MAPK3, P-ERK was measured. P-ERK is decreased in hyperproliferative but increased in hypoproliferative NPCs. While these P-ERK changes are not responsible for the phenotypes, P-ERK and bFGF response are inversely correlated with the defects. Finally, we analyzed iPSCs and discovered that 16pDel displays hyperproliferation, while idiopathic iPSCs were normal. These data suggest that NPC proliferation defects are common in ASD.Neural precursor cell (NPC) dysfunction has been consistently implicated in autism. Induced pluripotent stem cell (iPSC)-derived NPCs from two autism groups (three idiopathic [I-ASD] and two 16p11.2 deletion [16pDel]) were used to investigate if proliferation is commonly disrupted. All five individuals display defects, with all three macrocephalic individuals  exhibiting hyperproliferation and the other two I-ASD subjects displaying hypoproliferation. NPCs were challenged with bFGF, and all hyperproliferative NPCs displayed blunted responses, while responses were increased in hypoproliferative cells. mRNA expression studies suggest that different pathways can result in similar proliferation phenotypes. Since 16pDel deletes  •NPC proliferation defects are common in idiopathic and 16p11.2 CNV-deletion ASD•All macrocephalic I-ASD and 16pDel individuals have hyperproliferative NPCs•NPC proliferative responses to bFGF correlate inversely with P-ERK levels•Both NPCs and IPSCs derived from 16pDel individuals exhibit hyperproliferation In this article DiCicco-Bloom and colleagues show proliferation defects in neural precursor cells from idiopathic and 16p11.2-deletion forms of autism. Those exhibiting macrocephaly were hyperproliferative, while two idiopathic individuals displayed hypoproliferative phenotypes. Interestingly, 16pDel individuals also exhibited iPSC-hyperproliferative defects. These findings suggest that dysregulated proliferation may be a common phenotype in multiple forms of autism and span developmental stages. Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder characterized by difficulties with social interactions and communication and the presence of repetitive and restricted behaviors. Most ASD cases are idiopathic, having no known genetic cause, and only 15%–20% of ASD cases are caused by known mutations such as copy number variants (CNVs) or monogenic mutations . AlthougMAPK3, which encodes ERK1. Both the deletion and the duplication are correlated with increased autism risk  subgroups, while the other individuals exhibit hypoproliferation. Interestingly, the proliferation phenotype is inversely correlated with P-ERK levels and response to basic fibroblast growth factor (bFGF), a mitogen that stimulates the ERK pathway. In examining whether proliferation dysregulation is specific to the cells of the brain, we find that the I-ASD cohort shows no differences in their iPSCs. In contrast, iPSC proliferation in 16pDel ASD subjects parallels the increases seen in NPC proliferation. In aggregate, our findings suggest that idiopathic and 16pDel subgroups share a common phenotype of dysregulated NPC proliferation that for our datasets is inversely correlated with P-ERK levels and bFGF stimulation.To generate an I-ASD dataset, male autism probands and sex-matched, unaffected siblings from three families were chosen from the larger New Jersey Language and Autism Genetics Study (NJLAGS) A Bartle, 2014. NFor the 16pDel cohort, we selected from RUCDR Infinite Biologics the only two deletion males diagnosed with autism by the Simons VIP cohort for whom iPSCs were created see . Both maFor each subject, multiple iPSC clones were generated. To ensure pluripotency and quality control, iPSC lines were karyotyped and assessed for expression of molecular makers of pluripotency . A minim3H]thymidine labeling, S-phase labeling index, cell enumeration assays, and apoptotic marker cleaved caspase-3 immunocytochemisty (ICC). Using this strategy, we found that all I-ASD subjects display NPC proliferative differences compared with unaffected sibling controls. I-ASD individuals from two different families (families 1072 and 1012) exhibited reductions in NPC proliferation, whereas in the third macrocephalic ASD subject (family 1077), NPCs exhibited an increase in proliferation.To rigorously define proliferation in human NPCs, we employed a multi-tiered strategy consisting of simple and reproducible assays followed by more in-depth analyses to further characterize neurogenesis. We examined multiple measures of cell proliferation and cell death: total DNA synthesis by [3H labeling , comparing multiple separate derivations of NPCs derived from four sibling iPSC clones with those from five ASD iPSC clones. The ASD NPCs exhibited a significant 65% reduction in DNA synthesis at 48 h by labeling A. To seelabeling B. In exalabeling C, and AS at 24 h D. These Similar to family 1072, ASD-1012 NPCs also exhibited a decrease in DNA synthesis, by 20% at 48 h E, and a In marked contrast to these two hypoproliferative families, macrocephalic ASD-1077 NPCs displayed a 60% increase in DNA synthesis I that waIn sum, all I-ASD NPCs display a proliferation phenotype (two hypoproliferative and one hyperproliferative) compared with their respective sibling. Notably, all three I-ASD individuals are not related to one another and remain genetically undefined, yet they converge on proliferative defects.To inquire if NPC proliferation is also altered in the 16pDel cohort, we compared NPCs from the 16pDel individuals with two sex-matched genetically typical controls. Both 16pDel individuals are macrocephalic and exhibit a significant increase in DNA synthesis at 48 h A. Specif+ cells compared with NIH controls at 24 h , that is required for normal control of cortical progenitor proliferation . FollowiIn contrast, ASD NPCs that exhibited hypoproliferation (I-ASD-1072 and -1012) displayed an increase in DNA synthesis sensitivity and response to bFGF stimulation D and 4E.ID2 and METRN) compared with sibling controls . A multiplex, high-throughput Quantiplex panel was developed for 21 genes that are used as markers for human NPC proliferation. NPCs from the two I-ASD families with the greatest differences in proliferation (families 1072 and 1077) were examined. Family 1077 revealed no significant changes A, while controls B. In concontrols C and 5D. markers  and S3 aMAPK3, which encodes ERK1. Given that ERK signaling affects cell growth and proliferation, we measured levels of phosphorylated and total ERK1 protein. We first examined NPCs with a hyperproliferative phenotype. As expected, NPCs from both 16pDel individuals exhibited a 50% reduction in total ERK1 protein and P-ERK1 . Interestingly, the opposite signaling pattern was observed: a 50% increase in the levels of both total and normalized P-ERK1 . We titrFinally, we assayed for proliferation phenotypes in additional cell types such as iPSCs. Interestingly, none of the three I-ASD iPSCs displayed significant changes in DNA synthesis at 48 h A nor in In contrast, proliferation of the iPSCs from the two 16pDel individuals exhibited increased DNA synthesis at 48 h, with 16pDel-1 increasing by 360% and 16pDel-2 by 148% C. This iIn this study, we employed a rigorous approach to assess neurogenesis utilizing 53 NPC derivations from 24 distinct iPSC lines of 10 individuals, to compare the proliferation phenotypes between an idiopathic ASD subgroup and the genetically defined 16pDel model. Proliferation defects were observed in all five ASD NPC lines across both subgroups, which is interesting in light of their clinical and genetic heterogeneity. We also uncovered hyperproliferation defects in macrocephalic individuals from both subgroups, with this study being the first to report proliferative defects in 16pDel NPCs. These data suggest that dysregulation of proliferation is a common defect in ASD, manifesting as either “too little” or “too much.”Our proliferation results are supported by other studies. Proliferation defects have been reported previously using iPSC-derived NPCs for both idiopathic ASD and other CNVs, such as 7q11.23 . For exaWhile we observe consistent proliferation phenotypes, the cellular mechanisms are likely to be different between subgroups and ASD individuals. I-ASD NPCs from two families  with normal head circumference exhibited significantly decreased proliferation. Family 1072 NPCs demonstrated a much greater decrease in proliferation that was paralleled by a reduction in S-phase entry and an increase in cell death, while for family 1012, a smaller decrease in proliferation correlated with a significant increase in cell death, with no difference in cells engaged in S phase. Understanding the mechanisms responsible for the decrease is an issue for future study.Importantly, our results also add to the mounting evidence that autism NPCs from those with macrocephaly exhibit a hyperproliferation phenotype. While macrocephaly is well described in individuals who carry the 16pDel, this is the first evidence that their derived NPCs exhibit a proliferation phenotype. While both males have the same CNV deletion, they exhibit differing levels of increased proliferation, highlighting the heterogeneity of autism even within a genetically defined subgroup. Although the macrocephalic I-ASD family 1077 individual also displayed hyperproliferation, the mechanism is likely different. Indeed, compared with respective controls, family 1077 NPCs had no changes in the levels of P-ERK1 nor in NPC molecular markers, while 16pDel exhibited many mRNA differences and decreased P-ERK. Further, our findings of increased proliferation in 16pDel NPCs are in contrast to a previous study that did not find changes in proliferation or S-phase entry . While eThe idea that either too little or too much of a neurodevelopmental process may contribute to autism neuropathogenesis has broad support across multiple studies. For example, both deletion and duplication lead to neurodevelopmental phenotypes of Rett syndrome and MECP2 duplication syndrome ; the FXSNPCs from all idiopathic and 16pDel ASD cases displayed altered mitogenic responsiveness to bFGF, an important regulator of neurodevelopment . NotablyMAPK3 gene has been suggested to contribute to the head size changes in 16p11.2 individuals  and others have identified calcium and MAPK signaling as central nodes in ASD . Mutatioividuals .MAPK3/ERK1 deletion in the 16p11.2 CNV phenotype, we examined potential contributions to ASD NPC proliferation by measuring P-ERK1/2 protein levels. Our studies indicate that in four of the five ASD individuals, the absolute levels of P-ERK1 correlated inversely with the levels of NPC proliferation. In hypoproliferative I-ASD NPCs, we observed increased levels of P-ERK1 protein at baseline, a change that may underlie their increased sensitivity to bFGF stimulation. Conversely, in hyperproliferative NPCs from 16pDel individuals, we observed ∼50% reduction in P-ERK1 levels, as expected given the hemizygous genetic status of CNV deletion carriers. This reduction in absolute P-ERK1 protein levels may potentially contribute to the blunted responses to bFGF in the 16pDel cohort. It is notable that, unlike the 16pDel mouse model  as detailed in 3H labeling, cell counting, EdU, and activated caspase-3 ICC) were performed as detailed in Proliferation and apoptosis assays (6 cells/dish) from passages P3 to P8 were plated in 35 mm dishes and analyzed at 48 h  was designed and ordered through Invitrogen and used with standard protocols on triplicate samples see .Statistical Package GraphPad Prism version 7.0d  was employed. Statistical testing involved two-tailed Student’s t tests or two-way ANOVA with Sidak’s multiple comparison test where specified. Data are expressed as the mean ± SEM Outliers were determined with the ROUT test and removed from analysis. In each figure legend, the number of iPSC clones used, number of total NPC derivations, number of experiments, and number of wells are detailed for each individual. For all statistical tests, sample size was the number of individual data points including all technical and biological replicates .https://www.sfari.org/resource/simons-searchlight/) by applying at https://base.sfari.org.Approved researchers can obtain the Simons Searchlight population dataset described in this study (Conceptualization, J.H.M., E.D.-B., J.F., L.B., C.W.L., and Z.P.P.; methodology, J.H.M., P.M., P.L.Y., E.D.-B., and M.W.; formal analysis, M.W., R.C., S.P., P.M., P.L.Y., and M.M.; investigation, R.C., M.W., S.P., M.M., P.L.Y., C.W.L., A.M., C.P., X.Z., and C.R.M.; writing – original draft, M.W., R.C., M.M., J.H.M., and E.D.-B.; visualization, M.W., R.C., S.P., and M.M.; writing – review & editing, M.W., R.C., M.M., J.H.M., and E.D.-B.; funding acquisition, J.H.M. and E.D.-B.; resources, J.F., L.B., C.W.L., and P.L.Y. created and validated all I-ASD and sib iPSC lines used in this study, and NPC lines were generated by P.L.Y., P.M., M.M., S.P., M.W., and R.C.; supervision, J.H.M., E.D.-B., P.M., and C.W.L.The authors declare no competing interests.


A wide variety of transition metals, including copperand gold,have been successfully used as substrates for graphene growth. Onthe other hand, it has been challenging to grow graphene on silver,so realistic applications by combining graphene and silver for improvedelectrode stability and enhanced surface plasmon resonance in organiclight-emitting diodes and biosensing have not been realized to date.Here, we demonstrate the surface passivation of silver through thesingle-step rapid growth of nanocrystalline multilayer graphene onsilver via low-temperature plasma-enhanced chemical vapor deposition(PECVD). The effect of the growth time on the graphene quality andthe underlying silver characteristics is investigated by Raman spectroscopy,X-ray diffraction, atomic force microscopy, X-ray photoelectron spectroscopy(XPS), and cross-sectional annular dark-field scanning transmissionelectron microscopy (ADF-STEM). These results reveal nanocrystallinegraphene structures with turbostratic layer stacking. Based on theXPS and ADF-STEM results, a PECVD growth mechanism of graphene onsilver is proposed. The multilayer graphene also provides excellentlong-term protection of the underlying silver surface from oxidationafter 5 months of air exposure. This development thus paves the waytoward realizing technological applications based on graphene-protectedsilver surfaces and electrodes as well as hybrid graphene-silver plasmonics. For instance, it hasbeen challenging to produce highly oriented pyrolytic graphite toan areal dimension larger than ∼ (0.1 × 0.1) m2. In the case of graphene growth, chemical vapor deposition (CVD)is a common scalable approach to achieve large-scale high-qualitygraphene synthesis. Common transition-metal substrates such as Ni2 and Cu3 are used alongwith high processing temperatures to pyrolyze hydrocarbon moleculesto activate CVD graphene growth.4 Othertransition-metal substrates such as Ag,5 Au,7 Pt,8 Ru,9 and Co10 have beenreported for CVD graphene synthesis.The unique electronicand optical properties of graphene have stimulatedan extensive range of scientific research and technological applications.Additionally, multilayer graphene may be regarded as an alternativeto graphite in many graphite-related applications with the benefitsof much better scalability in the lateral dimensions11 In addition, substratesinvolving temperature-sensitive materials such as polymers would bedamaged under high-temperature processes, preventing their applicationsfor flexible materials.12 Moreover, thecurrent industrial trend of net zero carbon emission by 2050 makesthe high-temperature process unfavorable due to high energy consumption.Research progress has been made to reduce the growth temperature forCVD graphene. For instance, a growth temperature of 100 °C hasbeen reported through the use of a benzene precursor.13 Moreover, near-room-temperature growth has been demonstratedby utilizing liquid metal nucleation.14 Despite the fact that these novel approaches have greatly reducedthe growth temperature, they require sophisticated processing stepsthat are incompatible with industrial processes and large-area production.15One of the drawbacks ofCVD graphene growth is that it is a high-temperatureprocess, typically close to the melting points of the metal foils.Under such conditions, aged quartz furnaces and evaporated metal couldlead to contamination.16 have further loweredthe required temperatures for PECVD growth of graphene by forced convectionto increase the reaction probability of excited species or radicalson the substrate surface before their recombination. To date, thereported growth temperatures for various PECVD graphene synthesismethods are found to range from 160 to 700 °C on various substrates.22On the other hand, a scalable and industrially compatibleprocessfor low-temperature graphene growth is the plasma-enhanced CVD (PECVD)method. The key factor that allows PECVD to reduce growth temperaturelies in the plasma, which contains reactive species to promote growth.Recently, Kim et al.23 This problemmay be mitigated by the combination of graphene with silver, whichhas been reported to increase the stability of the silver nanowireelectrodes24 and theoretically predictedto enhance the SPR sensitivity while preventing oxidation.23 On the other hand, the inert nature of silverhas made it difficult to be used as a substrate for graphene growthwith the standard CVD techniques. Among the limited reports of graphenegrowth on silver, one approach involves evaporating atomic carbononto the surface of a single-crystalline Ag(111) substrate at elevatedtemperatures under ultrahigh vacuum conditions for in situ scanningtunneling microscopy studies.25 The otherapproach utilizes a high-temperature, atmospheric-pressure CVD processwith solid camphor as the carbon precursor and silver foil as thesubstrate in a gas mixture of Ar and H2.5 Despite these progresses, direct graphene growth on silverat low temperatures remains a challenging task.26Silver is commonly used as the electrode for organic light-emittingdiodes and in biosensing due to its strong surface plasmon resonance(SPR). However, silver is prone to oxidation, which would degradethe device performance.In this work, we show the viability of direct growthof nanocrystallinemultilayer graphene on a silver thin film via low-temperature PECVD,where the growth configuration involves flipping the substrate downwardso that the silver thin film faces away from the direct plasma. Thesuccessful growth of nanocrystalline multilayer graphene is confirmedby Raman spectroscopy. X-ray diffraction (XRD) studies of the silverthin films after the PECVD process further reveal the improvementof silver crystallinity. Studies by transmission electron microscopy(TEM) reveal that the resulting multilayer graphene is of turbostraticstacking. We further propose a growth mechanism of graphene on silverfrom studies of the X-ray photoelectron spectroscopy (XPS) and cross-sectionalannular dark-field scanning TEM (ADF-STEM) and demonstrate that XPSdata may be used as a nondestructive means to infer the average graphenethickness in agreement with the ADF-STEM results. Confirmed throughXPS, the surface of silver fully covered by the directly grown multilayergraphene exhibit no traces of oxidation after 5 months of ambientair exposure, which is in stark contrast to the XPS data of a controlledsilver surface without graphene coverage, implying perfect passivationof silver by the PECVD-grown multilayer graphene. The excellent protectionof silver from oxidation provided by PECVD-grown graphene and theimprovement of the underlying silver crystallinity after the PECVDprocess suggests that our approach paves ways toward realistic technologicalapplications of graphene-protected silver electrodes and surfacesas well as hybrid graphene-silver plasmonics.22.1The Ag substratein this work included a Si substrate covered with a 100 nm-thick Tiadhesion layer and a 500 nm-thick Ag thin film on top, both depositedvia an electron-beam (e-beam) evaporator.2.22SO4:H2O2 = 3:1 in volume) at room temperature. Afterward, O2 and H2 plasma were separately used to clean boththe quartz tube and the sample holders. The plasma system involveda microwave power source (Sairem) and an Evenson cavity. Prior toplasma ignition, CH4 of 1 sccm and H2 of 4 sccmwere added into the quartz tube with a total pressure of 100 mtorr.The Ag side of the Ag/Ti/Si substrate was flipped downward and placedon the sample holder before the PECVD process as shown in Before thePECVD process, the interior of the quartz tube was rinsed with nitricacid to remove potential silver residue, while sample holders werecleaned with piranha solution . Afterthe PECVD process, polymethyl methacrylate (PMMA) was spin-coatedon the silver samples covered with PECVD-grown graphene, which wasfollowed by silver etching with a gold etchant  inorder to transfer the graphene grown on silver to a SiO2.3α X-ray source and a hemispherical energy analyzer under a pass energyof 10 eV was used for the high-resolution scan, whereas a pass energyof 80 eV was used for X-ray induced Auger spectroscopy (XAES) of theC KLL Auger region. In addition, the XPS signal was used for estimatingthe graphene thickness, as elaborated in a later section. The instrumentwork function was calibrated with respect to the Ag 3d5/2 signal. Cross-sectional ADF-STEM and TEM plane view images wereacquired by aberration-corrected JEOL ARM-200F operated at 200 kV.Selected-area diffraction was performed using JEOL JEM2100F at 200kV. XRD (Rigaku Smartlab) was performed using Cu Kα radiation and a Ge (220) double-bounce monochromator for Kα2 elimination.Raman spectroscopiccharacterizations for graphene growth and quality confirmation weremade by using a Raman spectrometer  equipped witha 514 nm laser. Atomic force microscopy with PeakForce tapping mode was used for surface morphology characterization.XPS (Kratos Axis Ultra) with a monochromated Al K32 substrate with the transferred grapheneas shown in 2. Interestingly, despite very high I(D)/I(G) ratios for all samples, the distinct2D and 2D′ peaks indicated good graphene crystallinity of ourPECVD-grown graphene. The 2D and 2D′ peaks of graphene resultfrom intervalley and intravalley phonon scattering, respectively;neither has needs for defect activation.28 Therefore, one may expect that as the defect concentration increased,2D and 2D′ peaks would have become worse defined due to imperfectelectron dispersion, as demonstrated by Eckmann et al.29 In this context, had the high I(D)/I(G) ratios found in our graphene samples beenrelated to high defect concentrations, distinct 2D and 2D′peaks would not have existed.I(D)/I(G)]and the D-to-D′ Raman mode intensity ratio [I(D)/I(D′)], respectively.31 As shown in I(D)/I(G) ratio decreasedslightly with increasing growth time, which implied changes in thegrain size with growth time. The grain size of graphene L can be estimated by the I(D)/I(G) ratio through Elaser =2.41 eV. From I(D)/I(G) ratios suggest that the graphenegrown on silver thin films via our PECVD method was nanocrystalline.Meanwhile, as shown in I(D)/I(D′)ratio for different growth times was between 8 and 9, suggesting thatthe defect types were a mixture of sp3 bonds for I(D)/I(D′) = 13 and vacancy defectsfor I(D)/I(D′) = 7, althoughprimarily the vacancy type.30On the other hand, it is known that the grain sizeof grapheneand its defect types may be extracted by analyzing the D-to-G Ramanmode intensity ratio [I(2D)/I(G) may be used for determining the graphene thickness,with I(2D)/I(G) > 1 implyingmonolayergraphene. In addition, the full width at half-maximum (fwhm) of the2D peak may provide further information about the number of graphenelayers, with an fwhm of ∼24 cm–1 for single-layergraphene32 and of ∼50 cm–1 for bilayer graphene.34 However, turbostratic stackingof graphene could also give rise to Raman spectral characteristicssimilar to those of single-layer graphene. In this context, the I(2D)/I(G) ratio and the fwhm of the 2Dpeak for our PECVD-grown graphene on silver shown in A common perceptionin the Raman spectral analysis of grapheneis that the 2D to G peak intensity ratio 35 On the other hand, differentdoping levels of graphene could also lead to a spectral shift of thepeak position. As discussed by Lee et al.,36 the doping level and strain effect in graphene may be separatedby plotting the 2D peak position versus the G peak position. Followinga similar analysis, the 2D peak position versus the G peak positionof the PECVD-grown graphene on silver showed slightly hole-doped andslightly compressively strained, as demonstrated in Section S1.Besidesthe number of graphene layers and the grain size, Ramanspectroscopy could also shed light on the doping and strain of graphene.The spectral shift of either the 2D or G peak, along with the correspondingGrüneisen parameter, could be used to extract the strain effectof graphene.37To understand the effect of the flipped substrateconfigurationon the gas flow during the PECVD graphene growth, a computationalfluid dynamics (CFD) simulation was carried out. As shown in Figure S2 shows theAFM characterizationof the surface morphologies of a sample before and after the PECVDprocess. After PECVD graphene growth, the AFM image of the surfacemorphologies was still dominated by the underlying silver layer andrevealed a large coalesce of grains and facets, as shown in Figure S2a–c. In contrast, the silversurface before the PECVD process exhibited apparent granular structures,as shown in Figure S2d. The facet formationafter the PECVD process may be attributed to the stabilization ofgraphene on the metal surface.38 The increasedroughness after PECVD may be attributed to the coalescence of smallergrains, leading to a much larger grain boundary.Figure S3. The increased Ag (111) intensitycounts after PECVD for different growth times were all much greaterthan that before the PECVD graphene growth process. A similar peakintensity for all growth times was observed. Given that no activeheating source was involved in our PECVD and that the sample temperaturewas about 232–260 °C through plasma heating, which wasmuch lower than the melting point of silver (961.8 °C), the improvedcrystallinity of Ag (111) after the PECVD process when compared tothe reference sample may be attributed to the sufficient thermal energy12 provided by plasma activation. The improvedAg crystallinity could provide added benefits for silver plasmonicapplications.40Meanwhile,the changes in the crystallinity of silver after thePECVD process, as characterized through XRD measurements, are shownin 2 were also present. The Si 2pregion scans were carried out as shown in Figure S4, which confirmed the existence of SiO2 on thesurface. The origin of the SiO2 will be discussed in alater paragraph.The chemical changes inthe silver thin film quickly after thePECVD process were characterized via XPS as shown in Figure S5, which also confirmed the dominant contribution of sp2 carbon. Moreover, neither metal carbide formation41 nor bonding between the observed Si and graphene couldbe inferred from the absence of any apparent peak around the bindingenergy of 282–283 eV. The extent of hybridization of carbonmay be revealed by comparing the “D-parameter” in theC KLL Auger region, where the D-parameter is defined by the peak separationin the first derivative of the Auger spectrum;42 a larger D-parameter would indicate a higher sp2 hybridization percentage. Using the results from the XAES studies, we obtained the first derivativeof the spectra for different growth times shown in Figure S6e–g. In addition to the spectra of differentsamples before differentiation, the XAES of a graphitic referencehas been included in Figure S6d,h to validatethe data processing. As shown in Figure S6, the graphene sample with 15 min growth time exhibited the largest D value, suggesting a higher sp2 percentage thanthose samples with either 10 or 5 min growth time. This finding alsocorroborated the lower I(D)/I(D′)ratio shown in 3-like defects.The graphene formation on silver after PECVD wasfurther verifiedthrough XPS C 1s spectra as shown in I(D)/I(G) ratios in the Raman spectrum).ADF-STEM images as shown in 43 and by Cumpson et al. that suggested a “thickogram”graphical approach.44 Specifically, thegoverning equation for the thickogram is given byIo and Is are the integrated spectral area under thepeaks from the overlayer and the substrate, respectively; So and Ss are the corresponding relative sensitivity factors; Eo and Es are thekinetic energies of the overlayer element (carbon) and substrate element(silver), respectively; λo is the photoelectron inelasticmean free path, t is the overlayer thickness, and θ is the emissionangle. In this work, λo for graphene was 1.06 nm,42 and θ for the XPS system was 0°.Additionally, we note that the XPS spectra used for the thickogramanalysis must be recorded with the same number of scans and pass energy.Comparing the direct imaging of graphene layersby ADF-STEM withXPS characterizations, we investigate whether XPS studies may provideuseful information about the average number of graphene layers, similarto previous studies by Hill et al. that proposed to determine theoxide thin film thickness through XPSThe advancement of computational power since the graphical approachinitially proposed by Cumpson et al. has enabled the numerical computationof sample thicknesses based on the given XPS data. Using the XPS datataken on our PECVD-grown graphene on silver samples with differentgrowth times, we find that the extracted overlayer thicknesses for 15, 10, and 5 min growth times are 1.22, 0.55, and0.63 nm, respectively, which are in reasonable agreement with theADF-STEM imaging. Therefore, we have confirmed that XPS data may beused to infer the graphene thickness in addition to Raman spectroscopicanalysis and ADF-STEM imaging.46 the growth mechanism of graphene on silver is expectedto be similar to the surface adsorption mechanism on Au. In this contextand noting a previous work by Lu et al. on PECVD graphene growth onAu that demonstrated bilayer graphene growth for H2/CH4 ≥ 1 after 5 min,20 ourfinding of bilayer graphene growth on silver for the same growth time(5 min) with H2/CH4 = 4 appeared to be consistentwith the previous report.Based on the XPS and ADF-STEMdata, we hypothesize the PECVD graphenegrowth mechanism on silver, which is schematically shown in On the other hand, when the growth time was extendedto 10 and15 min, three or four graphene layers were observed. As shown in theADF-STEM images in 47 However, the penetration growth mechanism could not support graphenegrowth for more than bilayer due to the restricted penetration ofcarbon atoms,47 which contradicts our observation.It is worth noting that a penetration graphene growth mechanismhas been previously proposed by Wu et al.I(D)/I(G)ratios. However, higher plasma power would lead to more ejected Sispecies into the plasma and therefore the undesirable result of moreSi incorporation into the graphene layers during the growth. Thisconsideration therefore constrains the choice of plasma power duringthe PECVD growth of graphene on silver.Since the graphene growth temperature is dependent on the plasmapower, where higher power results in higher temperatures, it is conceivableto apply plasma power greater than 10 W to achieve larger graphenegrain sizes with smaller 45An important issue toaddress for the usefulness of our nanocrystallinemultilayer graphene is to evaluate its ability for surface passivationbecause the small grain sizes are accompanied by many grain boundaries,which may lead to compromised surface passivation because gas moleculescould pass through the grain boundaries and react with the underlyingsilver. Fortunately, we found that the multilayer nature and turbostraticstacking of our PECVD-grown graphene could compensate for the drawbackof many grain boundaries. 4In conclusion, we reporta low-temperature single-step method fordirect graphene growth on silver by PECVD for long-term surface passivation.Raman spectroscopic studies of the graphene-on-silver samples suggestedthat they consisted of nanocrystals with an overall good crystallinequality, underwent a slight compressive strain, and exhibited slighthole doping, with vacancies being the primary defects in the samples.Using CFD simulations, the benefit of using the flipped substrateconfiguration during the PECVD graphene growth was revealed. FromAFM and XRD characterizations, the silver surface morphology and crystallinityafter the PECVD process were found to differ from those before thePECVD process, with improved crystallinity after PECVD. The numberof graphene layers grown on silver was verified by cross-sectionalADF-STEM images, which varied from 2 to 4 layers depending on thegrowth time. The stacking order of the multilayer was confirmed throughTEM electron diffraction to be turbostratic. We further proposed amechanism for the PECVD growth of graphene on silver based on thefindings from the XPS and ADF-STEM studies and demonstrated that XPSdata may be used for the nondestructive thickness determination ofgraphene. Moreover, the multilayer graphene was found to protect theunderlying silver against oxidation for at least 5 months of ambientair exposure. The combined benefits of passivation and improved crystallinityof silver by PECVD-grown graphene imply that our approach paves theway toward scalable technological applications based on graphene-protectedsilver surfaces and electrodes as well as hybrid graphene-silver plasmonics.


Various prognostic biomarkers of disease progression  have been described and can potentially be used to determine the group of patients who will benefit from the use of corticosteroids/cytostatic drugs/biologics.Many factors confirm the autoimmune nature of sarcoidosis and help in determining the strategy of patient management and treatment initiation. However, the causes and the mechanisms of disease progression that result in fibrosis and insufficiency of the affected organ remain unclear. This narrative review aims to analyse the mechanisms and biomarkers of sarcoidosis progression, as well as the pathogenetic basis of sarcoidosis therapy. The following characteristics of progressive chronic sarcoidosis were revealed: the disease develops in patients with a genetic predisposition (SNP in genes GREM1, CARD15, TGF-β3, HLA-DQB1 According to the current concept, sarcoidosis occurs as a result of an exposure to various exogenous or endogenous antigens in subjects with a genetic predisposition to autoimmune disorders and is associated with the development of non-necrotizing granulomas in different organs .The search for etiologic factors of sarcoidosis has revealed various infectious agents  as well as non-organic factors  that could be associated with its development , 3. LongBased on the pathologic findings, sarcoidosis is currently believed to be a granulomatous disease that can be either acute, subacute or chronic and is most commonly (in >90% of cases) associated with the involvement of the lungs and mediastinal lymph nodes as well as other organs and tissues and the development of non-necrotizing granulomas , 5, 6. IThe aim of this review is to analyse the mechanisms and biomarkers of sarcoidosis progression, as well as the pathogenetic basis of sarcoidosis therapy.The data for the narrative review were collected from the studies published in international databases MEDLINE, Current Contents, PubMed, Elsevier between 2001 and 2021 using the search words of “sarcoidosis, progression, pathogenesis, genetic markers, laboratory markers, treatment.” Inclusion criteria were original research with observation of patients with progressive sarcoidosis and comparison group (group without progression or the studied group before the progression), review articles and research articles on pathogenesis of sarcoidosis.The key feature of the pathogenesis of sarcoidosis is the formation of granulomas in the lungs, mediastinal lymph nodes, skin and other organs. In patients who are genetically predisposed to this disease, a contact of antigen-presenting cells  with an unknown foreign antigen results in the dysregulated immune response that is manifested as granulomatous inflammation . The maiMacrophages, dendritic cells and epithelial cells are the first cells to react with the antigens due to the presence of Toll-like receptors. Long-term exposure of Toll-like receptors to foreign antigens results in activation and epithelioid differentiation of macrophages, which then start producing pro-inflammatory cytokines . Having bound to the antigen, dendritic cells migrate to lymph nodes, where they present the antigen to T-lymphocytes. Further activation of immune cells involves various chemokines such as CCL5 (for Th1) and CCL2 (for Th2). Much attention has been recently paid to the role of Th17 lymphocytes in the pathogenesis of sarcoidosis. These cells are a CD4+ lymphocyte subset that express IL-17A and have pro-inflammatory or anti-inflammatory properties as a result of the local inflammatory environment. Granuloma macrophages in patients with sarcoidosis were shown to express CCR20 thus attracting Th17 cells, while IL-23 expression causes a significant increase in IL-17A concentrations , 14. Sinin vitro and in vivo models have also demonstrated the presence of M2 at the initial stages of granuloma formation. There is growing evidence of the role of mTOR signaling pathway in the granuloma formation demonstrated in studies of molecular mechanisms of macrophage dysfunction. The activation of mTORC1 in murine macrophages was shown to result in the disease progression and formation of granulomas just as in humans with progressive sarcoidosis  should be the main objectives of disease profiling determining future personalized therapeutic approaches .The development of a chronic disease is associated with individual characteristics of the immune system indicating the existence of some genetic predisposing factors. Several candidate genes have been described in literature:GREM1 encodes gremlin, a highly conserved glycoprotein, a member of the BMP antagonist family (Members of the TGF-β superfamily), including TGF-βs and bone morphogenetic proteins (BMPs). Heron et al.  15 gene (nucleotide oligomerisation domain (NOD)2) encodes an intracellular protein CARD15 of the NOD family. These proteins are involved in innate immunity through recognition of muramyl dipeptide, a component of bacterial cell wall peptidoglycan . It was p = 0.04; corrected p = 0.2; OR, 2.9)  .*06:02, HLA-DRB1*07/14/15 genotypes were associated with chronic sarcoidosis  pulmonary disease compared with the self-limiting group. Among these processes most significant changes were revealed in intracellular signaling (NF-kB and JAK-STAT cascades) and cell life  .In some cases when it is impossible to eliminate the pathogen or chronic autoimmune inflammation develops , 24–26, The causes of fibrosis are unknown. However, some characteristics have been described in patients with chronic sarcoidosis.Biopsy specimens obtained in patients with a progressive disorder demonstrated Treg with BTNL2 and ANXA11 mutations whose gene products have anti-inflammatory and immune regulatory properties. It is possible that Treg downregulate the effective inflammation and contribute to the development of chronic sarcoidosis , 29.The role of Th2 in the progression of sarcoidosis is being actively studied. It has been suggested that a transition from a Th1 to a Th2 cytokine signature may occur in chronic sarcoidosis, perhaps as a response to persistent inflammation . The patM2 macrophage polarization, as reflected by an increased CD163 expression, is associated with an enhanced antigen presentation within a select subset of regulatory T cells, leading to lower concentrations of IFN-γ and higher concentrations of IL-10 upon stimulation. Moreover, IL-13 has anti-inflammatory properties, including suppression of TNF-α release.IL-13 can stimulate macrophages to release TGF-β . In patiIL-13 and CCL2 secreted by Th2 were implicated in the pathogenesis of lung fibrosis models, including in idiopathic pulmonary fibrosis , 38 Tab. CCL2 prCCL18 has been directly implicated in fibrosis . CCL18 pM2 polarized macrophages preferentially express Arg1, which results is an increased arginase. Arginase metabolizes arginine to ornithine, a precursor for the collagen substrate proline . Th2 cytCytokines released during the granuloma formation and macrophage transformation directly activate JAK-STAT pathway, which supports chronic inflammation . High prThe current understanding of sarcoidosis progression is shown in M. Tuberculosis peptides, P. Acne, carbon nanoparticles). In various studies, mice have developed multiple areas of granulomatosis , which may indicate the features of the immune response in healthy mice, allowing for more effective elimination of pathogens. The granulomas observed in the experiment were shown to exist only if there was a continuous exposure to the adjuvant. We believe that sarcoidosis patients are chronically exposed by one or several triggers that contribute development and persistence of granulomas  knockout gene described the development of granulomas showing epithelioid-like macrophages, M2 macrophage polarization, and mTORC1 pathway activation . TSC2 doSome pathogenesis-related changes in the pulmonary tissue are accompanied by alterations in peripheral blood and bronchoalveolar lavage fluid in patients with progressive sarcoidosis. Accordingly, the observed correlation between some parameters and the development of granuloma inflammation in the tissues has revealed markers for sarcoidosis progression, namely increased levels of neopterin molecule synthesized from macrophages, sIL-2R marker of T-cell activation, chitotriosidase secreted by active macrophages, neutrophil-derived collagenase and elastase , and a lIncreased number of Th17 in peripheral blood  and highSome clinical characteristics such as extrapulmonary manifestations , composite physiologic index (CPI) - the index of lung function variables, which correlates with the degree of interstitial disease and CT measurements - the degree of fibrosis, enlargement of the pulmonary artery and the measures of diffuse lung carbon monoxide (DLCO) percentages were shown as markers of severity of pulmonary sarcoidosis , 55, 56.The use of Scadding radiographic stages can also predict the prognosis, the stage I disease was associated with the spontaneous resolution in 60 to 90% patients, while with an increase of stages the percentage of resolution possibility decreased (up to 0% in stage IV fibrotic disease) .The whole list of possible laboratory and clinical markers predicting the progression of sarcoidosis is presented in In some patients, sarcoidosis may be associated with a failure in the affected organ, which requires treatment. However, a substantial proportion of patients are likely to subsequently develop a chronic or progressive sarcoidosis, an estimated percentage of 10% to 20% of patients will have sequelae, and a fatal outcome is observed in 6–7% of cases . The aimRegulation of the immune response, as well as prevention of fibrosis due to suppression of the overactive macrophages, T-cells and cytokine  production can be the main approaches of the sarcoidosis treatment , 61.TNF-α, INF-γ) that contribute to the development of granulomas  . The mecanulomas , 64. Theanulomas .Recent studies have showed evidence for a role for humoral immunity in the sarcoidosis development indicating the possible use of anti-B-cell therapy. Therapy with anti-CD20 monoclonal antibody rituximab was associated with clinical improvement in sarcoidosis patients, which is most likely due to the role of “naive” and/or B- memory cells in the development of sarcoidosis –67. The The prescribing factor for these therapy could be the finding of autoantibodies on BAL or blood serum, such as autoantibodies to mutated citrullinated vimentin (anti-MCV) , 69, andvia IL4-independent pathways. Thus, the desirable anti-inflammatory effects of MTX are leveled by an increased risk of fibrosis . The main objective of the use of this group of drugs is to overcome the adverse effects of corticosteroids, taper their dose up to complete discontinuation (steroid-sparing effect). Methotrexate is the most commonly used second-line therapy agent. Methotrexate is a folic acid antagonist, which suppresses the production of TNF-α through adenosine A2A receptors, simultaneously inducing IL-4 and IL-13 that are up-regulators of M2 polarization. MTX can also polarize M0 to M2 fibrosis , 71.Leflunomide, a dihydroorotase inhibitor, can be used alone or in combination with methotrexate, including in patients with generalized, progressive disease or with incomplete effect from other therapy. Leflunomide affects the inflammatory process by inhibiting Th17 cells and activating Tregs . AzathioThe third line includes the use of biological targeted therapy . TargeteBased on the hyper-expression of JAK-STAT pathway in granuloma cells the JAK-inhibitors were used in the treatment of patients with different types of sarcoidosis. The cases of efficient use of tofacitinib and ruxolitinib were described in pateints with cutaneous , 83, mulThe described therapies can be beneficial for patients with weakened immune response, which can be diagnosed with the BAL or blood biomarkers, such as increased levels of neopterin, sIL-2R, chitotriosidase, neutrophil-derived collagenase and elastase , increasThe described algorithm of the pathogenesis-based therapy of sarcoidosis is presented on *06.02, HLA-DRB1*07/14/15) as a result of granulomatous inflammation following an antigen presentation in the lungs. It either causes immune system impairment or activates autoimmune disorders. Thus, the antigen elimination becomes impossible, and immune cells change their differentiation  during the chronic inflammation process and start producing cytokines  and enzymes (Arginase) resulting in fibroblast activation and the development of fibrosis.Based on the results of this analysis of published data, the following characteristics of progressive chronic sarcoidosis were revealed: the disease develops in patients with genetic predisposition  have been described and can potentially be used to determine the group of patients who will benefit from the use of corticosteroids/ cytostatic drugs/biologics see .However, currently it is difficult to determine which prognostic markers can be considered indicators for the use of corticosteroids/cytostatic agents/biologics, therefore clinical studies are needed to evaluate the diagnostic significance of determining the described genetic and laboratory markers for selecting a management strategy.At the moment, one of the problems of studying sarcoidosis is the lack of a clear connection of pathogenesis, a specific biomarker and the choice of treatment option. The solution of this practical issue can be aimed at: determining a group of patients with a low probability of self-limitation; pathogenetic justification for choosing a group of the 2nd and 3rd line therapy, based on the specific laboratory markers; determining patients at risk of relapse after drug withdrawal. For progress in these areas, it is necessary to continue research in the field of animal models of sarcoidosis, the connection of laboratory markers with variants of the course of the disease and their changes against the various therapy options, conducting clinical trials. This may make it possible to form clearer criteria for prescribing therapy, which in turn will increase its effectiveness.AM and IK conducted analysis of the materials and wrote the manuscript. AS conducted analysis of the materials, wrote the manuscript, and was a coordinator of the project. DK, YZ, and AG wrote the manuscript. PY was a coordinator the project and wrote the manuscript. YS was a coordinator of the project. All authors have read and agreed to the published version of the manuscript.This work was supported by the grant of the Government of the Russian Federation for the state support of scientific research carried out under the supervision of leading scientists, agreement 14.W03.31.0009.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping researchers promote themselves alongside their papers. Maize Cao is first author on ‘ Maize CaoHow would you explain the main findings of your paper to non-scientific family and friends?TARDBP). TDP-43 binds to DNA and RNA, which are critical to gene expression. Every cell type has a special function that is determined by RNA transcripts. This is what makes cells unique from one another. When TDP-43 function is impeded, the interaction with RNA transcripts is lost, affecting the gene expression of the cell. This can lead to a change in biological function, which can be detrimental. By combining published studies of TDP-43 loss of function that have used RNA sequencing to analyse gene expression, we can determine what RNA transcripts (or parts of transcripts) have been changed in various cell types. Identifying these changes are essential for unravelling the functions of TDP-43 and shed light on the cause of disease.Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are debilitating neurodegenerative diseases. The causation of disease is difficult to untangle, but a common denominator is the dysfunction of a protein called TDP-43 (encoded by What are the potential implications of these results for your field of research?TARDBP mRNA targets drives pathogenesis and disease phenotype remains unclear. By assessing TARDBP/Tardbp mRNA targets in human and rodent models, in neuronal and non-neuronal cell types, we can then verify which of these targets are also regulated by TDP-43 in human ALS/FTD neurons. These mRNA targets act as markers that can reveal how TDP-43 loss of function is associated with neurodegeneration. Further, by identifying common markers, we can exploit them to probe for evidence of TDP-43 dysfunction in various cell models used in research. They may also serve as potential biomarkers of disease and/or targets for therapeutic drugs.The impairment of gene expression and splicing regulation are now widely accepted features of ALS with TDP-43 proteinopathy, but how the specific complement of What has surprised you the most while conducting your research?“The most significant challenge [in ALS research] is uncovering the mechanism of disease to determine effective targets for treatment.”This study is an amalgamation of published TDP-43 knockdown studies and was a pandemic ‘lockdown’ project that started when lab access became limited. As I didn't start my PhD versed in bioinformatics, it was a good opportunity to practice. What surprised me the most was how much I enjoyed it, despite the steep learning curve. When conducting the study, the component of comparing the differential exon usage from published studies was the most interesting to me. As my own research involves TDP-43 knockdown as well, it was always satisfying to see my findings validated in the literature. The findings of this paper have contributed to my research in more ways than I could have imagined, and I hope others in the field will find it useful too, particularly with the supplementary webpage that provides the results in one place.Describe what you think is the most significant challenge impacting your research at this time and how will this be addressed over the next 10 years?The heterogeneity in ALS/FTD is one of the biggest hurdles in the field. The increasing number of genetic mutations associated with ALS alone highlights the multitude of cellular processes that could become impaired in disease. Consequently, the most significant challenge is uncovering the mechanism of disease to determine effective targets for treatment. Dysfunctional TDP-43, being the pathological hallmark of disease, certainly holds a lot of promise for uncovering mechanisms. However, as we found, TDP-43 is quite heterogeneous in function too! Over the next few years, ALS will likely move into stratified medicine, where treatment is tailored towards the genetics of the individual.What changes do you think could improve the professional lives of early-career scientists?From my experience, early-career scientists experience a lot of self-doubt, even when they are motivated and passionate about their research. I think this is brought upon by uncertainty and imposter syndrome when trying to find their ‘niche’ in the space. That is why I think mentorship, career guidance and peer-support is incredibly important.What's next for you?Currently my most imminent goal is completing my PhD. After that I would like to continue as a postdoctoral researcher. I also have a keen interest in RNA biology and would like to explore that further in my work. Collaborating and working with overseas groups would be another goal of mine, as I enjoy travelling and learning from people with different worldviews.


Graphene has exceptional electronic properties, such as zero band gap, massless carriers, and high mobility. These exotic carrier properties enable the design and development of unique graphene devices. However, traditional semiconductor solvers based on drift-diffusion equations are not capable of modeling and simulating the charge distribution and transport in graphene, accurately, to its full extent. The effects of charge inertia, viscosity, collective charge movement, contact doping, etc., cannot be accounted for by the conventional Poisson-drift-diffusion models, due to the underlying assumptions and simplifications. Therefore, this article proposes two mathematical models to analyze and simulate graphene-based devices. The first model is based on a modified nonlinear Poisson’s equation, which solves for the Fermi level and charge distribution electrostatically on graphene, by considering gating and contact doping. The second proposed solver focuses on the transport of the carriers by solving a hydrodynamic model. Furthermore, this model is applied to a Tesla-valve structure, where the viscosity and collective motion of the carriers play an important role, giving rise to rectification. These two models allow us to model unique electronic properties of graphene that could be paramount for the design of future graphene devices. Graphene has been the focus of various research topics for the last two decades and is pushing toward the market ,2, and eThis article is structured as follows. The transport properties of graphene depend on many internal and external parameters, such as its deposition technique, impurities, type of materials in contact with graphene, operating temperature, etc. These aspects need to be accurately considered for the design of next-generation graphene-based electronic devices in order to account for the effects, such as nonlinear contact doping, due to metals. To exploit the full potential of graphene’s exotic properties, one should be able to access and manipulate the Fermi level of graphene. Making use of graphene in electrical devices requires shifting the Fermi level, by gating and making contacts with metals, which inherently dopes the graphene locally and results in Fermi level shifts. Both of these effects, gating and contact doping, can already be analyzed under static conditions where there is no net current flowing through the graphene. The key relations that determine the electrostatic charge and Fermi level distribution are the Thomas–Fermi equation and Poisson’s equation. These equations can be written for the analysis of charge distribution of graphene under electrostatic conditions. Using the equilibrium Fermi–Dirac function th order . Electro), as in . Gate poIn this work, Equation (2) has been discretized using the Finite Element Method (FEM) with a triangular mesh and with scalar  shape functions and is solved iteratively together with Equation (1), by using successive under-relaxation method as in ,23. EquaIn addition to the analysis of gating on graphene, the contact doping and Fermi level pinning due to the contact of metals on graphene  can alsoThe accurate analysis of semiconductor devices requires advanced models for the charge carrier transport. The traditional drift-diffusion models (DDM) start losing their validity as the operation frequencies increase , and theBy assuming the geometry is 2-D, and separating the components of the vectorial quantities, this set of HDM equations can be written in a more compact way: M in 1-D  and 2-D M in 1-D . HoweverThe hydrodynamic properties of charge transport in graphene have been demonstrated multiple times, such as its viscosity effects ,10, PoisAnother very important parameter for hydrodynamic transport is the momentum-relaxation-time constant that reveals itself in the right-hand-side collision term of the momentum-conservation Equation (5). This parameter is primarily dependent on the scattering mechanisms taking place, and for high-temperature applications, phonon scattering would be the primary scattering mechanism. The momentum-relaxation-time constant for phonon scattering in graphene as a function of Fermi level, phonon velocities, mass density, and acoustic deformation potential can be obtained by following the formalism in ,34. For 2 is used as the gate-insulator material.Moreover, carrier-saturation velocity also plays a significant role for the transport and can be written as ,36:(12)vThe last missing parameter for the HDM of graphene is energy-relaxation time that appears in the collision term of the energy conservation Equation (6). For this work, it is taken as 1 ps for high-temperature operation ,38. HaviIn order to test and analyze the developed 2-D hydrodynamic solver for graphene, a 2-D device geometry, as in The developed HDM solver for graphene is applied for the device geometry given in The narrow width, in the order of 10 nm, of the graphene device in ghost elements”, just outside the device domain, and their presence is felt through the numerical-flux term in the DGTD-FEM formalism [There are two types of boundary conditions used in this structure, the first one is the ohmic contact condition on the boundaries ‘a’ and ‘b’, to make sure the potential is applied on electrons, so they can enter and leave the structure without resistance. The other boundary condition is applied on the edges of the Tesla valve structure. For that purpose, an isolation boundary is used to eliminate the possible flow outside of the computational domain. This isolation condition is implemented weakly, by assuming zero velocity just outside of the graphene device. In other words, usage of the DGTD method allowed us to assign zero velocity for the carriers on the “ormalism . For theormalism , and timElectron flow directions and velocities can be seen in For the same level of bias applied from the contacts in both the easy- and hard-flow cases, the diodicity yields to ghost’ elements). Therefore, the velocity profiles in the narrow channels, as in The classical Poiseuille flow is a well-known phenomenon, due to hydrodynamic effects and the collective motion that occurs when fluids flow in a long duct, such as a pipe. It has also been shown that under certain circumstances, the electrons in graphene also demonstrate the Poiseuille flow, while carrying a current ,9. In orTwo separate solvers to model and simulate the charge behavior in graphene have been developed. The first simulator combines the nonlinear Poisson’s equation with the Thomas–Fermi equation in graphene, for the computation of the Fermi level and carrier distribution under the gate potential and contact doping, due to the metal layers. An example scenario is given in The developed solvers can be adopted for the development of next-generation graphene devices, especially for THz applications, by modeling and making use of the carrier inertia effects, viscosity effects, and possible ballistic charge transport. Furthermore, graphene nanoribbons (GNR) can also be modeled with this approach, by using appropriate carrier velocity and phonon-scattering times, emerging due to edge effects. Moreover, a parallel solver for the HDM can be implemented in the next generation, thanks to the employed DGTD algorithm.


Within implementation science studies, contextual analysis is increasingly recognized as foundational to interventions' successful and sustainable implementation. However, inconsistencies between methodological approaches currently limit progress in studying context and guidance to standardize the use of those approaches is scant. Therefore, this study's objective was to systematically review and map current methodological approaches to contextual analysis in intervention implementation studies. The results would help us both to systematize the process of contextual analysis and identify gaps in the current evidence.We conducted an evidence gap map (EGM) based on literature data via a stepwise approach. First, using an empirically developed search string, we randomly sampled 20% of all intervention implementation studies available from PubMed per year (2015–2020). Second, we assessed included studies that conducted a contextual analysis. Data extraction and evaluation followed the Basel Approach for CoNtextual ANAlysis (BANANA), using a color-coded rating scheme. Also based on BANANA and on the Context and Implementation of Complex Interventions (CICI) framework–an implementation framework that pays ample attention to context– we created visual maps of various approaches to contextual analysis.Of 15, 286 identified intervention implementation studies and study protocols, 3017 were screened for inclusion. Of those, 110 warranted close examination, revealing 22% that reported on contextual analysis.n = 15) and individual interviews (n = 13). Ten studies reported mixed-methods analyses. Twenty-two assessed meso-level contextual and setting factors, with socio-cultural aspects most commonly studied. Eighteen described the use of contextual information for subsequent project phases . Nine reported contextual factors' influences on implementation and/or effectiveness outcomes.Only one study explicitly applied a framework for contextual analysis. Data were most commonly collected via surveys (This study describes current approaches to contextual analysis in implementation science and provides a novel framework for evaluating and mapping it. By synthesizing our findings graphically in figures, we provide an initial evidence base framework that can incorporate new findings as necessary. We strongly recommend further development of methodological approaches both to conduct contextual analysis and to systematize the reporting of it. These actions will increase the quality and consistency of implementation science research.The online version contains supplementary material available at 10.1186/s12874-022-01772-w. Successful implementation of interventions in real-world settings depends on the dynamic, multi-dimensional, multi-level interplay between context, intervention and implementation strategies , 2. TherWithin an implementation scienceAlthough the importance of context has been widely emphasized regarding implementation, little attention has been paid to its assessment in studies partly driven by funding frameworks that do not normally recognize this phase's importance –15. Yet,No unifying definition of context yet exists. Instead, we see terms including setting—sometimes divided into inner and outer setting—environment, or system characteristics, with each signifying a slightly different perspective , 19, 20.Within a postpositivist paradigm, researchers tend to focus on single factors (commonly referred to as facilitators and barriers) to the exclusion of those occupying multiple levels and dimensions , 20, 21.Basel Approach for CoNtextual ANAlysis (BANANA) and applied it successfully in two implementation science projects [Based on a methodology reported by Stange and Glasgow  within aprojects –27. BANAprojects ), but altarget group, e.g., patients, family caregivers), who implement an intervention  or who decide on the implementation of an intervention  [To guide contextual analysis in implementation science projects, BANANA includes six components: (1) choosing a theory, model or framework (TMF) to guide contextual analysis. ; 2) reviewing empirical evidence about relevant contextual factor(s), including facilitators and barriers, as well as practice patterns related to the implementation and intervention; (3) involving relevant stakeholders in the contextual analysis. This includes implementation agents, i.e., individuals (or organizations) targeted or affected by the implementation of an intervention ( reviewinfunders) . To streTo draft an evidence gap map (EGM) we reviewed and categorized the methodologies applied to contextual analyses in the identified studies. This process was basically a systematic search that included surveying the current state of methodological approaches to contextual analysis. As the name implies, this was very useful to identify gaps in those approaches –30. As fAs a first step, to develop comprehensive, relevant research questions, this study's authors—all experienced implementation science scientists—discussed the scope and focus of the EGM , 33. As and acceptability). Further, only papers (i) written in English or German and (j) with available full texts were included. Because the level of detail of contextual analysis in study protocols is usually limited, we used the "cited by" function in PubMed to determine whether the intervention study had been already published and contained further information on contextual analysis. In cases where we identified the study protocol and related intervention implementation study, only the intervention study was included in the review. Further, we excluded studies reporting on context exclusively as part of the process evaluation or retrospectively.In step 1, we employed ten inclusion criteria to the prospective sample. We included (a) peer-reviewed articles or study protocols (b) concerning intervention implementation studies (c) if they employed experimental or quasi-experimental designs (d) to test intervention effectiveness (e) in real world settings. They also needed (f) to include at least one of Brown et al.'s "7 Ps" , i.e., pn = 81) or a validation set (n = 82). Second, using Pubmed ReMiner (https://hgserver2.amc.nl/cgi-bin/miner/miner2.cgi), we identified the search terms (keywords and MeSH terms) most commonly used in the development set articles. The identified search terms were used to develop a search string. In a third step, this string was tested against the validation set. The final search string consisted of 22 keywords (MeSH and free terms) and achieved a sensitivity of 95.1% . The fourth step consisted of documenting the search string development . Using a random number generator, we then selected a random sample of 20% of the articles identified from each year. No further filters were applied.https://rayyan.qcri.org), two reviewers  independently screened titles and abstracts of the randomly selected implementation science papers against the described inclusion criteria [For step 1, using the web application Rayyan . Guided by BANANA, specific characteristics of studies including contextual analyses were extracted, including general information , implementation agents involved in each analysis and methods applied to conduct contextual analysis . Further, we assessed the results of the contextual analyses, i.e., we noted how those results were used for subsequent study phases and whether the researchers had considered how contextual factors might influence implementation and summative outcomes . The structure of the tables follows the BANANA approach and provides a comprehensive overview of all relevant information. More detailed information on the assessed approaches can be found in the Additional files To provide an overview of contextual factors assessed, an EGM was developed using two software tools: EPPI-Reviewer Version 4.12.3.0  and EPPITo critically evaluate the methodological approaches to contextual analysis reported in the included studies, we grouped the extracted data via five of the six components described in the BANANA approach . The sixWe used a two-phase sampling process. In Phase 1, our PubMed search returned 15,286 records. After removing duplicates, we randomly sampled 20% of the remaining studies from each of the six selected publication years (2015–2020) (n = 3017). In Phase 2, we screened this sample via the inclusion criteria noted above. Figure n = 90); most (n = 82) were either from North America (n = 45) or Europe (n = 37) (Table n = 20), community care (n = 15), the health care system (n = 13) and schools (n = 12). Eighty-four of their designs were experimental; twenty-six were quasi-experimental. Further details of the studies are described in the Additional file Of the 110 extracted articles the majority were study protocols (rica n = 5 or Euron = 15) as well. Based on Curran et al.'s definitions [Of the sample's 110 studies, 24 (21.8%) reported conducting contextual analyses  referred more generally to their TMFs guiding their overall implementation process, with RE-AIM (n = 7) and the Consolidated Framework for Implementation Research (CFIR) (n = 3), cited most often. Four studies reported combining two TMFs, e.g., CFIR and RE-AIM. In addition, seven considered empirical evidence about relevant contextual factors , determinant frameworks , or classic theories  . One, thors Fig. .Fig. 2Chn = 19)  were established. These included, e.g., health care providers from various medical fields, people affected by specific illnesses or health conditions, leaders and administrators, and delegates for non-profit organizations or government departments  Fig. . In somen = 2) or qualitative (n = 8) methods alone , behavior mapping (n = 1), and retrospective use of national survey (n = 1) and surveillance (n = 1) data. Seven qualitative data collection methods were used: individual interviews (n = 13), focus groups (n = 13), observations (n = 2), as well as photovoice methodologyn = 2), telephone interviews (n = 1), yarningn = 1) and site visits (n = 1).Of the 24 studies that reported using contextual analyses, 23 clearly described their methods. Of these 23, while ten explicitly reported using mixed-methods analysis, we found that 13 applied combinations of quantitative and qualitative methods. The remaining ten applied either quantitative (n = 30) or setting factors (n = 13), then mapped them on an evidence gap map  were most commonly assessed, accounting for almost half of all mentions. The remainder were roughly equally divided between macro- (n = 13) and micro-level factors (n = 12). Fifteen studies considered context on at least two levels. We report a detailed overview of all assessed factors in Additional file We identified 43 separate factors. Following the CICI framework, we first categorized these as either context  (n = 20); no studies reported on legal aspects. In descending order of frequency, other contextual domains included political  (n = 12), geographic  (n = 5), epidemiological  (n = 5), socio-economic  and ethical , conflicts of interest (n = 2)). Seven studies described their assessment of inner or outer context or of facilitators and barriers, but did not further specify contextual factors in detail.Setting factors. In view of setting, most studies assessments focused on the work environment  (n = 15). Other setting aspects assessed included practice patterns  (n = 11) as well as the setting's physical characteristics  (n = 7).n = 17) and/or adapt interventions (n = 11), eight used contextual information to choose implementation strategies, and six used it to interpret study outcomes. Of these, ten described their processes for doing that. Both original study papers described the further use of contextual information; however, only one reported how it was used.Eighteen study protocols described further uses of contextual information to develop  and the involvement of implementation agents (n = 9). The latter was also described partly within most of the remaining studies (n = 15). The two least addressed components were the use of contextual information to interpret outcomes (n = 7) and the use of empirical evidence (n = 7).Our evaluation of the identified approaches to contextual analysis revealed that few studies addressed the key components of contextual analysis that are described in detail within BANANA Fig. . The comThis study provides an overview of the current methodological approaches to contextual analysis in intervention implementation studies and indicates gaps. Using EGM methodology, we applied a novel approach for summarizing and evaluating available evidence on contextual analysis to develop an initial evidence gap map on contextual analysis methodology. Based on a random sample drawn from 110 intervention implementation studies, we found that fewer than one-quarter of those studies (21.8%) reported on analyses of their projects' contexts and settings. The studies that did report on contextual analyses showed high variability in the methodological approaches they used. This was true both of the analyses and of how they were reported.Using the BANANA approach—one of the first frameworks for evaluating CAs—we found widespread significant methodological gaps. For example, few contextual analyses were theory based: only one study explicitly reported the use of a TMF for its contextual analysis; and fewer than half (8/22) provided information how they used findings from their CA to inform their project's subsequent steps.Building our understanding of context demands a stable theoretical basis. In addition to guiding our selection of multilevel contextual factors, this will enable operationalization both of context and of setting. Still, of the 24 studies we reviewed, only one provided both a specific description of its authors' use of a TMF to guide their contextual analysis and their rationale for using the one they did , 82. ConThe phenomenon of “underuse, superficial use or misuse”  of TMFs Consistent with other reviews' findings, the assessed studies' conceptualizations of context tended to be vague. For example, while a diverse range of factors were assessed at numerous levels, no definitions accompanied them. The resulting vagueness , hampered our efforts either to understand, to summarize and to compare those factors , 17, 18.We noted considerable differences regarding which levels' and domains' contextual factors were appropriate targets for investigation. In contrast to Rogers et al.'s review  of studiThis scarcity might also reflect a shortage of tools and frameworks focusing on the macro level , 90, 91.However, the choice of which contextual factors to study and which stakeholders to involve at which phases depends largely on the type of intervention. This may also explain why the recorded contextual factors differed so widely between studies.Furthermore, both the assessment of context and the reporting of contextual analysis might be biased by their analysts' level of pre-existing knowledge, i.e., researchers' inside knowledge may influence the quality or impartiality of their results. For example, researchers working in a specific setting may already be aware of certain contextual determinants  or may gather important information informally . While this information is not explicitly collected for the contextual analysis, it can lead to confirmation bias. I.e., it can leave "blind spots" in contextual analysis, exerting subtle pressure on analysis or interpretation to favor factors that support pre-existing hypotheses or beliefs .Both to enhance the quality of a project's research and to ensure appropriateness of intervention and implementation strategies through co-design, it is crucial to involve implementation agents in diverse positions , 98. ThiFor contextual analysis, either a combination of quantitative and qualitative methods, or, if possible, a mixed-methods approach is recommended. Merging, connecting or embedding data obtained via various means increases both the breadth and the depth of the analysis , 102. ItLikewise, surveys or interviews  were our sample's most common methods of capturing contextual details. However, recent studies increasingly emphasize the relevance of direct  observations in implementation research. These allow insider perspectives, including, for example, records of contextual aspects that implementation agents may take for granted and omit to mention, or tasks performed differently than generally reported –109.Problematically, as contextual analysis in implementation science is primarily done within a postpositivist paradigm, researchers' understandings of context are often mechanistic and reductionistic. Therefore, we recommend that they also consider constructivist perspectives, particularly rapid ethnographic methods. In addition to probing more deeply into the context , these require fewer resources than traditional methods. This efficiency makes them particularly useful for contextual analyses, which are rarely well-resourced , 111.As noted above, the reviewed studies showed significant gaps in their descriptions of how contextual information was later used. The results mainly informed intervention development. However, reporting gaps may have resulted from the fact that we assessed study protocols almost exclusively.Another factor influencing the reporting of contextual analyses in study protocols or journal articles is lack of space: a 5000-word article can adequately develop and describe its central topic, but very little more. Therefore, implementation scientists should consider publishing their contextual analyses as separate papers. This would allow detailed descriptions of their methods and results, as well as of how they used those results for further study phases. Detailed reporting guidelines for contextual analysis could help researchers to structure their findings and avoid the types of “blind spots” noted above.The current study's objective is to systematically review and map methodological approaches currently in use for contextual analyses, as well as to identify gaps in the identified approaches. In this regard, this paper's most notable strength is the empirical search string development. Given the reported challenges in finding implementation science literature, the string provides both high sensitivity and high specificity –114.Furthermore, we provide a novel framework for evaluating existing CA-related evidence by applying the BANANA approach . This frHowever, as we included primarily study protocols, the descriptions given of contextual analysis lacked adequate detail in some cases. This affects our analysis of how contextual information informed the studied projects' later phases. Although we searched study papers related to the protocol, we were unable to verify in every case the extent to which the planned approaches to contextual analysis were carried out in the project, or whether adaptations were made. We suspect that one major reason for the high number of identified study protocols was publishing bias. Considering that we only included articles reporting contextual analyses as part of intervention implementation studies, it is possible that many contextual analyses were reported in study protocols, then conducted as part of implementation projects but not published.The applied random sampling approach of study papers provided an opportunity to gain an initial overview of current evidence and its gaps. However, this approach may have excluded other relevant study papers that could have provided further insights into approaches to contextual analyses. Another possible weakness is that our strict inclusion criteria might have influenced our results. We focused on contextual analysis as a foundation for further study phases, i.e., prospective assessment of context and setting factors. As studies that conducted their contextual analyses retrospectively  would not enhance our understanding of contextual analysis in implementation science, we excluded them. For further research, it would be useful to adapt BANANA by planning a more comprehensive analysis—one that differentiates between the different implementation project phases . This wTo the best of our knowledge, this is the first study to provide a novel framework for evaluating and mapping methodological approaches to contextual analysis. Our evidence map provides a broad overview of methodologies applied in contextual analysis and shows which aspects of those studies can serve as models for other implementation science projects. The map is dynamic and can be updated as the literature on contextual analysis evolves.We found wide variation regarding which methods were used for contextual analysis, which contextual factors were assessed, and how the results were applied in later study phases. Such a high level of heterogeneity is a major barrier to inter-study comparison or to later scale-up efforts. To reduce it, we recommend conducting contextual analyses according to TMFs. In addition to providing clear, proven and repeatable methodologies, these both support stronger conceptualization of the assessed context and enhance the rigor of the entire analytical process. If the described gaps are left open, contextual analysis will become a "black box" in many cases, greatly reducing its contribution over the course of implementation projects. Therefore, the implementation science community needs to take concerted action to develop, test and improve straightforward, robust methodologies for contextual analysis and reporting.Across health care, researchers need to embrace contextual analysis as an essential element of every implementation science project; funding agencies need to develop specific opportunities to improve it; and journals need to demand full reporting on it. And every implementation science research team needs not only practical guidance on how to carry out contextual analysis, but also special guidelines on how to report their findings. Above all, we need to understand that, to achieve the quality and success that implementation science research promises, we will first need to break open the “black box” of contextual analysis.Additional file 1. Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist.Additional file 2. Research questions and screening tool inclusion-/exclusion criteria.Additional file 3. Empirical search string development.Additional file 4.STEP 1 - General characteristics of identified implementation intervention studies (n = 110).Additional file 5: STEP 2 - Study characteristics of implementation intervention studies that performed contextual analyses.Additional file 6. Evidence gap map.Additional file 7: Overview of contextual factors identified in implementation intervention studies (mapped according to the Context and Implementation of Complex Interventions (CICI) framework (1)).


There was an overall rainfall increase in Kampala (p = 0.029) by 0.241 mm, Mbale (p = 0.0011) by 9.804 mm, and Namutumba (p = 0.394) by 0.025 mm. On the other hand, humidity has decreased both in Kampala (p = 0.001) by 13.3% and in Namutumba (p = 0.035) by 13.2% while there was a no significant change in Mbale. The results of GLM showed that each variable, taken individually, had a direct effect on the pests' occurrence in all three districts. However, with all these climate variables taken together, the effect on the pests' occurrence varied with each of the three districts; Kampala, Mbale, and Namutumba. This study demonstrated that pest occurrence is different from one agroecology to another. Our findings suggest that climate change is a driver that favors bio-invasion of tomato invasive insect pests occurrence in Uganda. It calls for awareness to policymakers and stakeholders to consider climate-smart pest management practices and policies to deal with bio-invasion.The shift in the geographical spread of invasive pests in Africa has rarely been linked directly to climate change. However, it is predicted that environmental changes play a significant role in spreading and expanding pests. The occurrence of new tomato invasive insect pests has been increasing in Uganda during the past century. Assessing the impact of temperature, rainfall, relative humidity, and windspeed on the occurrence of invasive tomato insect pests, gives a better understanding of managing and limiting the bio-invasion process sustainably. We used the Mann Kendall trend Test to establish trends in climate variables from 1981 to 2020 and to document the trend in the occurrence of new invasive pests. The relationship between climate variables and pests occurrence is analyzed using Pearson's correlation and the Generalized Linear Model (GLM-quasi-Poisson) in R-software. The results showed that temperature and windspeed have significantly increased in both Kampala and Namutumba by 0.049 °C, 0.005 m  GlobaClimate change impacts result in variations in the earth's atmosphere parameters such as temperature, precipitation, wind, humidity, and soil moisture ,10. The As a result of these variabilities, there have been a plethora of invasive pests in sub-Saharan Africa (SSA) in the last decade. In Uganda, agriculture plays an essential role in the country's economy. Mbale and Namutumba are among the districts where most of the tomatoes produced in Uganda are grown ,15. TomaEstablishing the relationship between the biology of the pests and the relative humidity is crucial to understanding the fast invasion of a pest in certain regions . However22.1The study was conducted in Uganda. Uganda is located in East Africa  and is a2.2https://power.larc.nasa.gov/data-access-viewer/) at a spatial resolution of 0.5 × 0.625° latitude/longitude. The annual averages of these bioclimatic variables were used to match the number of invasive pests recorded annually [https://www.eppo.int/, the Global Biodiversity Information Facility (GBIF) (https://www.gbif.org/), and the Centre for Agriculture and Bioscience International (CABI) (https://www.cabi.org/) [o E, 0.32o N). This was expected as the Kampala agricultural produce markets are central to all the agricultural products originating across the country hence many pests also become introduced and centralized in Kampala. Additionally, this could be attributable to the fact that the capacity to identify, record, and report these pests could be higher in Kampala than in other parts of the country.The climate variables used in the study were temperature, rainfall, windspeed, and relative humidity obtained from the years 1981–2020 to capture changes in the climate observed within the forty years ,24. Thesbi.org/) . All thebi.org/) . The nat2.32.3.1The Mann-Kendall (MK) trend method, which is a non-parametric test, was used on the climatic variables to assess the negative or positive tendency in the level of temperature, rainfall, relative humidity, and windspeed over the years . The MK The statistical equation With:xb = the value of the climatic variable  data at time b xa = the value of the climatic variable  data at time a where equation a) the Sen's slope equation The Sen's slope estimator test is used to determine the statistical significance of the tendency in climatic variables over time ,36. For 2.3.2-log of the mean per unit change in X.βs represents the expected change in the -exp(β) is the effect of the independent variable on the mean.The effect of climate variables on pest invasion over the years was assessed using a Generalized Linear Model (GLM) because the data was ‘count data’ ,38. ThisThe Quasi-Poisson GLM that was run in R software is: glm data, family = quasipoisson(link = log)).Furthermore, Pearson's correlation analysis was used to determine the association between the tomato invasive insect pest occurrence and climate variables.33.1n = 38) (The MK test showed an increase in the occurrence of tomato new pests over the years (n = 38) .Fig. 2Oc3.2p < 0.0001) and Namutumba (p = 0.006) by 0.049 °C and 0.037 °C per year, respectively. Over the years, we observed little decrease (0.002 °C) with no significance in the trend variation of mean temperature in Mbale (p = 0.894)  . The hig= 0.894) .Table 1E3.3p = 0.029) and Mbale (p = 0.0011) by 0.241 mm and 9.804 mm per year, respectively.The MK and Sen's slope trends for the annual rainfall for the three districts are presented in p = 0.394). However, there was an increase in rainfall by 0.025 mm per year. Overall, there was an increase in rainfall amounts in the three districts  and Namutumba (p = 0.002) with increasing speed respectively, as little as 0.005 m s−1 and by 0.003 m s−1 per year  and by 13.2% RH in Namutumba (p = 0.035). In Mbale, there was no significant difference in the humidity trend (p = 0.395). In Mbale, there was no significant difference in the humidity trend (p = 0.395). All these trends are captured in The last climate variable considered to affect insect pest occurrence is humidity. The abnormalities in the annual relative humidity trends are clear evidence of an unpredictable moisture pattern in the three districts. The MK and Sen's slope test showed a trend in the time series data with a significant decrease in humidity over the years by 13.3% RH in Kampala (3.6p < 0.05) in the three districts, except for humidity in Kampala, windspeed, temperature, and humidity in Mbale.The results of the relationships among the occurrence of invasive tomato insect pests, windspeed, temperature, humidity, and rainfall assessed by Pearson's correlation, are presented in 2 = 31.331, degrees of freedom (df) = 1, p = 2.176e−08), relative humidity , rainfall  and wind component  at various pressure levels were all significant in the occurrence model. Rainfall, temperature, and windspeed affected positively pest occurrence counts while relative humidity affected the occurrence negatively. Even though in an agroecology, the climate variables influence pest occurrence, windspeed (p < 2.2e−16) is much more significant than rainfall (p = 8.310e−12) and temperature (p = 6.144e−08). The variations simultaneously in both windspeed and temperature are the major effect in the pest occurrence (p = 6.102e−11). The concurrent variations in all the variables are likely to have a low effect on the pest occurrence (p = 0.054)  .Table 5G2 = 11.877, df = 1, p = 0.0007). On the contrary there was no significant influence of temperature  windspeed  and relative humidity  on the invasive insect pests counts. In this case, rainfall is the only parameter that (p < 2.2e−16) affects the invasive insect pest occurrence when the variables are combined in one model. The variations in relative humidity, windspeed, and rainfall (p = 0.028) as well as the variations in relative humidity, windspeed, and temperature (p = 0.027) are the only cases of significant change in the pest occurrence.The significance of climate variables in Mbale on the occurrence of pest counts varied in the model . Signifi2 = 12.893, df = 1, p = 0.0003). Among other variables, temperature did influence pest occurrence  as well as windspeed  compare to relative humidity which had no significant influence pest occurrence . The new occurrence of invasive insect pests in Namutumba agroecology where all the climate variables interact together is highly influenced respectively by windspeed (p = 7.612e−06), rainfall (p = 0.002), humidity (p = 0.04) (p = 0.021).The effects of climatic variables on invasive insect pests in Namutumba have similar results in Kampala. Their shared trends could explain this similarity and effect . Similar = 0.04) . Similar4In this study, we provided information on climate trends and their effect on insect pest occurrence in Uganda specifically the cultivated tomato areas in Kampala, Mbale, and Namutumba. Our results have shown an increasing annual temperature trend in Kampala and Namutumba over the last forty years by 0.04 °C annually.. [Our study showed that the rainfall trends increased significantly in Kampala (0.24 mm) and Mbale (0.0011 mm), with a significant decrease in the humidity in Kampala and Namutumba confirming the findings of Ssentongo et al. , who reps−1 and 0.003 m s−1, respectively, in Kampala and Namutumba during the study period. The rise of invasive insect pests in Uganda is wind-aided due to the positive relationship between the wind and pest data. This validates previous assumptions that the migration of some pests is favored by strong winds, which propel them [The trends of windspeed amplified between 1981 and 2020, our data shows an increased rate of 0.05 m pel them .Chilo partelous propagates faster when relative humidity increases which might be the case of our study in Mbale where there is positive correlation between Rh and pest occurrence [The variations in the annual precipitation trends are evidence of unpredictable rainfall patterns in the study area. Therefore, it is evident that rainfall has a significant positive relationship with pests . The inccurrence . This st. [icipe) and the Centre for Agriculture and Bioscience International (CABI) have reported that climate change was a major driver of transboundary pests’ establishment in Eastern Africa. Besides, rainfall and temperature variability are susceptible to influencing pests occurrence thereby affecting tomato production [Overall, the rate of pest occurrence trend increased across the three districts. The changes in the temperature and windspeed patterns are associated with the rise of pests. This suggests that there is a meaningful trend in the association between increasing pests and climate variables. Our findings are similar to those reported by Haack et al.  who foun.  who conc. . Scientioduction ,49. Invaoduction . While toduction . The resoduction .Finally, the present study has utilized secondary data and encourages its re-use for addressing further research questions., Our study suggests that the increase in establishment rates of invasive insect pests in Uganda, during the past four decades can be partially explained by climate change given that temperature, rainfall, wind speed, humidity can facilitate bio-invasion.5This study examined the relationship between climate change and variability factors and invasive insect pests in Uganda. The study focused mainly on temperature, rainfall, relative humidity, and windspeed. The analysis was done using the MK trend and Sen's slope test to observe the trends in the climate variables over 39 years from 1981 to 2020. The results showed some significant increase in the temperature in Kampala, Namutumba and no significant variation in Mbale, a significant increase in windspeed in Kampala and Namutumba with no variation in Mbale. Also an increase in rainfall in the three districts  while a decrease of humidity . This is a clear indication that climate change has occurred in Kampala, Namutumba, and Mbale. The GLM analysis concluded that variation in both windspeed and temperature is the major effect in the pest occurrence in Kampala. For Mbale, simultaneous changes in humidity, windspeed, rainfall as well humidity, windspeed, and temperature caused a significant occurrence of invasive insect pests while for Namutumba, it is the interaction between windspeed and rainfall. It showed that changes in climatic variables impacted differently the proliferation and occurrence of tomato invasive insect pests in each district. This study confirms that the expected occurrence curve can increase in the changing conditions. So, changes in temperature, windspeed, and humidity climate patterns are more conducive to the survival and occurrence of the invasive insect pest in Kampala and Namutumba than in Mbale, which is another stress to crop production.6Because of the lack of information about the exact date of bio-invasion of a pest in Mbale and Namatumba, we assume the dates of recordings of pests' introduction in the regions are taken to be the same date the pests invade Mbale and Namutumba. Another limitation of this study is the nature of data, as mentioned above, we used secondary data which may contain noises that reduce the accuracy of the analysis.N'dakpaze Gno-Solim Ela: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Daniel Olago: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.Amwata Dorothy Akinyi: Conceived and designed the experiments; Analyzed and interpreted the data; wrote the paper.Henri E. Z. Tonnang: Contributed reagents, materials, analysis tools or data; Analyzed and interpreted the data; Wrote the paper.10.13039/501100006456Federal Ministry for Economic Cooperation and Development (10.13039/501100006456BMZ) commissioned and administered through the Deutsche Gesellschaft fürInternationaleZusammenarbeit (10.13039/501100011099GIZ) Fund for.International Agricultural Research (10.13039/501100015633FIA), grant number 18.7860.2–001.00.This work was supported by the German Data are available upon request to the corresponding author.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.