The metabolism of methane is an important part of the global carbon cycle. While deciphering the community function and the potential role of the different functional guilds is very difficult when considering native complex communities, synthetic communities, built of species originating from a study site in question, present a simplified model and allow specific questions to be addressed as to carbon, nitrogen, and other nutrient transfer among species in a controlled system. This study applies an ecophysiological approach, as a proof of principle, to an already well-studied model system, contributing to a better understanding of microbial community function and microbial ecosystem processes. Methylococcaceae methanotrophs and the Methylophilaceae methylotrophs, along with minor but persistent partners, members of Burkholderiales and Flavobacteriales. As a proof of concept, we established minimalist synthetic communities that were representative of the four functional guilds to demonstrate the dependency of the non-methane-utilizing species on the methanotrophs as the primary carbon-providing species. We observed that in communities consisting of multiple representatives of the key guilds, members of the same guild appeared to compete for resources. For example, when two methanotrophs of the same family were present, the two expressed similar key methanotrophy pathways and responded similarly to changing environmental conditions, suggesting that they perform a similar keystone function in situ. Similar observations were made for the Methylophilaceae. However, differences were noted in the expression of auxiliary and unique genes among strains of the same functional guild, reflecting differential adaptation and suggesting mechanisms for competition. At the same time, differences were also noted in the performances of partners with specific metabolic schemes. For example, a mutant of Methylotenera mobilis impaired in nitrate utilization behaved as a more efficient cooperator in methane consumption, suggesting that the loss of function may lead to changes in communal behavior. Overall, we demonstrate the robust nature of synthetic communities built of native lake sediment strains and their utility in addressing important ecological questions while using a simplified model.The factors and processes that influence the behavior and functionality of ecosystems inhabited by complex microbiomes are still far from being clearly understood. Synthetic microbial communities provide reduced-complexity models that allow an examination of ecological theories under defined and controlled conditions. In this study, we applied a multiphasic approach to study synthetic methane-oxidizing communities and species interactions as proxies to the natural communities. Our results confirm that, under selective pressures, natural-sediment communities of high complexity simplify rapidly, selecting for several major functional guilds, the major partners in methane oxidation being the  In this regard, a model synthetic system that links function to identity can help elucidate the relationships between microorganisms and ecosystem processes , suggesting that, likely, multicarbon substrates excreted by either Methylococcaceae or Methylophilaceae or both were supporting their growth. Several strains  of each species in cocultures steadily increased. The pure culture of Methylomonas sp. LW13 showed the highest optical densities compared to other coculture treatments (Methylomonas sp. LW13 to M. methylotrophus Q8 plus Acidovorax sp. 30 and Flavobacterium sp. 81) over the course of the experiment. As predicted, the nonmethanotroph partners  did not grow on methane as the sole source of carbon  DNA was observed over time  was employed. For this experiment, a four-strain coculture was established from ver time . Sequencn biases . Note thee bands  and C, ree bands . These DMethylococcaceae and some Methylophilaceae encode functions for either partial or complete respiratory denitrification  subunit  encodes functions for the  respiratory denitrification pathway , resulting in the loss of its ability to both grow with nitrate and to denitrify . Information on the strains employed in this study, along with the information on previously described Lake Washington sediment isolates, can be found in Burkholderiales nor Flavobacteriales encoded any known methylotrophy functions, and tests for growth on methane or methanol, respectively, were negative (data not shown).To test our concept of additional functional guilds as prominent members of methane-oxidizing communities, we carried out isolation experiments, aiming at obtaining pure cultures of mic data , 19. We mic data , 19, andmic data , 19. Dilmic data . Strains600) values. Prior to mixing, pure cultures were grown in either nitrate minimal salts (NMS) (4 was injected into the headspace. Tubes were incubated with shaking (200 rpm) at 18°C. The headspace gas composition was replenished every 24 h. The OD600 measurements were carried out using a Jenway 7300 spectrophotometer  every 4 to 6 h. The values presented represent the results of 3 measurements, reported with a standard error. For experiments involving Methylotenera mobilis JLW8, lanthanum (10 μM) was added to the medium, as this organism relies on lanthanides for growth on methanol   plus metts (NMS)  medium umethanol .To obtain real-time relative population abundances, cell numbers were determined by flow cytometry. The differences in cell shape and size allowed for separation of all four populations . Nine huMethylobacter tundripaludum 31/32, Methylophilus methylotrophus Q8, Acidovorax sp. 30s, and Flavobacterium sp. 81, were mixed at equal optical densities. Fifty-milliliter cocultures were grown in NMS medium in 250-ml vials, with 25% 13C-labeled methane/75% air (vol/vol) in the headspace for 24, 48, 72, and 96 h, using 3 vials per experiment. Their contents were mixed before DNA extraction. DNA was extracted using the MPBio FastDNA Spin kit for soil , following the manufacturer’s instructions. The heavy (13C-enriched) fractions of DNA were separated from the light (12C) fractions by CsCl-SYBR green dye density gradient ultracentrifugation, visualized under UV, and collected and purified following standard procedures, as previously described , samples were flushed with dinitrogen and supplemented with methane (25%), but not air, to create hypoxic conditions. Samples for RNA extraction were taken after 1, 25, and 49 h. Total RNA was extracted as previously described (Cocultures (50 ml) were assembled by mixing four or 14 strains at equal optical densities. These were grown in NMS medium with 25% methane/75% air (vol/vol) in the headspace, in duplicate. When cultures reached an ODThe transcript sequences have been archived with the IMG Genome Portal IMG genome identifiers (IDs) 1125205, 1125208, 1125211, 1125214, 1125217, 1125220, 1125223, 1125226, 1125229, 1125232, 1125235, and 1125238.


People high in negative affect (anxiety or depression) show cognitive distortions, specific thinking errors which contribute to the maintenance of their condition. It is thought that weak attentional control is a risk factor for negative affect and emotional disorders, because weak attentional control exaggerates the expression of attentional bias, another cognitive feature of emotional disorders. We wondered whether weak attentional control might similarly exaggerate the expression of cognitive distortions. In two samples of students from Turkey and the UK, we found that weak attentional control was indeed related to cognitive distortions, but this relationship was explained by both variables’ relationships with negative affect. This suggests that weak attentional control, while related to negative affect, does not necessarily exaggerate all of its cognitive features. There seems to be a limit on the affective consequences of poor attentional control, which may limit its clinical usefulness as a risk factor for emotional disorders. Disorders related to negative affect, i.e. anxiety and depression, are associated with certain cognitive distortions. In this article, we present evidence that the expression of such cognitive distortions is unrelated to attentional control.We studied the link between cognitive distortions and attentional control because attentional control has been implicated in the expression of another cognitive feature of emotional disorders and negative affect: attentional bias to threat (e.g.). StudieWe wanted to know whether the relationship between attentional control and attentional bias would generalise: whether attentional control would influence the expression of other cognitive features of negative affect or emotional disorders. There are two possibilities here: it is implied in the literature (e.g. ) that wecognitive distortions. These are habitual errors in thinking, not necessarily explicitly negative, but characteristic of emotional disorders [In the present studies, we tested whether attentional control was related to the expression of isorders –29. Muchisorders ), but thisorders , 32. Likisorders –37. StudStudy 1 was conducted with Turkish students studying at a small private university in Istanbul. We assessed their attentional control using the self-report scale commonly used in the attentional bias literature, their anxiety and depression, and their tendency to exhibit cognitive distortions.The study was approved by the Sabancı University Research Ethics Committee (FASS-2018-35), and participants gave their written consent.Mage = 19.47, SDage = 0.87). Data were collected in person by the last eight authors, as a class project.Eighty-two native Turkish speaking students from Sabancı University participated voluntarily. Six were excluded from analyses as they reported a current psychiatric diagnosis other than an anxiety or affective disorder, and another four were excluded for skipping too many questions. The final sample for analysis consisted of 72 students , but this correlation was somewhat smaller than anxiety’s and depression’s positive correlations with cognitive distortions (rs = .51 and .59).Descriptive statistics and correlations between variables are presented in R2 = .38, F  = 21.48, p < .001, and the standardised coefficient was significant for both anxiety, β = .25, p = .04, and depression, β = .43, p < .001. However, adding attentional control did not significantly increase model fit, R2 change = .02, Fchange  = 2.81, p = .10. Based on the literature summarised in the Introduction, the lack of a relationship between attentional control and cognitive distortions is surprising, and theoretically important. We studied this further by repeating our analysis as a Bayesian linear regression, using JASP [01, which indicates how much more likely the observed data are if the tested effect is absent in the population, relative to if the tested effect is present in the population. The BF01 for adding attentional control to our model was 1.24, representing weak evidence that attentional control did not have a unique relationship with distortions. The same was true when cognitive distortions in the achievement context replaced total distortions as the outcome, R2 change = .01, Fchange  = 1.62, p = .21, BF01 = 2.25; when distortions in the social context as the outcome, attentional control marginally increased model fit, R2 change = .03, Fchange  = 3.51, p = .07, BF01 = 0.81, but upon closer inspection this result was due to a strong correlation between attentional control and one particular distortion, the use of should statements  = 2.35, p = .13, BF01 = 1.38. This suggests that, with the exception of should statements in the social context, the relationship between attentional control and cognitive distortions is accounted for by anxiety and depression.Given that both attentional control and cognitive distortions correlated with both anxiety and depression, we wanted to check whether attentional control still predicted cognitive distortions independently from the effects of these variables. Total cognitive distortions were regressed on anxiety and depression, with attentional control entered in a second block. The first block model significantly predicted distortions, ents see . When thR2 change = .0002, Fchange  = 0.02, p = .88, BF01 = 3.91. We then checked whether attentional control moderated depression’s relationship with distortions; it did not, R2 change = .001, Fchange  = 0.15, p = .70, BF01 = 4.11. Finally, we repeated both moderation analyses with both social and achievement distortions as outcomes, no significant interactions emerged . These results suggest that attentional control does not moderate the relationship between negative affect (anxiety or depression) and cognitive distortions.We next checked to see whether attentional control moderated anxiety or depression’s relationships with cognitive distortions: as described in the Introduction, attentional control tends to moderate negative affect’s relationship with attentional bias. Total distortions were regressed on mean-centred attentional control and anxiety, with their interaction term entered in a second block. This interaction term did not improve model fit, Attentional control was related to the expression of cognitive distortions, but this relationship became nonsignificant when anxiety and depression were controlled, suggesting the relationship was explained by negative affect. Attentional control also did not moderate anxiety or depression’s relationships with cognitive distortions. These findings suggest that weak attentional control, while related to negative affect, does not necessarily exaggerate the expression of all negative affect’s cognitive features. There seems to be some limit on the psychopathological sequelae of poor attentional control, which may limit its clinical usefulness as a risk factor for emotional disorders.The Bayes factors for the moderation analyses above indicate quite convincing evidence that attentional control does not moderate negative affect’s relationship with cognitive distortions. However, the Bayes factors for the relationship between attentional control and distortions when negative affect is controlled do not indicate convincing evidence. In other words, Study 1 cannot completely rule out the possibility that there may be a unique relationship between attentional control and cognitive distortions. This is probably an issue of sample size; in Study 2 we were able to collect a much larger sample, and found more convincing evidence that no such unique relationship exists.r = -.40), which is rather stronger than the correlation between this distortion and depression (r = .28). This finding was not replicated in Study 2’s UK sample, reported below; so it is unclear whether this finding reflects a simple Type I error, or whether Turkish students are more likely to show such a relationship than are UK students. Özdel et al. [One complicating issue is that attentional control seemed to show a unique relationship with one particular distortion, the use of should statements in the social context. This is due to an unusually strong correlation between attentional control and this distortion . AlthouThe University of Kent’s Psychology Ethics Committee approved the study (201815180106374922), and participants gave their consent electronically before beginning the study.Mage = 19.59, SDage = 3.02). Participants were told the study was about “thinking styles, and which personal characteristics affect habits of thinking.” They completed the scales online.Two hundred and nine undergraduates from the University of Kent participated for course credit. Ten participants were excluded from analyses as they reported a current psychiatric diagnosis other than an anxiety or affective disorder , or chose not to disclose their diagnoses. The final sample for analysis therefore included 199 students . This pAgain, we first checked for a relationship between attentional control and cognitive distortions, and then whether attentional control moderated anxiety’s or depressions’ relationship with cognitive distortions.Correlations are presented in R = .54, R2 = .30, F  = 41.27, p < .001; the standardised coefficient was marginal for BAIT  and significant for BDI . However, adding attentional control did not improve model fit, R2 change = .003, Fchange  = 0.92, p = .34, indicating that the relationship between attentional control and cognitive distortions is accounted for by anxiety and depression: there is no unique relationship between these variables. This analysis was repeated as a Bayesian regression as before, comparing the full model to a null model including only BAIT and BDI. The BF01 for adding attentional control was 4.14, indicating good evidence that there truly is no unique relationship between attentional control and cognitive distortions. The hierarchical regression analysis was repeated for cognitive distortions in both the social and achievement contexts, and then all three analyses were repeated with STAI trait anxiety in the model in place of the BAIT. Attentional control did not improve model fit in any of these analyses .Again, we investigated the relationship between attentional control and cognitive distortions using hierarchical regression. Total cognitive distortions were regressed on BAIT and BDI scores in a first block, and attentional control was added in a second block. The first block model significantly predicted cognitive distortions, R2 change = .006, Fchange  = 1.34, p = .25. Bayesian linear regression showed that the evidence for the interaction’s being absent was somewhat stronger than the evidence for it being present, BF01 = 2.81. These analyses were then repeated with STAI trait anxiety in the model instead of the BAIT; again, adding the interaction term did not improve model fit, R2 change = .004, Fchange  = 1.12, p = .29, and the evidence for the interaction’s absence was stronger than the evidence for its presence, BF01 = 3.97. There was a significant interaction between depression and attentional control on total distortions, R2 change = .02, Fchange  = 6.76, p = .01, but this did not resemble the interaction of negative affect and attentional control on attentional bias reported in the literature: simple slopes calculated for low (mean– 1 SD), medium (mean), and high (mean + 1 SD) levels of depression revealed that attentional control only predicted distortions when depression was low , and not when depression was medium  or high , where distortions were more uniformly high. Essentially, attentional control’s relationship with cognitive distortions was only visible when depression was low; this simply shows that attentional control’s relationship with distortions was rather weaker than depression’s relationship with distortions. It was not the case that distortions were strongest when depression was high and control was low, as found in the attentional bias studies reviewed in the Introduction.Again, moderated regression was used to check for any interaction between trait anxiety and attentional control on total distortions. The interaction term did not significantly improve model fit, Again, attentional control showed a significant relationship with cognitive distortions; however, this relationship seems to be reliably and wholly accounted for by negative affect. The correlations between attentional control and STAI trait anxiety, BAIT trait anxiety, and depression were all very similar, suggesting that it is negative affect in general, not anxiety or depression specifically, which accounts for the relationship between attentional control and cognitive distortions.Unlike Study 1, this study found evidence for an interaction between depression and attentional control on cognitive distortions. However, this interaction was very unlike the interaction reported in the literature between attentional control and negative affect on attentional bias to threat. Instead, we found that attentional control’s relationship with distortions was only visible when depression was low; this is not consistent with the idea that low attentional control increases the expression of affect-related cognitive distortions. Rather, both these studies suggest that weak attentional control does not necessarily exaggerate all cognitive features of negative affect.In two studies, attentional control significantly predicted cognitive distortions, a key symptom–and presumably, a key cause–of negative affect and emotional disorders. However, more careful analyses showed that this relationship was essentially an artefact, resulting from the fact that both control and distortions correlate with negative affect .This could happen for two reasons. One possibility is that both attentional control and cognitive distortions are both independently related to negative affect, but not to each other. The other possibility is that people high in negative affect might have a response bias, so that they tend to respond in a negative way on any scale concerning their own performance or abilities, including both the ACS and the cognitive distortions scale; this could be considered a kind of common method bias , whsee also . Howeversee also . These rsee also , 14, it f2 = .03 Click here for additional data file.


BackgroundThe inhibition of programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) signaling pathway has been shown to be an effective targeted therapy in fighting both solid organ cancers and hematological malignancies. PD-L1 expression also serves as a prognostic marker in various cancers. However, the expression of PD-L1 and its prognostic significance in multiple myeloma remains largely unknown.MethodsImmunohistochemistry staining of PD-L1 was performed in bone marrow biopsy samples  in 32 myeloma patients receiving autologous stem cell transplant (ASCT) at various time points: before ASCT, post-ASCT, and/or at relapse after ASCT. More than 1% of myeloma cells with PD-L1 staining was considered a positive expression of PD-L1. A correlation analysis was performed between post-ASCT overall survival (OS) and the status of PD-L1 expression.ResultsIn this pilot study, a total of 11 patients (34%) out of our cohort (32 patients) were positive for PD-L1 expression at least once during the course of the disease. A dynamic change of PD-L1 expression was noted in three patients converting from negative (before ASCT) to positive (post-ASCT) and two patients converting from positive (before ASCT) to negative (post-ASCT). Patients with positive PD-L1 expression persisting or occurring post-ASCT had shorter post-ASCT overall survival than those with negative PD-L1 expression post-ASCT . No significant differences were detected in the known prognostic factors between these two groups at the time of ASCT. Pre-transplant PD-L1 expression status, however, showed no significant impact on post-ASCT overall survival. Furthermore, a few patients switching from positive PD-L1 expression before ASCT to negative PD-L1 expression post-ASCT had a relatively good post-ASCT overall survival .ConclusionImmunohistochemistry can be reliably used for measuring PD-L1 expression in decalcified marrow core biopsy materials. Our results suggest that positive PD-L1 expression persisting/occurring post-ASCT could be an adverse prognostic marker for post-ASCT OS. Additionally, PD-L1 expression appears to be dynamic and is subjected to change after ASCT. Our findings suggest that periodically monitoring PD-L1 expression in patients with multiple myeloma post-ASCT is warranted. Further studies are needed to confirm our initial observation and to evaluate if timely intervention with PD-L1 blockade can improve post-ASCT outcomes in myeloma patients. Multiple myeloma is a B cell malignancy producing monoclonal immunoglobulin and leading to organ damage, including kidney injury, lytic bone disease, hypercalcemia, and anemia . AccountThe membrane-bound molecules programmed cell death protein 1 (PD-1) and its ligand programmed death-ligand 1 or 2 (PD-L1 or PD-L2) belong to the immune checkpoint pathway . The binIn recent years, researchers have been studying and evaluating the PD-1/PDL-L1 blockade as a potential target for the treatment of multiple myeloma -8. StudiPatientsThis retrospective study was conducted on a total of 32 patients with multiple myeloma who received ASCT at the Florida Hospital Blood & Marrow Transplant Center from 2008 to 2013 and were deceased before December 2017. We were only able to obtain Institutional Review Board (IRB) of Florida Hospital approval for using samples of deceased patients without additional consent. All patients were diagnosed according to the International Myeloma Working Group (IMWG) criteria. All patients included in this study received similar chemotherapy regimens and underwent at least one ASCT, had a minimum of a three-month follow-up post-ASCT available for performing PD-L1 immunohistochemical staining.The clinical data and outcomes were obtained from the patients’ medical records and Florida Hospital Cancer Registry, including the patients’ age, gender, free light chain concentration/ratio, hemoglobin, platelet count, calcium level, albumin, b2-microglobulin, and survival time from stem cell transplant. These data were categorized according to the status of PD-L1 expression post-ASCT  were evaluated for this study. The hematoxylin and eosin (HE)-stained slides and syndecan-1 (CD138)-stained slides, which highlighted the plasma cells, of all samples were reviewed to confirm the diagnosis and the adequacy for the immunohistochemistry (IHC) staining of PD-L1. The PD-L1 stain was performed using Food and Drug Administration (FDA) approved monoclonal antibody  according to the manufacturer’s protocol, using decalcified formalin-fixed paraffin-embedded tissue sections. The PD-L1 stained slides of each case were scored by two pathologists independently without knowing any clinical data. The final score was the average score of the two pathologists. Greater than 1% of plasma cells with PD-L1 staining was considered positive for PD-L1 expression. The PD-L1 staining was mainly located in the cytoplasm and/or membrane. In addition to myeloma cells, PD-L1 expression was also detected in some tumor-infiltrating lymphocytes.Statistical analysisA T-test was performed to compare the difference in continuous variables, and χ2 was used to compare the difference in count numbers between PD-L1 negative and PD-L1 positive patients. A Kaplan Meier estimator was used to assess the survival time, and a log-rank test was used to compare the overall survival (OS) between PD-L1 negative and PD-L1 positive patients. All analyses were performed by using Stata version 14 . All 𝑃 values were two-tailed, and 𝛼< 0.05 was set as the level of statistical significance for all tests.PatientsIn this cohort, a total of 32 patients with relapsed multiple myeloma who underwent chemotherapy and stem cell transplant from Florida Hospital were studied . The median age of patients was 56 years (ranging from 44 to 70). The post-ASCT follow-up period ranged from five months to 88 months with a median of 23 months.PD-L1 expression during the disease coursePD-L1 expression was positive in five out of 26 (19.2%) patients (excluding six patients without pre-transplant staining) prior to the ASCT and nine out of 32 (28.1%) patients post-ASCT with the cutoff of 1% of plasma cells expressing PD-L1. Examples of PD-L1 expression via immunohistochemical staining are shown in Figure Prognostic significance of PD-L1 expression prior and post-ASCTThe median overall survival of this cohort post bone marrow transplant was 22 (18-27) months Figure . The mucOf note, the patients with positive PD-L1 expression post-ASCT showed shorter survival than those without PD-L1 expression  is a potent inducer and that multiple myeloma cells exhibited exaggerated upregulation of PD-L1 under INF-gamma induction as compared to normal plasma cells . More reOur findings suggest that patients with positive PD-L1 expression post-ASCT, but not prior to ASCT, have significantly shorter survival in our limited cohort. Additionally, we observed two patients who had a switch from PD-L1 positive expression prior to ASCT to PD-L1 negative expression. These two patients appear to have improved outcomes. Although it is difficult to assume any significance from two patients, nevertheless, taken together, these results suggest that PD-L1 expression in bone marrow has the potential to serve as a prognostic marker after ASCT. Monitoring PD-L1 expression in bone marrow for patients receiving ASCT could be employed to identify patients who are at high risk for poor outcomes, requiring additional treatment if these findings are confirmed by other, larger studies. Currently, no clinical trials have been published to demonstrate the efficacy of using PD-L1 inhibitors in treating patients with refractory multiple myeloma. Monitoring PD-L1 expression could be helpful to identify those high-risk patients during treatment.Similar to our findings, previous studies -12 showeSeveral mechanisms may attribute to the adverse outcomes of PD-L1 positive multiple myeloma patients post-ASCT. First, the PD-1/PD-L1 pathway negatively regulates T cell antigen receptor signaling. The expression of PD-L1 in multiple myeloma cells can serve as a mechanism for multiple myeloma cells to escape the host immune response -17. SecoOur study has two limitations. First, this study is a retrospective analysis with a limited sample size. Second, the study is further limited to only studying deceased patients after ASCT (as per our IRB policies). These limitations have likely biased the study toward a cohort of patients with high-risk disease and poorer outcomes. However, even within this group of patients, PD-L1 expression appears to be an important prognostic marker post-ASCT. Further studies are needed to confirm our initial observations, study if this observation is limited to post-ASCT patients or is generalizable to all MM patients, and evaluate if a checkpoint blockade may have a role in improving patient outcomes in the post-ASCT setting, particularly for those patients with positive PD-L1 expression.Immunohistochemistry staining can be reliably used for measuring PD-L1 expression in bone marrow core biopsy materials. Positive PD-L1 expression persisting/acquired post-ASCT could be an adverse prognostic marker for post-ASCT OS. Additionally, PD-L1 expression appears to be dynamic and is subjected to change after ASCT. Our findings suggest that periodically monitoring PD-L1 expression in patients with multiple myeloma post-ASCT is warranted.


Adsorption of Li and Na on pristine and defective graphene and graphene oxide (GO) is studied using density functional theory (DFT) structural and electronic calculations, quantum theory of atoms in molecules (QTAIM), and electron localization function (ELF) analyses. DFT calculations show that Li and Na adsorptions on pristine graphene are not stable at all metal coverages examined here. However, the presence of defects on graphene support stabilizes both Li and Na adsorptions. Increased Li and Na coverages cause metal nucleation and weaken adsorption. Defective graphene is associated with the presence of band gaps and, thus, Li and Na adsorptions can be used to tune these gaps. Electronic calculations show that Li– and Na–graphene interactions are Coulombic: as Li and Na coverages increase, the metal valences partially hybridize with the graphene bands and weaken metal–graphene support interactions. However, for Li adsorption on single vacancy graphene, QTAIM, ELF, and overlap populations calculations show that the Li-C bond has some covalent character. The Li and Na adsorptions on GO are significantly stronger than on graphene and strengthen upon increased coverages. This is due to Li and Na forming bonds with both carbon and oxygen GO atoms. QTAIM and ELF are used to analyze the metal–C and metal–metal bonds . The Li and Na clusters may contain both covalent and metallic intra metal–metal bonds: This effect is related to the adsorption support selection. ELF bifurcation diagrams show individual metal–C and metal–metal interactions, as Li and Na are adsorbed on graphene and GO, at the metal coverages examined here. Trations) . Currentrations) . The userations) ,9, C60, ectively . Graphenectively ,12,13. Gectively .Increased LIB graphene capacities relative to graphite seem indicative of increased Li atoms being adsorbed on graphene relative to graphite. However, the contrary has been observed via in situ Raman spectroscopy . Althoug6 clusters collapse during adsorption on 24 vacancies defective graphene [The main disadvantage of LIBs is Li cost, which has been rising steadily in recent years, with the exception of a price drop observed in the last quarter of 2018 . Li is agraphene .64 formation [+ size relative to Li+ (100 pm vs. 76 pm) and Na high ionization potential [Sodium ion batteries could serve as alternative batteries due to the abundance of Na. However, the NIB capacity of graphite is only 35 mAh/g, substantially lower relative to LIB . This isormation ,35 due totential . NIB capotential . DFT calotential . Specifiotential .adsE and cE, respectively) per metal, the metal–Carbon plane distances , and the electronic charge transfers from the metals to graphene and GO supports. The adsE and the n = 1, 2, …) on graphene and GO are defined asadsE is the minimum energy required to remove a single Li or Na atom during adsorption on graphene and GO, whereas cE measures the cluster-support interaction. For n = 1, adsE = cE. Single metals and their clusters form a bond with the support if cE < 0. The metal-support interaction is considered stable if the |adsE| is larger than the metal binding energy of its bulk. The metal-support interactions are also examined via electronic structure analyses.In this work, we examine Li and Na adsorptions on pristine and defective graphene, as well as on graphene oxide (GO), at various metal coverages, using periodic DFT, with and without long-range interactions. The metal-support interactions are elucidated the via changes in the adsorption and cohesive energies  and the electron localization function (ELF). QTAIM was developed by Bader and co-workers, who note that orbitals, much less wave functions, are unphysical in nature; thus, chemical bonds are not quantum observables ,39. Howe systems . ELF [41 systems ,43.n and Nan  adsorbed on pristine graphene, the Li and Na cE values, the heights h for the closest metal to the graphene plane, and the smaller of the metal–metal distances per configuration. The Lin/graphene and Nan/graphene  unit cells are built as follows: for n = 1, single metal atoms  are initially placed distant from graphene, followed by geometry optimizations; for n = 3, two metal atoms are placed distant from the previously optimized Li/graphene and Na/graphene structures, followed by geometry optimizations. A similar approach is followed for the Li5/graphene and Na5/graphene cases. n and Nan  adsorbed on single and double vacancy defective graphene, as well as the cE and geometrical parameters, in a similar fashion as in n and Nan  adsorbed on graphene oxide , with cE and geometrical parameters. Here, GO supports contain epoxides and hydroxyls at 25% and 18.75%, respectively (percentages relative to C). Our GO structure is in agreement with the proposed non-hydrated GO structure from Zhou and Bongiorno [Monolayer pristine graphene is modeled as a two-dimensional 4 ongiorno  . A large integration grid is used: this is a pruned grid with 75 radial and 974 angular points. The Crystal Orbital Overlap Populations (COOP) [For geometry optimizations, Brillouin zone integrations (Monkhorst-Pack grid) , the Ferc mixing , coupleds (COOP) , for sels (COOP)  and the s (COOP) , the lats (COOP) ,39. Hirss (COOP)  and smals (COOP) . The abss (COOP) , and thuadsE and cE calculations suffer from basis set superposition errors (BSSE) [adsE and cE values. Calculated adsE and cE values using the counterpoise correction are less negative in energy than corresponding uncorrected values [The stabilities of the final geometry conformations are secured via post-geometry optimizations. When Gaussian basis sets are used, s (BSSE) . The BSSd values ,49,67.The  program , which i density . Specifiectively ,71. Gattectively . Therefo2) serves as an additional criterion to classify non-polar covalent and metallic bonds [Ionic, covalent, and metallic bonds are classified via changes in nts r→cp . Ionic bp values . For metic bonds ,74. For nt bonds .The electron localization function (ELF) is an alternative to QTAIM for analyzing chemical bonding ,75,76,77 0≤ηr→≤1 . The ηr→vy atoms . Kohout Li to Sr . Savin eLi to Sr  interpreg basins . In QTAIins [ρr→ . Basins ins [ρr→ ). Only cdiagrams , with thg basins . The ELFg basins .3, Li5, Na3, and Na5, cE values is small. However, disagreements between the DFT calculations with and without D2 corrections are observed in the h values and the metal–metal closest distances  coverage, Li and Na are adsorbed on the hollow site, in agreement with past reports ,17,18,22ulations b,c. ThesadsE for Li and Na adsorptions on graphene and GO. At the same metal coverages, Li adsorption on pristine graphene is significantly stronger relative to Na, as shown by adsE, cE, and h values. This statement is extended for adsorption on defective graphene and GO, with the exception of Na3/GO calculations without D2 corrections (vide infra). Our Li and Na |adsE| values are smaller than the binding energies of bulk Li  and Na 63 eV/Li ): These 63 eV/Li  and Lian63 eV/Li . Althougguration , where ig energy . For adsexpected ,37. ThisadsE| values at all coverages are larger than the binding energies of their metallic bulk, in agreement with past reports [cE| from 3.44 eV to 2.14 eV. At 3/32 ML coverage, the presence of a single C vacancy causes the Li clusters to collapse, whereas in all other cases Li and Na form clusters, in a similar fashion as during adsorption on pristine graphene.Li and Na adsorptions on defective graphene are stable . Here, L reports ,37. The The strong adsorption of Na on defective graphene  is indicative of its possible use in NIB. Although, Na adsorption on graphene is not as strong as Li, its abundancy is a significant advantage over Li.3/GO geometry calculated without D2 corrections. 3/GO with and without D2 corrections. Therefore, for Na adsorption on GO, at coverages > 1/32 ML, it is important to include long-range interactions due to Na atoms’ large polarizations [3/GO, Na clustering is observed, in contrast to the Li3 case.Li and Na adsorptions on GO are also stable . Moreoveizations . For Na3cE| and, thus, the weakening of the metal-support interaction.Dobrota et al. showed that Na adsorption on graphene-OH compounds leads to Na being neutral . This is3 group [The metal–C bond strengths can be estimated via changes in their COOP values. The majority of the metal–C COOP values in 3 group . Our COO3 group . TherefoDE, DOS(DE) = 0) below the Fermi energy  and conduction bands (π*) meet in a linear dispersion fashion [DE downshift, due to Li and Na adsorptions on pristine graphene, is indicative of n-doped graphene support via charge transfer from Li and Na. At low Li and Na coverages, the DE is intact  and thus, partially occupied. The partial occupancy of these states decreases the Coulombic interaction between the metals and pristine graphene, as the metal coverage increases. This statement also applies for adsorption on defective graphene.At higher Li coverages, the s broken e,f, whercoverage f. HoweveD region f, turninDE above the Fermi energy and create band gaps. For example, the double vacancy graphene is a semiconductor [DE upshift is indicative of a p-doped graphene support [3 adsorbed on double vacancy graphene, the DE appears above the Fermi energy  . This vaED . Li and ED . The GO ion-VSCC ) and  point . In all cases here, with the exception of Li adsorbed on single vacancy graphene, QTAIM shows that metal–C bonds are ionic. Recall that for ionic bonding a et al. . Here n . The For Li/graphene, the bifurcation diagram reveals the Li and C core basins (C(Li) and C(C)) and the polysynaptic valence basin V a. For Nao et al. , who stugraphene . At highgraphene b and polgraphene c.At low metal coverages and for adsorption on single vacancy graphene, the first bifurcation point occurs at higher d region a. COOP, 3 clusters collapse during adsorption on single vacancy graphene and GO. The adsE calculations show that Li and Na adsorption on pristine graphene is unstable at all the metal coverages examined here, whereas the presence of defects in the graphene support stabilizes Li and Na adsorptions. Increased metal coverage weakens metal-graphene and strengthen metal-GO interactions. Adsorption on GO is strong due to simultaneous metal bonding with C and O GO atoms.Li and Na adsorptions on pristine and defective graphene and GO has been studied using periodic DFT structural and electronic calculations paired with COOP, QTAIM, and ELF analyzes. DFT optimized geometries show that increased metal coverage clusters Li and Na during adsorption on graphene, which lowers the efficiency of LIB and NIB. However, LiDE is pushed below the Fermi energy due to Li and Na adsorptions on pristine graphene and may be associated with small band gaps at higher metal coverages. The presence of defects on graphene is associated with DE shifting and bang gap openings.DOS calculations show that, at low metal coverages, Li and Na adsorptions on pristine does not affect the DOS spectra below the Fermi energy. This effect is indicative of Coulombic interactions between the adsorbates and the support. These interactions weaken as metal coverages increase, due to the downshift of the metal valances below the Fermi energy. The band calculations show that the The metal–C bonds are analyzed using COOP, QTAIM, and ELF. The COOP metal–C COOP values at the order expected for ionic bonding, for all cases except for adsorption on single vacancy graphene, at low metal coverages. QTAIM shows that only the Li-C bond contains covalent character for adsorption on single vacancy graphene, whereas in all other cases the metal–C bond is ionic. ELF partially agrees with QTAIM and shows that both Li-C and Na-C bonds are covalent for adsorption on single vacancy graphene. QTAIM and EFT are also used to study Li-Li and Na-Na bonds: Li and Na clusters may contain metallic and covalent metal–metal bonds depending on adsorption support .


Tribolium castaneum, while it is absent in honeybees, carpenter ants, and vinegar flies. In our study, we focus on cell proliferation in the adult mushroom bodies of T. castaneum. We reliably labelled the progenies of the adult persisting mushroom body neuroblasts and determined the proliferation rate under several olfactory conditions within the first week after adult eclosion. We found at least two phases of Kenyon cell proliferation in the early adult beetle. Our results suggest that the generation of Kenyon cells during the first three days after adult eclosion is mainly genetically predetermined and a continuation of the developmental processes (nature), whereas from day four on proliferation seems to be mainly dependent on the odour environment (nurture). Considering that the mushroom bodies are linked to learning and memory, neurogenesis in the mushroom bodies is part of the remodelling of neuronal circuits leading to the adaption to the environment and optimization of behaviour.Several studies showed adult persisting neurogenesis in insects, including the red flour beetle  Following development, when the nervous system first encounters environmental sensory input, it is crucial for the survival of an animal to adapt to the actual conditions and cues. Therefore, the genetically encoded wiring scheme of the nervous system must be altered. This plasticity typically occurs in special time windows of elevated susceptibility to sensory input 10 and song learning in birds13. However, several studies demonstrated the existence of critical periods also in insects19, with many focusing on the plasticity of the olfactory system in the vinegar fly Drosophila melanogaster22.The research on critical periods has mainly focused on vertebrates, where critical periods were among others found during the postnatal development of the visual system26. From there the information is relayed into the primary olfactory processing centres29 and further projected to the mushroom bodies (MB) and the lateral horn30, with the former being linked to olfactory learning and memory45.Many insects rely on their olfactory systems to accomplish various tasks, such as the localization of food sources, mating partners, and suitable habitats. The adult olfactory system of holometabolous insects is first confronted with environmental cues upon adult eclosion. These olfactory cues are mainly detected by the olfactory sensory neurons (OSN) housed in the chemosensory sensilla of the antennae and palpsApis mellifera and D. melanogaster, described synaptic plasticity, but no neurogenesis, in the mushroom bodies55. Since adult neurogenesis in the mushroom bodies was first described in the hemimetabolous house cricket Acheta domestica57, it has also been found in holometabolous insects, such as the noctuid moth Agrotis ipsilon58, as well as several beetle species59 including the red flour beetle Tribolium castaneum60.Studies in adult holometabolous insects, including T. castaneum is a member of the largest insect order (Coleoptera)62 and a major pest of stored cereal products64. With its annotated genome sequence67, an expanding genetic toolbox70 including strong systemic RNA interference (RNAi)73, and its relative longevity of up to two years74, T. castaneum represents an eligible beetle model for studying adult plasticity.T. castaneum26, to answer whether the adult persisting neurogenesis is genetically predetermined and continuation of developmental processes or if it is activity-dependent.We concentrated on olfaction which is supposed to be a main input into the mushroom bodies of 76. With this method, we reliably and exclusively labelled the mushroom body neuroblasts and their progeny, while neurogenesis is not present in other areas of the cerebral ganglion of the red flour beetle  methodtle Fig. . The neutle Fig. .Figure 1T. castaneum, adult-born Kenyon cells usually derive from two neuroblasts (NB) per hemisphere and send their axons into the core of the mushroom body peduncle (PED) and lobes as shown by Zhao and colleagues60  dependent behavioural response81.For enrichment of the olfactory environment we chose the beetles’ aggregation pheromone 4,8-dimethyldecanal (DMD)The mushroom body neuroblasts of beetles reared in mixed-sex groups of 20 individuals generate new Kenyon cells within the first four days after adult eclosion. During the first three days of the proliferation phase, the median daily proliferation rate of each neuroblast is 10 to 15 cells, with a reduction on day four Figs. . At day Isolating beetles as pupae lead to a proliferation phase lasting three days after adult eclosion. During this proliferation phase, each neuroblast gives rise to about 10 to 20 cells per day, decreasing on day three and significantly dropping at day four before from day five onwards no neurogenesis is detectable.Enriching the environment of group-reared beetles with DMD leads to a prolonged proliferation phase of five days. During the first three days of the proliferation phase, the median daily proliferation rate of each neuroblast equals to about 15 to 20 cells, with slight decrease on days four and five. At day 6 the proliferation rate drops significantly and afterwards no neurogenesis is detectable.orco injection) group-reared beetles with DMD leads to a proliferation phase lasting four days after adult eclosion. During the first four days each neuroblast’s median daily proliferation rate is 10 to 15 cells, with a high variation on day four  group-reared beetles with cis-3-hexenol leads to a proliferation phase lasting four days after adult eclosion. During the first three days of the proliferation phase, the daily proliferation rate per is 10 to about 15 cells, with a significant reduction on day four  differ in their response to the tested odours, compared to beetles injected with sham (DsRed) dsRNA .Electroantennographical recordings (EAG) were used to demonstrate that beetles with an RNAi-mediated systemic knockdown of Orco (dsRNA−8 − 10−6) of the beetle’s aggregation pheromone DMD leads to no obvious reaction of the antennae  of the food volatile cis-3-hexenol leads to no obvious responses. Stimulating with a dilution of 10−3 causes a slight response in the control group only. Whereas, at the highest tested concentration (10−2) both groups respond  as published for the red flour beetle by Zhao and colleaguessts Fig.  we studins Figs.  to answe79 prolongs the proliferation phase by one day, while stimulation with the food odour cis-3-hexenol leads to a prolongation of the proliferation phase for at least four days. This prolonging effect is mostly inhibited in beetles with a significantly reduced perception of the tested odorants , generated by RNAi mediated knockdown of orco.Rearing beetles in groups of 20 animals of both sexes leads to the generation of new Kenyon cells during the first four days after adult eclosion. This phase is shortened in isolated beetles by one day and prolonged by additional olfactory stimulation. Enriching the olfactory environment of group-reared beetles with the beetles’ aggregation pheromone DMD82. A second study based on these results asked what proportion of neurogenesis is caused by visual and olfactory stimuli83 and demonstrated that olfactory and visual deprivation is sufficient to decrease neurogenesis in adult crickets, regardless whether reared in groups or isolation. This clearly showed a link between sensory input and adult neurogenesis.A study in crickets already demonstrated that sensory deprivation by isolation results in significantly less neurogenesis among the Kenyon cells of young females, compared to group-reared femalesIn our study, we used a day-to-day analysis of adult neurogenesis together with the specific manipulation of the Orco dependent olfactory sensory neurons. This allowed us to temporally dissect adult neurogenesis into distinct phases and link them to olfactory activity.82, our data suggest that isolation reduces adult neurogenesis. We were able to demonstrate that this reduction is based on a shortened proliferation phase rather than an altered proliferation rate.Similarly to the results from cricketsIt seems that the generation of Kenyon cells during the first three days after adult eclosion is mainly genetically predetermined and a continuation of developmental processes rather than depending on sensory activity.orco knock-down in odour stimulated beetles is still longer than in isolated beetles. Since the beetles were kept under constant darkness, visual stimulation can be excluded, making tactile and gustatory cues the most likely triggers. This is partially in accordance with findings in crickets, were unilateral removal of the antennae, causing the loss of chemosensory as well as mechanosensory antennal input in the ipsilateral hemisphere, lead to less adult neurogenesis in the ipsilateral hemisphere83.The shorter proliferation phase in isolated beetles compared to group-reared beetles suggest an influence of social interactions. This is supported by the fact that the proliferation phase after The significant differences in the proliferation rate within the first three days might indicate an influence of odour stimulation. However, as the results for DMD and cis-3-hexenol are contradicting, we conclude that these differences are very likely artificial.orco injected beetles. Since after a RNAi mediated knockdown of orco the EAG response towards DMD is significantly reduced 26.This longer proliferation phase is massively shortened in dsRNAion Fig. , which s95. This could mean that continued exposure to high DMD concentration leads to a desensitisation of the OR/Orco complex, causing the shorter proliferation phase. Whereas, the response to cis-3-hexenol seemingly also perceived by IRs persists longer. Furthermore, there is the possibility of different pheromone vs. normal odour processing networks in the antennal lobes that provide the input to the MB as shown among others in Manduca sexta96, D. melanogaster97, and A. mellifera98. However, so far, such separate olfactory processing networks were not described in the red flour beetle and it remains unclear whether they exist.The different effects of DMD and cis-3-hexenol on the duration of the proliferation phase (five vs. eight days) might be explained already on the receptor level, as IRs, unlike ORs, do not exhibit adaptation to longer stimulationsorco injection, corresponding roughly to A4. Besides, we performed immunohistochemical staining against Orco in freshly eclosed (A0) and seven days old beetles (A7) using the cross-reactive Moth-R2 antiserum26. This resulted in no immunoreactivity after pupal injection of dsRNAorco  of the wild-type strains “San Bernadino”107 and “black”108, as well as the neuron labelling77 transgenic line EF1-B-DsRed  were used. The beetles were bred under constant darkness at about 28 °C and 40–50% relative humidity on organic whole grain wheat flour supplemented with 5% dried yeast powder and 0.05% Fumagilin-B  to prevent sporozoan infections109. For age determination, freshly eclosed beetles (A0) were collected and kept in mixed-sex groups of 20 in 68 ml Drosophila vials on about 20 g substrate.Red flour beetles . Injection was performed with glass micropipettes made from thin-walled glass capillaries  attached to a pressure ejection system . After injection, the beetles were transferred into a para-film sealed petri dish and incubated at 28 °C for 24 hours.Cold anaesthetized beetles were mounted ventral-up on microscope slides using double-faced adhesive tape and were subsequently injected with about 1 µl 5-ethynyl-2′-desoxyuridine (EdU) solution  and fixed in 0.01 M PBS containing 4% paraformaldehyde for 1–2 hours at room temperature or at 4 °C overnight. Fixation was stopped by rinsing 4 × 10 min in PBS supplemented with 0.3% Triton X-100 (PBS-TrX). Afterwards, the brains were transferred into a blocking solution  in PBS-TrX) and incubated for 3–4 hours at room temperature or overnight at 4 °C. The brains were then incubated with primary antibody solution  or 1 µl of the beetles aggregation pheromone 4,8-dimethyldecanal79 in a dilution of 1:1000 in silicone oil , on filter paper (1 cm²).For enhanced olfactory stimulation we reared 20 freshly eclosed adults in mixed-sex groups in para-film sealed 68 ml Orco dsRNA (dsRNAorco) and DsRed dsRNA (dsRNAsham) used as a control were synthesized as previously published26. Both dsRNAs were injected into pupae at about 30%–40% of total metamorphosis. The Orco knockdown was verified by immunohistochemistry against Orco  on cryo-sections of antennae as previously published26  and a confocal laser scanning microscope . Confocal image stacks were analysed with Amira 6 graphics software .For further processing, snapshots of single sections and projections generated in Amira were processed  in Adobe Photoshop CC . Final figure arrangements were performed in Adobe Illustrator CC.80. The tests were performed using five female beetles aged seven days after dsRNA injection, corresponding to about A4, with three repeated measurements per animal. The antennal response was recorded at 25 Hz via a custom-build amplifier attached to a data acquisition controller  using EAG 2000 software (Syntech). During the EAG recordings, the antennae were exposed to a constant flow (~3 l/min) of filtered and humidified air. The green leaf volatile cis-3-hexenol and the beetle’s aggregation pheromone DMD111 were used as test odours. They were presented as 1 s pulse via a stimulus controller  that looped in the odour diluted in silicone oil from a filter paper impregnated with 20 µl odour sample. Measurements were performed in minute-long intervals with increasing concentrations (10−8–10−2). Each repeat was preceded by the measurement of the reaction to DMD (positive control) and silicone oil, which was used as solvent)For electronantennographical recordings, we followed a previously published protocolwww.python.org) based custom scripts. Those scripts utilize the SciPy ecosystems (www.scipy.org) modules SciPy (version 1.2.1)112, Numpy (version 1.16.2)113, Matplotlib (version 3.0.3)114, and Pandas (version 0.24.2)115, as well as the additional modules scikit-posthocs 116, Seaborn (version 0.9.0)117, and localreg .Analysis of cell numbers and EAG responses including statistical comparison and plotting was performed using Python , Kruskal-Wallis test from SciPy and posthoc analysis by Dunn’s multiple comparison from scikit-posthocs with p-value correction using the Holm method119 were used.To test for significant differences between experimental groups, Scheirer-Ray-HareRaw EAG voltage trains exported from EAG 2000 were initially smoothed using robust LOESS  method from localreg to account for local spikes and subsequently normalized by subtracting the corresponding responses to silicone oil which was used as the solvent to dilute the test odours.Figures were plotted as vector graphics and final figure arrangements were performed in Adobe Illustrator CC.All experiments involving animals were performed in compliance with the guidelines of the European Union (Directive 2010/63/EU). As all experiments were on insects an approval of the study by an ethics committee was unnecessary.Supplementary information.Dataset 1.Dataset 2.


Idiopathic pulmonary fibrosis (IPF) is the most common of the idiopathic interstitial pneumonias. It is characterized by a chronic, progressive, fibrotic interstitial lung disease of unknown cause that occurs primarily in older adults. Its prevalence and incidence have appeared to be increasing over the last decades. Despite its unknown nature, several genetic and environmental factors have been associated with IPF. Moreover, its natural history is variable, but could change depending on the currently suggested phenotypes: rapidly progressive IPF, familial, combined pulmonary fibrosis and emphysema, pulmonary hypertension, and that associated with connective tissue diseases. Early recognition and accurate staging are likely to improve outcomes and induce a prompt initiation of antifibrotics therapy. Treatment is expected to be more effective in the early stages of the disease, while developments in treatment aim to improve the current median survival of 3–4 years after diagnosis. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease of unknown cause, often with characteristic imaging and histological appearances, which occurs primarily in older adults . The chaThe population burden and costs of IPF have grown due to higher rates of hospital admissions and deaths, the costs of which are secondary to those of disease progression . Its etiThis review is focused on epidemiological aspects, natural history, and phenotypes in IPF.Determining the epidemiology of IPF is a difficult task. Idiopathic pulmonary fibrosis shares problems and limitations with other chronic respiratory diseases, all suffering from blurred definitions, changing coding guidance and expert consensus, high under-diagnosis which often occurs very late in their natural history, as well as the status of rare disease occurrence. Overall, it makes determining and quantifying the distribution of IPF (and its determinants) in time and space a complex issue. We aim to summarize the available evidence on the incidence, mortality and burden of IPF globally and with other levels of granularity, notwithstanding the fact that the results will contain large degree of uncertainty.IPF accounts for 20% to half of all cases of interstitial lung disease (ILD), and represents the most frequent and severe of the idiopathic interstitial pneumonias (IIPs), a group of ILDs of unknown cause . Idiopathttps://vizhub.healthdata.org/gbd-compare). According to the GBD’s tools, there is wide heterogeneity in the distribution of this category of diseases by country, with prevalence ranging 30-fold from 214.5 per 100,000 in Guam to a low of 6.8 per 100,000 in Benin; mortality ranging 150-fold from 10.5 per 100,000 in Japan to a low of 0.064 per 100,000 in Burkina Faso; and the combined estimate of both morbidity and mortality, the so-called disability-adjusted life years (DALYs) ranging more than 60-fold from 173.1 per 100,000 in Guam to a low of 2.7 per 100,000 in Burkina Faso  , an initina Faso . The alrWithin ILD conditions, IPF accounts for 17–37% of all ILD diagnoses . HoweverA study of USA Medicare beneficiaries aged ≥65 years observed IPF rates higher than previously stated  [Beyond non-Caucasians, a Japanese study in Hokkaido explored IPF from 2003 to 2007, finding a prevalence of 10 per 100,000/year and an incidence of 2.23 per 100,000/year . They alSame as with prevalence and incidence rates, mortality statistics are subject to bias. As IPF is an under-recognized and misdiagnosed condition, it is likely to be underestimated at the time of death .Mortality from IPF appears to increase steadily worldwide, even considering the abovementioned overarching underestimation. In 2014, between 28,000 and 65,000 deaths in Europe and between 13,000 and 17,000 deaths in the United States were estimated [As with prevalence and incidence, male IPF mortality is higher than in females, increases with age, and appears to be increasingly common . Olson eTo conclude, IPF fulfils all determinants to be considered a rare disease, but compared with other respiratory diseases, studying IPF epidemiology is not easy for reasons including old studies with heterogeneous methodology and case definitions that have been sequentially modified by experts and consensus. Definitively, IPF prevalence and incidence trends are on the rise. IPF incidence is universally estimated to be around 10 per 100,000 per year, and is uncommon in those younger than 50 years old and more frequent from the sixth decade on. It is more frequent in males than females, and it can be observed in all races and ethnic origins, although in all likelihood with substantial variability. IPF natural history is relentlessly progressive, as is extensively discussed in the next chapter. Yet at the individual patient level, clinical variability is the rule and exacerbations cannot yet be predicted. More and better epidemiology might help us to better determine the burden and unmet needs of IPF, now and in the near future.Idiopathic pulmonary fibrosis is a disease of unknown cause, but there is an interaction between genetic and environmental risk factors .TERT, TERC, PARN, and RTEL1—genes involved in the maintenance of telomere length—are associated with an increased risk of IPF [DSP, AKAP13, CTNNA, and DPP9) responsible for cell adhesion, integrity, and mechanic transduction  also confer a predisposition to IPF [Different genetic alterations have been associated with an increased risk of IPF, such as shortened telomeres, oxidative injury, surfactant dysfunction , proteostatic dysregulation, endoplasmic reticulum stress, mitochondrial dysfunction leading to decreased alveolar epithelial cell proliferation, and the secretion of profibrotic mediators . Mutatiok of IPF . Variatin to IPF ,21.MUC5B substantially increases the risk of IPF. MUC5B codes for mucin 5B, a glycoprotein required for airway clearance and innate immune responses to bacteria. The rs35705950 minor allele leads to overexpression of mucin 5B in small-airway epithelial cells, a universal finding in patients with IPF (regardless of the MUC5B genotype) [A single-nucleotide polymorphism (rs35705950) in the promoter region of enotype) . Althougenotype) ,24.MUC5B, ATP11A, TOLLIP), and disease progression [There are also some studies that relate IPF risk, microbiome diversity, host defense pathways . Most importantly, risk factors that predispose to IPF—age, gender, cigarette smoke, and genetic variants—all influence epigenetic paths. An association of DNA methylation and histone modifications with the presence of disease and fibro-proliferation has been reported . The miRLet-7d, miR-200, and miR-32, which target the TGFβ pathway and other fibrosis-related pathways, are diminished, while miR-21, which promotes epithelial-mesenchymal transition, is increased in the alveolar cells of lungs of patients with IPF .Clinicians should consider ILD in the differential diagnosis for adults presenting with unexplained exertional dyspnea, chronic dry cough, or crackles on examination. Exertional dyspnea typically progresses over a period of months to years. Often, patients with ILD initially receive a diagnosis of heart failure or chronic obstructive pulmonary disease, suggesting that clinicians frequently fail to consider ILD in patients with dyspnea. In some cases, patients have presented with dyspnea and dry cough up to 5 years before ILD was diagnosed .There are several reasons why there may be a delay in referring patients with suspicion of IPF to a tertiary center. First, patients may wait for months or longer before consulting their general practitioner due to cough and dyspnea in exertion, the most common symptoms of IPF. Secondly, the suspicion of this “rare” disease might be missed by physicians working in primary health care, and recognition of its main clinical sign—crackles in lung auscultation—may be overlooked. Thirdly, a referral to tertiary care may be complicated by shortcomings in the referral letter and the absence of relevant clinical information such as tobacco use, occupational history, and information about previous medication, leading to delays in arranging an appointment and even to inappropriate examinations .Early recognition and accurate diagnosis are likely to improve outcomes through the avoidance of potentially harmful therapies  and prompt initiation of therapies (antifibrotics) that are effective even in the early stages of disease .The etiology of IPF is unknown, but different risk factors have been identified such as older age, male sex, and cigarette smoking ,30.Cigarette smoking, especially of more than 20 pack-years, is the most strongly associated environmental risk factor. This applies to both familial as well as sporadic IPF. Emphysema and pulmonary fibrosis are both related to smoking and they may even coexist in the same susceptible individuals ,32. HoweIncreased risk for IPF has been associated with several environmental exposures . Activities such as working with livestock and in agriculture have also been associated with IPF ,35.Several studies have investigated the possible role of chronic viral infection in the etiology of IPF . Currently, definitive conclusions about the role of infection in IPF cannot be made, despite the large number of related studies ,37. It iGastroesophageal reflux (GER) probably has a role in the pathogenesis of the IPF and its natural history because predisposition to aspirations or microaspirations of the gastroesophageal content could a constitute factor that triggers damage of the alveolar epithelium, which characterizes IPF. Abnormal GER is frequent in patients with IPF and, usually, is clinically silent. In addition, cases of exacerbation of the disease have been described in the context of aspiration. On the other hand, a decrease in cough has been observed in some cases of IPF in which anti-reflux measures are established. A retrospective study showed that patients with IPF and GER who received treatment for the latter presented lower mortality than patients without treatment ; howeverSome autoimmunity phenomena (large lymphocyte aggregates composed of CD3+ T cells and CD20+ B cells as well as the presence of autoantibodies) have been described in patients with IPF, suggesting a breakdown in immunological tolerance .Other risk factors such as diabetes mellitus  and obstIdiopathic pulmonary fibrosis is a disease with a poor prognosis. Retrospective studies suggest that the median survival time from diagnosis is 3–4 years . In mostThe natural history of the disease is variable and difficult to predict . Some paEarly referral to ILD specialty centers is important for accurate diagnosis and to start therapies that are effective (antifibrotics), even in the early stages of disease, and may be associated with improved outcomes .N-acetylcysteine interferon-γ, endothelin antagonists, and warfarin are ineffective or even harmful in patients with IPF [The ATS/ERS/JRS/ALAT (American Thoracic Society/European Respiratory Society/Japanese Respiratory Society/Latin American Thoracic Association) guideline for the treatment of IPF includes recommendations for the use of pharmacological and non-pharmacological treatments ,50. Two with IPF .Pulmonary rehabilitation improves exercise capacity and health-related quality of life  while loLung transplantation can prolong survival and improve lung function and quality of life for selected candidate patients with IPF ,59. WhetA phenotype is the outward manifestation of a gene or genes, may involve more than one organ system, and is dynamic, changing over time or in response to environmental factors . HoweverThere is compelling evidence that different phenotypes of IPF can be identified, with different rates of disease progression, including: rapidly progressive, familial, combined emphysema and pulmonary fibrosis, pulmonary hypertension, and that presenting with autoimmune features . SeveralTLR9, CCL18, antibodies against HSP70, and KL-6 [The course of patients with IPF is unpredictable and there remains prognosis heterogeneity. On the one hand, there are those patients who survive more than 5 years, usually with higher body mass indices with relatively preserved FVC, TLC, and DLCO . Howeverand KL-6 . Lung miand KL-6 Familial pulmonary fibrosis (FPF) is defined by the presence of at least two cases of pulmonary fibrosis within the same family . AlthougTINF2, TERT, TERC) that shorten telomere length; (2) Surfactant protein mutations in specific genes of surfactant proteins called SFTPA, SFTPB, SFTPC, and SFTPD [SFTPC ones; (3) Other syndromes with pulmonary fibrosis associated with monogenic disorders that are also associated with extrapulmonary manifestations have been described. The most typical are the Hermansky–Pudlak syndrome  and type I neurofibromatosis  [MUC5B, but other have also been related to IPF, i.e., TLR9 and TERT [It is believed that the transmission is autosomal dominant. Several genes have been linked with this phenotype: (1) Telomerase complex mutations (nd SFTPD . Among the iris) ; (4) In and TERT .Epigenetic changes have also been associated with FPF, such as methylation modifications of certain genes and microRNAs .MMP-1 or SFTPC genes), and shorter telomeres [Several studies have suggested that IIPs commonly associate with combined pulmonary fibrosis and emphysema (CPFE) and mostly (28–35%) with IPF ,72. It ielomeres ,71.Combined pulmonary fibrosis and emphysema is most frequent in males older than 65 years, and active or former smokers. It is characterized by severe dyspnea, very impaired transfer capacity for carbon monoxide, preserved lung volumes, and hypoxemia during exercise. Its diagnosis is based upon high-resolution computed tomography (HRCT) by the presence of fibrotic changes more evident in the lower lobes and the emphysematous component more present in the upper lobes .CPFE frequently (about 50%) presents with pulmonary hypertension, conferring worse prognosis than IPF alone, with a median survival time of about 25 months .There are no specific treatment recommendations for CPFE. It might be reasonable to treat both components—bronchodilators to treat the obstruction associated with the emphysematous component and antifibrotics for IPF . RegardiPulmonary hypertension (PH) is often found in patients with IPF, and its occurrence increases with IPF severity. Its prevalence remains unclear and estimates vary widely (from 3% to 86%), reflecting the heterogeneous subpopulations of patients studied, as well as the underlying disease severity, disease definition, and different diagnostic measures used . The preEchocardiography is the test of choice for the detection of PH, although its accuracy in patients with advanced respiratory disease is low. Definitive diagnosis of PH is established by RHC .At present, there is no specific therapy for PH associated with IPF. Published experience with targeted PH drug therapy is scarce, and so far there is no evidence from randomized controlled trials (RCTs) suggesting that PH drugs improve symptoms or other outcomes in patients with IPF . Hence, Connective tissue diseases (CTD) are a group of diseases characterized by circulating autoantibodies and systemic manifestations considered to be related to autoimmune-mediated organ damage. They include rheumatoid arthritis, systemic sclerosis, poly/dermatomyositis, systemic lupus, Sjögren’s syndrome, and mixed connective tissue disease . Most ofThe treatment of IPF in this phenotype should be the same as that of the CTD—mostly systemic steroids and/or immunosuppressive agents .Some authors proposed IPF acute exacerbators as a phenotype “per se”, but patients who develop acute exacerbations can merely be considered as an IPF complication . The difOther potential phenotypes might be the radiological phenotypes. However, nowadays some radiological features in IPF are considered to be related to the prognosis rather than to new phenotypes. On the one hand, the more extended interstitial infiltrates and/or honeycombing, the poorer prognosis; on the other hand, an atypical pattern confers a better prognosis . The appPulmonary function tests (PFTs) do not define distinct phenotypes, but certain characteristics can be associated with some phenotypes . For ins


Supramolecular hydrogels, obtained from small organic molecules, may be advantageous over polymeric ones for several applications, because these materials have some peculiar properties that differentiate them from the traditional polymeric hydrogels, such as elasticity, thixotropy, self-healing propensity, and biocompatibility. We report here the preparation of strong supramolecular pseudopeptide-based hydrogels that owe their strength to the introduction of graphene in the gelling mixture. These materials proved to be strong, stable, thermoreversible and elastic. The concentration of the gelator, the degree of graphene doping, and the nature of the trigger are crucial to get hydrogels with the desired properties, where a high storage modulus coexists with a good thixotropic behavior. Finally, NIH-3T3 cells were used to evaluate the cell response to the presence of the most promising hydrogels. The hydrogels biocompatibility remains good, if a small degree of graphene doping is introduced. Hydrogels have been extensively explored as promising biomaterials for biosensors, tissue engineering, and drug delivery systems ,2,3,4, bPeptide-based hydrogels ,14,15,16A strategy that has emerged for improving the functionality of supramolecular hydrogels is the incorporation of nanofillers ,21,22,23Recently, incorporation of graphene-based nanofillers in hydrogels has been used to tailor their properties ,39,40,41d-Oxd or d-pGlu moiety  in their skeleton [We recently reported several pseudopeptides that freeze water in low concentration and form hydrogels that possess high mechanical strength and transparency ,48,49. Askeleton ,51 that skeleton ,53 that skeleton ,55,56,57skeleton ,59, pH vskeleton ,61, enzyskeleton ,63, dissskeleton , and ultskeleton ,66. d-Oxd-l-Phe-CO(CH2)7CO-l-Phe-d-Oxd-OH A possesses two l-Phe-d-Oxd  dipeptide units coupled with an azelaic acid unit , and the hydrogels mechanical properties were tested and compared. The hydrogels biocompatibility was also tested.In this work, the pseudopeptide A (in 1% or in 2% w/w concentration) and an increasing amount of graphene in MilliQ water  and loss moduli (G″). All the analyzed hydrogels are characterized by a “solid-like” behavior, i.e., the storage modulus is approximately an order of magnitude higher than the loss component  that is compatible with cellular environment. Thus, we further analyzed all the hydrogels, with a particular interest in the properties of hydrogels 13–16.Considering the mechanical properties and the final pH of the 16 samples, we can gather that the most promising hydrogels for applications as biocompatible materials are hydrogels 1–16, multiple cycles composed of three steps were applied to the gels. During the first step, the sample was subjected to a strain value within the LVE region and was characterized by G’ values greater than G’’. When the applied strain was increased above the crossover point, the sample behavior switched from gel-like to sol-like, with G’’ values greater than G’. Finally, the sample was leaved at fixed strain within the LVE range to check the recovery of the gel-like behavior.Self-healing properties ,71,72,7313–16 are characterized by a great capability to recover the gel-like behavior and confirm at the molecular level their thixotropic properties, as they all fully recover the “solid-like” behavior when the strain level goes back within the LVE region , a significant toxic effect on fibroblasts is observed. However, after 48 h the cells continue to proliferate both in hydrogel 13 (without graphene) and in hydrogel 14–16, insensitive to the graphene doping concentration. This mean that the scaffold support cell growth. Supramolecular hydrogel, doped with graphene, may represent a promising building block for the development of smart materials for biomolecules and tissue engineering purposes. The degradation of the gel does not modify the graphene toxicity. We measured the cell viability after the destruction of the matrix, and the results, in Cell adhesion is strongly influenced by graphene doping. Hydrogel All chemicals and solvents were purchased from Sigma-Aldrich , VWR , or Iris Biotech GMBH  and used as received. Acetonitrile was distilled under inert atmosphere before use. MilliQ water  was used throughout. Solvent were dried by distillation before use. All reactions were carried out in dried glassware.d-Thr, l-Phe, and azelaic acid following a multistep procedure in solution, reported in [The compound was synthesized from orted in ,662 according to [Three dispersions of MilliQ water (20 mL each) containing edge-functionalized graphene,  were prepared and vigorously stirred for ~20 min with a magnetic stirrer. The graphene sample, produced by 24 h ball-milling in the presence of COrding to , was kinrding to .A  was placed in a test tube (diameter: 8 mm), then MilliQ water (0.5 mL) or a graphene solution  was placed in a test tube (diameter: 8 mm), then arginine  was added. 0.5 mL of MilliQ water or a graphene solution (see A portion of gelator gel was determined by heating some test tubes (diameter: 8 mm) containing the gel and a glass ball  on the top of it. When the gel is formed, the ball is suspended atop. The Tgel is the temperatures in which the ball starts to penetrate inside the gel. Some hydrogel samples melt, producing a clear solution, whereas in other cases, the gelator shrinks and water is ejected, as syneresis occurs.T1–16 were freeze-dried using a BENCHTOP Freeze Dry System LABCONCO 7740030  with the following procedure; the hydrogel was prepared into an Eppendorf test tube at room temperature. After 16 h, the samples were deepened in liquid nitrogen for 10 min, then freeze-dried for 24 h in vacuo (0.2 mBar) at −50 °C.Samples of hydrogels Scanning electron micrographs of the samples were recorded using a Hitachi 6400 field emission gun scanning electron microscope operating at 15 kV .−1. Once the LVE of each hydrogel was established, frequency sweep tests were performed (ω: 0.1–100 rad s−1) at a constant strain within the LVE region of each sample (γ = 0.04%). Step strain experiments were performed on hydrogels to analyze the thixotropic behavior of the material. The sample was subjected to consecutive deformation and recovery steps. The recovery step was performed by keeping the sample at a constant strain γ = 0.04%, i.e., within the LVE region, for a period of 400 s. The deformation step was performed by applying to the gel a constant strain γ = 100%, i.e., above the LVE region of the sample for a period of 300 s. The cycles were performed 3 times at a fixed frequency ω = 10 rad s−1.Rheology experiments were carried out on an Anton Paar Rheometer MCR 102  using a parallel plate configuration (25 mm diameter). Experiments were performed at constant temperature of 23 °C controlled by the integrated Peltier system and a Julabo AWC100 cooling system. Oscillatory amplitude sweep experiments (γ: 0.01–100%) were carried out to determine the linear viscoelastic (LVE) range at a fixed frequency of 1 rad sHigh quality infrared spectra (64 scans) were obtained with an ATR-FT-IR Bruker Alpha System spectrometer (64 scans) .−1 glucose), supplemented with 10% (v/v) Fetal Bovine Serum, 2 mM l-glutamine, 0.1 mM MEM Nonessential Amino Acids (NEAA), 100 U mL−1 penicillin, and 100 U mL−1 streptomycin in a humidified incubator set at 37 °C with 5% CO2. For cell culture, the samples were sterilized with ultraviolet (UV) radiation for 20 min before use. The gels were incubated for 30 min at 37 °C with 5% CO2 with complete medium. Cells were seeded on gel in 24-well plates at a density of 105 cells per mL.Mouse embryonic fibroblast (NIH-3T3) cells were cultured under standard conditions in Dulbecco’s modified Eagle’s medium (DMEM) . We included a negative control of only medium without cells to determine the background signal and a positive control of 100% reduced resazurin reagent without cells.Cell viability was determined by resazurin reduction assay; the reagent is an oxidized form of the redox indicator that is blue in color and nonfluorescent. When incubated with viable cells, the reagent is reduced and it changes its color from blue to red becoming fluorescent. Briefly, cells were seeded on gels with complete medium. After incubation times, the resazurin reagent was added directly to the culture medium with 10% volume of medium contained in each sample and incubated for 4 h at 37 °C with 5% CO1–8  proved to be very strong but quite rigid, as testified by the analysis of the values of storage moduli and loss moduli variation during step strain experiments. In contrast, hydrogels 13–16  are very strong. SEM analysis of the corresponding aerogels demonstrates that the intimate structure of these hydrogels is formed by fibrous networks lacking in the other aerogels, which are characterized by locally oriented long strips that cross on the large scale. We also tested the biocompatibility of the most promising hydrogels 13–16 by assessing the bioavailability of mouse embryonic fibroblast (NIH-3T3) cells in the hydrogels, where pure water was replaced with complete medium serum. Cell adhesion on the hydrogel is strongly influenced by graphene doping. The hydrogel scaffold support cell growth at all the concentration of graphene doping.We report here the preparation of strong supramolecular hydrogels that owe their peculiar properties to the introduction of graphene in the gelling mixture. These materials are strong, stable, thermoreversible, and elastic. The concentration of the gelator, the degree of graphene doping and the nature of the trigger are crucial to get hydrogels with the desired properties, where a high storage modulus coexists with good self-healing properties. Indeed, the hydrogels In conclusion, in this paper, we demonstrated that the rheological properties and the biocompatibility of these graphene-hybrid hydrogels may be easily tuned selecting (i) the degree of the graphene doping and (ii) the nature of the gelator in the gelling mixture.


With the application of a recently developed deposition method called initiated chemical vapor deposition (iCVD), responsive hydrogel thin films in the order of a few hundred nanometers were created. When in contact with humid air, the hydrogel layer increases its thickness considerably. The measurement of the thickness change was realized interferometrically with a laser and a broadband light source in two different implementations. The relative change in thickness with respect to humidity can be described with the Flory–Huggins theory. The required Flory–Huggins interaction parameter was determined for the actual hydrogel composition. The setup was designed without electric components in the vicinity of the active sensor layer and is therefore applicable in harsh environments such as explosive or corrosive ones. The implemented sensor prototype delivered reproducible relative humidity ( Water is involved in many processes in life and technology and plays an important role in the climate system. The term moisture refers to the amount of liquid water in a gas in the form of small droplets, whereas by humidity the amount of gaseous water is meant ,2. HumidDevices for measuring humidity are called hygrometers, which quantify the amount of water vapor in the air. There are several principles ranging from ancient methods based on the elongation of a hygroscopic material , measuring the temperature difference between a dry and wet bulb (psychrometer) to optical detection of the condensation on a chilled surface (dew point hygrometer) ,6. A verof water . Since tNew materials and measurement techniques are under investigation for the development of novel humidity sensors. A promising class of materials are hydrophilic polymer networks that can absorb water in their meshes, the so-called hydrogels. Electronic properties of these hydrogels are related to the humidity of the surrounding atmosphere , but theThe correlation of the relative thickness change and the relative humidity was used ,15. The Developing a new sensor principle involves contributions from different fields of science. Certainly an important part is the hydrogel itself and its deposition, which is well-explored in the field of polymer, surface and thin-film physics ,18,19,20Variation of Equation  12]. from a hot wire suspended above the substrate. The created radicals react with the vinyl bonds of the monomers, which are vaporized and fed into the vacuum chamber. The substrate is kept at temperatures between 10 and 60 ications ,17.In the present study, the chosen deposition conditions were the following. HEMA was heated up to 75 r et al. . With thr et al. . The refl. [1.44 , a transThe optical detection was realized in two implementations, which are both based on interference phenomena. The first implementation consists of a laser and a photo detector and is able to measure relative thickness changes with a time resolution of The optical path and the principal detection setup are sketched in Equation .(2)Ir=|Equation  using thIf the wavelength Equation  leads toThe experimental setup was implemented with the substrate mounted on the sidewall of a measurement chamber shown in In another implementation of the optical setup, the hydrogel was deposited onto the tip of a fiber probe, which was illuminated and detected with the same components used for the tabletop solution connected with a fiber beam splitter. This setup was more robust in terms of optical misalignment and therefore easy applicable for future field measurements. The optical path can be described with the same model a and an The basic outcomes of the performed measurements were different intensities and raw spectra, which vary with the hydrogel thickness depending on the humidity in the chamber. To evaluate the model shown in The total increase of the hydrogel thickness can be observed in Equation ), which The Flory–Huggins theory was evaluated over a humidity range from 3% RH to 97% RH. Thus, the data from the spectral reflectance measurements were used for this evaluation. The initial thickness Equation ) was derEquation  for the  solvent . The sma process . Such cr% to 90% ,24. In p% to 90% . Since tBy applying the derived Equation , the corWith the approval of the Flory–Huggins model function and the determination of the corresponding interaction parameter surement a as wellsurement b. The laThe response time is commonly defined as the  sensors .The response times of the hydrogel sensor are much shorter than expected from previous experiments . It was In the presented work, hydrogel thin films produced by initiated chemical vapor deposition were characterized and their capability as humidity sensor was investigated. The theory underlying the sensor by Flory–Huggins was evaluated and the corresponding interaction parameter