Recent studies demonstrated that the Golgi reassembly stacking proteins (GRASPs), especially GRASP55, regulate Golgi-independent unconventional secretion of certain cytosolic and transmembrane cargoes; however, the underlying mechanism remains unknown. Here, we surveyed several neurodegenerative disease–related proteins, including mutant huntingtin (Htt-Q74), superoxide dismutase 1 (SOD1), tau, and TAR DNA–binding protein 43 (TDP-43), for unconventional secretion; our results show that Htt-Q74 is most robustly secreted in a GRASP55-dependent manner. Using Htt-Q74 as a model system, we demonstrate that unconventional secretion of Htt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as starvation and endoplasmic reticulum stress. Mechanistically, we show that GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and subsequent lysosome secretion and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the lumen of the endoplasmic reticulum–Golgi intermediate compartment. Moreover, we found that GRASP55 levels are upregulated by various stresses to facilitate unconventional secretion, whereas inhibition of Htt-Q74 secretion by GRASP55 KO enhances Htt aggregation and toxicity. Finally, comprehensive secretomic analysis identified novel cytosolic cargoes secreted by the same unconventional pathway, including transgelin (TAGLN), multifunctional protein ADE2 (PAICS), and peroxiredoxin-1 (PRDX1). In conclusion, this study defines the pathway of GRASP55-mediated unconventional protein secretion and provides important insights into the progression of Huntington’s disease. Neurodegenerative diseases are characterized by increased neuronal cell death and decreased cognitive abilities of patients. Different diseases, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease (HD), and amyotrophic lateral sclerosis, are caused by different protein-based aberrations that affect different pathways; yet all cause neuronal cell death by forming inclusion bodies , 2, 3. TIn the conventional secretory pathway, transmembrane and secretory proteins, such as the amyloid precursor protein, are synthesized in the endoplasmic reticulum (ER) and transported to the Golgi, from where they are targeted to their final destinations. However, many neurotoxic proteins, such as Htt, SOD1, α-synuclein, tau, and TDP-43, are synthesized as cytosolic proteins. Secretion of these proteins is independent of the traditional secretory pathway in which the Golgi functions as a trafficking center. For example, mutant Htt exists in two forms, an intracellular form in patient neurons and an extracellular form in CSF  exportedDictyostelium discoideum and Saccharomyces cerevisiae  , 14, 15.revisiae , 21. In elopment . In mammelopment , 24. HowIn this study, we report that mutant Htt is unconventionally secreted in a GRASP55-dependent manner. Htt secretion is elevated under stress conditions, including energy deprivation and nutrient starvation, inhibition of proteasomal degradation, and induction of ER stress, all of which also upregulate GRASP55 expression. GRASP55 facilitates Htt secretion through two actions: one is to accelerate autophagic flux by facilitating autophagosome and lysosome fusion, the other is to stabilize p23/TMED10, a protein channel in the ER–Golgi intermediate compartment (ERGIC) that translocates cytosolic proteins into the ERGIC lumen for unconventional secretion. Finally, we performed systematic proteomic and secretomic analysis of WT and GRASP55 KO (55KO) cells and identified a list of cytoplasmic proteins whose secretion depends on GRASP55.A–D), but Htt-Q74 secretion was the most robust and GRASP55 dependent (A). Therefore, we used GFP-Htt-Q74 as a main marker to study the mechanism of GRASP55-dependent unconventional secretion.To determine if GRASP55 is required for the secretion of cytosolic proteins related to neurodegeneration, we expressed a number of neurotoxic proteins, including Htt exon 1 fragment with a long polyglutamine (Q74) stretch (GFP-Htt-Q74), WT SOD1 (GFP-SOD1), TDP-43 (TDP-43-GFP), and tau (FLAG-tau), in WT and 55KO HeLa cells we established earlier . For Httependent A. TherefA). Interestingly, these Htt aggregates were often found in the perinuclear region where the Golgi was localized. This prompted us to look at the effect of Htt expression on Golgi morphology. While soluble Htt did not seem to affect Golgi morphology, Htt aggregates induced Golgi defects as previously reported . For comparison, we also tested GFP-Htt-Q23 in parallel, which did not form aggregates or significantly affect the Golgi morphology (E). Similarly, expression of SOD1, TDP-43, or tau in WT cells had no significant impact on the Golgi structure (F).Since both GRASP55 and GRASP65 have been shown to be involved in unconventional secretion , we exprFig. 1reported , 31, andrphology E. Similatructure F.Figure B and C), indicating that Htt secretion depends on GRASP55 but not GRASP65. Secretion and processing of clusterin, a cargo molecule secreted by the conventional secretory pathway, were not affected by GRASP KO (B). In this assay, actin and two additional cytosolic proteins, hexokinase (HK1) and glucokinase (GCK), were not detected in the conditioned media (B and S1G). In addition, GFP-Htt-Q74 expression did not cause cell death as determined by the lactate dehydrogenase (LDH) cytotoxicity assay (H). These results indicate that Htt was secreted rather than nonselectively released from dead cells. Taken together, GFP-Htt-Q74 expression causes Golgi defects especially in 55KO cells, whereas 55KO reduces GFP-Htt-Q74 secretion.Next, we tested whether Htt secretion is GRASP dependent. After transfection of GFP-Htt-Q74 in WT, 55KO, 65KO, and double KO cells for 24 h, we cultured the cells in serum-free medium for 4 h and determined the level of Htt in the conditioned media by Western blot. Htt was readily detectable in the conditioned media, and the level was significantly reduced in 55KO but not 65KO cells , B and CGRASP KO B. In thied media B and S1Gty assay H. These D and E, BFA treatment diminished clusterin in the conditioned media, indicating a block of conventional secretion. Htt secretion was unaffected by BFA treatment, confirming that it is secreted via a Golgi-independent unconventional secretory pathway.To confirm that Htt is secreted through an unconventional Golgi-independent pathway, we blocked ER-to-Golgi trafficking by brefeldin A (BFA) treatment. As shown in F and G, when GFP-Htt-Q74 and GFP-Htt-Q23 were coexpressed, the level of GFP-Htt-Q74 in the conditioned media was higher than that of GFP-Htt-Q23. This indicates that the selectivity for GFP-Htt-Q74 secretion relies on the polyQ tract. Indeed, when Q80-GFP (in the absence of Htt exon 1) was expressed in HeLa cells, it was secreted at a comparable rate as GFP-Htt-Q74 (∼10%), and Q80-GFP secretion was also robustly reduced in 55KO cells (A and B).The toxicity of Htt is associated with the length of the polyQ tract within the exon 1 fragment; Htt with more than ∼35 glutamines in the stretch is regarded as a disease mutant , 7. To dKO cells , A and BH and I). More importantly, STS treatment increased Htt secretion, whereas OA treatment reduced its secretion (J and K), indicating that the dephosphorylated aggregate-prone form of Htt is preferentially secreted compared with the phosphorylated soluble form of Htt. Taken together, these results demonstrate that unconventional secretion is selective, at least to a certain degree, and may serve as a clearing mechanism for cells to expel toxic proteins.Interestingly, Htt exhibited two bands, and only the lower band was detected in the conditioned media; we were curious about the difference between the two bands. Htt is highly modified by post-translational modifications, such as phosphorylation, acetylation, and cleavage . Among tecretion , J and KIt has been demonstrated that unconventional secretion is elevated under different stress conditions when the autophagy level is high , and thaA and B). A similar effect was observed in 55KO cells, although the overall secretion was significantly lower than WT cells. In addition to starvation, inhibiting proteasomal degradation with MG132, or inducing ER stress with tunicamycin (Tu), both enhanced Htt secretion (C–F). Under these experimental conditions, no cell death was detected by the LDH cytotoxicity assay (C). These results confirm that Htt secretion is elevated under stress conditions.These observations prompted us to test the effect of energy deprivation and nutrient starvation on Htt secretion. As expected, both glucose starvation and amino acid deprivation  increased the level of Htt in the conditioned media compared with Dulbecco's modified Eagle's medium (DMEM) (D) , A and Becretion , C–F. Unty assay C. These A and B). The elevation of GRASP55 expression was not limited to amino acid starvation, as it also applied to ER stress. When cells were treated with ER stress inducers, including Tu, thapsigargin, and DTT, the level of GRASP55 but not that of other Golgi proteins increased (C and D).The fact that GRASP55 localization and function are regulated by cellular stressors suggests that GRASP55 may serve as both a stress sensor and an effector . In addincreased , C and DD and E). To validate this observation in mice, we took advantage of a previously established knock-in mouse model that expresses full-length Htt-Q200 in the brain  (E and F). Taken together, GRASP55 level is upregulated by cellular stress and Htt expression, possibly to meet the high demand of unconventional secretion.Since mutant Htt forms aggregates that could induce proteostasis stress, we tested the effect of Htt-Q23 and Htt-Q74 on GRASP55 expression. Both Htt-Q23 and Htt-Q74 expression increased GRASP55 level in cells, although Htt-Q74 exhibited a stronger effect , D and Ehe brain , 42. TheE and F, , D and ED and E) , E and FA). Htt secretion was reduced in ATG7 KO cells to an almost undetectable level (A and B). In addition, inhibition of autophagosome–lysosome fusion by bafilomycin A1 (BafA1) treatment also decreased Htt secretion in WT but not 55KO cells (C and D).The fact that GRASP55 expression, autophagy, and unconventional secretion are all elevated under stress conditions implies that these events are related. To test if Htt secretion depends on autophagy, we expressed GFP-Htt-Q74 in the ATG7 KO mouse embryonic fibroblast (MEF) cell line established by Dr Masaaki Komatsu  in whichFig. 4via controlling autophagosome maturation. If this conclusion is correct, we would expect to see more colocalization of Htt with the autophagosome marker LC3 in 55KO and BafA1-treated cells. Consistent with this idea, Htt partially colocalized with LC3 in WT cells, whereas 55KO and BafA1 treatment enhanced their colocalization (E and F). Taken together, these results demonstrated that Htt is released through an autophagy-dependent mechanism.The results described previously are consistent with the role of GRASP55 in autophagosome–lysosome fusion, suggesting that GRASP55 regulates Htt unconventional secretion lization , E and FG and H). Consistent with the notion that GFP-Htt-Q74 is secreted via lysosome exocytosis, long exposure of the gels reveals an intermediate form of GFP-Htt-Q74 and free GFP in the cell lysate (G), indicating a possibility that some GFP-Htt-Q74 molecules might be partially processed in lysosomes prior to their secretion.It has been indicated that unconventional secretion could occur through lysosomal secretion . Indeed,l lysate G, indicaA–D, both starvation and proteostatic stress conditions strongly enhanced the secretion of both Htt and mature cathepsin D. We also induced ER stress by Tu treatment, which has been previously reported to preferentially increase the precursor but not mature cathepsin D secretion , whereas Htt was only found in the supernatant (lane 4). To confirm that Htt was not secreted in membrane-bound structures such as exosomes, we treated the conditioned media of Htt-expressing cells with proteinase K with or without Triton X-100 (TX-100). The results showed that Htt was degraded by proteinase K regardless of whether TX-100 was included (J). Taken together, these results demonstrate that Htt secretion depends on autophagy and lysosome exocytosis but not exosome secretion.It has previously been shown that Htt is secreted in exosomes , which iFigure 4included J. Taken A). In contrast, expression of the F37A mutant of GRASP55, which abolishes GRASP55–LC3 interaction (B and C). The 5A mutant of GRASP55, whose O-GlcNAcylation is disabled and thus its role in autophagy is constitutively active, also rescued Htt secretion similar to WT GRASP55. In this experiment, GRASP55 itself was not secreted with Htt; and we did not detect an interaction between Htt and GRASP55 by coimmunoprecipitation. These results demonstrate that GRASP55 facilitates Htt secretion by promoting autophagosome maturation.GRASP55 facilitates autophagosome maturation by tethering autophagosomes and lysosomes through the interactions with LC3-II on autophagosomes and LAMP2B on lysosomes . To confFig. 5eraction , failed A). Considering that some members of the p24 family proteins interact with GRASP65 (B and C). In addition, p23 coimmunoprecipitated with GRASP55 but not GRASP65, whereas known interactions between GRASP65 and GM130 and between GRASP55 and Golgin-45 were readily detectable in the same experiment . These results support the idea that GRASP55 interacts with p23 to facilitate Htt unconventional secretion.Recently, it has been reported that an ERGIC protein p23, also known as TMED10, functions as a protein channel for cytosolic proteins to be translocated into the ERGIC lumen before they are transported to autophagosomes and secreted to the extracellular space . Indeed,Fig. S4 GRASP65  and that GRASP65 , inhibitD). Expression of WT GRASP55 and its 5A mutant, but not the autophagy-inactive F37A mutant, reduced Htt aggregation in 55KO cells (E), consistent with their roles in Htt secretion. In this experiment, a significant amount of GRASP55 and GM130 was found in the pellet (E), consistent with the idea that they form a detergent-insoluble Golgi matrix (F–H). These results demonstrate that GRASP55-dependent Htt secretion reduces its aggregation and toxicity in the cell.It is generally believed that HD is caused by the accumulation of toxic mutant Htt protein in the cell. To determine the functional significance of Htt secretion, we assessed Htt aggregation in WT and 55KO cells. We lysed GFP-Htt-Q74–expressing cells in detergent and separated Htt aggregates from its soluble form by centrifugation. Western blot analysis of the two fractions showed that Htt formed more aggregates in 55KO than WT cells D. ExpresKO cells E, consise pellet E, consisi matrix , 49. We i matrix , F–H. ThA). Similar to HeLa cells, GRASP55 depletion also increased GFP-Htt-Q74 aggregation in N2A cells (B). Second, to determine if full-length Htt-polyQ stably expressed under the endogenous promoter is also secreted in a GRASP55-dependent manner, we assessed Htt-polyQ secretion using the striatal-derived cell line STHdhQ111/Q111 (C). Furthermore, GRASP55 depletion also enhances the aggregation of full-length Htt-polyQ in striatal cells (D). These results validated our finding that GRASP55 facilitates mutant Htt secretion in neuronal cells.As Htt aggregation causes neurodegeneration, we tested Htt secretion in neuronal cells. First, in Neuro2A (N2A) neuronal cells, transfected GFP-Htt-Q74 was detected in the conditioned medium, and its level was reduced by GRASP55 depletion A. Simila2A cells B. Second111/Q111 . Indeed,111/Q111 C. Furtheal cells D. These versus WT secretome (A). Significantly changed cargoes  without predicted signal peptides are shown in black. On this plot, a majority of the unconventional targets were reduced in 55KO.To identify native proteins that are secreted in a GRASP55-dependent mechanism, we analyzed the conditioned media of WT and 55KO cells by tandem mass tag (TMT) labeling and LC–MS. With a 1% false discovery rate, we identified 1696 proteins in the media . We plotecretome A. Signifp < 0.05) , whereas 276 proteins did not appear to have ER signal peptides (B), consistent with our previous findings that GRASP depletion accelerates protein trafficking (C). It is worth mentioning that a GRASP55-dependent secretome has recently been reported  and without signal peptides, are included in unconventional secretion, we further differentiated the proteins into two separate groups and showed different effects by 55KO. Taken together, these results support our conclusion that GRASP55 plays essential roles in unconventional protein secretion.Next, we systematically analyzed the impact of GRASP55 depletion on the secretion of both conventional and unconventional pathway cargoes. Among the 1696 proteins, we identified a total of 445 proteins whose secretion was highly affected by 55KO  . Within peptides . Within peptides B, consisfficking . In contfficking C. It is reported . But unlD and To identify the pathways affected by 55KO, we performed Gene Ontology (GO) analysis of the significantly changed proteins. GO term pathway analysis of the 169 proteins with predicted ER signal peptides identified the following pathways as significantly changed in 55KO: lysosomal enzymes and structural proteins, extracellular matrix organization, glycosaminoglycan metabolism, cell adhesion and proliferation, cellular response to growth factor stimuli, immune response and tissue remodeling, and response to amyloid-beta, unfolded protein response, and aging , including transgelin 1 (TAGLN), multifunctional protein ADE2 (PAICS), and peroxiredoxin-1 (PRDX1), using the secretion assay we established for Htt. These proteins were selected because they are predicted to be highly secreted by the SecretomeP 2.0 server (http://www.cbs.dtu.dk/services/SecretomeP), an online tool to predict the probability of unconventional protein secretion, although their secretion and the GRASP55 dependence in the secretion have not been experimentally confirmed previously. Indeed, these proteins were all detected in the conditioned media, and their secretion was all reduced in 55KO cells (F–K). In summary, the unbiased secretomic analysis provided new evidence that GRASP55 depletion accelerates conventional protein trafficking and secretion but inhibits unconventional secretion. It also identified a list of bona fide candidates that are secreted by GRASP55-dependent unconventional secretion.To confirm that the identified proteins without ER signal sequences are indeed secreted through a GRASP55-dependent mechanism, we tested a few top candidates A, includKO cells , F–K. Invia direct sequestration by phagophores. Because of lysosomal dysfunction, undegraded proteins in autolysosomes and lysosomes could be released by lysosome exocytosis (L). GRASP55 enhances Htt secretion through two actions, one is to facilitate autophagosome–lysosome fusion by interacting with LC3 and LAMP2, and another is to stabilize p23, the translocon that allows cytosolic proteins to enter the lumen of the ERGIC, from where they are transported to autophagosomes. Finally, using systematic secretomic analysis, we identified and cataloged new proteins in GRASP55-dependent unconventional secretion.In this study, we used GFP-Htt-Q74 as a model protein to study the role of GRASP55 in unconventional secretion and cytosolic aggregation of neurotoxic proteins. We discovered that mutant Htt is secreted through a GRASP55- and autophagy-dependent but Golgi-independent pathway. Our results also revealed a link between enhanced Htt secretion, elevated autophagy, and increased GRASP55 expression under various stress conditions and confirmed a novel role of GRASP55 in stress response . Htt mayocytosis L. GRASP5via direct translocation across the plasma membrane, which is regulated by heparan sulfate and PtdInsP2 P2 . So far,sP2 . TDP-43,exosomes . While iIn HD, mutant Htt with a long polyQ stretch tends to form toxic protein aggregates that cause cellular toxicity and neurodegeneration , 6, 58. Autophagy is an energy and nutrient deprivation–induced lysosome-mediated degradation pathway essential for organelle turnover and degradation of aggregated proteins. Compared with non-neuronal cells, neurons need to maintain high basal autophagy for survival , 60. AutD–H and Htt secretion could be a double-edged sword to the neuronal cells and the disease. On the one hand, the aggregation-prone dephosphorylated Htt with a long polyQ stretch is selectively secreted, which reduces the amount of Htt aggregates in the cells and subsequently ameliorates its toxicity in the cell. In this sense, Htt secretion is beneficial to the cell. Our results support this hypothesis as GRASP55 depletion increases overall Htt aggregation in the cell D–H and . On the All reagents were purchased from Sigma–Aldrich, Roche, Calbiochem, and Fisher unless otherwise stated. The following complementary DNA constructs have been described previously: pEGFP-N1-GRASP55 , pEGFP-N1-GRASP65 (WT), and pEGFP-N1-Q80 , 64. OthAntibodies used in this study include monoclonal antibodies against β-actin , clusterin , FLAG , GFP , Myc , and PolyQ ; polyclonal antibodies against Atg7 , BiP , cathepsin D , GCC88 , GCK , Golgin-97 , Golgin-160 , GRASP55 , human GRASP65 , HK1 , α5 integrin , LC3 , and p62 .2 incubator. STHdhQ111/Q111 cells  were cultured in the same medium with the addition of 400 μg/ml G418. For Htt expression, GFP-Htt-Q74 was transfected into WT or GRASP KO HeLa cells in 3.5 cm dishes when the cells reached the confluency of 50 to 60%. For drug treatment, 5 μg/ml BFA, 500 ng/ml nocodazole, 100 μg/ml cycloheximide, 100 μM MG132, 5 μM vacuolin-1, 1 μM STS, 1 μM OA, or 5 μg/ml Tu was directly applied to the medium for the indicated times.For cell culture, HeLa, N2A, and MEF cells were grown in DMEM (Invitrogen) containing 10% super calf serum  and glutamax at 37 °C in a 5% COg for 10 min at 4 °C and 2000g for 20 min at 4 °C. About 0.9 ml of precleared conditioned medium was then mixed with 0.11 volumes of ice-cold 100% trichloroacetic acid  and placed on ice. After 10 min, 500 μl of ice-cold 10% TCA were added to the sample–TCA mix. After 20 min, samples were centrifuged at 20,000g for 30 min at 4 °C. Pellet was washed with cold acetone (−20 °C), dried, and dissolved in 1× SDS loading buffer for Western blot analysis. The cells were washed with cold PBS twice, scraped, and lysed in 20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% TX-100, and protease inhibitor cocktail (Thermo) for 30 min on ice. Lysates were cleared by centrifugation . The cell lysate and medium precipitate were loaded at 1:8 ratio. After electrophoresis and transfer, nitrocellulose membranes were incubated with corresponding primary antibodies overnight at 4 °C. The membranes were extensively washed and further incubated with horseradish peroxidase–conjugated goat anti-rabbit or goat antimouse secondary antibodies for 1 h at room temperature and exposed to a FluorChem M machine (Proteinsimple).WT or GRASP KO HeLa cells grown in 3.5 cm dishes were transfected with GFP-Htt-Q74 (4 μg DNA for a 3.5 cm dish) when the cells reached the confluency of 50 to 60% and incubated in full growth medium for 24 h. Cells were washed with PBS five times and further incubated in 1 ml DMEM without serum for 4 h. Conditioned media were collected and cleared by centrifugation at 500To quantify the efficiency of protein secretion, the intensity of the bands of the cargo protein in the conditioned medium and cell lysate on the same gels were quantified using ImageJ , and the percentage of protein secretion was calculated as signal in the conditioned medium/ × 100%, with 1:8 lysate to medium protein loading ratio applied in the equation.Cells were grown on sterile glass coverslips and rinsed with PBS before fixation. To stain total cellular proteins, cells were fixed in 4% paraformaldehyde (PFA) for 10 min and permeabilized with 0.2% TX-100 in PBS for 10 min. Cells were incubated with primary antibodies overnight at 4 °C, washed, and probed with the appropriate secondary antibodies conjugated to tetramethylrhodamine (for 45 min). To stain cell surface LAMP1, cells were fixed with 1% PFA for 10 min without permeabilization. Cells were incubated with an anti-LAMP1 antibody  overnight at 4 °C, washed, and probed with the appropriate secondary antibodies conjugated to tetramethylrhodamine for 45 min at room temperature. To analyze LC3 and Htt colocalization, cells were prepermeabilized in 125 mM potassium acetate, 25 mM Hepes, pH 7.2, and 2.5 mM magnesium acetate buffer containing 0.1% saponin (prefiltered) for 2 min on ice to release the cytosol and washed by 125 mM potassium acetate, 25 mM Hepes, pH 7.2, and 2.5 mM magnesium acetate buffer for 5 min at room temperature. Cells were then fixed with 4% PFA, permeabilized in 0.2% TX-100, and incubated with primary and secondary antibodies as described previously. DNA was stained with Hoechst 33342 (Thermo Fisher Scientific) in PBS for 5 min. Coverslips were rinsed with PBS and mounted with Mowiol onto slides. Images were taken with a 20× air objective or a 63× oil objective on a Nikon ECLIPSE Ti2 confocal microscope and shown as max projections.Protease K protection assay of the conditioned media was performed as previously described . Brieflyg for 3 min at 4 °C, and lysed in lysis buffer  for 20 min on ice. The supernatant and pellet were collected after centrifugation at 55,000 rpm  for 1 h in a TLA55 rotor. The supernatant concentration was adjusted to 1.5 mg/ml (the volumes of the samples are different) and supplemented with 1/5 volume of 6× SDS loading buffer with DTT . The pellet was dissolved in the same amount of 2× SDS loading buffer with DTT. Both samples were boiled, and the pellet and supernatant were loaded onto the gel at a 6:1 ratio.WT or GRASP KO cells were transfected with GFP-HttQ74 for indicated times. Cells were washed three times with cold PBS, scraped in cold PBS, centrifuged at 1000LDH cytotoxicity assay was performed following the manufacturer’s instruction . Briefly, 50 μl of conditioned medium was mixed with 50 μl of 1× reaction mixture and incubated in dark at room temperature for 30 min. Then 50 μl of stop solution was added, and the absorbance at 490 and 680 nm (background) was measured. The percentage of LDH cytotoxicity was calculated by dividing the LDH activity of the conditioned medium by the maximal LDH activity of the cell lysate as the kit protocol suggested.g for 10 min at 4 °C followed by 4000g for 15 min at 4 °C, and filtered with a 0.45 μm filter. The media then were concentrated with a 3 kDa cutoff ultrafilter , and the protein concentration was determined with Bradford assay . Cells were lysed in Pierce radioimmunoprecipitation buffer  with a protease inhibitor cocktail, and the protein concentration was tested with Bradford assay. About 75 μg of each sample was provided to the Mass Spectrometry–Based Proteomics Resource Facility at Department of Pathology, University of Michigan for TMT labeling, LC–MS/MS, and bioinformatics analysis.WT and 55KO cells were cultured in full growth medium in 15 cm dishes in triplicates till they reach 80% confluency. The medium was exchanged to 20 ml DMEM only (without serum) for 12 h; cells and conditioned media were collected. The media were cleaned by centrifugation at 5005; maximum injection time [IT] = 100 ms) followed by data-dependent “Top Speed” (3 s) MS2 scans . For multinotch-MS3, top 10 precursors from each MS2 were fragmented by higher energy collisional disociation followed by Orbitrap analysis .For TMT labeling, cell and medium samples were proteolyzed and labeled with TMT 10-plex essentially by following manufacturer’s protocol . For LC–MS analysis, as previously described . An OrbiData analysis was performed using Proteome Discoverer . In data analysis, MS2 spectra were searched against SwissProt human protein database  with the search parameters: MS1 and MS2 tolerance were set to 10 ppm and 0.6 Da, respectively; carbamidomethylation of cysteines (57.02146 Da) and TMT labeling of lysine and N termini of peptides (229.16293 Da) were considered static modifications; oxidation of methionine (15.9949 Da) and deamidation of asparagine and glutamine (0.98401 Da) were considered variable. Identified proteins and peptides were filtered to retain only those that passed ≤1% false discovery rate threshold. Quantitation was performed using high-quality MS3 spectra .t test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.In all figures, the quantification results are expressed as the mean ± SEM from three independent experiments, unless otherwise stated. The statistical significance of the results was assessed using a two-tailed Student’s L was drawn with BioRender (BioRender.com).All figures were assembled using Adobe Illustrator ; Figure 6The data that support the findings of this study are available from the corresponding author upon reasonable request.This article contains supporting information. It includes four supplemental figures and two supplemental tables. The authors declare that they have no conflicts of interest with the contents of this article.


We elucidate that the CuPc HTL,when employed in an “inverted” p–i–n solarcell configuration, attains a solar-to-electrical power conversionefficiency of up to 13.9%. Importantly, unencapsulated devices aslarge as 1 cm2 exhibited excellent long-term stability,demonstrating no observable degradation in efficiency after more than5000 h in storage and 3700 h under 85 °C thermal stressing inN2 atmosphere.Vacuumdeposition is a solvent-free method suitable forgrowingthin films of metal halide perovskite (MHP) semiconductors. However,most reports of high-efficiency solar cells based on such vacuum-depositedMHP films incorporate solution-processed hole transport layers (HTLs),thereby complicating prospects of industrial upscaling and potentiallyaffecting the overall device stability. In this work, we investigateorganometallic copper phthalocyanine (CuPc) and zinc phthalocyanine(ZnPc) as alternative, low-cost, and durable HTLs in all-vacuum-depositedsolvent-free formamidinium-cesium lead triodide [CH(NH Furthermore,materials such as Spiro-OMeTAD and PTAA are prone to degradation underenvironmental stressors such as temperature and humidity, thus curtailingthe operational lifetimes of PSCs when incorporated into the devicestructure.30 Moreover, while the evaporated hole transportingMoO3/N4,N4,N4″,N4″-tetra--4,4″-diamine(TaTm) structure has been successfully utilized in multiple high-efficiencydevices by Bolink and co-workers, it is hindered by thermal instabilityassociated with the MoO3 layer.31 Hence, it is paramount to find alternative HTLs that can be readilyevaporated, are inexpensive, and, most critically, are stable understrict storage and operational conditions.32PSCs with, at minimum, a thermally evaporated MHP layer haverecentlyachieved impressive progress in their photovoltaic performance.3NH3PbI3 (MAPbI3), formed by the vacuum codeposition of methylammonium iodide (MAI)and lead iodide (PbI2), has so far been the most commonlychosen photoactive semiconducting layer.36 For example, Abzieher et al.recently presented an all-evaporated MAPbI3 solar cell,which gave an impressive PCE as high as 19.5% by utilizing a p–i–ndevice architecture of ITO/2,2″,7,7″-tetraamino-9,9-spirobifluorene(Spiro-TTB)/MAPbI3/C60/Bathocuproine (BCP)/Au.35 However, the properties of MAI, such as itsvolatility, low molecular mass, and low sticking coefficient to manyunderlying materials of interest, make it very difficult to preciselycontrol its deposition rate, potentially limiting the viability ofvacuum-deposited MAPbI3.38 To date, the adhesion characteristics of MAI vaporhave proven to be dependent on a diverse array of factors, such asdeposition-chamber pressure, precursor purity, composition, and thetemperature of the underlying substrates, hence complicating the coevaporationprocess.40 Furthermore, MAPbI3 degrades rapidly under thermal stress,where, for example, decomposition and emission of NH3 andCH3I gas were observed at temperatures as low as 80 °C.41 However, replacing the MA+ cationwith CH(NH2)2+ (FA+) significantlyimproves resistance to thermal decomposition.25 Unfortunately,neat FAPbI3 perovskites are also susceptible to structuralphase transitions.25 In particular, theradius of the FA+ cation is significantly larger than thatof the MA+, which leads to FAPbI3 being onlymetastable in its black perovskite α-phase, with its yellowhexagonal δ-phase being thermodynamically favored at room temperature.42 However, alloying FA+ with Cs+  forms the stable FAx1–CsxPbI3 in its perovskite α-phase for a range of alloy fractionsfrom x = 0.1 to x = 0.5.44 Nevertheless, there are only a few reports of vacuum codepositionof such multication FACs-based perovskites.45Meanwhile, although a variety of all-vacuum-deposited PSCstackswith different ETLs and HTLs have been reported, methylammonium leadtriiodide CH46 They are desirable HTL candidates for an all-vacuum-processed PSCdue to their acceptable hole mobility , well-aligned energy levels, relatively low cost, andexcellent chemical and thermal stability.50 Even when CuPc derivatives are solution-processed,devices exhibit enhanced long-term stability under thermal stressingand cycling, especially when compared to PTAA or Spiro-OMeTAD.52 While evaporated PSCs with ZnPc HTL are rarely investigated,53 CuPc has been successfully employed with all-evaporated MAPbI3 stacks in both n–i–p and p–i–nconfigurations.54 However, the difference between CuPc and ZnPc is not yet clear,and neither has been used with vacuum-deposited FACs-based perovskite.More importantly, the long-term stability and degradation pathwaysare so far not well-understood for an all-evaporated stack.CuPc and ZnPc are organometallic semiconductorsfrom the classof metal phthalocyanine and can be deposited through thermal evaporation.0.83Cs0.17PbI3 (bandgap of approximately 1.56 eV) in the p–i–n configuration.We also uncover the varying adhesion behavior of the organic FAI precursoron underlying substrates, which influences the stoichiometry of thedeposited FACs perovskite films. We further optimized the perovskitecomposition on CuPc by introducing a 10% increase in PbI2 evaporation rate from the stoichiometric rate of 0.3 Å/s andimproved the all-vacuum-deposited device up to a PCE of 13.9%. Finally,we thoroughly examined their long-term stability under a range oftesting conditions and demonstrated that devices are intrinsicallystable for more than 3700 h (154 days) under 85 °C thermal stressing.Herein, we elucidate the different optoelectronic properties anddevice performance between CuPc and ZnPc as the HTL, when employedwith coevaporated FA0.83Cs0.17PbI3/C60/BCP/metal electrodes (Ag or Au). Reference devices werefabricated using solution-processed PTAA as HTL. 56 Both CuPcand ZnPc are direct-gap semiconductors with a bandgap of approximately1.7 eV.46 Similar toPTAA, previous studies have suggested CuPc has a highest occupiedmolecular orbital (HOMO) energy of −5.2 eV,47 which is well-aligned with that of the evaporated FA0.83Cs0.17PbI3 perovskite and would facilitateefficient hole extraction with minimal loss in the open-circuit voltage(Voc). Indeed, transmission-reflectionmeasurements of CuPc and ZnPc thin films of various thickness depositedon z-cut quartz substrates indicate similar transmission and absorptioncharacteristics . Thicker 300nm films of both CuPc and ZnPc absorb strongly in the green to redregion , which is expectedfrom their comparable bandgaps and is consistent with previous reports.48We investigatedboth evaporated CuPc and ZnPc as potential HTL candidates in an all-evaporatedp–i–n stack with the following configurations of ITO/HTL(CuPc or ZnPc)/FA0.83Cs0.17PbI3. 57 we believe thatthis observation is consistent with a higher nonradiative recombinationcenter density at the ZnPc–perovskite interface as comparedto the PTAA–perovskite control. This notion is further supportedby findings from photoluminescence quantum yield (PLQY) measurements,which quantify the degree of nonradiative recombination. kb is the Boltzmannconstant and T is approximatedto room temperature.5900 As the perovskite bandgap isunchanged on each HTL, this bandgap-independent analysis deduces thatthe expected decrease in QFLS, or equivalently Voc, is much more significant between PTAA and ZnPc than thatbetween PTAA and CuPc, by more than 30 meV.We first examined solar-cell“half stacks”,whichconsisted of ITO/HTL/FAFigure S4. We analyzed the transient PL decay with the method previouslyreported by Oliver et al.58 A double exponentialdecay was used for reproducing the transients and accounting for differentelectron–hole recombination processes.60 We then calculated the differential lifetime by taking the numericalderivative of the fitted photon flux (Φ(t))according toFurthermore, a similaroptoelectronic response is identified fromtime-resolved PL measurements of these half stacks using the time-correlatedsingle photon counting (TCSPC) technique 1d, with As shown in 0.83Cs0.17PbI3 perovskite. Thecurrent–voltage (J–V) curves from reverse scans of the best devices are plotted in Figure S5. These results illustrate that solar cells with solution-processedPTAA HTL performed best with PCE as high as 15.3%, mainly as a resultof a higher Voc, which may be attributedto its lowest unoccupied molecular orbital being higher than thoseof CuPc and ZnPc . Fromcomparison between devices with CuPc and ZnPc, we find a small meandifference of 0.05 V in Voc, as expectedfrom our optical measurements. Interestingly, there is a significantimprovement in short-circuit current density (Jsc) of almost 5 mA/cm2 for CuPc devices as comparedto ZnPc devices. This implies that the difference between CuPc andZnPc is more nuanced than just interfacial properties and is likelyto be also related to the reduced optical transmission of ZnPc  as well as a lack of charge selectivity.Indeed, poor external quantum efficiency (EQE) is seen for full solarcells with ZnPc HTLs, indicating increased parasitic absorption fromthe ZnPc HTL as well as poor electron extraction from these devices.We fabricated complete p–i–nsolar-cell devices toexamine the compatibility of these three HTLs with our vacuum codepositedFA0.83Cs0.17PbI3 as the wavelength is reducedresults in the bluer light generating electron–hole pairs veryclose to the HTL interface in solar cells with a p–i–narchitecture.22 This means holes are morereadily extracted due to the short distance to the electrode, whereaselectrons have to traverse most of the device depth. The drop in EQEat wavelength <500 nm for solar cells with ZnPc HTLs as comparedagainst those with CuPc HTLs indicates that electrons recombine rapidlyat the FA0.83Cs0.17PbI3–ZnPcinterface or are extracted into the ZnPc and recombine there. Nevertheless,these results suggest that CuPc would be a more proficient HTL thanZnPc, and hence we focused on optimizing an all-evaporated devicewith CuPc as the HTL.The strongly increasingoptical absorption coefficient of FA3.61 Yet, there have been few investigationsof the deposition mechanism of organic FAI vapor to date, which, asfor MAI, may prove pivotal in controlling perovskite-film growth.Therefore, we first endeavor the understanding of the growth of FA0.83Cs0.17PbI3 on the CuPc HTL. We vacuumcodeposited FA0.83Cs0.17PbI3 perovskitesimultaneously on substrates of PTAA and CuPc and investigated theirrespective crystalline phases via X-ray diffraction (XRD). 2 peak at 2θ of 12.7°is absent for perovskite formed on CuPc. Additionally, a small peakat 32°, which corresponds to the (210) peak, is only seen fromthe perovskite formed on CuPc, suggesting a slightly disparate orientationof the perovskite as a result of different templating from underlyingtransport layers.Precise control of deposition parameters is criticalin forminghighly crystalline perovskite films with correct phases in vacuumcodeposition and hence achieving optimal solar-cell performance. Inparticular, multiple past reports have demonstrated that the underlyingsubstrate is crucial in influencing the growth of perovskite films,especially with coevaporated MAPbI20 The measuredresults are summarized in Table S1. Here,we define the tooling factor as the ratio of the actual film thicknessmeasured via a stylus profiler to the registered thickness from thequartz crystal microbalance near the substrate. 2, are essentially unaltered on different HTLs, as reflected by theconsistent tooling factors and similar thicknesses measured acrossdifferent substrates. These data are shown in Table S1.To probe the difference in thecrystalline phasesof coevaporatedperovskite, we first measured the tooling factor of all precursorson different HTLs, which can convey important understandings of theiradhesion characteristics.S8). The different FAI thicknesseswere controlled by its tooling factor on PTAA. While it is evidentthat FAI adopts an “island growth” mode on both PTAAand CuPc, contrasting nucleation dynamics are observed. On PTAA, FAInucleates across the film forming a significant number of small islands,which is reflected by the smooth film with no considerable topologicalvariation  on FAI thin films of 5 and 25 nm deposited on CuPc and PTAA. However,as seen in the XRD data in 2 peak in the XRD pattern for the perovskite layerdeposited on CuPc . Figure S12 shows that the intensity of the PbI2 XRD peak at 12.7° increases with increasing PbI2 deposition rate, indicating that unreacted PbI2 has beenintroduced into the perovskite film.Hereon,we refer to FAic” case 2a. From 0.83Cs0.17PbI3 (520 nm)/C60 (23 nm)/BCP (2 nm)/Ag (100nm). Solar cells with stoichiometric FA0.83Cs0.17PbI3 perovskite deposited on CuPc exhibit a large spreadin device performance and have an undesirably low Voc of around 0.9 V, which limits the PCE output to between9% and 11%. With more PbI2 evaporated, a constant improvementin Voc can be achieved, and there is analmost 100 mV boost between coevaporating perovskite with the stoichiometricparameters and when the PbI2 rate is increased by morethan 20% excess. This trend of Voc boostis consistent with a previous report of coevaporated FACs-based perovskites.19 However, the measured Jsc quickly deteriorates when the PbI2 rate is increasedbeyond 15% excess. We found that the best devices are attained withan increment of 10% excess PbI2 rate from the stoichiometriccase, which resulted in a Voc boost, inconjunction with a small enhancement in both Jsc and fill factor (FF).J–V scansunder simulated solar conditions of the champion all-evaporated device (0.25 cm2 activearea) are plotted in Jsc of 21.1 mA/cm2, Voc of 0.93 V, and FF of 0.72 were observed. Notably, minimal hysteresisis observed in the champion 0.25 cm2-area all-evaporateddevice with CuPc HTL. The inset of 2-area device is slightly lower than that of the 0.25cm2-area device due to further loss in the FF. The best1 cm2-device has a PCE of 12.9% and a stabilized outputof 12.5% .Forward and reverse 2-area device andthe corresponding integrated Jsc. TheEQE spectrum demonstrates a strong peak in the blue region, indicatingelectrons are well-extracted. We conclude that the dip at 600 nm wavelengthis associated with the CuPc absorption , as expected for a solar cell in the p–i–nconfiguration. An integrated Jsc of 21.4mA/cm2 was calculated from EQE of the photovoltaic device,in excellent agreement with the measured Jsc of 21.1 mA/cm2 from the J–V scan.62 Remarkably, however,similar rigorous benchmarks are scarcely reported for evaporatedperovskites and devices.63 Here, we examineda range of storage conditions for the optimized all-evaporated stackwith different metal contacts of Ag or Au.The stability ofsolution-processedPSCs has been studied previously under a range of environmental andoperational conditions.2 atmosphere of the 0.25 and 1 cm2 champion devices, fabricated in the same batch, was tracked overtime .Regular testing identified that the PCE of the photovoltaic deviceshad improved slightly over the first 720 h (30 days), before maintaining104% and 101% of their initial efficiencies after 4296 h (179 days),respectively. A second EQE measurement was also taken on the champion0.25 cm2-area devices after 3192 h (133 days), revealingonly a slight change in the spectra and a similar integrated Jsc .We further demonstrate a consistent device stability trend by measuringadditional 0.25 and 1 cm2-area devices made in the sameand in separate batches and stored in the same environment . Figure S17 highlights that all devices were able to maintain theirinitial performance output for a period in excess of 5000 h (208 days),with the longest tested devices retaining more than 90% of the initialPCE exceeding 7200 h (300 days), demonstrating excellent batch-to-batchreproducibility. We also note that the larger 1 cm2-areadevices exhibit shelf stability behavior similar to that of all 0.25cm2-area devices, underlining the outstanding uniformityand homogeneity of this all-vacuum-deposited stack. The spread indevice performance increases after 5500 h, after which a few devicesceased to operate, which is likely a result of corrosion of the Agcontacts. The stability of the PTAA devices within a N2 atmosphere is also studied as a reference . PTAA devices also demonstrated an excellent shelf-lifestability, with all 0.25 cm2-area devices retaining morethan 90% of their respective initial efficiency after 2800 h (117days). Nevertheless, the larger 1 cm2-area device quicklyfailed only after 648 h (27 days), which may have succumbed to reactionswith the presence of residual solvent.First, the shelf-lifestability in NS20a, and S21 demonstrate the impressivethermal stability of these unencapsulated devices when placed insidean 85 °C oven in N2 at atmospheric pressure in darkness.Devices with active areas of 0.25 and 1 cm2 capped withAu contact retained more than 100% of their original PCE after morethan 3710 h (154 days), despite experiencing an early dip in theirperformance. These findings suggest that there is minimal thermal-induceddegradation of any element of the device stack.In parallel, 2. For solution-processed PSCs, it hasbeen reported that I– ions can migrate through theETL and react with Ag electrodes to form AgI, undermining the stabilityof the devices.65S20b depictthat the majority of Ag devices maintained a minimum 95% of initialperformance for the first 200 h, before experiencing a gradual decay,and after 1 month in the oven, only recorded between 60% and 75% oftheir initial efficiencies. Hence, this operational stability testingaccentuates the excellent thermal stability of devices with Au contacts,which we attribute to the use of CuPc HTL and MA-free FACs perovskite.In particular, the high sublimation temperature (>300 °C)anddecomposition temperature (>530 °C) of CuPc have resultedinits enhanced thermal durability as a stand-alone material and in perovskitephotovoltaic devices.66We also thermallystressed devices capped with Ag metal contactsat 85 °C in NFigure S22), before eventuallysuccumbing to moisture- or oxygen-related degradation. In comparison,devices with Ag contacts failed much quicker. XRD patterns  illustrate that device degradationwas most likely caused by a phase transition into the yellow hexagonalδ-phase for the perovskite layer.Furthermore, unencapsulated devices were also kept in a darkambientcondition (30–40% relative humidity) to probe their responseto a combination of air and moisture. After nearly 1500 h, the majorityof devices with Au electrodes maintained more than 80% of their initialPCE, with the smaller active-area devices outperforming the largerarea ones  and on a control solution-processed hole transportlayer (PTAA). In particular, we found striking differences in thesticking, adhesion, and nucleation of the perovskite precursor, FAI,to the metal phthalocyanines layer in comparison with the PTAA layer.This highlights the impact of sticking coefficients on the stoichiometryof vacuum codeposited perovskite films and hence the importance ofoptimizing growth parameters of FA-based MHPs by vacuum processeson different materials. We also demonstrate that the incorporationof a small amount of PbI2 excess in the coevaporation ofFA0.83Cs0.17PbI3 can enhance deviceperformance, in particular, with Voc improvement.Wehave investigated the use of metal phthalocyaninesas cost-effectiveand stable hole-transporting layers in all-vacuum-deposited MA-freep–i–n perovskite solar cells. We studied the nucleationand growth of the vacuum-deposited FA0.83Cs0.17PbI3 semiconducting thinfilms with CuPc as hole-transporting layers consistently outperformedthose based on ZnPc despite their similar electronic structure. Weconclude that the higher rates of electron–hole recombinationin the vicinity of the ZnPc–perovskite interface are the likelycause of the inferior performance of solar cells with ZnPc as thehole transport layer.We find that all-vacuum-deposited solar cells based on intrinsicFA0.83Cs0.17PbI3 as the intrinsic semiconductor. These solar cellsexhibited excellent durability of more than 150 days under thermalstressing and long-term storage conditions, with reasonable PCEs ofjust below 14%. In addition, a similar stability was observed forlarger 1 cm2-active-area devices, epitomizing that CuPcis a viable, cheap, and scalable hole transport layer. Looking forward,CuPc is a promising thermally stable component for more complicatedsolar-cell architectures, such as tandem and multilayer solar cells,which are well suited to fabrication by all-vacuum deposition.Significantly, we demonstrate an impressivethermal stability forthe all-vacuum-deposited p–i–n solar cells with CuPcas the hole transport layer and FAITO substrates were gently brushedin Decon-90 detergent solution (1% volume in deionized water) andthen sonicated for 5 min each in fresh Decon-90 solution, deionizedwater, acetone, and isopropyl alcohol sequentially. Before deposition,the substrates were further treated with UV-Ozone for 15 min.60, and the bufferlayer BCP were all evaporated in the same custom-built thermal evaporator.The chamber was pumped down to a base pressure between 8 × 10–7 and 2 × 10–6 mbar for alldepositions. The walls of the chamber were maintained at 17 °Cand the rotating substrate at 20 °C through two separate chillers.Rates were monitored through gold-plated quartz crystal microbalances(QCMs) and a customized control software. During all depositions,QCM readings at each source and at the substrate were cross-checked.Each precursor material was individually calibrated on cleaned ITOsubstrates  to determinethe tooling factor, and hence the actual deposition rate.To fabricate solar-cell devices, the HTLs of CuPc and ZnPc, the perovskitelayer, the electron transport layer CCuPc was evaporated at a rate of 0.08 Å/s at temperatures between320 and 340 °C until a layer thickness of 7.5 nm was achieved.ZnPc  was evaporated at a rate of0.08Å/s at temperatures between 300 and 330 °C until a layerthickness of 7.5 nm was achieved.2 atmosphere. The PTAA powder (Xi’an Polymer LightTechnology) was dissolved in toluene solvent at a concentration of1.5 mg/mL, stirred overnight, and spin-coated statically with 100μL of solution at 6000 rpm for 30 s with an acceleration of2000 rpm, followed by 10 min annealing at 100 °C on a hot platein the same N2 atmosphere.The PTAA transport layer wassolution-processed in a glovebox underN2 , and CsI  were coevaporated to form the FA0.83Cs0.17PbI3 composition. To form the precise stoichiometric FA0.83Cs0.17PbI3 composition on CuPc, FAIwas evaporated at 0.2 Å/s (155–170 °C), PbI2 was evaporated at 0.3 Å/s (270–290 °C), and CsIwas evaporated at 0.04 Å/s (400–430 °C). For eachdeposition, FAI powder in the crucible was topped up to 1.1 g, andfor a 520 nm perovskite layer, 0.19–0.21 g of FAI was typicallyevaporated. All as-deposited films were further annealed at 135 °Cfor 30 min on a hot plate under N2 glovebox conditions.For the perovskite layer,FAI , PbI60  was depositedat 0.1 Å/s to form a 23 nm thick layer for devices with Ag contact,and a 30 nm thick layer for devices with Au contact. Subsequently,a 2 or 5 nm thick BCP  layer was deposited at0.07 Å/s for devices with Ag contact or Au contact, respectively.Fullerene CThe Ag top contact with a thickness of 100 nm was evaporated ina separate Lesker Nano36 chamber. Using QCM readings, we maintainedthe initial rate at 0.2 Å/s for the first 10 nm, before rampingup to 1.5 Å/s. The Au top contact with a thickness of 100 nmwas evaporated in the same Lesker Nano36 chamber. The initial ratewas maintained at 0.1 Å/s for the first 10 nm, before rampingup to 0.7 Å/s.2, generated by a Wavelabs Sinus-220 solar simulator and aKeithley 2400 source meter. The solar simulator was calibrated withrespect to a KG-3 filtered silicon reference photodiode (Fraunhofer)prior to the measurement. Devices were characterized in ambient airconditions at room temperature with relative humidity between 25%and 40%. The Voc was first measured for3 s. Reverse and forward scans between −1.2 and 0.2 V at aconstant scan rate of 0.13 V/s were sequentially performed. Steady-statecurrent and voltage were further probed for 30 s under continuousillumination, keeping the device close to its maximum power point(MPP) by actively tracking the maximum power point with a gradientdescent algorithm. Finally, Jsc was measuredfor 3 s. A mask was used for each substrate to separate the activearea for each device to either 0.25 or 1 cm2.Deviceswere measured understimulated AM1.5G sunlight with an equivalent irradiance of 100 mW/cm2 beam splitter, and a silicon diode detector.Transmission–reflectionmeasurements were performed on a Bruker Vertex 80v Fourier transforminterferometer, with a tungsten-halogen near-infrared source, a CaFα 1.54 Å set at40 kV and 40 mA).XRD patternswere measured with a Panalytical X’pert powder diffractometerwith copper X-ray source  and measured with an ICCD camera .PL measurements were performed through photoexcitationof ITO/HTL/perovskitethin films with a 398 nm continuous wave laser with a power density of 6.38 W/cmTCSPC measurements were carried out throughphotoexcitation ofITO/HTL/perovskite thin films with a 398 nm pulsed semiconductor diodelaser  with a repetition rate of 10 MHz fromthe perovskite side. The emitted PL was coupled into a grating monochromator and collected by a photocountingdetector (PDM series from MPD). Timing was controlled by a PicoHarp300event timer.–4 mbar.AFM measurements were carried out using an AsylumMFP3D (AsylumResearch and Oxford Instruments Co.) in AC (tapping) mode. OlympusAC240-TS-R3 silicon tips were used for topography measurements. FEIQuanta 600 FEG was used to take all SEM images. Prior to all measurements,the chamber was pumped down to high vacuum with a pressure of lessthan 2 × 10


In addition, the liver sample from the 1000 μg/L ATZ−treated group was used for GC/MS metabolomic analysis. The principal component analysis (PCA) model showed significant separation of the 1000 μg/L ATZ group from the control group, indicating that ATZ exposure altered hepatic metabolism in male adult zebrafish. A total of 29 significantly (p < 0.05) different metabolites were observed and identified in the ATZ−treated group. Moreover, the most disturbed pathways by ATZ were the arginine and proline metabolic pathways, followed by the glutathione metabolic pathway. Three and two metabolites were significantly altered in the arginine and proline metabolic pathways and glutathione metabolic pathway, respectively. Based on these results, we suggested that ATZ was capable of altering liver metabolism in zebrafish and that its ecological risk to aquatic organisms cannot be ignored.Atrazine (ATZ) is a herbicide used in agricultural production and has been detected in surface water due to its widespread use worldwide. This may pose a threat to the health of aquatic animals. To explore the ATZ−induced hepatic metabolism disorder, male zebrafish were exposed to 300 and 1000 μg/L ATZ for 21 days, respectively. The results revealed that ATZ exposure significantly reduced hepatic triglyceride (TG) levels, while significantly ( In the last decades, pesticide have been widely used to improve the production in agriculture all over the world. Widely used pesticides are present in our daily life . The harAtrazine  has been extensively used as an effective herbicide for the past 50 years. Over the past few decades, 64–80 million pounds of ATZ have been reported to be used annually for agriculture and residential purposes in the United States . ATZ in In some studies, metabolomics have been found to be a good explanation for environmental chemical and pesticide toxicity ,24,25. MAtrazine  was purchased from TCI and used as received. ATZ was dissolved with dimethyl sulfoxide (DMSO) to prepare stock solutions at concentrations of 300 and 1000 µg/L. The stock solution was kept in reserve at 4 °C until use.Danio rerio) were used for the present experiment. Experimental zebrafish were acclimatized in tap water  for at least 1 week, with water temperature controlled at 28 °C and a dark/light photoperiod of 10:14 h [Mature male adult AB strain zebrafish  and ATZ−H (1000 μg/L). Each group of 18 adult males was housed equally in 3 tanks with 3 L of solution in each glass tank. After 21 days exposure, livers from 3 fish were collected together as one sample, resulting in 6 pooled samples per group for further analysis.To explore the metabolomic analysis of zebrafish liver, experimental exposures were manipulated with reference to our previous studies ,34. BrieThe liver and PBS were ground at a ratio of 1:9, then centrifuged at 2000 rpm for 10 min and the supernatant was finally collected. Kits for the determination of hepatic glucose (GLU), PYR, TG and TC levels were purchased from Nanjing Jiancheng Biotechnology Research  and quantified according to the manufacturer’s designation instructions.v/v/v) with 5 µL 3 mg/mL Myristic acid-d27 and homogenized at low temperature using Tissuelyser-192 . The mixture was then placed in an ice bath for 1 min and then centrifuged at 14,000 rpm for 5 min at 4 °C. Then, 800 μL of supernatant was added to the GC vial and evaporated to dryness. A further 20 µL of methylhydroxylamine hydrochloride (40 mg/mL in pyridine) was added and placed in an oscillating incubator at 30 °C for 90 min. Next, 90 µL of MSTFA (N−methyl−N− (trimethylsilyl) trifluoroacetamide)  was added and the mixture was incubated at 37 °C for 30 min, followed by centrifugation at 14,000 rpm for 5 min at 4 °C. Finally, 60 µL of supernatant was removed from each sample for GC/MS analysis.About 25 mg liver tissue with 1000 µL of acetonitrile/isopropanol/water . Data analysis was carried out using SIMCA-P+14.0 software .Next, the samples were injected into the Agilent 7890A/Agilent 5975C MSD GC/MS system equipped with an Agilent DB5-MS  column. The injection temperature, ion source temperature and transmission line temperature required for the experiment were all set to 250 °C. The helium carrier gas flow rate is set at a constant rate of 1.1 mL/min. Started with a setting of 60 °C and held for 1 min, then increased the speed to 3000 rpm and ran for 10 min. Full scan monitoring was performed over a mass slope range of p) value set at 0.05 was deemed to be significant. Graphing was performed using SPSS  13.0  and GraphPad Prism Version 8.0 .All differences between the control and ATZ treatment groups were assessed using a one-way ANOVA. A probability (p < 0.05) altered in ATZ−exposed zebrafish compared to the control group. Compared with the control group, TG level in the ATZ-H group decreased significantly (p < 0.05), while the PYR level increased significantly (p < 0.05). The TC level notably increased after ATZ treatment. All these results indicated that ATZ could cause disorders of hepatic energy metabolism in zebrafish.As shown in p value < 0.05). A significant deviation of metabolites occurred in the ATZ−H group compared to the control group (p < 0.05) different between the control group and the treated group, there were many substances that were not identified. All these changed substances were enriched in many different metabolic pathways (p < 0.05) different metabolites in total. The results showed that for the group treated with 1000 μg/L ATZ, the levels of Glycerol−2−phosphate, Hypoxanthine, Glucose (1MEOX) (5TMS) MP, Neuraminic acid, N−acetyl− (6TMS), Fumaric acid (2TMS), Docosahexaenoic acid (1TMS), similar to Pentasiloxane, dodecamethyl, Cholesterol (1TMS), Hexadecanoic acid (1TMS), Hexadecanoic acid (1TMS), Threonine, allo− (2TMS), Proline (1TMS), Isoleucine (1TMS), Inosine (4TMS), Inositol−2−phosphate, myo− (7TMS), Inositol−2−phosphate, myo− (7TMS), Putrescine (4TMS), Putrescine (4TMS), Saccharic acid (6TMS), Xanthine (3TMS), Glycine (2TMS) and Octadecenoic acid, 9− (E)− (1TMS) were significantly higher than those of the control group, while 1-Pyrroline−2−carboxylate (1TMS), Noradrenaline (5TMS), Leucine (2TMS), Mannose−6−phosphate (1MEOX) (6TMS), Tocopherol, alpha− (1TMS), Sarcosine (2TMS) and Tagatose (1MEOX) (5TMS) MP were obviously lower compared to the control group. These results suggested that ATZ significantly (p < 0.05) altered the hepatic metabolism in zebrafish.The hepatic metabolic profile in male zebrafish was also influenced in zebrafish after 21 days of exposure to ATZ . The PCAol group B,C. The pathways D. Differpathways E. There p < 0.05), while 1,4-Butanediamine and L-Proline decreased notably. Meanwhile, after 1000 μg/L ATZ exposure, the 1,4-Butanediamine and Pyroglutamicacid levels of the glutathione metabolic pathway were elevated (ATZ exposure induced alterations in hepatic metabolic pathways in zebrafish. The greatest effects were observed on the arginine and proline metabolic pathways A. In addelevated C.By analyzing the changes in metabolites, we found that ATZ caused changes in glycolipid metabolism, amino acid metabolism and some other related metabolic pathways . We founIncreasingly more studies have suggested that environmental chemicals including pesticides had the potential to influence the hepatic metabolism in animals ,36,37. TMetabolomics is becoming a popular approach in the study of organismal metabolism . It can The hypothesis that environmental chemical pollutants may cause metabolic abnormalities in the liver has been confirmed by many studies ,41,48. PIn this study, male adult zebrafish were exposed to 300 and 1000 g/L of ATZ for 21 days, respectively. We observed that 21 days of ATZ exposure could cause abnormal liver metabolism in zebrafish. These results showed that several different pathways related to metabolism could be influenced by ATZ, and the potential effects of ATZ on fish or other aquatic animals should be extensively studied. We thought that the results made in this study could provide new insights into the toxicity of ATZ in the aqueous environment.


An. stephensi mosquitoes in the Horn of Africa represents a significant regional threat, which may jeopardise malaria control, particularly in urban areas which were formally free from disease transmission. Novel vector surveillance methods are urgently needed, both agnostic to mosquito larval morphology, and simple to implement at the sampling stage. Using new multiplex TaqMan assays, specifically targeting An. stephensi and Ae. aegypti, we validated the use of environmental DNA (eDNA) for simultaneous vector detection in shared artificial breeding sites. Study findings demonstrated that An. stephensi and Ae. aegypti eDNA deposited by as few as one second instar larva in 1L of water was detectable. Characterization of molecular insecticide resistance mechanisms, using novel amplicon-sequencing panels for both vector species, was possible from eDNA shed by as few as 16–32 s instar larvae in 50 ml of water. An. stephensi eDNA, derived from emergent pupae for 24 h, was remarkably stable, and still detectable ~ 2 weeks later. eDNA surveillance has the potential to be implemented in local endemic communities and at points of country entry, to monitor the spread of invasive vector species. Further studies are required to validate the feasibility of this technique under field conditions.The invasion and establishment of  In sub-Saharan Africa, malaria, transmitted by Anopheles mosquitoes, remains the leading cause of morbidity and mortality ; despite the widespread deployment of effective vector control measures, especially the use of insecticide-treated nets (ITNs) and indoor residual spraying (IRS), which have averted more than 1.5 billion cases and 7.6 million malaria-related deaths since 20002. Concurrently, approximately 51 million individuals are infected with lymphatic filariasis3, a severe cause of disability and stigma, and many arthropod-borne viruses (arboviruses) including, dengue, chikungunya, Rift Valley fever, yellow fever, Zika, o’nyong’nyong and West Nile, circulate unabated among humans, wildlife and livestock across the continent5. Over 27,000 cases of viruses transmitted by Aedes mosquitoes have been reported in West Africa since 2007, but seroprevalence surveys, indicative of prior disease exposure, suggest this is a gross underestimation of the actual arboviral disease burden6. While there is growing recognition that parts of Africa are at serious risk for arbovirus outbreaks, the understanding of the current distribution of these diseases is inadequate, as regional VBD surveillance is focused primarily on malaria, detection is usually reactive in response to outbreaks7, and robust evidence for efficacious control tools targeting arbovirus vectors is lacking8.Vector-borne diseases (VBDs) account for 17% of all infectious diseases worldwide and are responsible for more than 700,000 deaths annuallyAnopheles stephensi is a highly competent malaria vector whose distribution until 2011 encompassed the Indian subcontinent, parts of South-East Asia and the Arabian Peninsula. Recently it has become an invasive vector species in the horn of Africa, recognised by the WHO as a “biological threat”9. An. stephensi was first reported in Djibouti in 201210 and since then this species has spread to neighbouring Ethiopia (2016)12, the Republic of Sudan (2019)14, Somalia (2019)15, Yemen (2021)15 and most recently Nigeria (2020)15. The invasion of An. stephensi in Djibouti was accompanied by an increase in malaria cases from 25 in 2012 to 14,810 in 201716, with outbreaks reported in Djibouti City, including historically malaria-free areas that were considered unsuitable for anopheline breeding sites10. Unlike An. arabiensis, the main malaria vector species in this region, An. stephensi often breeds in urban areas in man-made water containers, buckets, discarded tyres, and water storage tanks for domestic use and construction. These are sites that may not be under routine surveillance by the National Malaria Control Programme (NMCP); such larval habitats are often used by Aedes species, vectors responsible for dengue outbreaks and transmission of other arboviruses, including yellow fever, in Ethiopia18. An. stephensi and Ae. aegypti have already been observed in the same larval habitats in eastern Ethiopia19. Given the propensity of the former vector species to colonize urban environments quickly, breeding mainly in man-made water containers, mathematical models estimate over 126 million people in cities across Africa are at risk of malaria transmitted by An. stephensi20 and that annual Plasmodium falciparum cases could increase by 50% if no vector control interventions are implemented21.An. stephensi poses a significant problem to urban populations in Africa, the WHO recommends targeted surveillance to monitor existing populations, dispersal dynamics and to characterize their insecticide resistance profiles to inform prospective vector control strategies22. Moreover, dengue has been recognised as a significant public health threat in Ethiopia, where a need for improved vector surveillance and control has been recognised17. Novel vector surveillance methods are urgently required, which demand little prior knowledge of mosquito larval morphology and are quick and easy to implement at the sampling stage, with the ability to simultaneously detect the presence of multiple species of interest. Recent advances in molecular techniques are providing new opportunities for surveillance and prevention of VBDs, which can be used to better target vector control strategies. Detection of the traces of genetic material that organisms leave behind in their environment is one possible approach. Environmental DNA (eDNA) found in collected samples may allow us to indirectly confirm the presence of a species of interest and its abundance even when there are no obvious signs of the organism being present24. eDNA can be extracted from environmental samples, such as water from potential mosquito larval habitats, and can be detected using sensitive molecular techniques such as qPCR. eDNA decay and degradation are environmentally-dependent and due to its nature, detection of eDNA is an indicator of recent presence of the species of interest25. This approach can be especially useful for detection of invasive species, including mosquitoes27.As the spread of An. stephensi and Ae. aegypti from the same larval habitats and characterization of their molecular insecticide resistance mechanisms, under controlled laboratory conditions.This study assessed the suitability of using eDNA for simultaneous detection of An. stephensi and Ae. aegypti eDNA sampled from shared breeding sites, we designed a multiplex TaqMan assay based on single-nucleotide polymorphisms (SNPs) in COX1. Initially the analytical sensitivity, linearity, and dynamic range of the qPCR assay for eDNA detection was estimated using serial dilutions of An. stephensi and Ae. aegypti gDNA derived from colony strains, tested in technical triplicate. Both qPCR assays produced good linearity (R2 = 0.99 for both An. stephensi and Ae. aegypti assays) and efficiencies of 90.01% and 94.62%, respectively  with good linearities (R2 = 0.99 for simultaneous detection of An. stephensi and Ae. aegypti gDNA). The LOD/LOQs were determined to be 0.0000154 copies/reaction for simultaneous detection of An. stephensi and Ae. aegypti : 31.35–32.42]; 2 larvae: 27.36 [95% CI 24.72–29.99]; 4 larvae: 26.74 [95% CI 25.75–27.73]; 8 larvae: 25.42 [95% CI 23.77–27.07]; 16 larvae: 21.96 [95% CI 21.09–22.83]; 32 larvae: 21.01 [95% CI 20.09–21.92] , both species were still detectable, with higher average Ct values for the minority species (An. stephensi); the average Ct value for An. stephensi was 37.20 [95% CI 36.43–37.97] and for Ae. aegypti was 29.72 [95% CI 29.08–30.36] .A longitudinal cohort of 1L artificial breeding sites were monitored for 2 weeks, following the emergence of 32 ime Fig. .The averAn. stephensi using amplicon-sequencing, was undertaken with eDNA extracted from 50 ml artificial breeding sites. Amplicons covering the voltage-gated sodium channel (vgsc), glutathione-s-transferase 2 (GSTe2) and acetylcholinesterase-1 (ace-I) genes, which contain genetic variants associated with insecticide resistance, were tested28.Characterisation of published insecticide resistance mechanisms in Of the 6 eDNA samples tested , no PCR amplicons were generated for either eDNA-L1 or -L2 samples. Average read depth per amplicon for eDNA-L4, -L8, -L16 and -L32 is presented in Table Ace-1 gene in amino acid 177; this SNP was present in the eDNA-L8 and eDNA-L16 samples, with eDNA-L8 genotyped as heterozygous and eDNA-L16 as the homozygous alternate. This SNP has been previously reported in both colony and wild-caught samples28. No SNPs associated with insecticide resistance were identified.A total of 40 variants were identified, accounting for 33 single nucleotide polymorphisms (SNPs) and 7 insertions or deletions (indels). Only one missense mutation was identified in the Ae. aegypti 50 ml artificial breeding sites containing 4 and 8 larvae did not amplify using amplicon-seq primers, targeting the vgsc and ace-1 genes29. PCR reactions using eDNA from Ae. aegypti artificial breeding sites containing 16 larvae (eDNA-L16) were able to amplify 5 targets: ace-1 and four amplicons for the vgsc 19. With such variation in breeding site environments, it is anticipated that the rate of eDNA persistence will differ quite considerably, with respect to water turbidity, velocity, UV exposure, salinity/pH, pollution, and co-occupancy . In our study, An. stephensi eDNA, derived from emergent pupae for 24 h, was remarkably stable, and still detectable almost two weeks later. Similarly, eDNA from 15 Ae. albopictus larvae, reared for 12 days in 500 ml water, was demonstrable up to 25 days after live material was removed27. Our study may have in fact underestimated the sensitivity of eDNA detection, by only allowing larvae to remain in artificial breeding sites for 24 h, as well as the rate of eDNA degradation, due to highly controlled insectary conditions .An. stephensi28 and Ae. aegypti29, fragments of key insecticide resistance genes (including portions of the voltage-gated sodium channel and acetylcholinesterase) were able to be amplified from eDNA shed by as few as 16–32 s instar larvae in 50 ml of water; as expected, no insecticide resistance mutations were identified in our insecticide-susceptible insectary colonies. The detection sensitivity of insecticide resistance genes in natural breeding sites may be greater than in our laboratory study, particularly as larval densities can be higher and vector populations will deposit eDNA throughout their ~ 2 week lifecycle in breeding sites. Conventional insecticide resistance monitoring is based on larval dipping from known, productive breeding sites, rearing of vectors to the adult stage, followed by phenotypic characterization in bioassays and/or genotypic analysis using various molecular techniques37. eDNA detection offers a new avenue to explore molecular insecticide resistance at the community-level, circumventing some of the laborious sampling requirements and potentially allow wider-scale surveillance of water bodies which may be less productive sites or entirely unknown to local entomologists. This strategy could be combined with additional analytical techniques, such as high-performance liquid chromatography, to survey the extent of breeding site contamination with agricultural pesticides, a known driver of insecticide resistance selection in mosquito vector populations39.In addition to vector species identification, this study investigated the potential to exploit eDNA for molecular insecticide resistance surveillance. Using recently developed amplicon-seq panels for An. stephensi specifically, eDNA detection could be considered for surveillance of seaports in countries at greatest risk of introduction of this invasive species40. Following laboratory validation in this study, further work is required to assess the feasibility of sampling eDNA of An. stephensi/Ae. aegypti in situ, including an assessment of environmental risk factors which impact eDNA detection levels and comparison of eDNA quantification versus larval density per breeding site.Water sampling for mosquito eDNA detection represents a cheap, low-technology tool, which requires virtually no entomological skills or training, and thus has the potential to be easily integrated into citizen science initiatives. Furthermore, with regards to An. stephensi and Ae. aegypti in shared artificial breeding sites. Study findings demonstrated that An. stephensi and Ae. aegypti eDNA deposited by as few as 1 s instar larva in 1 L of water was detectable. Characterization of molecular insecticide resistance mechanisms, using novel amplicon-seq panels for both vector species, was possible from eDNA derived from as few as 16–32 s instar larvae in 50 ml of water. eDNA surveillance has the potential to be implemented in local endemic communities as part of citizen science initiatives, and/or in cargo ports at points of country entry, to monitor the spread of invasive mosquito vector species. Further studies are required to validate the feasibility of this technique under field conditions.This study validated the use of eDNA for simultaneous detection of An. stephensi (SD500 strain) and Ae. aegypti (AEAE strain) larvae were used in all experiments. An. stephensi SD500 was colonised from Pakistan in 1982 and is insecticide susceptible41. Ae. aegypti AEAE was colonised from West Africa before 1926 and is insecticide susceptible . Larvae were reared in sterile, distilled water, in plastic bowls (20 × 18 × 7 cm) under controlled insectary conditions  and fed once daily with NISHIKOI staple fish food pellets .Laboratory-reared second instar An. stephensi or Ae. aegypti larvae . We performed three biological replicates with 6 different larval densities: 1, 2, 4, 8, 16 and 32 of either An. stephensi and Ae. aegypti were evaluated initially in separate 1L breeding sites and then in mixed breeding sites. The mixed breeding site experiments used 24 different conditions, comprising three biological replicates with 5 larval densities: 2, 4, 8, 16 and 32 larvae of An. stephensi and Ae. aegypti in equal proportions (50:50), three biological replicates with larvae of the two species in different proportions: 4:40 An. stephensi:Ae. aegypti larvae and three negative control breeding sites.In the second experiment, 21 different conditions were tested by adding 1L of sterile, distilled water to 21 sterile plastic rectangular containers. We performed three biological replicates with 6 different larval densities: 1, 2, 4, 8, 16 and 32 larvae. Three negative control experimental habitats with no larvae were run in parallel for each condition. Larvae were left in each 1L breeding site for 24 h, prior to manual removal, using a sterile pipette. An. stephensi pupae added to each container. Three negative control experimental habitats with no pupae were run in parallel for each condition. Pupae were left in each 1L breeding site for 24 h, prior to manual removal of all emerged adults, remaining pupae, and pupal skins, using a sterile pipette. Breeding sites were then sampled daily for 14 days, following the removal of adults and pupae.In the third experiment, the rate of eDNA degradation in artificial breeding sites was assessed. One litre of sterile, distilled water was added to 9 sterile plastic rectangular containers. We performed three biological replicates with 32 eDNA from water samples were concentrated prior to extraction. For each breeding site replicate, 15 ml of water was added to a sterile 50 ml falcon tube  and 1.5 ml of 3 M sodium acetate solution (pH 5.2)  was immediately added, followed by 33 ml of absolute ethanol and stored overnight at − 20°C. Samples were then centrifuged at 8000 rpm at 6 °C for 30 min. The supernatant was discarded, and the pellet was washed in 20 ml of absolute ethanol by centrifuging at 8000 rpm at 6 °C for 10 min. The supernatant was discarded, and ethanol allowed to evaporate at 65°C. The pellet was dissolved in 720 μl ATL buffer and 80 μl proteinase K  and incubated overnight at 56 °C. eDNA was extracted using a Qiagen DNeasy 96 Blood and Tissue kit , according to the manufacturer’s protocol, with minor modifications.An. stephensi (ASTE016222) and Ae. aegypti (AAEL018662) eDNA based on a fragment of cytochrome oxidase I (COX1). Forward (5ʹ-CAGGAATTACWTTAGACCGAMTACC-3ʹ) and reverse (5ʹ-TCAAAATAARTGTTGRTATAAAATRGGGTC-3ʹ) primers and two TaqMan fluorescent-labelled probes (An. stephensi probe 5ʹ-/5HEX/ATTACTATA/ZEN/TTACTTACAGACCG/3IABkFQ/-3ʹ and Ae. aegypti probe 5ʹ-/56FAM/ATTACTATG/ZEN/TTATTAACAGACCG/3IABkFQ/-3ʹ) were designed using Geneious Prime® 2021.1.1 to amplify a 202 bp region of COXI  to assess PCR efficiencies. Genomic DNA concentrations were determined using the Qubit 4 fluorometer 1X dsDNA HS assay . Standard curve reactions were performed in a final volume of 10 μl containing 2X PrimeTime® Gene Expression Master Mix , 250 nM of forward and reverse primers and 150 nM of each probe and 2 μl genomic DNA. Reactions were run on a Stratagene Mx3005P Real-Time PCR system (Agilent Technologies) at 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All assays were run in technical triplicate alongside PCR no-template controls (NTCs).Standard curves of Ct values for each probe were generated individually and then multiplexed using a ten-fold serial dilution of control An. arabiensis, An. gambiae s.s., An. funestus and Cx. quinquefasciatus.To confirm primer and probe specificity, the multiplex assay was used to assess amplification of stock genomic DNA from other medically important sympatric vector species: Following eDNA extraction, eDNA detection was performed in a final volume of 10 μl containing 2X PrimeTime® Gene Expression Master Mix , 250 nM of forward and reverse primers, 150 nM of each probe and 4.2 μl eDNA. Reactions were run on a Stratagene Mx3005P Real-Time PCR system (Agilent Technologies) at 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All assays were run in technical triplicate alongside PCR no-template controls (NTCs).42, implemented in R version 4.0.243. All other statistical analyses were conducted in GraphPad Prism v9.4.0.Stratagene MxPro qPCR software  was used to produce qPCR standard curves. qPCR assay limits of detection (LOD) were determined using the “Generic qPCR LoD/LoQ calculator”An. stephensi, briefly, three genes of interest were identified, and a total of 7 amplicon primer pairs were designed to target SNPs, previously associated with insecticide resistance28. To enable sample multiplexing, each primer had a 6 bp barcode attached to the 5ʹ end to allow for sample identification in downstream analysis. Amplicons were generated using the eDNA extracted from experiment #1 . PCR reactions contained 5X Q5 reaction buffer , Q5® High-Fidelity DNA polymerase , 250 nM of forward and reverse primers and 2 µl of eDNA. PCR reaction conditions were an initial 30 s denaturation at 95 °C, followed by 35 cycles of 98 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s, and a final elongation step at 72 °C for 2 min. PCR products were visualized on SYBR Safe 1% agarose gels , prior to purification using Agencourt AMPure XP magnetic beads , using a ratio of 0.7:1 (µl of beads to DNA).To characterize molecular mechanisms of insecticide resistance in Ae. aegypti, briefly, sequences for portions of the voltage-gated sodium channel  and acetylcholinesterase-1  were extracted from publicly available assemblies for Ae. aegypti (LVP AGWG)29. Forward and reverse primers were designed using PrimerBLAST software to amplify regions of 450–500 bp that contain known SNP loci or regions of interest, with 6 bp inline barcodes and partial Illumina tails that allow the samples to be sequenced on an Illumina sequencing platform. PCR reactions contained 5X Q5 reaction buffer , Q5® High-Fidelity DNA polymerase , 250 nM of forward and reverse primers and 2 µl of eDNA extracted from experiment #1 . PCR reaction conditions were 98 °C for 30 s, followed by 35 cycles of 98 °C for 10 s, 57 °C for 60 s and 72 °C for 90 s. PCR products were visualized on SYBR Safe 1% agarose gels , prior to purification using Agencourt AMPure XP magnetic beads , using a ratio of 0.7:1 (µl of beads to DNA).To characterize molecular mechanisms of insecticide resistance in The concentration of purified PCR products was measured using the Qubit 4 fluorometer 1X dsDNA HS assay  and samples with unique barcode combinations were pooled in equal concentrations to create an overall pool of 20 ng/µl in 25 µl total volume. A second PCR to insert Illumina adaptors (no further library preparation required) and amplicon sequencing was performed at Genewiz (Illumina-based Amplicon-EZ service).29. Sequences were trimmed, aligned to reference (Ae. aegypti LVP AGWG for Ae. aegypti or UCI_ANSTEP_V1 assembly for An. stephensi) and data were quality checked using FastQC and Samclip. Paired-end reads were mapped against the reference sequences using the BWA-MEM algorithm. Variants were then called using three packages: Freebayes, GATK, and an in-house naive variant caller. Identified variants were then compared and normalised against each other to reduce false negatives and positives, and then filtered and annotated using BCFtools; for An. stephensi, variants were annotated using snpEFF, based on a manually built database for the UCI An. stephensi genome assembly. SNPs were then manually filtered to ensure that they appeared in more than one sample, with an allele depth of > 50.Samples were demultiplexed based on the in-line barcodes in each forward and reverse primer using an in-house python scriptSupplementary Information 1.Supplementary Information 2.


Although many efforts have been made to elucidate the pathogenesis of COVID-19, the underlying mechanisms are yet to be fully uncovered. However, it is known that a dysfunctional immune response and the accompanying uncontrollable inflammation lead to troublesome outcomes in COVID-19 patients. Pannexin1 channels are put forward as interesting drug targets for the treatment of COVID-19 due to their key role in inflammation and their link to other viral infections. In the present study, we selected a panel of drugs previously tested in clinical trials as potential candidates for the treatment of COVID-19 early on in the pandemic, including hydroxychloroquine, chloroquine, azithromycin, dexamethasone, ribavirin, remdesivir, favipiravir, lopinavir, and ritonavir. The effect of the drugs on pannexin1 channels was assessed at a functional level by means of measurement of extracellular ATP release. Immunoblot analysis and real-time quantitative reversetranscription polymerase chain reaction analysis were used to study the potential of the drugs to alter pannexin1 protein and mRNA expression levels, respectively. Favipiravir, hydroxychloroquine, lopinavir, and the combination of lopinavir with ritonavir were found to inhibit pannexin1 channel activity without affecting pannexin1 protein or mRNA levels. Thusthree new inhibitors of pannexin1 channels were identified that, though currently not being used anymore for the treatment of COVID-19 patients, could be potential drug candidates for other pannexin1-related diseases. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected more than 518 million people worldwide, resulting in over 6.25 million coronavirus disease 2019 (COVID-19)-related deaths, according to the COVID-19 case tracker of the Johns Hopkins University ,4. Infla10) were retrieved from published data  or 10-fold of the Cmax was used to ensure a physiologically relevant concentration range [10, Cmax, or Cmax x10 of each drug . All other reagents were obtained from various suppliers at the highest analytical grade possible. Each drug was dissolved in the respective solvent , ribavirin (R9644), lopinavir (SML0491), ritonavir (SML1222), hydroxychloroquine sulphate (HO915), chloroquine diphosphate (C6628), and azithromycin dihydrate (A9834) were supplied by Sigma-Aldrich . In the case of the latter 3 drugs, the salt forms were purchased to avoid limitations due to the low solubility of the drugs. Remdesivir (30354-10) was purchased from Sanbio , and favipiravir (FF29069) was obtained from Biosynth Carbosynth . The peptide mimetic  solvent .2) using Dulbecco’s modified Eagle medium (DMEM) containing GlutamaxTM , supplemented with 10% foetal bovine serum (FBS) , streptomycin, and penicillin . Cells were maintained at 37 °C in an incubator (5% CO2).Transduced Dubca cells stably overexpressing human Panx1 (a 20-fold increase in Panx1 expression in comparison with the Dubca wild-type cell line) were thawed and seeded in a T75 flask (75 cm2). Then, 24 h after seeding, cells were exposed to the drugs dissolved in 200 µL of phenol red-free DMEM , supplemented with GlutamaxTM , 10% FBS , streptomycin, and penicillin  for 24 h or in classic buffer  for 45 min to a predetermined concentration range of each drug -2,5-diphenyltetrazolium bromide (MTT)  assay. Transduced Dubca cells overexpressing human Panx1 were seeded in flat-bottom, 96-well culture plates  at a cell density of 12,000 cells/well  in flat-bottom, 96-well culture plates  and incubated at 37 °C overnight (5% CO2). Drugs were dissolved in classic buffer  and osmotic buffer . Following a washout period of 30 min using a classic buffer, cells were preincubated with the drugs in a defined concentration range (2). Next, 50 µL of each solution was transferred into white, opaque, 96-well plates , and extracellular ATP release was measured using an ATP Bioluminescent Assay Kit, following the manufacturer’s instructions , in a VICTOR3 Multilabel Plate Counter .Transduced Dubca cells overexpressing human Panx1 were seeded at a cell density of 12,000 cells per well  and sonicated for 30 s at 50% pulse. Subsequently, samples were shaken on a rotator for 15 min at 4 °C and centrifuged at 14,000× g for 15 min at 4 °C. Finally, supernatants were transferred to tubes and stored at −80 °C. Protein content was quantified via the PierceTM Bicinchoninic Acid Protein Assay Kit . Next, 20 µg of protein of each sample was loaded onto 10% Mini-PROTEAN® TGX Stain-Free™ precast gels . After electrophoreses and blotting, nitrocellulose membranes  were blocked using a blocking buffer containing 5% w/v non-fat milk  in a Tris-buffered saline solution (20 mM Tris and 135 mM sodium chloride) containing 0.1% v/v Tween-20 . Membranes were incubated overnight at 4 °C, with primary antibodies directed against Panx1  diluted in blocking buffer (1:1000), followed by an additional incubation for 1 h at room temperature with polyclonal goat anti-rabbit secondary antibody (1:1000) . Proteins were detected using enhanced chemiluminescence, and Image Lab 6.1 software was used for densiometric analysis. For semi-quantification purposes, a normalisation method based on total protein loading was used to overcome the drawbacks associated with housekeeping proteins [Following 24 h of exposure to the drugs dissolved in DMEM culture medium , transduproteins . Panx1 sTM Mammalian Total RNA purification Miniprep Kit  and the On-column DNase I digestion Set  according to the manufacturer’s instructions. Isolated RNA was spectrophotometrically measured using a NanoDrop® 2000 Spectrophotometer  to assess RNA yield and purity. A cut-off ratio between 1.8 and 2.1 for the absorption at 260/280 nm was set. Synthesis and amplification of cDNA were performed via an iScriptTM cDNA Synthesis Kit , using a MiniAmp Plus Thermal Cycler . Samples were purified using a GenElute™ PCR Clean-Up Kit  according to the manufacturer’s protocol. Samples were tested on an Applied Biosystems QuantStudio 3 Real-Time PCR system  using TaqMan® Gene Expression Assays . TaqMan probes and primers specific for the target and reference gene used for RT-qPCR analysis are depicted in Cell pellets for RNA extraction were collected following exposure of transduced Dubca cells overexpressing human Panx1 to the drug panel for 24 h . PelletsN = 3). The number of technical replicates (n) is specified in the figure legends. Data are presented as means ± standard deviation. The normal distribution of the data sets was assessed by means of a Shapiro–Wilk test. Depending on the degree of normality, results were analysed with a parametric one-way analysis of variance (ANOVA), followed by a Dunnett’s post hoc test or non-parametric Kruskal–Wallis test, followed by a Dunn’s multiple comparisons test. Significance levels are indicated according to the following symbols: * p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001 and **** p ≤ 0.0001. Statistical analysis was performed in GraphPad® Prism 9 software .All experiments were performed in 3 different passages of transduced Dubca cells overexpressing human Panx1 (