerations as the liver is considered to be the prime target for toxicity of pharmaceutical drug candidates, while human skin is the target tissue for cosmetics. As a critical need of incorporating barrier tissue into MOC devices has been increasingly recognized , the nexThis article describes a series of systems biology insights extracted from Caco-2 models cells grown in a 2D layer and in a microfluidic chip. The aim was to chart a way for integration of Caco-2 component into multi-organ platforms, and in validating existing intestine on-the-chip models.To assess the state of the Caco-2 cells’ monolayer during the differentiation process, the basic electrical parameters of the monolayer were evaluated using impedance spectrometry Fig. . The graIn a subsequent experiment, the pre-formed confluent monolayers composed of 98 differentiated Caco-2 cells were incubated either under conventional conditions or in a 99 three-well microfluidic chip . One of Nevertheless, more detailed investigations showed that the impression that everything is fine with the monolayer grown in conventional conditions is incorrect.p-value < 0.0002). It is noteworthy that the mentioned genes encode the proteins responding to 57 different small molecule drugs, with the most abundant activity classes being anticholesteremic and antineoplastic ones.Metabolic properties of cells cultured under different flow conditions and the inherent variation in drug response heavily contribute to commonly reported fluctuations in predictive power of preclinical model system. Additionally, depending on cultivation conditions, the selection for the growth of particular subpopulations of cells may result in a variety of cellular model systems with properties that may differ from the original cell line . BecauseThis study shows that the druggable gene expression profiles of Caco-2 cells grown in microfluidic chip resemble ones observed in Caco-2 formed tumors, while substantially differing from that in conventional monolayer conditions. This observation indicates that conventional Caco-2 studies may overestimate the power of particular drug candidate to modulate a molecular target, thus, emphasizing the necessity of expanding the use of microfluidic devices chip in a drug discovery process.LAMA1 gene, encoding laminin-α1, was detected in the former (p = 0.022) . Af. Af59]. The TEER measurements were carried out at room temperature using an STX100C96 electrode (World Precision Instruments) and an EVOM volt-ohm meter instrument (World Precision Instruments) .Impedance spectra were measured in the frequency range from 20 Hz to 20 kHz using an impedance spectrometry system (SRC BioClinicum) and STX100C96 (World Precision Instruments) electrode at room temperature.


Staphylococcus aureus growth, can inhibit the catalytic activity of SrtA in fibrinogen (Fg) cell-clumping and immobilized fibronectin (Fn) adhesion assays. Molecular dynamics simulations and mutagenesis assays suggest that QEN binds to the binding sites of the SrtA G167A and V193A mutants. These findings indicate that QEN is a potential lead compound for the development of new anti-virulence agents against S. aureus infections.Sortase A (SrtA) is a cysteine transpeptidase of most Gram-positive bacteria that is responsible for the anchorage of many surface protein virulence factors to the cell wall layer. SrtA mutants are unable to display surface proteins and are defective in the establishment of infections without affecting microbial viability. In this study, we report that quercitrin (QEN), a natural compound that does not affect  Staphylococcus aureus (S. aureus), is the most commonly isolated human bacterial pathogen, which persistently and asymptomatically colonizes up to 20%–30% of humans and intermittently colonizes up to 50%–60% . The binding constants (KAs) of QEN to the binding sites on WT-SrtAt-test. Differences were considered statistically significant when p < 0.05.The statistical significances of all assays were calculated using the 2-tailed Student’s


Lung cancer is a major cause for cancer-related deaths. The detection of pulmonary cancer in the early stages can highly increase survival rate. Manual delineation of lung nodules by radiologists is a tedious task. We developed a novel computer-aided decision support system for lung nodule detection based on a 3D Deep Convolutional Neural Network (3DDCNN) for assisting the radiologists. Our decision support system provides a second opinion to the radiologists in lung cancer diagnostic decision making. In order to leverage 3-dimensional information from Computed Tomography (CT) scans, we applied median intensity projection and multi-Region Proposal Network (mRPN) for automatic selection of potential region-of-interests. Our Computer Aided Diagnosis (CAD) system has been trained and validated using LUNA16, ANODE09, and LIDC-IDR datasets; the experiments demonstrate the superior performance of our system, attaining sensitivity, specificity, AUROC, accuracy, of 98.4%, 92%, 96% and 98.51% with 2.1 FPs per scan. We integrated cloud computing, trained and validated our Cloud-Based 3DDCNN on the datasets provided by Shanghai Sixth People’s Hospital, as well as LUNA16, ANODE09, and LIDC-IDR. Our system outperformed the state-of-the-art systems and obtained an impressive 98.7% sensitivity at 1.97 FPs per scan. This shows the potentials of deep learning, in combination with cloud computing, for accurate and efficient lung nodule detection via CT imaging, which could help doctors and radiologists in treating lung cancer patients.  I.•A novel automated clinical decision support system for lung detection based on a 3D Deep Convolutional Neural Network (3DDCNN) architecture. In order to leverage 3-dimensional information from CT scans, we applied novel median intensity projection and introduced a novel multi-Region Proposal Network (mRPN) in our architecture for automatic selection of potential region-of-interest.•To further improve the efficiency and performance of our proposed model, we integrated cloud computing into our CAD system. Proposed computer-aided decision support system is used for nodule detection and for assistance of radiologists in clinical diagnosis at Shanghai Sixth Peoples Hospital.•A comprehensive experimental evaluation of our CAD system done on four different datasets with varying CT imaging parameters with existing state-of-the-art CAD systems for lung cancer detection demonstrated that our system outperformed the existing systems and obtained an impressive 98.7% sensitivity at 1.97 FPs per scan.Among different types of cancer, pulmonary cancer also refer to as lung cancer is considered to be one of the most deadly cancers. In 2018, there were approximately 2.2 million new pulmonary cancer cases and about 1.8 million deaths in U.S. within a year. Pulmonary cancer is an uncontrollable abnormal lung cells growth, referred to as nodules, whose detection in early stages is highly crucial to the effective control of disease progression and thus potentially increase the survival rate of the patient. Commonly used manual lung nodule delineation by radiologists on high-resolution and high-quality chest Computed Tomography (CT) is complex, time consuming and extremely tedious The rest of the paper is organized as follows. II.CAD system is one of the most common means to improve the accuracy of cancer diagnosis done by the radiologists and decrease the time required for interpretation of the CT images. CAD systems are further categorised as: Computer Aided Detection (CADe) systems and Computer Aided Diagnosis (CADx) systems. CADe systems assist in finding the locality of nodules in CT images acquired from different imaging modalities while on the other hand the CADx systems characterize and classify these detected lesions as malignant or benign tumors. In general, a CAD system designed for the detection of pulmonary lesions (nodules) has two steps namely candidate nodule detection and FP Elimination. Firstly, the Regions Of Interest (ROIs) are selected in the input CT image, then the lung nodule candidates are extracted. Teramoto and Fujita et al.et al.In the past few years, researchers have presented deep learning based CAD systems for cancer detection with promising results III.A.For the training of our proposed method for nodule detection, we used LUng Nodule Analysis (LUNA16) dataset B.CNN models have a tendency to overfit data in case of limited labeled training dataset C.1)Multi-scale ROI patches were generated by zooming in or out of the CT image in coronal, axial and sagittal views see . The mot2)Multi-angle ROI patches were generated by rotation of obtained multi-scale patches by small angle 3)In a two dimensional lung CT scans, most of the FPs are caused by the trachea and the blood vessels having tubular spatial structure. To reduce the noise caused by trachea and the blood vessels, we used a three dimensional bilateral smoothing filter D.Our proposed architecture uses the basic framework of the Faster R-CNN 1)To use the training ability of CNN model for lung cancer, the model should be both end-to-end and trained frequently trainable for ROI detection and classification such as, Faster R-CNN The proposed anchor in RPN Nodules detection carried out using the different levels of RPN results in improvement of the nodule detection since both diameter and volume are considered. Volumetric values (3D input) are in correlation with the diameter values (2D input), therefore the combination of both volume and diameter provides divergence for non-nodules.2)False Positive reduction is carried out using by novel 3DDCNN which is inspired by the ResNet-101 network In case of 3DDCNN, the feature map is defined as:According to radiologist annotations, we add a E.In the training process with multiple RPNs providing the region proposals, RPN loss function is applied for each RPN and Fast R-CNN loss function in an iteration. Our loss function can be defined by merging box regression and the cross-entropy loss:F.In this paper, we have proposed a two stage computer-assisted decision support system for lung cancer detection. To further improve the performance of our proposed method, we integrated cloud computing (Infrastructure as a Service (IaaS) by providing Virtual Machines, and Software as a Service (SaaS) by giving our 3DDCNN model) into our CAD system. The first stage for our proposed CAD system is the training of 3DDCNN model for the nodule candidate screening. The second stage is the reduction of false positive results from the first stage in order to improve the overall diagnosis decision making by our CAD system as shown in The proposed CAD system uses body area network (BAN) comprising of sensors attached to patients body to record physiological information and CT-scan for chest CT which are stored on the cloud storage and undergo pre-processing. Furthermore gateways are used to forward that data to storage cloud for further processing. We deployed 12 VMs, and 24 processing units in our dedicated cloud back-end. For each case the complete processing time is about 219 ± 25.47 seconds. HTCondor tool was used for real-time optimization and monitoring of computing resources, thus the radiologists and the physicians have updated responsive CAD system. 3DDCNN CAD system SaaS model was used to provide supportive decision support system for assistance of the radiologists whereas IaaS provided the GPU-acceleration, fast computation as well as storage resources. This model provides automatic support for on demand scalability of computing and storage resources. Moreover, Cloud-based 3DDCNN CAD system is more efficient and provides cost-effective solution since CAD results can be reviewed in real-time by multiple radiologists while a cloud back-end is taking care of computations. On the other hand, traditional stand-alone CAD systems have low performance and high computational cost with no feedback from multiple radiologists in real-time.IV.In this research work, we used two phase validation. The first phase is nodule detection without classification as done by other researchers A.Our 3DDCNN model was trained for 300 epochs during each fold of cross-validation. After approximately training 100 epochs, the loss on the validation set became more stable. The result for each fold was selected to be the one with lowest malignancy prediction loss on the validation dataset. If nodule malignancy is B.1)et al.et al.For evaluation of our proposed method’s nodule classification, we compared our results with the two state-of-the-art published methods, i.e. Dou C.1)We used a holdout validation set from LIDC dataset The effectiveness of 3DDCNN is verified by comparing with CNN 2)Performance comparison between our proposed CAD system versus state-of-the-art CAD systems on LIDC-IDR dataset is shown in We calculate FP rate between the number of non-nodule samples falsely predict as nodule with a certain level of malignancy and the total amount of non-nodule samples. In the following we give the definition of FP rate:3)We investigated the performance of our 3DDCNN system in comparison to Cloud-Based 3DDCNN system on 120 cases from Shanghai Sixth People’s Hospital using sensitivity and average FPs/Scan as parameter shown in D.As mentioned before the performance evaluation metric include Sensitivity, Specificity, AUROC, and false positives per scan (FPs/Scan). For further quantitative evaluation of our proposed work with the existing RCNN based methods, we used the statistical performance evaluation based on error calculation metrics which were used to determine the error rate on the testing dataset i.e. Clinical Dataset . To calculate the average difference between the radiologists nodule detection and the proposed method nodule detection we proposed the Detection Error Rate (50, AP75 and APm.Since, we obtained the detection results on three malignancy levels To quantitatively evaluate the results for our proposed method, we have measured Mean Detection Error Rate (Mean E.Qualitative results from V.For the detection of lung cancer, CAD systems are developed to assist the radiologist in the process of nodule detection by providing a reference opinion. From the performance comparisons it is evident that our proposed model 3DDCNN attained the highest results against other state-of-the-art systems for sensitivity and FPs per scan. Although the current tested performance metric of 3DDCNN is relatively high, it could be further improved. The performance was relatively less accurate in detecting micro nodules, therefore future work will investigate the detection of micro nodules whose diameter is less than 3 mm. To ensure our solution is scalable, future work will consider extending the training stage to include data from hospitals worldwide. Integrating more data-augmentation methods to increase the training sample in order to achieve more robustness and reduce the overfitting problem brought by local optimal. Another future direction for lung cancer CAD system is to propose CAD system that performs well on all nodule types maintaining good performance in terms of sensitivity and FPs/Scan, even if the dataset contains relatively less amount of such nodule types samples.