CH4 is oxidized by methane oxidizing bacteria (MOB), and subsequently utilized by chironomid larvae, which may exhibit low δ13C values. This has been shown for chironomid larvae collected from lakes, streams and backwater pools. However, the relationship between CH4 concentrations and δ13C values of chironomid larvae for in-stream impoundments is unknown. CH4 concentrations were measured in eleven in-stream impoundments located in the Queich River catchment area, South-western Germany. Furthermore, the δ13C values of two subfamilies of chironomid larvae (i.e. Chironomini and Tanypodinae) were determined and correlated with CH4 concentrations. Chironomini larvae had lower mean δ13C values (−29.2 to −25.5 ‰), than Tanypodinae larvae (−26.9 to −25.3 ‰). No significant relationships were established between CH4 concentrations and δ13C values of chironomids (p>0.05). Mean δ13C values of chironomid larvae  were similar to those of sedimentary organic matter (SOM)  and tree leaf litter . We suggest that CH4 concentration has limited influence on the benthic food web in stream impoundments.Methane gas (CH Kiyashko et al. 13C values for some chironomid larvae than for particulate organic matter in lakes. The observed differences between δ13C values of chironomid larvae and potential food resources led to the conclusion that the chironomid larvae might have fed on MOB, which has very low δ13C values. The carbon isotope composition of consumers (e.g. insects) is determined by their diet and usually potrays an enrichment by about 1 ‰, even though the δ13C can deviate from −3 ‰ to +3 ‰ These quantitative estimates are based on the stable carbon isotope signature  of chironomid larvae in such systems has not been examined. Globally, there exist millions of small impoundments  the relationship between CH4 concentrations and δ13C values of chironomid larvae in impoundments located in the Queich River catchment area, South-western Germany. We hypothesized that CH4 would have a significant influence on the δ13C of chironomid larvae.In shallow aquatic systems such as rice paddies and small lakes, CHThis study was conducted in the Queich River catchment area (see coordinates below) and was not conducted in an area requiring research permit  or private land. This study did not involve endangered or protected species.o10′6oN 7o50′48oE) and flows  through the upper Rhine Valley to its confluence with the Rhine River in Germersheim (49o13′39oN 8o23′4oE). The catchment (area: 271 km2) is primarily covered by sandstone and is between 100 m and 673 m above sea level. The Rhineland-Palatinate region has dry climate conditions in summer.The study was conducted in the Queich River catchment area, Rhineland-Palatinate State, South-western Germany. The Queich River (length: 52 km) originates from the Palatinate forest  have been constructed on the main stem and tributaries of the Queich River, South-western Germany, for various purposes such as hydropower generation and flood control. Here, we selected eleven study sites . Average water depth was computed from three measurements taken on a transect across the river channel and current speed was estimated by timing a float over a distance of 5 metres Field measurements and sampling were conducted between 9T is the water residence time, V is the volume of water stored in the impoundment, and Q is the water discharge 4 in stream and pore-water were measured at the impoundments. Water samples were collected from each study impoundment using 20 mL serum bottles. The samples for stream water CH4 analysis were collected by filling water to the sample bottles from the bottom to top, and overflowing the sample bottles several times over. Three bottles were completely filled with water at each sampling site and several drops (250 µL) of mercuric chloride were added to each bottle as preservative 4 analyser . A closed loop was created between the gas inlet and outlet of the analyser. A gas tight syringe was then used to inject 0.5 mL gas sample into the closed loop. The CH4 concentrations were averaged over 30 seconds before and after gas injection. Concentration of the injected gas was computed as:samplec is the mol fraction of the sampled gas in parts per million, LosGatosΔc is the change in mole fraction before and after gas sample injection, LosGatosV = 92.5 mL and InjectionV = 0.5 mL.Concentrations of dissolved CH4 analysis were obtained at the impoundments, where fine sediment accumulated. Pore-water CH4 concentrations were assessed from sediment cores (1 core per site). Cores were taken at location of soft sediment using a piston corer and analyzed for porosity, carbon:nitrogen (C:N) ratio, total organic carbon (TOC) content and pore-water CH4 concentration. Cut-off syringes (3 mL) were used to extract sediment sub-samples which were immediately placed into crimp capped 20 mL vials containing 3 mL of 2.5% NaCl solution for conservation of the CH4. Pore-water was sampled in the cores from the homogenized upper (0–10 cm) sediment layer, where chironomid larvae are found 4 samples were measured as described for water samples. For C:N ratio, TOC and porosity, three sediment sub-samples were extracted from the cores (0–10 cm) and placed into glass tubes before analysis in the laboratory.Sediment samples for pore-water CH13C variability from 9 replicates. Second instar larvae were discarded as they were too small for identification and their mass was insufficient for isotope analysis. Before isotope analysis, chironomids were placed into glass tubes, oven dried at 60°C for 24 hours and subsequently stored in a desiccator.Chironomid samples were collected from the deepest point in each impoundment using an Ekman grab sampler . Sediments were sieved by passing them through two metal sieves (mesh size: 500 µm and 2 mm). Materials such as stones and large pieces of organic matter (>5 cm) were removed and chironomid larvae were picked from a sorting tray using forceps and placed into 500 mL sample bottles containing river water. In some sites only few chironomids (<5 chironomids) were found. The chironomid samples were transported to the laboratory and transferred to sample bottles containing clean tap water for 24 hours to allow gut clearance. Faecal materials were periodically removed to prevent ingestion by chironomids 13C of sedimentary organic matter (SOM), TOC and C:N ratios. They were rinsed with a 2.5% HCL solution for four hours to remove carbonates 13C of potential allochthonous food resources, were collected from trees near the impoundments, washed with demineralised water, rinsed, oven dried and ground before analysis.Three replicate sediment samples, from each site were analysed, for δSediment samples for C:N ratios and TOC were weighed into tin cups (15–20 g) and analysed using a Vario Microcube elemental analyser . The chironomid, SOM and leaf litter samples for stable isotope analysis were also weighed (approximately 0.5–1.0 mg for chironomids and 5–20 mg for SOM and leaf litter) into tin cups before their combustion in an isotope ratio mass spectrometer . Stable isotope ratios were expressed in per mille (‰).4 concentrations in pore and stream water, and between δ13C values of chironomids, and leaf litter and SOM were done using paired t-test. A value of p<0.05 was considered as statistically significant. Homogeneity of variances was examined with Bartlett's test and data were square root transformed to improve normality. Statistical analyses were done using the R statistical package Relations between variables were tested using Spearman's rank correlation test 3 L−1, and from 0.1 to 0.3 mg PO4 L−1. Water temperature ranged from 10.2 to 18.6°C, whereas electrical conductivity, dissolved oxygen concentrations and water discharge ranged from 61 to 380 µS cm−1, 8.5 to 11.9 mg L−1 and 0.2 to 7.8 m3 s−1  higher than the stream water CH4 concentrations. Some pore-water CH4 measurements at sites 3 and 6 differed greatly from the other measurements, either due to disturbance during sampling or gas leakage during analysis, and were therefore taken to be unreliable and excluded from further analysis.Average values of dissolved CH Site 10 . Pore-wa13C values of SOM ranged from −29.3‰ at site 7 to −27.1‰ at site 3  and fourth (−26.3±0.14 ‰) instar Tanypodinae, whereas slightly lower mean δ13C values were measured in fourth (−27.2±0.16 ‰) and third (−26.9±0.25 ‰) instar Chironomini. δ13C values did not differ significantly between third and fourth instar Chironomini larvae . The analysis of 9 replicates of fourth instar Chironomini larvae from site 4 had a standard deviation as low as 0.19 ‰ for δ13C  (13C of chironomid larvae (−26.8 ‰) was more similar to that of the SOM (−28.4 ‰) than leaf litter (−29.8 ‰) and there were significant differences (p<0.05) between δ13C values of chironomids, and SOM and leaf litter.The chironomids were identified as Chironomini, Tanypodinae and ronomids , Table 24 concentrations measured in this study are comparable to those measured in other aquatic ecosystems 4 concentrations showed a highly variable pattern among the impoundments (4 concentrations to be influenced by sediment heterogeneity. The authors reported that enhanced retention of sediment by macrophytes (Ranunculus penicillatus) increased pore-water CH4 production in streams. Significantly higher mean CH4 concentrations were recorded in pore than stream water. This can be explained by the fact that CH4 is usually produced in sediments, where anoxic conditions are likely to develop, whereas the stream water is rather well oxygenated  than those of potential photoautotrophic food resources, indicating ingestion and assimilation of MOB, which had oxidized isotopically light CH4. This was also demonstrated for lake invertebrates 4 concentrations and δ13C values of some invertebates indicated ingestion and assimilation of MOB.Few studies examined CHtes e.g. . In thes13C of chironomid larvae ranged from −27.2 (fourth instar Chironomini) to −25.3 ‰ (fourth instar Tanypodinae). The mean δ13C values of potential food resources ranged from −28.4 (SOM) to −29.8 ‰ (leaf litter) and were significantly correlated to those of the chironomids. The similarity of δ13C of Chironomini larvae and SOM can be attributed to the fact that Chironomini larvae are either filterers or gathering-collectors, feeding on fine particulate organic matter in aquatic systems 13C values may be difficult to interpret when compared with the other chironomids. The differences between the δ13C values of chironomids, SOM and leaf litter were within the reported range (±3 ‰) of δ13C values for consumers and their food resources Mean δ4 concentrations in stream and pore-water and δ13C values of chironomid larvae. In the UK, Trimmer et al. −1) CH4 concentrations than pore-water (0.07 µmol L−1). Highly depleted mean δ13C values (<−42 ‰) of Trichoptera larvae (Agapetus fuscipes), relative to those of potential food sources (−38 ‰), were only measured in areas with low pore-water CH4 concentrations. In comparison to Trimmer et al., we recorded higher mean CH4 concentrations in stream and pore-water and the mean δ13C values of chironomid larvae were not low. The type of food consumed by chironomids and presence of MOB influence the δ13C values of chironomid larvae. The short water residence times and the shallow nature of the studied impoundments could have enhanced water turn over rates, mixing of the entire water column and supply of organic matter or periphyton into the benthic zone. Thus, in the case of chironomid larvae feeding on sedimenting organic matter, we would anticipate their δ13C to be similar to that of their food resources. Mixing of the water column may also increase oxygen concentration and CH4 oxidation rates, and reduce CH4 production and the biomass of MOB available to chironomids −1 O2) of a stratified lake than in the well mixed and oxygenated waters (9 mg L−1 O2) of a polymictic lake. Additionally, the contribution of MOB to chironomid larvae biomass was higher in the anoxic than oxygenated waters. Jones et al. −1). Within the above mentioned studies, the isotopic values of chironomids were more similar to those of the SOM, when they were collected from sediments overlaid by well oxygenated waters. Use of MOB was particularly pronounced under hypoxia or post mixing following on from stratification.In the current study, no significant relationships were established between CH13C values of chironomid larvae  were similar to those of SOM  and leaf litter . Additionally, δ13C values of chironomid larvae were significantly correlated to each other. Given that the δ13C values varied little between consumers and their food sources δ13C values of chironomid larvae did not indicate utilization of methane derived carbon, other invertebrates could have used it as a source of energy. For example, Kohzu et al. 13C values than the other invertebrates , suggesting increased utilization of methane derived carbon. In summary, this study reveals that methane derived carbon did not contribute substantially to chironomid larval biomass in small impoundments, rather that allochthonous organic matter was the main source of energy. Future studies assessing the role of methane derived carbon in stream impoundments should include MOB community characterization, CH4 oxidation rates and fluxes, and δ13C values of other invertebrates.Although the δFile S1Supporting figure and tables. Figure S1, Example of a weir that impounded the studied rivers. The white arrow shows the direction of water movement. Table S1, Location and environmental characteristics recorded from the studied impoundments. Table S2, δ13C values (‰) of chironomid larvae from the studied impoundments. For each of the impoundments, between 1 and 9 replicates (n) were made from pooled samples.(DOC)Click here for additional data file.


Drosophila melanogaster (the fruit fly) is a valuable experimental platform for modeling host–pathogen interactions. It is also commonly used to define innate immunity pathways and to understand the mechanisms of both host tolerance to commensal microbiota and response to pathogenic agents. Herein, we investigate how the host response to bacterial infection is mirrored in the expression of genes of Imd and Toll pathways when D. melanogaster strains with different γCOP genetic backgrounds are infected with Pseudomonas aeruginosa ATCC 27853. Using microarray technology, we have interrogated the whole-body transcriptome of infected versus uninfected fruit fly males with three specific genotypes, namely wild-type Oregon, S057302γCOP/TM6B and 14aγCOP/14aγCOP. While the expression of genes pertaining to Imd and Toll is not significantly modulated by P. aeruginosa infection in Oregon males, many of the components of these cascades are up- or downregulated in both infected and uninfected S057302γCOP/TM6B and 14aγCOP/14aγCOP males. Thus, our results suggest that a γCOP genetic background modulates the gene expression profiles of Imd and Toll cascades involved in the innate immune response of D. melanogaster, inducing the occurrence of immunological dysfunctions in γCOP mutants. Pseudomonas aeruginosa is a metabolically versatile opportunistic pathogen, with a very wide host spectrum that includes plants, nematodes, insects, and vertebrates. P. aeruginosa can cause a wide range of severe human infections, with high mortality rates, particularly in immuno-depressed patients. A diverse arsenal of P. aeruginosa virulence factors, which can have different expression profiles depending on the infection localization and the host immune response, contribute to its high competitiveness [P. aeruginosa is included in the lists of the most threatening antibiotic resistant pathogens, such as ESCAPE  and the WHO list of priority one, critical pathogens [P. aeruginosa has a high capacity to form biofilms, which are known for their high phenotypic resistance or tolerance to different antimicrobial agents including antibiotics, antiseptics, or host effectors [tiveness . Moreoveathogens ,3. Besidffectors .P. aeruginosa infection triggers an intense innate host response in mammals, mainly mediated by neutrophils, resulting in significant host tissue damages. A higher incidence of severe P. aeruginosa infections in patients with acquired and primary immunodeficiencies and the better prognoses of the infections with P. aeruginosa for patients at the mature age reiterate the importance of the innate immune responses [esponses ,6,7.P. aeruginosa infections.Therefore, a better understanding of the interactions among the bacterial and host factors is expected to contribute to the development of improved therapeutic strategies for Drosophila melanogaster and mammals have many similarities [D. melanogaster as a powerful experimental platform for investigating the host’s innate immune response [D. melanogaster are regulated by either one or several signaling pathways [D. melanogaster [The bacterial infection process and the development of an innate immune system in larities . An apparesponse ,10,11,12nogaster . These pP. aeruginosa on fruit fly strains with different genetic backgrounds. A goal of this study was to evaluate the impact of normal and disturbed expression of the γCOP gene on Toll and Imd pathways consecutive to the infection of males. γCOP is an essential, highly conserved gene in eukaryotes and is involved in retrograde vesicular transport between endoplasmic reticulum (ER) and Golgi [γCOP is important for the ER homeostasis and hence for the innate immunity, but also for the adaptive immunity as revealed in humans [In our study, we have harnessed microarray technology in order to investigate the non-lethal effects of experimental infection with nd Golgi . Perturbnd Golgi ,16,17,18n humans .P. aeruginosa infection is affected by their genetic background, which critically influences the extent of the early cell-mediated innate immunity activation [D. melanogaster has an impact on commensal microbiota diversity and argued that the natural resistance to the external microbiota colonization could be compromised [Our inquiry was relying on a set of particular facts that support a link between the genetic background and the severity of infections. For example, the gut microbiome displays distinct features in complex human diseases and some microbial groups have significant heritability estimates . Noticeativation ,22,23. Lpromised .γCOP.The microarray experiment generated expression data for a total number of 14,437 targets employed by FlyChip procedure based on FL003 arrays. The microarray probes that do not match the target transcripts or match in multiple locations can introduce inconsistency in quantifying gene expression . Therefop < 0.05) modulated in at least one category of uninfected and infected S057302γCOP/TM6B or 14aγCOP/14aγCOP mutant males .An overview of the 51 genes significantly  and four are downregulated . Both categories of S057302γCOP/TM6B males share only seven modulated genes, as dl, cact, Tep4, and wntD are upregulated, while SPE, BaraA2, and Ulp1 are downregulated. On the other hand, three upregulated genes  and two downregulated genes (SPE and BaraA2) are common for both homozygous and heterozygous mutant males.No less than 10 genes from the Toll pathway have an affected expression pattern in both infected and uninfected imd, Fadd, and Plc21C) and are downregulated in all four categories of mutant males. When considering the 14a/γCOP14aγCOP infected and uninfected males, in a group of seven shared genes we found four of them to be upregulated  and three downregulated . On the other hand, there are 11 genes displaying a similar expression pattern in both categories of S057302γCOP/TM6B males, where 1 gene is upregulated (hep) and 10 genes are downregulated .A similar analysis of Imd/Imd-JNK pathways revealed that, out of 27 considered genes, only 3 of them are mutual , suggesting that the 14a/γCOP14aγCOP males set up an enhanced response to a commensal microbiota challenge. When scrutinizing inside of the same genetic background, there are only very small numeric differences (Δ) of the affected genes in infected versus uninfected flies . Remarkably, six of the genes modulated in the infected S057302γCOP/TM6B males are also affected in the infected 14a/γCOP14aγCOP flies. Only wntD and Ulp1 genes are specifically responsive to infection with P. aeruginosa in heterozygous flies.In the Toll pathway, the number of genes with impacted expression is bigger for the default (uninfected) condition in 14a/γCOP14aγCOP males seem to be less reactive, as long as they exhibit by default only 9 affected genes comparative to 14 genes modulated in the uninfected S057302γCOP/TM6B flies (Δ = −5). Conversely, there is a double increment in the number of the affected genes in the infected 14a/γCOP14aγCOP mutant males  comparative to the infection response of S057302γCOP/TM6B mutant males . Coincidentally, 19 genes (Δ = 0) from Imd/Imd-JNK pathways are modulated in both infected 14a/γCOP14aγCOP and infected S057302γCOP/TM6B males. Out of these 19 affected genes, the two categories of infected males share six upregulated genes  and six downregulated genes . It is somehow difficult to avoid noticing the symmetry of this numbers.Concerning the Imd/Imd-JNK pathways, uninfected 14a/γCOP14aγCOP mutants comparative to the heterozygous males, but for both considered genetic pathways, the experimental infection impacts more on gene modulation in the 14a/γCOP14aγCOP mutant males. However, the upregulation of some genes encoding for pathway inhibitors invite careful interpretation of these aggregate data S057302 chromosome [l(3)S057302 chromosome is a specific third chromosome of D. melanogaster obtained by insertional mutagenesis [SP{lacW}057302 in the 5′UTR region of the γCOP gene [SP{lacW}057302—proof that there is no other hidden mutation in the l(3)S057302 chromosome accountable for the lethal phenotype [S057302γCOP fails to complement the lethality of ∆114γCOP S057302), confirming data of Jayaram et al. [γCOP is indeed an essential gene, being also involved in fruit flies’ fertility [14a/γCOP14aγCOP males and females are always sterile, but both homozygous males and females are fertile when crossed with flies having at least one wild-type (WT) copy of γCOP (WTγCOP).In our experiments, we used the fruit fly strain romosome . The lC, which is also present in many natural populations but not in the Oregon strain [In(3R)C are 92D and 100F; γCOP is located in 100C, close to the distal breakpoint of this inversion. Detailed data concerning the multiple inversions’ effects on the expression of genes located in fruit fly balancer chromosomes were reported [TM6B balancer contains multiple inversions and rearrangements, but also recessive lethal genetic markers causing the homozygous individuals to die as embryos. These chromosomal aberrations prevent recombination by meiotic crossing over with the normal homologous chromosome 3. Herein, the term “normality” refers only to the natural linkage order of the genes, but not to alleles and or to other small-scale mutations. A balanced chromosome is actually a haploblock, which keeps in cis configuration not only the lethal recessive alleles, but also other known or hidden mutations, considering that the balancer is not leaky . TM6B con strain . The brereported . Misexprreported .D. melanogaster genome. The same authors prove that trans-interactions at the Malic enzyme (Men) locus were eliminated by inversions of two different chromosomal fragments that contain Men [Men allele is upregulated by the enhancers of a null Men allele, and the relative quantity of the specific mRNA and enzyme recovers to those of homozygous wild-type organisms. Bing et al. [Men upregulation of the wild-type was indeed a transvection, as inversions containing Men wild-type locus prevents its compensatory upregulation. The temperature was also an impeding factor, pointing to an environmental influence on the plasticity of transvection aspects.As revealed by Bing et al. , the imptain Men . The autg et al.  further S057302γCOP/TM6B strain contains a null allele and a relocated copy of WTγCOP, impairments of the anterograde and retrograde transport between Golgi and ER may be reasonably considered if the WTγCOP allele’s physiology is disturbed by position effects or by defective trans-interactions. On the other hand, 14a/γCOP14aγCOP males are homozygous for two hypomorphic alleles [γCOP gene comparative to the Oregon control flies.Since the γCOP in 14aγCOP/14aγCOP males does not necessarily result in an overproduction of γCOP protein since the 14aγCOP allele proved to be functionally hypomorphic. The qRT-PCR and microarray data show a compensative overexpression of an abnormal mRNA encoded by the 14aγCOP allele, actually conducting a net deficit of the protein comparative to Oregon males [S057302γCOP is an embryo lethal null allele, so an upregulation is not expected for it, but it is for the wild-copy allele, by a transvection phenomenon similar to the one reported for the Men gene [γCOP by trans-regulation of the normal allele. Alternatively, this inversion may also expose γCOP to the action of local non-specific suppressors, resulting in a slight downregulation of gene expression [We suppose that alternative compensatory mechanisms are acting in the mutant background of the two strains. The overexpression of on males . On the Men gene . Hence, pression .14aγCOP/14aγCOP males harbored the lowest bacterial load as compared to both 14aγCOP/TM6B and Oregon flies, the latter having the highest microbial charge [14a/γCOP14aγCOP and Oregon individuals. These differences may be a consequence of impaired intracellular vesicle cargo protein transport that could impact protein secretion and the assembly of receptors involved in microbial recognition.In a previous work focusing on a commensal microbiota load of our mutant strains , we founl charge . LowerinγCop alleles [γCOP. If commensal load data was inconsistent with the severity of γCOP alleles, we would have suspected a hidden mutation in l(3)S057302 chromosome.The data fit the same hierarchy of the allelic severity scale that we previously reported for lethality and sterility of these  alleles . TherefoK652ECopg1 mice are affected by combined immunodeficiency (CID), defined by disturbed humoral and cell-mediated immunity. Therefore, the authors demonstrate that p.K652E in the homozygous condition is responsible for CID and reveal that the COPG1 gene is required for a normal adaptive immune response.A recent paper  reports P. aeruginosa ingestion, the γCOP heterozygous or homozygous mutants exhibit a different expression profile of many genes encoding for highly evolutionarily conserved innate immune receptors, signaling pathways, and effector molecules, which might affect the response to both pathogenic agents and normal microbiota. When focusing on microarray results for genes from Toll and Imd pathways involved in the innate immunity, we noticed intriguing results. Significant gene expression modulations in either infected or uninfected flies are more frequent in 14a/γCOP14aγCOP males as compared to S057302γCOP/TM6B ones, and are more frequent in Toll rather than in Imd pathways . We previously detected a comparative level of overexpression in testes and the oocytes of 14a/γCOP14aγCOP individuals, but also in 14a/γCOP14aγCOP embryos [γCOP mutant background is, at least partially, responsible for the genes’ expression snapshot noticed in Toll and Imd/Imd-JNK pathways.Our microarray data reveal that the transcription patterns of Toll and Imd/Imd-JNK pathways in  embryos . These cγCOP alleles, hidden or known mutations are to be considered when making conclusions about the results. Sometimes, more or less gratuitous genetic markers are employed in crosses of fruit flies in order to ease the trace back of mutant alleles of the genes of interest. In our case, both 14aγCOP/14aγCOP and S057302γCOP/TM6B males have constitutively hemizygous mutations for white, a gene located in the X chromosome and recently reported to be involved not only in red eye pigmentation, but also in the response to some metabolic and cellular stresses [white is involved either in the immune response of flies or in interactions with γCOP or COPI. Nevertheless, in order to exclude a potential contribution of white to the activation of Toll and Imd/Imd−JNK pathways in our experiments, we took advantage to the fact that S057302γCOP/TM6B males contain a single functional copy of mini-white (+mCw) allele in the SP{lacW}057302 transposon [+mCw is a key genetic marker used to confirm a P{lacW} insertion in the genome of fruit flies with white defective genetic background. Since all of the S057302γCOP/TM6B males have deep orange eyes instead of white ones, it is evident that +mCw is functional in these transgenic organisms. Our microarray data reveal that the expression level of white in the uninfected and infected heterozygous males is indeed the same as in the Oregon ones, which contain a wild-type copy of white. The 14aγCOP allele was obtained by inducing an imprecise excision of SP{lacW}057302, therefore 14aγCOP/14aγCOP males no longer contain the +mCw allele. As expected, the microarray data show a significant similar downregulation of white expression in both uninfected and infected 14aγCOP/14aγCOP, but the behavior of Toll and Imd/Imd−JNK are still similar to the +mCw, S057302γCOP/TM6B males. Altogether, these genetic and molecular data reasonably rule out a significant contribution of the white background in the activation of Toll and Imd pathways in the S057302γCOP/TM6B males, or in keeping these pathways in a dormant state in the wild-type white background of Oregon flies. Although over a thousand specific alleles are reported in FlyBase, white is not an essential gene, as no lethal alleles of it are documented so far. Conversely, excepting for εCOP, lethal alleles are reported for 7 out of 8 genes encoding for COPI complex in D. melanogaster.The genetic background of the organisms used in biology experiments are always a sensible aspect of data interpretation. Although we rely on complementation tests for assessing the stresses ,37. To dansposon . The w+mγCOP background is the genetic driver that modulates the expression of many Toll and Imd genes in the uninfected mutant homozygous and heterozygous males, but not in the Oregon ones. Our findings are consistent with the essential role of γCOP proteins in the assembly of COPI vesicles; γCOP is the main interactor of the ADP-ribosylation factor at 79F (Arf79F), the fruit fly ortholog of mammalian ARF-1, which, in its turn, is a key protein for both assembly of the COPI complex and for anchoring it to the membrane of Golgi. The other interactor of Arf79F is βCOP (encoded by βCOP in D. melanogaster) and disruption by RNAi of either gene influence the immune system response of D. melanogaster [Drs and Mtk [S057302γCOP/TM6B males, where Mtk and Drsl1 are constitutively downregulated, but dl, cact, Tep4, and wntD are upregulated.It is remarkable that none of the genes scrutinized in this paper are either up- or downregulated in infected Oregon males. Plausibly, the nogaster ,39. A ve and Mtk  even in Arf79F, βCOP, and γCOP represent the key triplet involved in COPI assembly. Since Arf79F and βCOP are associated with the immune system GO term in FlyBase, it is logical to learn that γCOP is also involved in the immune response of D. melanogaster.γCOP. Oregon males respond to per os infection with P. aeruginosa but without involving genes of Toll and Imd pathways. The Oregon males exhibit an immune response to these bacteria, as ventral veins lacking, Listericin, Turandot A and Turandot C genes are modulated consecutively with infection. On the other hand, experimentally infected Oregon strain may not activate some genes involved in the innate immune response, most probably if a distinct infection is interpreted as a relatively mild one. As an example, a per os infection with the flagellate parasite Crithidia spp. activates CecA1 and Diptericin genes from the Imd pathway and Def and Drs from the Toll pathway only in the ILL97 wild-type strain but not in the Oregon [Our experiments revealed that Toll and Imd/Imd-JNK pathways are not activated in the infected Oregon males mainly because they are wild-type for e Oregon  flies.γCOP  is that disturbances in the normal physiology of γCOP impact Toll and Imd/Imd-JNK pathways and induce modulations of the transcriptional profiles for many of the constituent genes. Albeit the homozygous mutants present a robust overexpression and the heterozygous display a questionable downregulation of γCOP, it appears that both conditions have an impact on the pathways. Since misregulation of 14aγCOP is stronger and significant, it is expected to have a greater impact on the transcriptional landscape of both pathways. Overexpression of 14aγCOP in 14aγCOP/14aγCOP males determines a more consistent perturbation of the Toll pathway and a bigger difference between infected versus uninfected homozygous males.The biological significance of the mutant background of We inquire further about the genes from the Toll and Imd/Imd-JNK pathways that had a significantly modulated gene expression in our experiment.D. melanogaster anti−infectious defense is represented by highly conserved, complex, and interrelated innate immunity mechanisms, which are activated by the recognition of pathogenic viruses, bacteria, fungi, and parasites [P. aeruginosa, previous studies have shown that besides the well-known pro-inflammatory effects of LPS, many other virulence factors, including proteins involved in protein folding, quorum sensing, transcriptional regulators, efflux (multidrug transporters) systems, biosynthesis of redox active compounds (periplasmic thiol-disulfide oxidoreductase), toxins , and proteins of unknown function are essential for maximum pathogenicity in D. melanogaster, triggering the activation of an innate immune response and secretion of proinflammatory cytokines [In contrast to most vertebrate species, the arasites . Regardiytokines ,43,44.Toll and Imd are the major innate immunity pathways regulating the production of AMPs and other immune responses in fruit flies .The Toll/Cactus signaling pathway is mainly triggered by fungal infections, while the Imd cascade, including TAK1, Dredd, IKK complex, and Rel, is activated in bacterial infections ,47. The Drosophila, although their involvement in the upregulation of AMP genes is not fully understood. However, regarding Toll-9, it was demonstrated that vertebrates express multiple Toll-like receptors (TLRs) resembling Drosophila Toll-1 receptor. They are involved in the regulation of different immune responses upon recognition of different pathogen-associated molecular structures, such as Gram-negative bacterial lipopolysaccharides, peptidoglycans, or flagellin [Eight other Toll receptors (Toll 2–9) are described in lagellin .SPE was downregulated in all four mutant lines, irrespective to the infection, demonstrating that the genetic background inhibits the production of spz cytokine, the ligand of Toll-9. Moreover, the constitutive expression of Toll-9 is inhibited in the infected and uninfected homozygous mutants, as well as in the infected heterozygotes, proving the role of the genetic background in the innate immune response. Previously, it was demonstrated that Toll-9 is functionally homologous to Toll-1 inducing a strong dl expression [Pathogen recognition is dependent on the interaction between the extracellular modSP and GNBP1, GNBP3 or PGRP-SA, and PGRP-SD , leadingpression .cact is upregulated. To bind to Toll-1, the circulating zymogen (pro-spz-1) is cleaved by the SPE into spz [cact could block the nuclear translocation of the dl transcription factor. However, in our study the dl expression was increased in all experimental groups.Regarding the Toll cascade inside the nucleus, we have observed that in both infected and uninfected experimental conditions, into spz . Subsequinto spz . Thus, oγCOP mutant background is also associated with increased expression of the wntD. It was previously shown that wntD acts as a feedback inhibitor of dl/NF-κB, blocking dl nuclear accumulation, independently of the I-κB homologue cact. The wntD loss-of-function (LOF) was associated with immune defects and increased levels of Toll/dl signaling [wntD and dl expression levels were increased. Thus, our results suggest that in γCOP mutants, dl expression levels are increased, even though two of the inhibitors of this pathway, i.e., cact and wntD, are upregulated. This might suggest the involvement of other regulatory pathways.The ignaling . HoweverUlp1, correlated with decreased expression of the β-arrestin krz, was noticed in the heterozygous γCOP mutants. The Ulp1 downregulation could also account for the dysregulation of the Toll signaling pathway and impairment of the immune system homeostasis. Previous studies have shown that Ulp1 or krz LOF were accompanied by an increased inflammatory response in Drosophila larvae [The downregulation of the SUMO protease, a larvae .γCOP mutants via the modulation of the events upstream of the Toll receptor, i.e., downregulation of GNBP1 and upregulation of PGRP-SA, PGRP-SD, and ModSP. ModSP is a modular serine protease that was shown to interconnect the circulating recognition molecules with the grass-SPE-spz extracellular pathway upstream of the Toll receptor [γCOP mutants is additionally impaired by the downregulation of the highly conserved pll (coding for a serine/threonine protein kinase), a positive regulator of the nuclear translocation of the NF-κB transcription factors Dif and dl [Gprk2 was inhibited only in homozygous mutants. This kinase has an evolutionarily conserved role in normal dl expression through a NF-κB regulation mechanism, which is unrelated to cact degradation. Noticeably, the cytokine expression consecutive to Escherichia coli infection was impaired in zebrafish embryos for the orthologous GRK5, and in D. melanogaster, the susceptibility of infections with Enterococcus faecalis infection is increased by RNAi silencing of Grpk2 [The Toll pathway seems to be more severely affected in homozygous receptor . Going df and dl . Interesof Grpk2 .γCOP alter the proper function of this conserved NF-κB signaling regulation pathway. Consequently, the fruit fly’s capability to respond adequately to Gram-negative bacterial infections is also perturbed.Our results demonstrate for the first time that mutations in γCOP background interferes with the Toll signaling pathway and with the innate immunity of Drosophila. Most probably, this interaction affects the fruit fly’s ability to recognize the pathogen-associated molecular patterns and to further activate the signal transduction cascades and the transcription of effector genes.Overall, our data suggest that the The second major cascade involved in the host–pathogen crosstalk is the Imd pathway, whose main role is the production of AMPs through the NF-κB transactivator Rel.The Imd pathway  shares mmeso-diaminopimelic acid (DAP)-type peptidoglycan (PGN) and the PGRP-LC transmembrane receptor [Imd pathway-mediated pathogen recognition is dependent on the interaction between LPS and other Gram-negative bacterial wall components, such as the receptor ,62. Addireceptor . The intreceptor .Upon receptor activation, an intracellular complex is recruited at the receptor site, consisting of imd, Fadd, and Dredd (caspase-8 homolog Death-related ced-3/Nedd2-like protein). Ubiquitination of Dredd by Diap2, Uev1A, ben, and eff complex activates Dredd for the cleavage of an imd N-terminal site, followed by ubiquitination of cleaved imd . AlternaγCOP infected and uninfected homozygous mutants, as well as infected heterozygous mutants exhibited an increased expression of the PGRP-LC. While this upregulation was expected for P. aeruginosa infected flies, the expression profile change in the uninfected homozygous males suggests that the γCOP mutant background induces a dysregulation of the humoral immune response in Drosophila. The same upregulation profile was recorded for PGRP-LB, an amidase that specifically degrades the Gram-negative PGN. This process downregulates the Imd pathway and provides a fine negative feedback regulation of the immune response to infection, including the modulation of the immune reactivity to ingested bacteria in the gut of the fruit flies [γCOP homozygous flies also supports the hypothesis of an immunodeficiency condition in these mutants. However, an alternative explanation could be that the fruit flies distinguish the low virulence of the infecting strain and thus an exaggerated immune response is prevented. This hypothesis is sustained by a significant upregulation of PGRP-LB in infected versus uninfected homozygous γCOP mutants.In our study, both it flies . The uprFadd and imd expression was inhibited in both infected and uninfected flies with different γCOP mutant genotypes, suggesting a disturbance of the innate immunity response in these mutants. The association of Fadd with imd is required for further activation of the Imd signaling pathway and previous studies have shown that loss of Fadd function was associated with high susceptibility of flies to Gram-negative bacteria infections [Fadd is strongly downregulated in all of the experimental conditions, and this could severely impair the proper activation of the Imd pathway in γCOP mutants. Moreover, P. aeruginosa infection upregulates the expression of pirk, a negative regulator of the Imd signaling pathways, in both mutant genotypes, thus enhancing the immunodeficiency condition in these flies. Previous studies have also shown that pirk overexpressing fruit flies were indeed more susceptible to Gram-negative bacterial infection than WT ones [POSH, which is essential for the properly timed activation and termination of Imd-mediated immune response [γCOP mutation induces a dysregulation of the innate immune response against Gram-negative bacterial infection [fections . Our res WT ones . Anotherresponse . These rnfection .Rel was significantly upregulated in the homozygous infected males and downregulated in uninfected heterozygous flies. This different expression pattern is visible when all the experimental conditions are considered . These data could suggest: (i) that the lack of functional γCOP impairs the regulation of D. melanogaster response intensity to a non-virulent P. aeruginosa strain or/and (ii) particular interaction between the Toll and Imd pathways could synergistically act to overcome some defects affecting one of the two pathways  expression [Atf-2 was downregulated in the infected experimental groups, as well as in the uninfected heterozygous males. The decreased expression levels of Atf-2 could be linked to a lower production of ROS, as a result of a decreased level of the ROS-producing enzyme Duox [Duox exhibited a FC of −0.145 in infected 14aγCOP/14aγCOP, almost reaching the significance threshold with a p value of 0.088).As already shown in  factors ,67. Both factors , which i factors , are linpression . The p38yme Duox  . These strains are: 1. Oregon, standing for the standard WT strain; 2. S057302γCOP/TM6B harboring the insertion of S057302P{lacW}γCOP transposon in the 5′UTR region of γCOP gene (GenBank accession number AJ492220) and causing embryo lethality; 3. The homozygous 14aγCOP/14aγCOP males were harvested from 14aγCOP/TM6B strain and contain the hypomorphic 14aγCOP allele (GenBank accession number DQ279402). Both mutant lines have white defective genetic background.To perform the experimental infection and subsequent microarray experiment we employed young adult males from three strains of P. aeruginosa ATCC 27853 strain was cultivated on Cetrimide agar at 37 °C and following 24 h of cultivation, bacterial suspensions of 9 × 109 CFU/mL were obtained.The S057302γCOP/TM6B, and 14aγCOP/14aγCOP males were starved for several hours prior to being placed in vials containing 5% sucrose agar covered with sterile paper blotted with either 230 µL of 5% sucrose solution containing 9 × 109 CFU of P. aeruginosa suspension (infected replicates) or 230 μL of 5% sucrose (uninfected replicates).WT Oregon, S057302γCOP/TM6B and 14aγCOP/14aγCOP infected and uninfected experimental variants four biological replicates were used, as well as for the infected Oregon males. Every biological replicate comprised of 25 to 27 individuals.A total of 20 Oregon uninfected replicates were considered as reference; for each of the The males were allowed to feed for about 63 h at 25 °C, then, a total of 25 males/replicate were placed in 1.5 mL Eppendorf tubes, covered with 300 mL TRIzol, and shipped to FlyChip Facility .D. melanogaster samples  were processed at FlyChip according to their standard protocol. Briefly, medium-scale RNA extraction was performed in the first step; then, the 20 control Oregon RNA samples were pooled together and converted into double-stranded DNA used as a reference to compare all experimental conditions against the control. All the samples were submitted to Klenow labelling of 1 µg double-stranded DNA derived from total RNA and subsequent hybridization to amino-modified long oligonucleotide microarrays, using the FL003 (FlyChip_long_oligonucleotide_003—INDAC) array format. The fluorescent spots were analyzed with a GenePix Axon scanner (5 μM pixel resolution).The vsn [limma [2 ratios of sample/control .Microarray data are available online at NCBI’s Gene Expression Omnibus (GEO), accession number GSE80084. Normalization and quality control steps were performed using vsn  and limmn [limma  softwareγCOP mutants, were generated with GraphPad Prism 5.03 software  using the log2FC values.Heatmaps summarizing the up− and downregulated genes associated with Toll and Imd pathways, as well as the overview of the differential expression of immune genes in D. melanogaster males with specific γCOP mutant backgrounds revealed that the expression of at least 52 genes of the Imd and Toll pathways and their interactors were significantly modulated upon infection with P. aeruginosa. Remarkably, in both infected and uninfected mutant males, the expression pattern of a vast majority of the genes from both pathways is affected when compared to the uninfected Oregon control. A very interesting aspect is that none of these genes are significantly modulated in infected Oregon males. This led us to hypothesize that natural microbiota might act as an environmental trigger in γCOP mutant background, a condition that signals an alert state. The peculiar microarray pattern may suggest that genes from Toll and Imd pathways respond to this alert by adjusting their transcription rate even in the absence of experimental infection with P. aeruginosa. One scenario is that, in the absence of enough functional γCOP product, the host increases the stringency of the feedback control of commensal microbiota, by triggering an erratic immune response.This insight into the whole-body transcriptome of P. aeruginosa bacterial strain with low virulence administered by ingestion, in order to evaluate the ability of adult flies to adequately sense and respond to the presence of Gram-negative ubiquitous bacteria relative to their γCOP genotype. In natural conditions, fruit flies seem to be able to detect Gram-negative components, such as LPS, which is sensed through the transient receptor potential (TRP) channels , and thus avoid eating food contaminated with LPS and bacteria [P. aeruginosa strains administered by direct injection in the hemolymph [γCOP mutations in the innate immune response to Gram-negative infections. The γCOP mutations are affecting the fruit fly’s ability to recognize the pathogen-associated molecular patterns and to further activate the signal transduction cascades and the transcription of effector genes in the Toll cascade. Both inhibitory  and activating  effects were noticed, suggesting a dysregulation of this important innate immunity pathway. The same dual effects were recorded for the Imd cascade, whose expression profiles suggest either inhibition  or activation (increased expression of the PGRP-LC and downregulation of POSH). The variable expression profile of transcriptional factors and AMPs in different experimental groups demonstrates the complex interactions between Toll and Imd, as well as with other signaling pathways.Further studies are needed to confirm and refine the contribution of both host’s genetic background and different virulence factors to bacterial pathogenicity and host immune response. In our experimental design, we have selected a laboratory bacteria . Howeverbacteria . Complememolymph  could he14a/γCOP14aγCOP males are also unable to compensate for the conditional sterility of 14a/γCOP14aγCOP females in the respective crosses [γCOP is the most probable genetic determinant herein, since comparative data we obtained for viability, fertility, and infection tests points to a similar behavior of the inquired γCOP alleles. Basically, 14aγCOP behaves as a hypomorph, and therefore is a less severe allele than the S057302γCOP null, but heterozygous S057302γCOP/TM6B exhibit less severe phenotypes of sterility and immune sensitivity compared to 14a/γCOP14aγCOP males.Interestingly,  crosses . We arguγCOP background interferes with the Imd and Toll signaling pathways at different levels. The γCOP mutations disturb the fine regulation of the innate immune response and hence the fruit fly’s capability to adequately respond to Gram-negative bacterial infections.Overall, our data suggest that the in premiere that mutations of the γCOP gene impair not only the adaptative immune response, as in humans and mice, but also the innate immune response of D. melanogaster, reinforcing the power of this organism for modeling infections.Our results reveal γCOP mutations, as inferred from recent medical research data [Purportedly, the prognostic of some bacterial infections should be estimated not only by the classical clinical procedures, but also by a quick screening of patients for rch data .


The appearance of alterations in normal metabolic activity has been increasingly considered a risk factor for the development of sporadic and late-onset neurodegenerative diseases. In this report, we induced chronic metabolic stress by feeding of a high-fat diet (HFD) in order to study its consequences in cognition. We also studied the effects of a loss of function of isoforms 1 and 3 of the c-Jun N-terminal Kinases (JNK), stress and cell death response elements.Animals were fed either with conventional chow or with HFD, from their weaning until their sacrifice at 9 months. Before sacrifice, body weight, intraperitoneal glucose and insulin tolerance test (IP-GTT and IP‑ITT) were performed to evaluate peripheral biometrics. Additionally, cognitive behavioral tests and analysis of spine density were performed to assess cognitive function. Molecular studies were carried out to confirm the effects of metabolic stressors in the hippocampus relative to cognitive loss.Jnk3−/− lead to synergetic responses. Loss of function of JNK3 led to increased body weight, especially when exposed to an HFD and they had significantly decreased response to insulin. These mice also showed increased stress in the endoplasmic reticulum and diminished cognitive capacity. However, loss of function of JNK1 promoted normal or heightened energetic metabolism and preserved cognitive function even when chronically metabolically stressed.Our studies demonstrated that HFD in Downregulation of JNK3 does not seem to be a suitable target for the modulation of energetic-cognitive dysregulations while loss of function of JNK1 seems to promote a good metabolic-cognitive profile, just like resistance to the negative effects of chronic feeding with HFD.The online version contains supplementary material available at 10.1186/s10020-022-00471-y. The disruption of physiological energetic metabolism can be the cause for the development of cognitive deficits in the context of neurodegenerative diseases  are a subfamily of the Mitogen Activated Protein Kinases (MAPK) that have been studied for their role in the regulation of a wide myriad of physiological and disease-related mechanisms, making them appealing targets for the prevention and amelioration of many pathological states . Feeding of a HFD was chosen as a model of metabolic stress and potential contributor to the appearance of sporadic forms of neurodegeneration based on preclinical, clinical and epidemiological reported data  and Mapk10−/−  mice were used in this study , RNA and cDNA were obtained as previously described from hippocampal samples    were anaesthetized through an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) before intracardiac perfusion with 4% paraformaldehyde. Posterior brain fixation, sectioning, and labelling have been previously described . The, they were blocked in a solution containing 10% fetal bovine serum (FBS) and 1% Triton X-100 diluted with PBS-T five times for 5 min each and incubated with the Glial fibrillary acidic protein (GFAP) antibody (Dako-Z0334). On the 2nd day, slices were washed with PBS-T 5 times for 5 min and incubated with the pertinent secondary antibody for 2 h at room temperature. Finally, sections were treated with 0.1 µg/mL Hoechst  during 8 min in the dark at room temperature and washed with PBS 0.1 M. All reagents, containers and materials exposed to Hoechst were properly managed and processed to avoid any cytotoxic contamination. Finally, brain slices were mounted in gelatin-coated slides using Fluoromount G (EMS) and were left to dry O/N. Image acquisition was obtained using an epifluorescence microscope . Comparative analysis of fluorescence intensity was done as described in Busquets et al. .To carry out the spine density analysis, five animals per group were used which were sacrifice by cervical dislocation. After, brain was isolated and the directions of the Kit purchased from FD Neurotechnologies, Inc.  were followed. Images were obtained by BX61 Laboratory Microscope (Melville NY-Olympus America Inc.). The quantification was carried out by selecting five neurons per animal in the dentate gyrus (DG) of the hippocampus. Measurement was done at least 50 μm from the soma along consecutive 10 μm on secondary branches starting 10 μm after branching from the primary dendrite. Spine density was calculated by dividing the number of spines per segment by the length of the segment and was expressed as the number of spines per 10 μm of dendrite.The Novel Object Recognition Test was used to evaluate hippocampal-dependent recognition memory of mice . Data from the TaqMan® array was presented in a heat map indicating increased expression in red and decreased in green. Other data was presented in bar graphs and/or violin plots showing all data points. All data showed normality and was analyzed using two-way ANOVA and Tukey’s. Significance was indicated as *−/− animals by 9 months of age was observed after HFD . These animals also showed decreased response to insulin . In contrast, Jnk1−/− animals showed significantly decreased body weight when compared to WT CT , just like heightened response to insulin . WT and Jnk3−/− HFD animals also showed reductions in their discriminatory capacity when tested through the NORT. Jnk3−/− animals presented decreased discriminatory capacity independently of whether they had been metabolically stressed with HFD or not .Regarding the weight and response to insulin in each experimental group, a significant weight gain in WT and Jnk3Results derived of the array were presented in a Heat Map Fig.  in whichSlc2a1), insulin receptor (Insr), insulin receptor substrate 1 (Irs1), protein kinase Akt 2 (Akt2), as well as the protein phosphatase Ptpn1. Overall, data indicated that the combination of the loss of function of JNK3 induced mild downregulation of Slc2a1 which, was aggravated with long-term HFD feeding. Also, HFD feeding promoted significant upregulation of the expression of Ptpn1 both in WT and Jnk3−/− HFD conditions. Jnk1−/− animals showed increased expression on Slc2a1, Insr and Akt2 . In the mitochondrial-related subset of genes, HFD was observed to have downregulating effects in some of the genes coding for the subunits of the OXPHOS complexes in WT and Jnk3−/− conditions. The most significant effects caused by HFD were detected on the Atp5b subunit of complex V, in which all experimental groups exposed to HFD showed downregulation. Jnk1−/− CT animals showed control like levels of expression in most cases except on Uqcrc1 (subunit of complex III) which was significantly upregulated . Finally, detection of changes of antioxidant genes enzymes Sod1 and Gpx1 indicated an expression increased on Jnk1−/− CT animals .The first subset of targeted genes in the array that showed significant differences in the expression profile were involved in energetic metabolism, including glucose transporters, glycolysis Fig. A, and miNtrk2 in all experimental groups, which was paired with mild decreases in the expression of Bdnf in WT and Jnk3−/− HFD groups. Meanwhile, Jnk1−/− CT animals showed mild increases in these same genes, as well as in Ppp1r9b, gene that codes for neurabin-2. Also, loss of function of JNK1 favored increases in the different isoforms of neurexin (Nrxn) and neuroligin 3 (Nlgn3). Nrxn3 was downregulated by HFD in WT and Jnk3−/− conditions .The second subset of genes included in the array were responsible for the control and maintenance of proper cognitive activity in neuronal and synaptic structures Fig. A, B. It p < 0.001 WT CT vs WT HFD, WT CT vs Jnk3−/− CT, WT CT vs Jnk3−/− HFD, WT HFD vs Jnk1−/− CT, WT HFD vs Jnk1−/− HFD, Jnk1−/− CT vs Jnk3−/− CT, Jnk1−/− CT vs Jnk3−/− HFD, Jnk1−/− HFD vs Jnk3−/− CT, Jnk1−/− HFD vs Jnk3−/− HFD and Jnk3−/− CT vs Jnk3−/− HFD). Animals in the Jnk1−/− experimental groups showed control-like responses in both analyses , P-EIF2/EIF2 (Ser51) , ATF4  and P-IRE1α/ IRE1α (Ser724)    (FD) Fig. A. DetectFD) Fig. B.Fig. 6LJnk1−/− and Jnk3−/− mice .HFD increased the reactivity of astrocytes in WT conditions, but not in Published data on the loss of function on JNK3 has described its beneficial effects in scenarios of cellular death and cytotoxicity, just like its importance in brain development in the embryonic stages  and, combined by a loss of function of JNK3 or JNK1 which, in a previous study had shown to be highly efficient metabolically and to be resistance to the consequences of HFD  was reported in the past by our research group (Petrov et al. In this process of oxidative phosphorylation at the mitochondrial level, ROS are also generated as a by-product. However, as we have previously discussed, Observation and quantification of the reactive profile of astrocytes reproduced the increased reactivity of these cells in WT animals when fed chronically with HFD (Busquets et al. Loss of function of JNK3 does not seem to be a suitable target for the development of therapeutic approaches due to its detrimental effects in the control of metabolism and cognitive function, despite its described effects in the prevention of activation of inflammatory and apoptotic pathways. Yet, the downregulation of JNK1 for the modulation of energetic metabolism proves to be a very attractive approach for the amelioration of cognitive-neurodegenerative states linked to insulin resistance and increased tissue inflammation. This study may allow a better understanding of the role of both isoforms in the brain and their potential role in the treatment of neurological diseases (Additional files Additional file 1: Additional Material 1. Specific descriptions of the genes included in the TaqMan® array.Additional file 2.Sup. Fig 2. Hematoxylin-eosin stain in different hippocampal areas.Additional file 3.Sup. Fig 3. Annex is show that no significant differences were observed in the levels of GADPH and tubulin in any of the experimental conditions.Additional file 4.Sup Fig 4.Determination of the levels of Superoxide dismutase, Catalase and 4-Hydroxynonenal in the hippocampus of JNK3-/- mice treated with a control diet and a high-fat dietAdditional file 5.Sup Fig 5. Expression of JNK1 and JNK3 levels in the hippocampus of mice treated with a standard diet and treated with a high-fat diet.Additional file 6. Immunoblot membranes.


Of the 119,600 designs, 104,972 were successfully built in to the AlphaSeq library and target binding was subsequently measured with 71,384 designs resulting in a predicted affinity value for at least one of the triplicate measurements. Data include antibodies with predicted affinity measurements ranging from 37 pM to 22 mM. To our knowledge, this dataset is the largest, publicly available dataset that contains antibody sequences, antigen sequence and quantitative measurements of binding scores and provides an opportunity to serve as a benchmark to evaluate antibody-specific representation models for machine learning.The dataset presented here contains quantitative binding scores of scFv-format antibodies against a SARS-CoV-2 target peptide collected via an AlphaSeq assay that can be used in the development and benchmarking of machine learning models. Starting from three seed sequences identified from a phage display campaign using a human naïve library, four sets of 29,900 antibodies were designed   Such approaches have been taken to engineer enzymes2, fluorescent proteins3, and antibodies4 among others6. Successful examples of machine learning-enabled protein engineering have relied on access to large, labelled datasets of protein sequences that are typically generated as part of high throughput experimental campaigns. When datasets and models have been made publicly available, the entire field has benefited from ensuing comparisons and benchmarks7.Protein modelling is an area of machine learning research that has attractive potential benefits to protein engineering. Given the magnificent size of the design space for a given protein, it is not feasible to measure phenotypic properties of all possible designs empirically. Machine learning methods can help constrain the design space and serve as a basis for recommending designs to test in the lab to save time and reduce cost8. Other efforts have resulted in datasets with close to one thousand antibodies with labels – either target sequences9 or neutralization values10. Additional work in antibody binding prediction reports a subset of the data generated and used4. Outside of antibody binding, one group has published multiple manufacturability measurements for over 100 antibodies that have completed or advanced through the FDA approval pipeline13.Although these approaches have been demonstrated for the engineering of antibodies, there remains a scarcity of labelled data available in the public domain to advance this area of research with respect to antibody binding. Large-scale curation efforts have resulted in databases of well over a billion antibody sequences without target or binding affinity values6 designs15. This type of data is unsuitable for training models because poor binding sequences are unknown and no quantitative binding measurements are generated. Methods that do provide quantitative measurements, such as enzyme-linked immunosorbent assays16 and surface plasmon resonance17, rely on isolation of individual antibodies and therefore are significantly lower throughput and more expensive.This lack of labelled data may be the result of differences in data requirements for training machine learning models compared to finding a design with the target phenotype in the absence of machine learning. Phage display, for example, is intended to provide the researcher with information on a small number of the top binders from a pool of >1019. We utilized such a technique, referred to as AlphaSeq, to generate the dataset described here. Importantly, we first conducted a phage display experiment to identify three candidates that bind to our target, a conserved peptide in coronaviruses. Those candidates were the seed sequences and in addition to designing all single mutants, we performed in silico randomization to introduce two or three random mutations in the complementary determining regions (CDRs) for 119,600 designs. As a result, we generated a dataset that covers a considerable breadth of sequence space and has a wide range of binding measurements that is suitable for training machine learning algorithms.Recent methods using engineered yeast expression systems and next generation DNA sequencing overcome some of these challenges and can generate large scale datasets of quantitative protein-protein binding interactions including antibody-antigen interactionsFigure 20. Additionally, this sequence is reported to have low variability across coronaviruses and could maintain therapeutic value against viral variants21. The exact amino acid sequence targeted was PDVDLGDISGINAS.Antibodies were targeted against a peptide in the HR2 region of the SARS-CoV-2 spike protein to which neutralizing antibodies have been observed10 and is in Fab format.A phage display panning experiment was performed by GenScript USA Inc. to identify candidate binders. A biotinylated target peptide, LCBiot-PDVDLGDISGINAS-OH,  was provided to GenScript USA Inc. The human naïve phage library used by Genscript USA Inc. is marketed to be derived from 300 healthy human donors, has a size of 1.1 × 10D values below 1 nM. The best chain orientation was selected for each antibody; HL was selected for Ab-14, LH was selected for Ab-91 and HL was selected for Ab-95.A total of five antibody sequences in scFv format were evaluated in a proof-of-concept AlphaSeq experiment; three of those five sequences bound to the target and were carried forward. All five antibody sequences were tested in both heavy-light (HL) and light-heavy (LH) chain orientation. In general, chain orientation had little to no effect on binding affinity with all but Ab-91-HL (3.39 nM) resulting in predicted KK-point mutations, where k = 1, 2, and 3, were produced for the CDRs of each chain. Point mutations were limited to amino acid substitutions; indels were avoided to ensure the amino acid sequence was constant length. Up to k = 3 mutations were chosen to guarantee there was at least one instance where one amino acid substitution occurred in all CDRs of a given chain at a given time. Martin Lab’s CDR rule set was applied to extract the CDRs from their approximate positions in each chain. The number of variants for k = 1 mutations was determined based on the combined number of amino acid positions in the CDRs of a given chain and the total number of possible amino acid substitutions in each position. All k = 1 variants were kept, ensuring that duplicates and original chain sequences were removed. Using the number of sequence variants for k = 1, a scaling factor of ~6 was applied to determine the number of variants to sample from the total number of k = 2 and k = 3 possible sequence variants, as shown in Table Two heavy chains and two light chains were arbitrarily selected from the three antibody seed sequences listed in Table Yeast peptone dextrose (YPAD), yeast peptone galactose (YPAG), and synthetic drop out (SDO) media supplemented with 80 mg/mL adenine were made according to standard protocols. Suppliers used for our yeast media are as follows: Bacto Yeast Extract (Life Technologies), Bacto Tryptone (Fisher BioReagents), Dextrose , Galactose (Sigma-Aldrich), Adenine (ACROS Organics), Yeast Nitrogen Base w/o Amino Acids (Thermo Scientific), SC-His-Leu-Lys-Trp-Ura Powder (Sunrise Science Products), Yeast Synthetic Drop-out Medium Supplements (Sigma-Aldrich), L-Histidine (Fisher BioReagents), L-Tryptophan (Fisher BioReagents), L-Leucine (Fisher BioReagents), Uracil (ACROS Organics), and Bacto Agar (Fisher BioReagents).18. Yeast transformations were performed with Frozen-EZ Yeast Transformation Kit II (Zymo Research) according to manufactures instructions. Yeast were plated on SDO-Trp plates and grown at 30 °C for 2–3 days. Successful transformants were struck out onto YPAD plates and grown overnight at 30 °C.AlphaSeq compatible plasmids encoding yeast surface display cassettes were constructed by Twist Bioscience and resuspended at 100 ng/µL. 100 ng of plasmid was digested with PmeI enzyme for 1 hr at 37 °C to linearize, leaving chromosomal homology for integration into the ARS314 locus at both the 5′ and 3′ ends as previously describedYeast were inoculated in YPAD and grown overnight at 30 °C. Yeast were labelled with FITC-anti-C-myc antibody  in PBS (Gibco) + 0.2% BSA (Thermo Fisher Scientific) for 30 minutes at RT. Yeast were pelleted and resuspended in PBS + 0.2% BSA and read on a LSRII cytometer.A 300 bp oligonucleotide pool synthesized by Twist Bioscience was resuspended at 20 ng/µL in molecular grade water. Libraries were PCR amplified from the oligonucleotide pool using KAPA DNA polymerase (Roche). The oligonucleotide amplification fragment was inserted into the seed scFv backbone using Gibson isothermal assembly (NEB), as well as a second DNA fragment containing a randomized DNA barcode. The assembled barcoded antibody DNA library was PCR amplified. Fragments were run on a 0.8% agarose gel and extracted using Monarch Gel Purification kit (NEB).18. All spin steps were performed at 3000 RPM for 5 minutes. Yeast were spun down and washed once in 50 mL 1 M Sorbitol (Teknova) + 1 mM CaCl2 solution. Washed yeast were resuspended in a solution of 0.1 M LiOAc/1 mM DTT and incubated shaking at 30 °C for 30 minutes. After 30 minutes, yeast were spun down and washed once in 50 mL 1 M Sorbitol + 1 mM CaCl2 solution. Yeast were resuspended to a final volume of 400 µL in 1 M Sorbitol + 1 mM CaCl2 solution and incubated with DNA for at least 5 minutes on ice. Yeast were electroporated at 2.5 kV and 25 uF (BioRad). Immediately following electroporation, yeast were resuspended in 5 mL of 1:1 solution of 1 M Sorbitol:YPAD and incubated shaking at 30 °C for 30 minutes. Recovered yeast cells were spun down and resuspended in 50 mL of SDO-Trp media and transferred to a 250 mL baffled flask. 20 µL of resuspended cells were plated on SDO-Trp to determine transformation efficiency. Both the flask and plate were incubated at 30 °C for 2–3 days. After 2–3 days, transformation efficiency was determined by counting colonies on the SDO-Trp plate.MATa AlphaSeq yeast were grown for 6 hours in YPAG media to induce SceI expression, as described previouslyin silico antibody library using BLASTN22 to determine the mapping between DNA barcodes and antibody sequence; only DNA barcodes with at least 2 reads observed were considered, and each DNA barcode was matched to the most common BLASTN antibody match among its constituent reads.Genomic DNA from yeast libraries was extracted using Yeast DNA Extraction Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. A single round of qPCR was performed to amplify a fragment pool from the genomic DNA containing the gene through the associated DNA barcode. qPCR was terminated before saturation to minimize PCR bias, generally between 15–20 cycles. The final amplified fragment was concentrated with KAPA beads, quantified with a Quantus (Promega), prepped with a SQK-LSK-110 ligation kit (Oxford Nanopore) and sequenced with a Minion R10 flow cell (Oxford Nanopore) following the manufacturer’s instructions. Each sequencing read was aligned to the set of expected antibody sequences from the 18 were included in each experiment to act as a set of standards for which BLI-derived interaction affinities were known a priori.Two mL of saturated MATa and MATalpha library were combined in 800 mL of YPAD media and incubated at 30 °C in a shaking incubator. Three technical replicates were performed for each assay (Table Genomic DNA was extracted using Yeast DNA Extraction Kit (Thermo Fisher Scientific) following manufacturer’s instructions. qPCR was performed to amplify a fragment pool from the genomic DNA and to add standard Illumina sequencing adaptors and assay specific index barcodes. qPCR was terminated before saturation to minimize PCR bias, generally between 23–27 cycles. The final amplified fragment was concentrated with KAPA beads, quantified with a Quantus (Promega), and sequenced with a NextSeq 500 sequencer (Illumina).Sequencing data were analyzed to identify the MATa and MATalpha barcode pairs present among diploid yeast. The observed number of sequencing reads for each MATa/MATalpha combination were normalized according to frequency among haploid yeast to account for uneven distribution of the input populations. Each aα pair was then assigned a score representing the ratio of observed sequencing reads to expected sequencing reads assuming random mating. A linear regression was performed comparing these normalized sequencing scores to known affinities for the control yeast strains and this regression was utilized to assign estimated affinities to all other aα pairs for each mating replicate.23 (10.5281/zenodo.5095284). The dataset contains the variables listed in Table A single dataset was generated during this study. This data set contains the output of two AlphaSeq assay performed as part of a single study and is deposited at Zenodo23.The data are stored in a single.csv file. All data can be downloaded from Zenodok mutations per library  while variance increases with each additional mutation . In other words, each added mutation increases the probability of breaking the antibody but there is also more room for improvement over the wild type.To further validate the assay results by identifying expected patterns, AlphaSeq-derived binding affinities were compared for all antibody sequences, binned by the number of mutations separating each antibody from its seed sequence . Results are shown in Fig. 10 nM to 7.14 log10 nM for assay 1 and 1.35 log10 nM to 7.32 log10 nM for assay 2. As these values are higher than the distribution of values for on-target binding, they serve as an additional confirmation that the pred_affinity measurements are resultant of on-target binding. The binding affinities measured against negative controls represent some combination of nonspecific yeast mating and molecular artifacts introduced to the barcodes during PCR and sequencing and can act as an empirical readout of the limit of blank for this dataset. Note that given the increase in technical variation observed with increasing pred_affinity values, it is not recommended to background subtract these values from the on-target pred_affinity measurements.The inclusion of MATα yeast expressing no POI serves as an opportunity to identify MATa yeast with non-specific binding. These negative control yeast strains: AlphaNeg1, AlphaNeg2, and AlphaNeg3 are expressing AGA2 with an N-terminal HA epitope tag and C-terminal Myc tag without a POI. As such, many of the entries within the dataset represent interactions between MATa yeast with a MATα yeast expressing a negative target. The values associated with these measurements range from 1.03 logEach assay and replicate contains each of the three seed sequences and can be used to normalize the data among the assays or replicates. These sequences can also be included in future assays to allow for integration of additional data. Additionally, the regression used to transform sequencing abundances to predicted affinity values is performed once for each replicate and then applied to the entire replicate; relative ranking of interactions within a replicate are insensitive to any technical variation in that calculation, but such error will propagate to all quantitative predicted affinity measurements in that replicate.Data entries in which a sequence and target pair is specified but does not have a pred_aff value indicate a poor binding interaction. These antibody sequences are observed in DNA sequencing of the MATa haploid yeast population, but not among diploid yeast, affirming the sequence is present in the MATa library but no mating was observed. There are multiple options for how to treat these entries in downstream applications, including removing them from the dataset. While not conclusive, absence of diploids is strong evidence of poor binding affinity; imputing affinity values to indicate as such may be advantageous. Values could be imputed, for example, as the maximum pred_aff value or as the median pred_aff value of sequences not having measurements in all replicates.


Background: There has been a significant increase in the volume of gender-affirming surgical (GAS) procedures over the past decade. The objective of this paper is to use online search data from Google Trends (GT) to describe national search trends for GAS procedures.health.com. RSVs were analyzed by metro area to determine the relationship between search demand and personal income. State Medicaid policies for transgender health services were also collected.Methods: GT was queried for search terms relating to GAS from January 2004 to February 2021. The 19 selected keywords covered a broad range of GAS topics. United States (US) search interest was collected as relative search volumes (RSVs) and then analyzed by geographic region. The number of plastic surgery providers offering GAS and academic surgery centers was collected from the World Professional Association for Transgender Health (WPATH) and Trans-Results: All search terms demonstrated a positive increase in RSVs over time except “sex reassignment surgery” and “penectomy”. The Mountain/Pacific and East South Central/West South Central had the greatest search volume for GAS and most providers offering care. The East South Central/West South Central region ranked last for providers offering care, despite the relatively high search interest. This region also had no states with explicit Medicaid policies covering gender-affirming care. Metro areas in the top five for RSV but bottom quartile for per capita personal income were identified.Conclusions: Online search interest for GAS-related terms has increased. Search interest for GAS has regional variation and did not show a specific pattern with provider availability. Approximately 1.4 million transgender and non-binary (TGNB) adults and 150,000 TGNB adolescents and young adults live in the United States (US) . GAS hasSearch engines such as Google can provide valuable information about healthcare-related search trends. The largest keyword search engine, Google Trends (GT), is a free, accessible tool that allows individuals to analyze geographic and temporal trends as relative search volumes (RSVs) for search terms . GT is gGT has not been utilized to analyze US interest in GAS. The purpose of this study is to describe US trends in internet searches for GAS-related keywords by region and over time. We hypothesize that there will be discrepancies across various national regions between GT search demand for GAS such that areas with more surgical providers and gender centers will generate higher search interest. Data sourceGT was used to assess search volumes and trends over time ,20. GT eData acquisitionGT search parameters were set to Geographic Location: “United States,” Time Period: “1/1/2004-2/18/2021,” Category: “All Categories,” and Type of Search: “Web Search” to capture all US queries for the designated terms. Specific dates were used to ensure the replicability of the study. The terms used in this analysis were among the core procedure types defined by the World Professional Association for Transgender Health (WPATH) [Temporal dataMicrosoft Excel  was used to conduct a trend line analysis for each search term’s RSV. Regional dataRSV values were recorded by state for each of the 19 search terms. States were categorized according to regions designated by the American Society of Plastic Surgeons (ASPS) Annual Reports . Figure To determine the specific region in which each search term was most popular, the top 10 RSVs were examined. The region composed the largest percentage of RSVs for each search term’s top 10 RSVs was deemed to have the greatest regional interest for that term. For search terms where there was insufficient data to report on 10 states, the RSV for all available states was reported. In the event that multiple regions made up equal percentages , the region with the larger average RSV for that search term was determined to have the greatest interest. Metropolitan area based on income dataA metropolitan analysis of RSV for each of our search terms was also conducted for all of our search terms. GT search parameters were set to Geographic Location: “United States,” Time Period: “1/1/2004-2/18/2021,” Categories: “All categories,” and Type of Search: “Web Search.” For each of our search terms, we analyzed any metro area that was in the top five for RSV but bottom quartile according to the US Bureau of Economic Analysis . Metro aProviders and academic medical centers offering GAS dataTo quantify the number of plastic surgeon providers in the US offering GAS as of February 2021, the World Professional Association of Transgender Health’s (WPATH) website was utilized. The website lists plastic surgery providers in each state, and the search was subsequently filtered for these providers . AdditioMedicaid coverage dataAs of February 2021, the states that have an explicit policy stating that TGNB health-related services are covered under Medicaid were determined . We alsoTrends in interest in the United States from 2004 to 2021Overall, national trends in search terms for gender affirming surgery revealed “transgender surgery,” “gender reassignment surgery,” “gender affirming surgery,” “gender confirming surgery” “top surgery,” “bottom surgery,” “upper surgery,” “lower surgery,” “male to female/female to male surgery,” “MTF bottom surgery,” “FTM bottom surgery,” “facial feminization surgery,” “metoidioplasty,” “FTM phalloplasty,” “tracheal shave,” “vulvoplasty,”, and “penile skin inversion.” all showed positive increases in RSV from 2004 to 2021. The two terms that showed a decrease in RSV overtime were “sex reassignment surgery” and “penectomy.” Interest in GAS by regionBased on GT data for all selected search term to identify interest by state and region, 41/159 25.8%) of the top ten RSV values fell into Region 5 (Mountain/Pacific), 38/159 (23.4%) RSVs fell into Region 4 , 34/159 (21.4%) of RSVs fell into Region 1 (New England/Middle Atlantic), 25/159 (15.7%) fell into Region 2 , and 21/159 (13.2%) RSVs fell into Region 3 (South Atlantic)  had the greatest interest for the search terms: “sex reassignment surgery,” “gender reassignment surgery,” “tracheal shave,” “penectomy,” “facial feminization surgery,” “metoidioplasty,” and “penile skin inversion.” Region 4  had the greatest interest for the search terms “bottom surgery,” “male to female/female to male surgery,” “vulvoplasty,” “lower surgery.” Region 1 (New England/Middle Atlantic) had the greatest interest for “transgender surgery,” “top surgery,” “FTM phalloplasty.” Region 3 (South Atlantic) had the greatest search interest for “MTF bottom surgery,” “FTM bottom surgery,” and “upper surgery.” Region 2  had the greatest interest for the search terms “gender affirming surgery” and “gender confirming surgery.”Metropolitan data based on incomeMetro areas in the top five for RSV but bottom quartile for per capita personal income included: Richmond, Virginia, Las vegas, Nevada, Spartansburg, South Carolina, Grand rapids-Kalamazoo-Battle Creek, MI, Jacksonville, Florida, Columbus, Ohio, Hartford, Connecticut, and Bowling Green Kentucky.Providers and academic medical centers offering GAS dataThe WPATH website provided 101 plastic surgery providers across the US. A national distribution of these providers can be seen in Figure A total of 46 academic medical centers with TGNB surgery programs or dedicated TGNB surgery sections on their websites were found. A national distribution of these institutions can be seen in Figure Medicaid coverage dataOf the 22 states and Washington D.C. in which Medicaid programs cover gender affirming care, Region 1 (New England/Middle Atlantic) has the most states with this policy (nine states). Region 5 (Mountain/Pacific) follows with seven states, Region 2  has four states, Region 3 (South Atlantic) has three states. Region 4  has no states with this explicit policy. Figure We used GT to describe US search trends for various GAS procedures according to geographic region. As past studies have done, we aimed to use GT search data as a proxy for national interest in GAS ,16-19. GOur results highlight growing US search interest in GAS, as 17 of 19 included search terms had positive increases in RSVs from 2004 to 2021. The only terms that decreased in popularity were “sex reassignment surgery” and “penectomy.” This may be due to a shift in language used by TGNB individuals -27. TGNB patients report that the lack of knowledgeable TGNB providers is a large barrier to care . Using GEach region had certain search terms that were more widely used. For example, Region 5 (Mountain/Pacific) had the highest demand for terms such as “tracheal shave”, “penectomy”, and “facial feminization surgery” whereas Region 4  had the highest demand for “vulvoplasty”. Variation in search interest for specific terms may indicate potentially popular procedures in certain regions and/or regional differences in language. Providers in these areas can use this knowledge to include popular searches in their internet presence or increase awareness about other available procedures that patients might appreciate but not be exposed to in their region.Additionally, certain states  did not have top 10 search volumes for any of the selected terms. Of the states that did not appear in the top 10 for RSV volumes, only Wisconsin and Virginia had available providers to perform GAS. These can be targeted as areas where increasing awareness of potential procedures may be of significant value to TGNB individuals. GT can be used to track increased interest, and providers can be recruited to practice in areas of increasing interest. We identified eight low-income metro areas with significant search interest for GAS procedures. To ensure equitable access to care, providers can be incentivized to practice in these areas. Our results demonstrate that Region 4  had the lowest rates of Medicaid coverage and the fewest providers. Increasing coverage may be another means to draw providers to certain areas. Ultimately, regional variations of demand for gender-affirming care are complex and are often the result of local and state legislation, Medicaid coverage, and culture .There are several limitations to this study. Search volume values can be skewed by population size. A state with a larger population may have a higher absolute search volume but a lower proportion of total search results compared to a state with a smaller population. This limitation is minimized as numbers are taken in the context of RSVs and there are no comparisons made between absolute search volumes. In addition, only search terms with sufficient interest to generate an RSV value were included. The authors also recognize that not every term relating to GAS may have been included in this analysis. Patients may also be searching terms not specifically analyzed in this study or search terms in another language. Additionally, this study is limited in not providing information or outcomes for those actually undergoing GAS: not differentiating between the number of insured versus uninsured patients undergoing GAS, complication rates, or the types of surgeries people are getting. This offers the future-direction of questionnaire-based studies to elucidate this information. The GT algorithm generates RSV values based on a random sample of Google searches, meaning that reproducibility of our exact search is not guaranteed. It is also important to acknowledge that not all patients have internet access. Our sample only represents patients who can search online. Nevertheless, the 2018 United States Census shows that 92% of households in the US own a computer and 85% had an internet subscription .  AlthThis paper reports on the utility of GT in relation to transgender health and GAS. We describe national trends in online search interest based on region, city, and metropolitan area. Our results highlight a strong, growing national interest in GAS. Search interest for GAS has regional variation and did not show a specific pattern with provider availability. By improving our understanding of temporal and geographical search interest, we can identify areas where patients may seek GAS.


Initially, this investigation was focused on the effect that carbon-linking (pendant–type) units exert on a microporous structure and how this affects the gas-permeation properties of pendant–type organosilica membranes. Gas permeation results were compared with those of bridged–type organosilica membranes (C1–C8). Network pore size evaluation was conducted based on the selectivity of H2/N2 and the activation energy (pE) of H2 permeation. Consequently, pE (H2) was increased as the C/Si ratio increased from C1 to C8, which could have been due to the aggregation of pendant side chains that occupied the available micropore channel space and resulted in the reduced pore size. By comparison, these permeation results indicate that pendant–type organosilica membranes showed a somewhat loose network structure in comparison with bridged–type organosilica membranes by following the lower values of activation energies (pE). Subsequently, we also evaluated the effect that fluorine doping (NH4F) exerts on pendant−type  and bridged-type  organosilica structures with similar carbon numbers (C1 and C3). The gas-permeation properties of F–doped pendant network structures revealed values for pore size, H2/N2 selectivity, and pE (H2) that were comparable to those of pristine organosilica membranes. This could be ascribed to the pendant side chains, which might have hindered the effectiveness of fluorine in pendant–type organosilica structures. The F–doped bridged–type organosilica (BTESM and BTESP) membranes, on the other hand, exhibited a looser network formation as the fluorine concentration increased.A series of pendant–type alkoxysilane structures with various carbon numbers (C Activation energy (pE) increased as the carbon number increased in both types of membranes, which indicates increased space occupation by pendant side chains as the C/Si ratio increased, and increased network flexibility in bridged–type organosilica membranes. However, permeation results revealed that pendant–type organosilica membranes demonstrated a larger pore size in comparison with bridged–type organosilica membranes, due to the lower values of activation energies (pE) which is evidence of a loose network formation.The pendant-type organosilica membranes showed a slightly looser network structure with a decrease in H2/N2) and pE (H2). Those results are ascribed to the existence of pendant side chains, which could have restricted the effectiveness of fluorine and allowed the pore size to remain unchanged. Conversely, fluorine significantly improved the permeation properties of bridged–type organosilica membranes (H2/N2); the F/Si ratio was increased due to the formation of a loose network formation caused by the Si–F and C–F groups present in the network structure. Fluorine–doped pendant–type organosilica membranes showed pore sizes similar to those of undoped organosilica membranes with comparable selectivity (HAfter several experiments, all membranes continued to exhibit considerable stability in characteristics such as molecular size dependence and temperature dependence of gas permeance. These good results notwithstanding, long-term separation performance of these membranes will be conducted in our future work.