Saccharomyces cerevisiae and in some species of the filamentous fungus Aspergillus. We elaborate on the physiological importance of tightly regulated endocytosis for cellular fitness under dynamic conditions found in nature and highlight how further understanding and engineering of this process is essential to maximize titer, rate and yield (TRY)-values of engineered cell factories in industrial biotechnological processes.Plasma membrane transporters play pivotal roles in the import of nutrients, including sugars, amino acids, nucleobases, carboxylic acids, and metal ions, that surround fungal cells. The selective removal of these transporters by endocytosis is one of the most important regulatory mechanisms that ensures a rapid adaptation of cells to the changing environment . At the heart of this mechanism lies a network of proteins that includes the arrestin‐related trafficking adaptors (ARTs) which link the ubiquitin ligase Rsp5 to nutrient transporters and endocytic factors. Transporter conformational changes, as well as dynamic interactions between its cytosolic termini/loops and with lipids of the plasma membrane, are also critical during the endocytic process. Here, we review the current knowledge and recent findings on the molecular mechanisms involved in nutrient transporter endocytosis, both in the budding yeast  The expSaccharomyces cerevisiae or Aspergillus nidulanstrans-Golgi network (TGN) and not to an early endosome, as in mammalian cells or as in the “traditional” yeast model. From the TGN, cargo is sorted to the prevacuolar endosome (PVE) Endocytic processes have been thoroughly characterized in model fungi such as S. cerevisiae and A. nidulans, ubiquitylation is catalyzed by a major NEDD4In S. cerevisiae, 14 α-arrestins were identified: Ldb19/Art1, Ecm21/Art2, Aly2/Art3, Rod1/Art4, Art5; Aly1/Art6, Rog3/Art7, Csr2/Art8, Rim8/Art9, Art10, Bul1, Bul2, Bul3 and Spo23 A. nidulans, 10 putative ARTs were already described: CreD ARTs are phylogenetically conserved, from yeast to humans. In ARTs are activated and recruited in response to distinct physiological or stress signals, including nutrient excess, limitation or depletion, alkali-, heat- or hypo-osmotic shock, as well as by hydrophilic compounds and other drugs that disturb the PM structure (mostly by increasing its fluidity) or affect nutrient signaling pathways ARTs are functionally redundant: a given α-arrestin can bind one or more transporters, or a particular transporter can be ubiquitylated by multiple ARTs, under distinct stress or environmental conditions S. cerevisiae, protein kinases that were found to directly phosphorylate (inactivate) ARTs are Snf1 (the yeast homologue to the human AMP-activated protein kinase - AMPK), Npr1 (nitrogen permease reactivator 1), Pho85, Yck1/Yck2 (yeast casein kinase 1/2) and Ypk1 (a serine-threonine protein kinase). Examples of protein phosphatases that directly or indirectly control the activity of ARTs include calcineurin, Glc7 ) and Sit4 (a serine-threonine phosphatase)  via apical extension and maintenance of polar growth In A. nidulans, apical PM proteins and nutrient PM transporters were also shown to follow different secretory pathways. Polar cargo traffic to the apical membrane via the conventional Golgi/TGN-dependent secretory/trafficking route, in contrast to de novo synthetized nutrient transporters that traffic to the PM via a recently discovered Golgi-independent route, apparently directly from the ER The continuous process of internalization and recycling of apical proteins ensures the maintenance of the polar growth of the tip S. cerevisiae. We also highlight what is known about these processes in two different species of the filamentous fungus Aspergillus (A. nidulans and A. oryzae). Furthermore, we underline the physiological importance of these processes for adaptation to dynamic cellular environments and illustrate how engineering of these systems has the potential to improve titer, rate and yield (TRY) in industrial biotechnological processes.In the different sections of this review, we highlight the main regulatory circuits required for the dynamic turnover of specific transporters, induced by distinct signals. We will focus on a set of important macro- (carbon (C) and nitrogen (N)) and micro-  nutrient transporters known to be regulated by endocytosis in the budding yeast 2S. cerevisiae, transport of these hexoses is mediated by some members of the Hexose transporter family (Hxt), composed of 20 PM proteins  S. cerevisiae cells, glucose is sensed (via glucose sensors Snf3 and Rgt2) over a wide range of concentrations and acts as the most important regulator, affecting the expression of these transporters at both transcriptional and post-transcriptional levels  from A. oryzaeS. cerevisiae, endocytosis of sugar transporters in Aspergilli remains poorly studied.The genome of 2.1The high-affinity glucose transporters Hxt6 and Hxt7 Specifically, under glucose-limited conditions . When glucose becomes depleted, these transporters are targeted for endocytosis, which requires their ubiquitylation by Rsp5 and the inactivation of the Ras/cAMP-PKA glucose signaling pathway S. cerevisiae Jen1 lactate transporter, a mechanism which, in this case, was found to be associated with alkali stress CRS2 is transcriptionally induced, as Mig1 and Mig2 repressors are inhibited by AMPK kinase Snf1. Upon protein synthesis, Crs2 becomes activated by ubiquitylation and acts as an adaptor protein for Rsp5 to mediate cargo ubiquitylation. In contrast, the presence of glucose inhibits the transcription of CRS2 and promotes Crs2 phosphorylation, its association with Bmh1/Bmh2 (14-3-3) proteins and its deubiquitylation. Importantly, PKA kinase appears to contribute to Crs2 inactivation, revealing an unexpected crosstalk between Snf1/AMPK and PKA pathways The endocytosis of medium- to high-affinity glucose transporters, including Hxt2, Hxt4, Hxt6 and Hxt7, was shown to be triggered when cells are grown for a prolonged time in medium containing lactic acid , as the sole C source, a condition that, the authors claim, also mimics glucose starvation +)-coupled α‐glucoside transporters belonging to the maltose permease subfamily +-coupled transport of glucose +-coupled re-uptake via these α‐glucoside transporters and ultimately result in a net translocation of H+ into the cell S. cerevisiae H+-ATPase Pma1 results in a net energetic expense of ATP and this cycle would ultimately deplete cellular energy Tightly coordinated sugar transporter expression and endocytosis could have evolved in environments where both mono- and disaccharides are present to prevent futile cycling of these nutrients across the PM. For example, disaccharides such as maltose are transported by proton . However, in this case, the phosphorylation state of CreD does not seem to affect transporter endocytosis In 3S. cerevisiae: Ato1/Ady2 Two distinct transport systems for monocarboxylates have been reported in Ato1 (acetate transport ortholog 1), recently reclassified + symporter (SHS) family 379NXX[S/T]HX[S/T]QDXXXT391, located in the TMS-7, seems to be involved both in transport capacity and in substrate affinity Jen1 is a monocarboxylate/proton symporter that belongs to the MFS superfamily, specifically to sialate:HS. cerevisiae on lactic acid Jen1 transporter is endocytosed and degraded in the vacuole in the presence of rich C sources, such as glucose Briefly, in the presence of lactate (0.5% (v/v), pH 5.0), as a sole C source, Jen1 is localized at the cell surface and Rod1/Art4 is phosphorylated (inactive) and bound to 14–3-3 proteins by the action of Snf1 kinase (yeast homologue of AMPK kinase). Upon glucose addition, the phosphatase complex Glc7/Reg1 dephosphorylates Snf1 and Rod1. While Snf1 becomes inactive, de-phosphorylation of Rod1 releases it from the phosphor-dependent binding with 14-3-3 proteins, allowing its ubiquitylation by the E3 ligase Rsp5 Glucose induced endocytosis of Jen1 also seems to involve the Yck1 kinase, directly or indirectly + symport.The endocytosis of Jen1 can also be triggered by other signals, such as the addition of rapamycin, cycloheximide or alkali stress, in a process involving Bul1 arrestin, but independent of Rod1/Art4 In alkali- and cycloheximide-induced Jen1 endocytosis, the requirement of TORC1 is supported by several lines of evidence by Talaia and co-workers It is not completely clear how TORC1 complex stimulates Bul1-mediated endocytosis, in response to physiological signals, although some insight has been gained, over the past few years, into this process. Despite observed similarities between Bul1-dependent endocytosis of Gap1 and Jen1, for the latter, Bul1 remained partly phosphorylated in the absence of Npr1, in the tested physiological conditions (addition of cycloheximide and prolonged growth in lactate) A. nidulans, homologs of S. cerevisiae Ato1 (AcpA and AcpB) and Jen1 (JenA and JenB) transporters were identified In filamentous fungi, little is known about carboxylic acid transporters regulation. In 4S. cerevisiae cells harbor many fungal AA transporters (fAATs) that differ according to substrate specificity, cellular location, regulation and protein fold Escherichia coliAmino acids (AAs) are a major N and/or C source for fungi. S. cerevisiae, 22 PM AA transporters, with known function, were already identified  are clustered in specialized PM domains called membrane compartments containing the arginine permease Can1 (MCCs) Depending on the nutrient status of the cell, transporters can move from MCCs to other regions/domains of the PM 4.1S. cerevisiae, different AA transporters undergo ubiquitin-dependent endocytosis in response to N availability or excess of substrate, including: Gap1, the General AA permease In Gap1, Can1 and Mup1, currently, have the best characterized endocytic mechanism , Fig. 5.4+), Gap1 is ubiquitylated on the K9 and K16 residues, located at the cytosolic N-terminus , Gap1 is localized at the PM. However, upon addition of a favored N source, that can be easily converted into the main AA precursors, like ammonium , converted into glutamate, by the Gdh1 and Gdh3 glutamate dehydrogenases, which is ultimately converted into AAs. The increase in intracellular AAs will then stimulates TORC1 signaling probably via EGO complex  and Put2 (pyrroline carboxylate dehydrogenase) enzymes. The pool of glutamate resulting from proline conversion is less efficient, resulting in a lower concentration of intracellular AAs, being unable to activate TORC1 + influx coupled with AA import. Importantly, TORC1 activation in response to increased cytosolic H+ also requires the ATP-dependent proton pump Pma1 responsible for maintaining H+ gradient across the PM to avoid acidification of the cytoplasm and maintain the proton-motive-force. Replacement of Pma1 by an equivalent and catalytically active plant H+-ATPase in S. cerevisiae does not result in the stimulation of TORC1 in response to increased cytosolic levels of H+. These results suggest that Pma1 may modulate TORC1 via signaling, however, the molecular mechanisms connecting H+ to TORC1 activation remain elusive In the second pathway, Gap1 alters its conformation during substrate transport, transiently shifting from a substrate-free outward-facing (OF) conformation to an inward-facing (IF) conformation. This alteration induces the remodeling of its cytosolic regions, allowing its recognition by Bul1/2 adaptors and subsequent Rsp5-mediated ubiquitylation and degradation Additionally, O'Donnell and colleagues As mentioned above, some transporters of the APC superfamily were shown to be localized at MCC domains. It is the case of Can1 that preferentially localizes inside MCCs in the absence of its substrate. Can1 downregulation, triggered by excess of arginine, also relies on the TORC1-Npr1 cascade and is dependent on the transport activity of Can1 A. Under NCE102 was not sufficient to abolish Can1 MCC clustering The molecular mechanism that triggers transporter partitioning and exit from MCCs is not known yet. The Nce102 protein was proposed to be necessary for sequestration of Can1 into MCCs Endocytic downregulation of Lyp1, Mup1 and Tat2 transporters by excess of substrate  also depends on the TORC1-Npr1 signaling, requiring the α-arrestin Ldb19 The aspartic acid/glutamic acid permease Dip5 is also endocytosed by substrate excess. O’Donnell and colleagues S. cerevisiae is likely related to AA biosynthesis pathways. In this microorganism, all N containing compounds are synthesized using either glutamate or glutamine as N donor S. cerevisiae is cultivated in any other N source, this has first to be converted to ammonia and glutamate, a process that can be limiting for cellular growth S. cerevisiae to achieve the highest growth rates in the presence of multiple N sources Tight regulation of AA uptake via endocytosis in Aspergilli, in particular in A. nidulansThe endocytic regulation of AA transporters in 4+-induced downregulation 4+ addition, these transporters are internalized and targeted for vacuolar degradation Both AgtA and PrnB are subjected to NHA. oryzae, the AoCan1 transporter (homologue of S. cerevisiae Can1) is localized at the PM, mainly in the basal region, when cells grow in arginine- starvation conditions. However this transporter is internalized and targeted to the vacuole for degradation, after a shift to a medium containing excess of arginine (5 mM) S. cerevisiae, the ARTs and signalling complex(es) involved in arginine-induced downregulation of AoCan1 is/are still under investigation.In 4.2+ starvation by a molecular mechanism distinct from the one involved in endocytic degradation triggered by excess of substrate + starvation is a slower process (requiring 3–6 h); it depends on the general amino acid control (GAAC) pathway and requires specifically the Ecm21/Art2 arrestin. Additionaly, while Ldb19 recognizes an acidic patch localized at the N-terminus of the transporters, Ecm21 interacts with a C-terminal acidic degron ECM21 and of genes involved in de novo AAs synthesis, preparing the cells for non-selective nutrient acquisition, if nutrients become accessible again Can1, Mup1, Lyp1 and Tat2 fAATs are also degraded in response to AAs or NH4ARTs gene expression  endocytosis induced by starvation conditions seem to rely on the interplay of several nutrient signaling pathways , culminating in the modulation of pression A; 5B.4.3Cycloheximide, rapamycin or PM structure disrupters, also induce the endocytosis of several AA transporters . Lyp1 do5In Fungi, pyrimidines and purines nucleobases transporters are divided in three distinct families, namely the Nucleobase/Cation Symporter 1 (NCS1) family S. cerevisiae and A. nidulans and the purine transporters UapA, UapC and AzgA from different Aspergillus species.Transporters belonging to these families also undergo endocytic downregulation in response to different environmental signals. Examples include the pyrimidine Fur-like transporters from 5.1S. cerevisiae uracil:cation symporter Fur4, belonging to the NCS1 family, is one of the most extensively-studied transporters at the level of regulation of cellular expression The Moreover, Fur4 was also found to be enriched in MCCs + flux  Internalization of Fur4 is induced by both intracellular and extracellular uracil Particularly, under uracil starvation conditions, Fur4 is stable at the PM and preferentially localizes at MCCs + starvation was also reported, but this process does not depend on Ecm21 arrestin, in contrast to what was described for AATs Fur4 endocytosis triggered by AA/NH4A. nidulans, Fur-like transporters, homologues of S. cerevisiae Fur4 and other members of the NCS1 family, have been extensively studied in relation to function, specificity, cellular expression and evolution In 4+ or excess of their substrates In particular, FurA , FurD (high-affinity uracil transporter) and FurE  show endocytic turnover in response to NH5.2+ symporters: UapA from A. nidulansA. nidulansA. oryzaeA. nidulansPurine transporters that are known to be regulated by endocytosis include the high-affinity purines HS. cerevisiae does not possess protein members of the NAT/NCS2 In contrast to filamentous fungi, + symporter, containing 14 TM segments organized into two domains, a gate domain  and a core domain  UapA is a high-affinity, high-capacity uric acid/xanthine HA. nidulansRsp5 E3 ubiquitin ligase in response to excess of substrate (xanthine or uric acid) or presence of preferred N sources (NH4+ or glutamine). This requires a highly selective ubiquitin acceptor residue (K572) within the C-terminal region of UapA 4+-elicited endocytosis A. nidulans, was proved to be clathrin-dependent but independent of AP2, which is considered to be the standard partner of clathrin The endocytosis of UapA transporter has been extensively characterized in A. nidulans and A. oryzae species, UapC is localized at the cell surface in cells growing in urea as sole N source, but if NH4+ is added to the medium, the transporter is removed from the PM and targeted to the vacuole, after a few minutes UapC is a high-affinity, moderate-capacity, uric acid-xanthine transporter, but also imports hypoxanthine, adenine, and guanine with lower affinity + symporter that contains 14 putative TMSs and cytoplasmic N- and C-termini 4+, however, this transporter undergoes substrate-induced downregulation with the requirement of the ArtA adaptor AzgA is an adenine-hypoxanthine–guanine HAspergillus species, still more studies are required to understand the signaling pathways and proteins involved in this process.The reported studies advanced the knowledge on the endocytosis of purine transporters in 6Metal ions such as copper (Cu), iron (Fe), zinc (Zn) and manganese (Mn) are essential micronutrients for all living organisms. They function as co-factors for different enzymes that participate in crucial biological processes with impact on growth, metabolism and physiology of yeast. Nevertheless, when present at high levels, these micronutrients are toxic  over the PM is established S. cerevisiae, this Δψ results in an intracellular environment that is negatively charged compared to the outside S. cerevisiae. Regarding Aspergilli, to the best of our knowledge, the endocytic regulation of metal ion transporters has not been reported in these organisms.To maintain a constant proton-motive force (pmf) with changes in extracellular pH, in many organisms a charge difference , and a cytosolic C-terminal domain Cu import is mediated by the high-affinity Ctr1 transporter. Studies based on human Ctr1 4), Ctr1 is rapidly endocytosed and delivered to the vacuole for degradation rsp5/npi mutant. The authors propose, instead, that, in the presence of excess copper, Ctr1 is rapidly inhibited upon copper binding, by a structural remodeling mechanism, dependent on the C-terminal cytosolic tail of Ctr1 CTR1 native promoter TEF2 promoter In the presence of Cu-limiting concentrations (medium lacking Cu or containing Cu-chelating compounds), Ctr1 is localized at the PM, however, upon extracellular Cu addition  uptake complex, Fet3-Ftr1, consists of the multicopper oxireductase Fet3 (which oxidizes ferrous Fe2+ to ferric iron Fe3+) and of the ferric iron permease Ftr1, responsible for the subsequent uptake of Fe3+. The protein structure of Ftr1 is predicted to contain 7 TMSs, while the Fet3 only contains 1 TMS. Both proteins contain an extracellular N-terminus and a cytosolic C-terminus, with both C-termini in close proximity In This heterodimeric complex is constitutively internalized when yeast cells grow under intermediate concentrations of Fe (10–100 µM); however, a dynamic population of Fet3-Ftr1 is kept at the PM via endocytic recycling 6.3In Zn shock conditions, Zn homeostasis is reported to depend mostly on the increased expression of the Zrc1 vacuolar Zn transporter but also on the regulated endocytosis of the PM high-affinity Zn transporter Zrt1. This transporter contains 8 predicted TMSs, with both N- and C-termini localized in the extracellular space and a very large intracellular loop connecting the TMS3 with TMS4 6.4Smf1 is a divalent metal ion transporter responsible for the uptake of manganese, but also Fe, Cu and other metals like cadmium, cobalt and nickel. This high-affinity transporter is present at the PM in manganese starvation conditions Toxic levels of Mn (>5 mM) also lead to Smf1 degradation Cellular exposure to toxic cadmium ions (0.1 mM cadmium chloride) also induces Smf1 downregulation. This process is dependent on Smf1 ubiquitylation, which is mediated by the Rsp5 ubiquitin ligase and Crs2 or Ecm21 arrestin proteins. Phosphorylation of Smf1 at the N-terminus is required for binding of Ecm21, although this post-translational modification does not seem to be triggered by Cd stress. After binding, Ecm21 recruits Rsp5 Ub ligase, which leads to Smf1 ubiquitylation, at K33 and K34 residues, and its subsequent targeting for vacuolar degradation Besides the substrate-induced internalization of metal transporters, AA and N starvation conditions have also been shown to trigger endocytosis of Ctr1 and Ftr1 metal transporters 7The concept of the cell factory is central to microbial biotechnology. Cell factories have been designed for the production of both bulk and high value products, food processing, and the production of pharmaceuticals. In the past decades, different strategies have been progressively developed to improve the design of these cell factories   but also from an applied perspective, considering their potential for the biotechnological field.There is surely much untapped potential for the continued engineering of post-translational regulation of nutrient transporters and a strong motivation for the characterization of the proteins involved.8Endocytosis has a physiological role in the homeostasis of nutrients, and the mechanisms underpinning the reciprocal regulation of endocytosis and metabolism are now being unveiled.The multitude of nutrient transporters known to be regulated by endocytosis highlights the critical importance of this regulation for the adaptation of cells to their environment and, consequently for their existence.Endocytosis is regulated on several levels, including posttranslational modifications, recruitment of adaptor proteins, substrate transport, conformational changes, dynamic interaction between cytosolic termini and/or their loops, as well as the proper interaction and organization of the transporters within specific domains  of the PM lipid bilayer . The manThe dysregulation of genes involved in the endocytic pathway is associated with a wide array of growth defects, connected to the loss of ability to quickly respond and adapt to nutrient fluctuations or stresses.Further investigations are necessary to clarify the roles of each regulatory circuit and how they communicate with each other and possibly with yet unidentified regulators. The continued study of the function and regulation of PM eukaryotic nutrient transporters is invaluable for their broad application as targets in biotechnological and medical fields, including their use as potential drug delivery systems in future therapies.Cláudia Barata-Antunes: Writing - original draft, Writing - review & editing, Conceptualization, Visualization. Rosana Alves: Writing - original draft, Writing - review & editing, Conceptualization. Gabriel Talaia: Writing - original draft. Margarida Casal: Writing - review & editing. Hernâni Gerós: Writing - review & editing. Robert Mans: Writing - original draft, Writing - review & editing, Conceptualization. Sandra Paiva: Writing - original draft, Writing - review & editing, Conceptualization, Supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Drosophila SLC22s—including transporters homologous to those previously identified by us in mammals such as the “Fly-Like Putative Transporters” FLIPT1 (SLC22A15) and FLIPT2 (SLC22A16)—have shown modest protection against oxidative stress. However, these fly transporters tend to be broadly expressed, and it is unclear if there is an organ in which their expression is critical. Using two tissue-selective knockdown strategies, we were able to demonstrate much greater and longer protection from oxidative stress compared to previous whole fly knockdowns as well as both parent and WT strains . Expression in the Malpighian tubule and likely other tissues as well  appear critical for managing oxidative stress. These four Drosophila SLC22 genes are similar to human SLC22 transporters , SLC22A13, SLC22A14)—many of which are highly expressed in the kidney. Consistent with the Remote Sensing and Signaling Theory, this indicates an important in vivo role in the oxidative stress response for multiple SLC22 transporters within the fly renal system, perhaps through interaction with SLC22 counterparts in non-renal tissues. We also note that many of the human relatives are well-known drug transporters. Our work not only indicates the importance of SLC22 transporters in the fly renal system but also sets the stage for in vivo studies by examining their role in mammalian oxidative stress and organ crosstalk.Several SLC22 transporters in the human kidney and other tissues are thought to regulate endogenous small antioxidant molecules such as uric acid, ergothioneine, carnitine, and carnitine derivatives. These transporters include those from the organic anion transporter (OAT), OCTN/OCTN-related, and organic cation transporter (OCT) subgroups. In mammals, it has been difficult to show a clear in vivo role for these transporters during oxidative stress. Ubiquitous knockdowns of related  Solute carrier proteins (SLCs) are membrane-bound transporters that manage a wide array of substrates such as antioxidants, signaling molecules, metabolites, hormones, nutrients, and neurotransmitters . ThroughThe connections between SLC22 transporters, other SLC transporters, ABC transporters, and DMEs appear to be particularly important in the regulation of the gut-liver-kidney (GLK) axis where inter-organ crosstalk occurs via transporters and other genes, to mediate signaling, cross-tissue metabolism and ultimately maintain homeostasis, such as after oxidative injury ,7. Drug Drosophila melanogaster (fruit fly) has emerged as a viable model organism for the investigation of ADME genes, due to the similarities between human and fruit fly drug metabolism and excretion physiology [While knockout mouse models have been useful for understanding the in vivo biology of SLC22 transporters, the overt phenotypes have for the most part been modest, and only after detailed biochemical and omics analyses, or additional experimental manipulations, have clear endogenous functional abnormalities been revealed ,15,16,17ysiology ,19,20. Tysiology . Due to ysiology ,24,25,26D. melanogaster putative SLC22 orthologs  display a modest increase in resistance to paraquat-induced oxidative stress when knocked down ubiquitously [Previous work has shown that four uitously . In suchuitously . The impDrosophila equivalent of the “gut-liver-kidney” (GLK) axis—the gut, fat body, and the Malpighian tubule [To evaluate this hypothesis, we created two sets of tissue-specific RNAi knockdowns using two different tissue specific drivers c42-Gal4 (Driver A) and c591-Gal4 (Driver B). Driver A has been employed to model and investigate the fly renal system ,31,32. Dn tubule ,34,35,36Thus, if both of the two drivers result in oxidative-stress resistance for one or more of the four aforementioned SLC22 transporters, it supports the view that SLC22 transporter expression in the Malpighian tubule is critical for protection from oxidative stress. As shown, we validate this for several of the SLC22 transporters studied, and the tissue-specific knockdowns were found to provide greater protection than previously reported for the whole fly. The results strongly support the view that SLC22 transporters in the Malpighian tubule are crucial for survival in the face of oxidant stress—though for some transporters, other tissues are likely to also be involved. The study also raises the possibility that the in vivo tissue-specific regulation of oxidative stress response requires one or more fly renal SLC22s, working together with non-renal transporters in the regulation of the redox state ,37,38.It has been shown that ADME  genes not only handle many drugs, but also many endogenous molecules. These include a broad range of metabolites and signaling molecules ,39,40. TDrosophila transporter CG6006 is deemed equally orthologous to SLC22A1 (OCT1), A2 (OCT2), A3 (OCT3), A6 (OAT1), A7 (OAT2), A8 (OAT3), A11 (OAT4), A12 (URAT1), A13, and A14. Although CG6006 does not have a single mammalian SLC22 counterpart, the 10 associated human SLC22s are all believed to transport some sort of antioxidant-like molecule as shown in Recently, a reclassification of the SLC22 family of OATs, OCTs, and OCTNs has resulted in their classification into eight subgroups based on sequence, evolutionary history, and functionality . These hAs described in the Remote Sensing and Signaling Theory, multispecific, oligospecific and monospecific SLC and ABC transporters maintain homeostasis through inter-organ and inter-organismal communication mediated by antioxidants, metabolites, signaling molecules, and gut microbiome products ,4. The rDrosophila transporters may form a putative GLK  axis. However, the broad expression pattern of these genes  to oxidative stress at these time points, when compared to control lines [p < 0.05) when compared to the four controls (Paraquat (PQ) is commonly used on fruit flies to determine if an altered resistance to oxidative stress exists ,52,53,54il 168 h . Moreoveol lines . In thiscontrols . In addicontrols , demonstp < 0.01, CG4630: p < 0.01, CG16727: p < 0.0001 and CG6006: p < 0.01) when compared to the four other control lines . Further statistical analysis shows that there are at least two individual time points for each F1 group, where F1-knockdown survival demonstrates a greater ability to survive under oxidative stress and is statistically significant  when compared to that of the four controls. This information can be found in p < 0.05, 48H: p < 0.05, 60H: p < 0.05) [p < 0.05, 36H: p < 0.05, 48H: p < 0.05, 60H: p < 0.05, 72H: p < 0.05, and 84H: p < 0.05), and Driver B yielded four  , a carnitine and ergothioneine (EGT) transporter. Classification of all SLC22s into subgroups places the closely related transporters A16 (FLIPT2) and A15 (FLIPT1) into the OCTN/OCTN-related subgroup. Previously, the orphaned transporter A15 had no confirmed ligands but both a phylogenetic and transporter assay analysis reveals that A15 shares A16′s affinity for EGT and carnitine, while also transporting carnosine ,59. Carn < 0.05) . However < 0.05) . For Dripa stage . Moreovep < 0.05) [p < 0.0001. Human A7 is known to be expressed within the liver and kidney, where it transports organic anions and zwitterions, including antioxidants such as uric acid [Drosophila Malpighian tubule led to an increased tolerance to ROSs such as hydroxyl radicals and hydrogen peroxide. We found that CG16727 is most orthologous to human SLC22A7 (OAT2) which forms its own SLC22 subgroup OATS2 in the eight subgroup classification of SLC22 [ric acid ,63. In hDIOPT identifies CG6006 as being equally orthologous to human SLC22A1 (OCT1), A2 (OCT2), A3 (OCT3), A6 (OAT1), A7 (OAT2/NLT), A8 (OAT3), A11 (OAT4), A12 (URAT1/Rst), A13, and A14 . FurtherDrosophila intestine system [p < 0.05) [p < 0.05). Likewise, driver B-specific knockdowns had markedly enhanced survival in the face of oxidative stress  . In verye stress . Driver Drosophila brain and/or Malpighian tubule. Given the expression patterns  [p < 0.05) and Driver B yielded two (36H and 72H: p < 0.05) , a member of the human OCT subgroup that is most expressed in the brain, liver, and kidney . Human S < 0.05) . On the  < 0.05) . DespiteDrosophila and mammalian models, the fruit fly can be a useful tool in the characterization of understudied transporters. By using Drivers A and B to pinpoint knockdown into specific tissues, we were able to achieve F1 generations of RNAi knockdowns with a much greater resistance to oxidative stress when compared to previous ubiquitous RNAi knockdown results. Although the results strongly support the view that fly renal system SLC22 transporters play a key role in the response to oxidative stress, we note that the tissue-selective drivers we used affected non-renal tissues as well. Having obtained a more robust phenotype, using tissue-selective drivers, the next step might be to evaluate whether levels of antioxidant metabolites are altered in one or more of the four knockdowns. It would also be useful to evaluate changes in gene expression resulting from the knockdown. While the assumption is that the phenotype might be due to altered transport of one or more antioxidants, it is possible that this is not the case for all four of the knockdowns. For example, rather than the altered transport of a specific antioxidant in one or more of the knockdowns, there may be multiple downstream effects on metabolism due to changes in expression of key enzymes or other genes which, collectively, could result in protection from oxidative stress. In other words, while it is attractive to explain the mechanism underlying the phenotype in terms of altered antioxidant transport, multiple possibilities may need to be explored.To summarize, given certain similarities in physiology between Our study sets the stage for a better understanding of the role of the human and murine relatives of these four fly transporters in protection from oxidative stress. The Remote Sensing and Signaling Theory proposes that SLC22 transporters (as well as other “drug” transporter families) work together to promote the homeostasis of endogenous small molecules, by mediating communication between organs and organisms as well as at multiple scales, including cells and organelles ,4. The tDrosophila melanogaster data were acquired following the data collection procedure published previously [SLC22 human and eviously ,27. Ortheviously . Human gDrosophila melanogaster Gal4 and RNAi lines were purchased from the Bloomington Drosophila Stock Center  [GAL4/UAS drivers c42-Gal4 (BDSC stock #30835) and c591-Gal4 (BDSC stock #30843) were used to downregulate expression of the following lines: CG6126, CG4630, CG16727, and CG6006 in selected tissues as noted in the Results and Discussion. Driver A  altered expression in the ellipsoid body, pars intercerebralis, fan shaped neurons, large field neurons, and Malpighian tubule. Driver B  altered expression in the trachea, salivary gland, dorsal head, antenna anlagen, gut, fat body, and Malpighian tubule. Male RNAi Drosophila SLC22 lines were crossed with virgin female tissue-specific Gal4 drivers to express the downregulation of listed transporters within an F1 generation [Drosophila expression data was collected from FlyAtlas2, the Drosophila gene expression atlas [https://biorender.com (accessed on 1 November 2021).IN, USA) . Wild-tyon atlas . FlyAtlaN,N′-dimethyl-4,4′-bipyridinium dichloride, Sigma) (PubChem CID: 15938) in combination with 10% sucrose with fresh PQ added every 24 h. Survival was recorded every 12 h. Flies were never exposed to non-room-temperature conditions. The statistical analysis was performed using GraphPad Prism and Microsoft Excel.All flies were observed throughout their life cycle to account for any unwanted developmental abnormalities. F1 male offspring were aged two to seven days before being exposed to paraquat for resistance to oxidative stress. F1, Gal4 parent, RNAi parent, YW wildtype and CS wildtype were all exposed to PQ simultaneously. Three replicates of each line were tested. Flies were fed on 3 mm Whatmann paper soaked with 10 mM paraquat (


With its expanding list of approved and emerging therapeutic indications, NSCLC is the exemplar tumor type requiring upfront assessment of several biomarkers to guide clinical management. Next-generation sequencing allows identification of different types of molecular alterations, each with specific analytical challenges. Library preparation using parallel DNA and RNA workflows can overcome most of them, but it increases complexity of laboratory operations, turnaround time, and costs. We describe the performance characteristics of a 15-gene RNA panel on the basis of anchored multiplex polymerase chain reaction for combined detection of clinically relevant oncogenic fusion transcripts and hotspot small variants.Formalin-fixed, paraffin-embedded NSCLC clinical samples (N = 58) were used along cell lines and commercial controls to validate the assay’s analytical performance, followed by an exploratory prospective cohort (N = 87).The raw assay sensitivity for hotspot mutations and fusions was 83% and 93%, respectively, reaching 100% after filtering for key assay metrics. Those include quantity and quality of input of nucleic acid and sequencing metric from primers on housekeeping genes included in the assay. In the prospective cohort, driver alterations were identified in most cases (≥58%).This ultrafocused RNA–next-generation sequencing assay offers an advantageous option with single unified workflow for simultaneous detection of clinically relevant hotspot mutations and fusions in NSCLC, focusing on actionable gene targets. EGFR, ALK, ROS1, BRAF, RET, ERBB2, MET, KRAS, and NTRKs) and consist of various single nucleotide variants (SNVs), insertions and deletions (indels), copy number variations, and oncogenic fusions and isoforms.,Lung cancer has the highest cancer-related mortality rate among solid tumors, and NSCLC is diagnosed at advanced stages (III–IV) in approximately 75% of patients.,Although next-generation sequencing (NGS) is increasingly used to address multigene testing in routine molecular diagnosis, the capacity to capture every type of genomic alteration using a single chemistry remains challenging. NGS assays within reach of hospital-based laboratories are mostly amplicon based and require managing DNA and RNA workflows separately to ensure proper detection of mutations and fusions, respectively. Hybrid-capture panels offer the possibility to deal with a single analyte, but even large comprehensive panels are subject to miss fusions and alternative splicing events, as intron length and repetitive regions are factors complicating the capture of relevant genomic regions.EGFR, ALK, and ROS1 for patients with NSCLC persists an unsatisfied challenge in several countries and community settings.,Currently, the availability and funding of NGS for standard diagnostic evaluation remain poorly described,Here, we present the evaluation of the performance characteristics of a custom-designed anchored, multiplexed, polymerase chain reaction (PCR)-based (AMP) RNA-NGS targeted panel allowing simultaneous detection of clinically relevant hotspot mutations and fusions in NSCLC.ERBB2 (fusions and hotspot mutations in exons 8 and 20). The assay allows detection of both known and unknown fusion partners and relevant actionable SNV/indel from a single workflow on the basis of RNA sequencing (RNA-seq), using total nucleic acids (TNAs) extracted from formalin-fixed, paraffin-embedded (FFPE) tissue as a starting material.The assay used is the ArcherDx Fusion Plex Lung  with custom additional targets . It consALK, ROS1, RET, or NTRK1 probes , EGFR RGQ PCR kit ran on a Rotor-Gene Q , or digital droplet PCR BRAF V600 or KRAS G12/G13 screening kits performed on a QX200 system . A subset of the case was also sequenced using QIAseq-targeted DNA scan technology . Hematoxylin and eosin-stained slides were reviewed for tumor content evaluation by a pathologist before extraction. Decalcified specimens were excluded from this set. Samples selected within the prospective cohort were tested by single-gene assay, including concomitant EGFR RGQ and IHC for ALK with clone 5A4  and ROS1 with clone D4D6  performed on a Dako Autostainer (Agilent), followed by FISH in equivocal cases.All samples were from patients who underwent interventional bronchoscopy or surgical resection for NSCLC followed by biomarker testing at the request of referring physician at the Institut Universitaire de Cardiologie et de Pneumologie de Québec—Université Laval . The study was approved by the Institut Universitaire de Cardiologie et de Pneumologie de Québec—Université Laval ethics committee (#2019-3183-21730). Representative FFPE specimens were selected for this validation set, including cytology cell blocks, small biopsies, and surgical resections. All samples had known alterations characterized previously by another established molecular method. Those consisted of fluorescence in situ hybridization (FISH) with ELM4-ALK Fusion-A549, LC-2/ad, and U118) or Leibniz Institute . Cell lines were used either alone or admixed with each other in formalin-fixed and paraffin-embedded cell blocks prepared similarly to clinical cytology specimens.Reference material included commercial FFPE tumor fusion control  in addition to a collection of cell lines from ATCC . Samples were quantified using the Qubit RNA HS Assay Kit on a Qubit 3.0 fluorometer . When possible, the maximum input material recommended by the manufacturer was used (250 ng TNA on the basis of RNA quantification), but samples under this threshold were also included to explore this factor on the assay’s performance and sensitivity.Libraries were prepared using the Archer FusionPlex reagent kit for Illumina  according to the manufacturer’s instructions. Briefly, cDNA is synthesized from the RNA using random priming and then undergoes end repair, dA tailing, and adapter ligation with Illumina molecular barcode adapters, which allow for read deduplication and quantitative analysis. Magnetic beads  are used for cleanups after every enzymatic step. The ligated fragments are processed through two rounds of PCR amplification using two sets of GSP and universal primers complementary to the Illumina adapters. The GSP2 pool in PCR2 consists of a nested pool designed 3’ downstream of GSP1. After completion of the two PCR steps, libraries are quantified using NEBNext Library Quant Kit for Illumina  and run on a Bioanalyzer high-sensitivity DNA chip . They are pooled at equimolar concentration and sequenced on Illumina MiniSeq at 2 × 150 base pair. The FASTQ files generated by the bcl2fastq conversion software  are then uploaded into the Archer data analysis pipeline .CHMP2A, GPI, RAB7A, and VCP). The Fusion QC score represents the average number of RNA unique start sites calculated per control GSP2. It must be superior to 10 to support enough RNA quality on the assay targets. The Variations QC score represents the average number of DNA or ambiguous unique start sites calculated per GSP2 across the entire panel. Other raw metrics that were monitored included percentage of RNA reads. A combined score integrating preanalytical factors and assay quality metrics on the basis of the observations herein and other unpublished familiarization data was developed to increase the likelihood of assay success: it integrates factors of minimal TNA input (≥100 ng), sample age of FFPE specimen (<4 y old), Pre-Seq Ct score (<25), and Fusion QC score (≥10).In this assay, the quality of first-strand cDNA synthesis is used as an indicator of RNA quality before library preparation and sequencing. It is evaluated using the Archer PreSeq RNA quality control (QC) Assay (Pre-Seq Ct score), a quantitative PCR-based method. Another key assay metrics is provided on the basis of a pool of GSPs included in the design targeting four control gene transcripts (https://software.broadinstitute.org/software/igv/).The criteria for calling a positive fusion were five or more unique supporting RNA reads and three or more unique starting sites (SSs) among the reads and an in-frame sequence. The minimal criteria for calling a positive hotspot variant were 5% or more variant allele frequency (VAF), five or more alternate observation, three or more unique start site supporting the variant, and 100 or more read depth; all known hotspot variants were reviewed manually on the Archer Analysis portal and on bam files using Integrative Genomics Viewer .Correlation, contingency analysis with Fisher’s exact test, chi-square test, and A.The performance of this customized Archer Fusion Plex Lung panel was evaluated on commercial reference material, characterized cell lines, and 58 individual FFPE NSCLC samples with known SNVs, indels, rearrangements, or splice variant relevant to NSCLC and covered by the panel design . Most NSFigure 1B). The sensitivity for fusion detection and SNV/indel reached 94% and 95% when this metrics was met but fell to 25% and 86% when failed, respectively (B). Samples with hotspot mutations in EGFR, KRAS, or BRAF were used as negative controls for fusions and vice versa, to capitalize on known mutual exclusivity of key oncogenic drivers in NSCLC; the specificity of the assay was 100% for both types of alterations. In complement, a commercial control containing a mixture of characterized alterations allowed testing the assay’s capacity to detect a wider variety of gene fusion partners, targets, and oncogenic isoforms not already covered with validation samples (C).As expected, the accuracy of the assay for fusion or SNV/indel detection on archival specimens was affected by the Fusion QC, the assay’s main sequencing metrics B. The seectively B. Sample samples C.Concordantly, a Pre-Seq Ct score above 25 was associated with lower proportion of samples revealing success of Fusion QC and ability to detect the targeted alteration . SupplemB for both types of alterations, the distribution of unique reads per GSP2 primers was relatively balanced between exons intended to cover hotspot SNVs/indels, fusions, and control genes (A), being slightly higher when averaged on exons targeting mutations than fusions . Supplementary Figure 2B and C reveals the range of read depth and VAF observed for targeted hotspot SNVs and indels. Known SNVs were detected with a mean read depth of 754 (SD = 1217) and mean VAF of 41 (SD = 30), whereas known indels had mean read depth of 1083 (SD = 1984) and mean VAF of 34 (SD = 23), not significantly different . For a subset of cases where VAF was available from another method , there was a fair correlation (R2 = 0.86) with the VAF provided by the FusionPlex Lung assay (D). The samples with VAF estimates larger than 5% between methods had a significantly lower proportion of DNA reads for supporting the call on the corresponding GSP2 when compared with samples with a less than 5% difference  (E).Although a high percentage of unique RNA reads on the target region was constantly observed using this assay , efficiency of primers specific to target exons for clinically relevant hotspot SNVs/indels and fusions was also evaluated. As expected from the similar accuracy rate inferred from Figure 1ol genes A, being ng assay D. The sactively) E.To determine the limit of detection of the assay, we used a mixture of known fusion and SNV-characterized cell lines fixed in formalin and processed in paraffin cell blocks as routine cytology specimens in our laboratory. First, libraries were prepared with decremental input of TNA . FusionsLibraries were also prepared by serial dilutions of samples along with previously tested sample TNA negative for the targeted known alterations  and 4. NTo determine the reproducibility of this assay, we analyzed fusion- and SNV-positive samples as triplicate in the same run (intrarun reproducibility) and in three to five different runs (interrun reproducibility). The concordance of both fusion and SNV detection was perfect across the replicates. There was also high reproducibility in the numbers of SS or unique reads for fusion transcripts and for the coverage, VAF, and numbers of reads supporting the variant for SNVs/indels .B). The first set included 53 samples negative for EGFR-activating mutations and ALK or ROS1 fusions, as evaluated by single-gene testing (SGT), enriched in tumors that underwent ROS1 FISH on the basis of equivocal IHC staining for ROS1 . Another set consisted of 29 samples reflex-tested by NGS in parallel to SGT. These were completed with five additional samples with known EGFR, ALK, or ROS1 alterations. Driver alterations in key NSCLC genes were found in 31 of 53 (58%) and 23 of 29 (79%) unknown cohorts, respectively . All EGFR, ALK, and ROS1 results obtained with SGT were concordant, with one case of ALK fusion revealing a concomitant KRAS mutation additionally. As expected from the known prevalence in NSCLC, hotspot driver mutations in KRAS were the most often found, where p.G12C represented 44% of KRAS variants . Other potentially actionable variants outside of EGFR, ALK, and ROS1 included BRAF p.V600E (n = 1/3 BRAF mutations), ERBB2 exon 20 insertion (n = 1), or S310F (n = 1), and MET exon 14 skipping alteration (n = 1). Altogether, potentially actionable variants were found in 26 of 82 unknown cases (32%), namely 14 of 29 (48%) when done reflex in parallel to SGT and 12 of 53 (23%) in the cohort of cases negative for ALK, EGFR, and ROS1 alterations.The validity and added value of using a highly focused panel for identification of clinically relevant driver alterations were further evaluated on prospective routine NSCLC samples . Those cFig. 2This study reports the analytical performance of a 15-gene customized version of an NGS assay using AMP technology and focused on NSCLC targets. This ultratargeted assay on the basis of RNA is intended for first-line simultaneous detection of clinically actionable oncogenic fusions/isoforms and hotspot SNVs/indels in key genes for NSCLC, with a single workflow for library preparation. The data presented establish the performance of the assay across different types of clinical FFPE specimens, providing that key metrics regarding RNA quality are monitored. More specifically, the maximum recommended TNA input (250 ng) should be used when possible (not less than 100 ng). In addition, use of complementary testing is warranted when interpreting a negative result if RNA quality, on the basis of the PreSeq Ct score and the sequencing Fusion QC assay, is under optimal threshold values.The data obtained during the course of this technical validation reveal some characteristics relevant to RNA-based NGS assays. First, RNA quality and quantity are crucial to obtain success as reflected by the importance of the Fusion QC metrics in the Archer’s design. Although not absolutely precluding detection of fusions or mutations, failure to meet this metrics is associated with a much lower probability to detect an alteration when present. Consequently, samples negative for alterations in the context of suboptimal metrics or coverage should be considered uninformative and undergo additional testing with complementary method, similarly to a DNA-based NGS assay. Interestingly, the use of TNA as starting input provides an additional indirect appreciation of RNA quality, as large differences in the number and percent of reads obtained from RNA and DNA can be identified in a sample with low RNA quality for each target.Second, the validation of fusion detection with RNA-NGS assays is challenging owing to variable expression levels found in tumor samples. Large differences in the level of expression of fusion transcripts affect the ability to determine the limit of detection as linearly as SNV/indels in DNA mutation testing. This factor can attenuate the impact of tumor content and concentration, limiting the ability to evaluate some validation parameters. The dilution experiments found here highlight the importance of using fusion samples with variable starting expression levels in the validation process of such assays. Similarly, the detection of fusions despite failure of the main RNA quality parameter (Fusion QC) suggests an impact of elevated expression.EGFR amplification, could presumably affect the VAF observed on an RNA-based assay. Nevertheless, this cannot be clearly highlighted as the assay is not intended to use the subset of DNA reads captured to provide amplification data. Furthermore, however, as RNA, DNA, and ambiguous reads captured for every GSP2 are used to call a variant, we noted that the RNA-based estimated of VAF seemed to be closer to the DNA-based one when the proportion of DNA reads, or DNA/RNA reads ratio, was higher.Third, despite the good correlation observed on a subset of samples matched with DNA-based estimate of VAF for SNV/indels, some samples exhibited larger differences. Factors affecting gene expression, per example owing to ,,,The performance of AMP-PCR chemistry in solid tumors has been abundantly described for fusion detection,Thus, some factors must be reminded considering the good concordance observed for SNV/indel detection using the current assay. First, although the design covers several exons, the assay is intended to detect only selected hotspot oncogenic driver or resistance mutations. The capacity to detect unique mutations outside of the selected genomic regions interrogated has not been evaluated and is presumably lower than a DNA-based assay given the expected variation on sequencing depth and coverage across the genome for RNA-seq data. This was observed here indirectly with the large spread of read depth on target exons, although the use of molecular indexes in the design likely compensates this factor, abrogating PCR artifacts and allowing detection of SNV/indels at lower coverage (<500 reads). Although similar GSP2 primer coverage was observed across the targeted exons for fusions versus SNVs/indel, suggesting good primer performance overall, only restricted gene regions limited to driver mutations are analyzed. On the samples failing the Fusion QC but correctly calling SNV/indels (n = 6), the proportion of DNA reads was high (72% DNA reads in contrast to 13% in RNA reads on average) regardless of read depth, suggesting DNA reads can partly secure such call when RNA quality is poor. On samples where the SNV/indel were missed (n = 3), one had lack of coverage of the hotspot region (<20 reads), one had no RNA or DNA reads (only ambiguous reads), and another was older than 5 years old (data not shown). Apart from these observations, data from single-cell RNA-seq support that oncogenic or actionable SNVs/indels are detectable in RNA-seq from lung adenocarcinoma, notably, but the selection of proper bioinformatics alignment tools for indel detection is essential.MET exon 14 skipping mutations when using DNA-based NGS only is emerging.,The NGS panel used here offers several advantages regarding its implementation in a clinical laboratory over its ability to replace multiple sequential or parallel PCR-based or in situ assays on the same material. The limited design focuses on the detection of clinically actionable set of gene alterations in NSCLC within a million reads per sample. When combined with programmed death-ligand 1 IHC, it allows to cover all oncogenes either with Food and Drug Administration–approved therapy or recommended by the National Comprehensive Cancer Network guidelines,Among other advantages of this assay, factors simplifying laboratory workflow are of consideration. These include the manufacturer’s user-friendly design and reagent configuration  and simplifying handling and training of laboratory technologists for a high-complexity wet-bench procedure. In addition, avoiding the simultaneous or sequential preparation of libraries from DNA and RNA increases overall laboratory handling efficiency and favors delivery of results within a shorter turnaround time. Although NGS reimbursement is not warranted in several jurisdictions, detection of the drug-related relevant hotspot driver mutations and fusions in a single workflow offers an interesting compromise to amalgam appropriate genomic testing within restrictions present in public health systems. Nevertheless, this strategy does not resolve the complexity generated by the companion diagnostic landscape and how some authorities will cope with results coming from assays outside of selected companion assays.EGFR, ALK, and ROS1) in samples not meeting the assay metrics. Another unaddressed question by this validation set is the potential of this assay to detect gene amplification, which would need further evaluation of expression data generated within the assay. Despite growing interest, MET and EGFR amplification detection in NSCLC is mostly found in acquired resistance setting,Some of the limitations of this assay, outside its low number of targets, are intrinsically related to the use of RNA as the main analyte. Although not evident from the current limited set including old retrospective samples with rare alterations, RNA is a substrate more labile and sensitive to preanalytical factors than DNA in clinical samples.EGFR/ALK/ROS1-negative cases is not surprising owing to the design focused on highly relevant targets in NSCLC. A rate of driver mutations at approximately 60% has been identified when using DNA/RNA-NGS panel in NSCLC,Overall technical considerations for which most considerations were directed in this study, the small prospective cohort offers an interesting perspective on the clinical use of this panel. The high percentage of driver alterations identified in both reflex-tested and In conclusion, the data presented in this study describe the performance characteristics of an ultrafocused AMP NGS assay allowing simultaneous detection of SNVs, indels, fusions, and oncogenic isoforms from a single unified RNA-based workflow. It provides important insight in the feasibility to detect hotspot mutations from RNA in a clinical setting. The prospective set of tumors found here does not provide a true incidence of driver alterations in our population owing to the sampling bias. Nevertheless, it provides insight on the high success rate and potential for added value in identification of driver and actionable alterations in NSCLC, even when using a small panel focused on genes with high clinical relevance. The assay used has the potential to replace multiple tests in a unified, single workflow and to provide genomic profiling in a timely manner for patients with NSCLC, especially in administrative settings where molecular testing relevance is tied on therapeutic drug approval scheme.Patrice Desmeules: Conceptualization, Funding acquisition, Investigation, Writing - original draft, review, and editing.Dominique K. Boudreau: Data curation, Formal analysis, Methodology, Writing - review and editing.Nathalie Bastien: Data curation, Methodology, Resources, Writing - review and editing.Marie-Chloé Boulanger: Data curation, Formal analysis, Resources, Project administration.Yohan Bossé, Philippe Joubert, Christian Couture: Writing - review and editing.


Science. With the recent FDA approval of four siRNA-based drugs, the potential of RNA therapeutics to become the third milestone in pharmaceutical drug development has become a reality. However, the field of RNA interference (RNAi) therapeutics still faces challenges such as specificity in targeting, intracellular processing, and endosome trapping after targeted delivery. Dicer-substrate siRNAs included onto RNA nanoparticles may be able to overcome these challenges. Here, we show that pRNA-based nanoparticles can be designed to efficiently harbor the Dicer-substrate siRNAs in vitro and in vivo to the cytosol of tumor cells and release the siRNA. The structure optimization and chemical modification for controlled release of Dicer-substrate siRNAs in tumor cells were also evaluated through molecular beacon analysis. Studies on the length requirement of the overhanging siRNA revealed that at least 23 nucleotides at the dweller’s arm were needed for dicer processing. The above sequence parameters and structure optimization were confirmed in recent studies demonstrating the release of functional Survivin siRNA from the pRNA-based nanoparticles for cancer inhibition in non-small-cell lung, breast, and prostate cancer animal models.Small interfering RNA (siRNA) for silencing genes and treating disease has been a dream since ranking as a top Breakthrough of the Year in 2002 by   siRNA for silencing genes and treating diseases has been a popular dream but not fully realized. Peixuan Guo and colleagues report the rational design of siRNA to RNA nanoparticles for efficient delivery while avoiding endosomes, thus providing guidelines for siRNA design on RNA nanoparticles to overcome previous roadblocks. Science ranked this technology as a top ten Breakthrough of the Year in 2002.C. elegans and in mammals in 2001.,,RNA interference (RNAi) is a post-transcriptional gene regulation pathway used by different classes of small RNAs.siRNA is synthetically created, short RNA with matching sequence to its corresponding messenger RNA (mRNA). When internalized into cells, siRNA is incorporated into the RNA-induced silencing complex (RISC), like microRNA (miRNA).in vivo, thus demanding stability against nucleases. To increase nuclease stability and reduce immunogenicity, nucleotide sugar modifications can be used ; however, Dicer-substrate siRNAs do not tolerate sugar modifications at each position, since these modifications can interfere with Dicer recognition. Therefore, careful consideration must be taken to increase siRNA stability. Researchers have created nuclease-stable siRNA while remaining sensitive to Dicer by only modifying key nucleotide sites against nucleases and reducing off-target effects.For RNA to be considered for clinical applications it must be stable ,,,The Dicer enzyme belongs to the RNase III family, which can cleave long double-strand RNAs (dsRNAs) into small RNAs.in vivo gene knockdown, another key is efficient delivery of RNAi into cancer cells. Although many nanocarriers have been developed, our studies,,in vivo applications.,,,,,,,,in vivo.,,,To achieve RNA nanotechnology has gained significant interest in the development of novel nanostructures.,LUC2) siRNA was designed to be incorporated into RNA nanoparticles. To demonstrate the Dicer processing, a molecular beacon was designed and constructed to monitor the activity. Our findings suggest that integration of length, nucleotide components, structure, and RNA nanotechnology into the designs of RNAi achieves efficient silencing of targeted genes in an in vivo cancer therapy setting. As such, our findings provide design parameters for siRNA incorporation into RNA nanoparticles for efficient gene silencing.In the present study we examine whether Dicer-substrate siRNA can be incorporated onto pRNA-based RNA nanoparticles and their required design parameters to retain Dicer processing. We integrate and test factors affecting Dicer processing, which directed us to design siRNA-loaded RNA nanoparticles that can function as a Dicer substrate to generate a robust gene regulation tool. Dicer-substrate siRNA is used in these studies because of the ease of inclusion into RNA nanoparticles by simple helical extension, increased efficacy in gene knockdown over short siRNA, and reduced immune responses over short siRNA.in vitro transcription by T7 RNA polymerase and transfected into KB cells. The gene-silencing efficiency of pRNA-Lamin25 was first demonstrated by qRT-PCR . Lamin A/C was chosen as a model gene to demonstrate the specific cleavage of mRNA by a 5′ rapid amplification of cDNA ends (RACE) technique to prove that the gene-silencing activity of pRNA nanoparticles is mediated by RNA interference. qRT-PCR . A RNA l qRT-PCR B. The mR qRT-PCR B, lane 2LUC2-siRNA). The pRNA-3WJ motif was used in these studies because the ease of construction and the multivalent nature of the pRNA-3WJ allow for inclusion of siRNA on one helical branch while targeting ligands, such as RNA aptamers, can be added to other branches to fulfill specific and favorable cancer targeting.,,,,Based on the above results, it was determined that siRNA sequences can release from RNA nanoparticles within the cells. However, confirmation of Dicer processing of siRNA from RNA nanoparticles has not been observed, nor is the release mechanism of siRNA from the RNA nanoparticles understood. pRNA-3WJ RNA nanoparticles were constructed, incorporating 24-bp 3′-UU overhang luciferase siRNA oligo . The Cy5 fluorescence was restored when the nanoparticles underwent processing by Dicer and the short fragment with Cy5 fell off of the nanoparticle, creating distance with the BBQ650 (“ON” status) (see LUC2-siRNA-Cy5 and 3WJ/LUC2-siRNA-Cy5/BBQ650 (molecular beacon), we have a better understanding about the processing of the siRNA in the cell cytosol and its processing efficiency.This 3WJ-tus) see A. By com,,LUC2-siRNA beacon nanoparticles into exosomes to be delivered to HT29 cells with continuous luciferase expression (HT29-luc). Additionally, the exosomes were decorated with previously developed RNA nanoparticles harboring folate (FA) to provide specific targeting and binding to the FA receptor-expressing HT29 cells.LUC2-siRNA/Cy5) to the HT29-luc cells  (LUC2-siRNA/Cy5 (S3) nanoparticles were also tested as a total Cy5 signal control. 3WJ/LUC2-siRNA beacon (S2) as well as control nanoparticles were delivered by the FA-displaying exosomes. The processing of siRNA from 3WJ scaffold was reflected by Cy5 fluorescence gradual increase in signal over time, as shown in confocal microscopy studies  A. 3WJ/LU studies B–3D. As in vitro. Rational design of siRNA should be illustrated when considering incorporation to RNA nanoparticles to ensure the intracellular Dicer processing of siRNA. Therefore, different factors affecting Dicer processing including siRNA length, 5′-phosphate, 3′-2-nt overhang, and 2′-F modifications were tested based on the Luciferase 2-siRNA used above. The base pairing length is a key factor, as Dicer processes siRNA according to the loop-counting rule.,,LUC2-siRNAs were designed and constructed ,Since its discovery in 1998,,,in vivo. Moving forward, these studies will be implemented.In the presented study, the pRNA-based RNA nanoparticles resulted in specific knockdown and gene silencing via siRNA delivery. Our cell microscopy studies involving siRNA beacons conjugated to RNA nanoparticles demonstrate a time release study of siRNA from the RNA nanoparticles by Dicer processing. As a result of the siRNA release and incorporation into RISC, target Lamin A/C mRNA was cleaved as demonstrated in in vivo delivery platform of RNAi, where target tissue specificity can be enhanced via conjugation of tumor-targeting ligands in addition to their rubber-like properties.,N-acetylgalactosamine-conjugated siRNA ,,,,,RNA nanoparticles provide a stable BRCAA1 siRNA to gastric cancer tumors in mice using the pRNA-3WJ labeled with folic acid.BRCAA1, leading to tumor growth inhibition. In a similar fashion, Lee et al. delivered luciferase siRNA as a proof of concept to glioblastoma tumors using folic acid targeting to the overexpression of FA receptors on the tumor model.MED1 siRNA to breast cancer tumors in mice to overcome tamoxifen resistance.MED1 by the RNA nanoparticles led to tumor inhibition as well as resensitization to tamoxifen, thus decreasing lung metastasis and cancer stem cell content. Xu et al. demonstrated the silencing of Delta-5-Desaturase (D5D) by pRNA-3WJ nanoparticles alongside the dihomo-γ-linolenic acid (DGLA) treatment for colon cancer suppression.Additionally, RNA nanoparticles have been used to deliver dicer-substrate siRNAs to tumor sites, resulting in specific gene silencing and tumor inhibition. Cui et al. delivered LUC2 beacon nanoparticles proved intercellular Dicer cleavage, RISC incorporation, and specific mRNA cleavage. Furthermore, our in-depth studies of Dicer processing of siRNA and RNA nanoparticle/siRNA conjugates not only proved that the RNA nanoparticle does not interfere in Dicer’s ability to read and process siRNA sequences but also provided design principles of incorporation of Dicer-substrate siRNA onto the nanoparticles. It can be concluded that the siRNA must be longer than 21 nt in length and allows for flexibility of chemical modifications and the inclusion of phosphate and linker nucleotides, albeit at a slight efficiency penalty. We have thus proven here, along with previous studies, that RNA nanoparticles serve as a powerful platform for the efficient delivery of siRNA.Through the completed studies we aimed to demonstrate the efficient processing of siRNA from RNA nanoparticles for high gene silencing while providing a comprehensive, mechanistic understanding of siRNA Dicer processing from RNA nanoparticles. As such, our pRNA-Lamin25 and 3WJ-in vitro by T7 RNA polymerase using previously described procedures in the Guo lab.,pRNA-lamin25 nanoparticles were designed with the lamin-targeting siRNA extended off the 3′/5′ proximate end of the phi29 pRNA. The sequence of the single-stranded RNA nanoparticle is shown in LUC2-siRNAs were designed to study factors affecting Dicer processing. 3WJ/LUC2-siRNA molecular beacon was designed to study intracellular Dicer processing. The sequences are listed in LUC2-antisense strand with Cy5-phosphoramidite, while BBQ650 was conjugated to 3′-3WJ-c with BBQ650-CPG column. All strands were synthesized, desalted, and purified before use.Different DS/2), heated to 90°C for 5 min, and slowly cooled to 37°C on a thermocycler.To assemble into DS or 3WJ nanoparticles, equal molar ratios of each component strand were mixed together in 1× Tris-magnesium sulfate (TMS) buffer  containing 10% FBS in a 37°C incubator under 5% CO2 and a humidified atmosphere.KB cells were cultured in RPMI-1640 medium (Life Technologies) containing 10% fetal bovine serum (FBS) in a 37°C incubator under 5% COLamin gene-specific 3′ primer: 5′-CCAGTGAGTCCTCCAGGTCTCGAAG-3′Lamin gene-specific 3′ nested primer: 5′ CCTGGCATTGTCCAGCTTGGCAGA-3′100 nM pRNA-lamin25 was transfected into KB cells with FuGENE HD . Cells were harvested 48 h after transfection. Total mRNA was isolated with the Poly(A)Purist Kit . The FirstChoice RLM-RACE Kit  was used in the 5′ RACE experiment, also known as single-sided PCR. With primers for the middle of the unknown sequence, mRNA is amplified in the full length, therefore either amplifying the cleaved and shortened mRNA from the siRNA or amplifying the much longer uncleaved mRNA. Thus, the PCR amplification allows for easy differentiation of cleaved product. The sequences of primers used in this study are:To examine the Dicer processing, 1 μg of RNA nanoparticles was incubated with 2 μL of Recombinant Human Dicer Enzyme (Genlantis), following the manufacturer’s instructions. After 6-h incubation at 37°C, 20% native PAGE was used to check the product before and after the Dicer processing. The gel was run in Tris-borate magnesium (TBM) buffer at 150 V for 1.5 h, stained with ethidium bromide (EB), and imaged on a Typhoon FLA7000 (GE) for EB and Cy5 signal.LUC2-siRNA were studied with procedures previously described.,The serum and thermodynamic stability of 3WJ/,,Exosomes were purified by a modified differential ultracentrifugation method as previously described.Pre-processed media were thawed slowly at 4°C overnight and then loaded into a pre-conditioned Pall Minimate tangential flow filtration (TFF) system with a 100 kDa MWCO capsule (OA100C12), preconditioned according to standard operation by the manufacturer. 500 mL of exosome-enriched medium was processed at 6 mL/min by setting pump speed at 40 mL/min. When volume reduced to ∼5 mL, 200 mL of sterile Dulbecco's phosphate buffered saline (DPBS) was added as a washing step, the system continued to run until the volume reduced to ∼5 mL again, and medium was then collected. Two 15-mL DPBS wash steps were performed, and a total 30-mL wash was collected and combined with the 5-mL sample from the last step.,,The 35-mL post-TFF media were then further purified by 100,000 rcf ultracentrifugation using a SW28 rotor (Beckman Coulter) for 90 min at 4°C. 200 μL of 60% iodixanol (Sigma) was added to the bottom of each tube to serve as iso-osmotic cushion as previously reported.Nanoparticle tracking analysis (NTA) was carried out with the Malvern NanoSight NS300 system on exosomes re-suspended in phosphate buffered saline (PBS) at a concentration of 10 μg of protein per milliliter for analysis following published methods.Purified exosomes were assayed for exosomal protein markers via western blot. Exosomes were loaded onto TDX FastCast SDS-PAGE gels , run at 100 V for 2 h, and transferred from gel to membrane. Membranes were blocked by 5% fat-free milk at room temperature for 1 h and incubated overnight in primary antibody. Protein bands were detected with an ECL system (Pierce) after incubating in the horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature and exposed to film for autoradiography. Primary antibodies used for western blot analysis were rabbit anti-human TSG101 , rabbit anti-human integrin α4 , rabbit anti-human integrin α6 , rabbit anti-human integrin β1 , rabbit anti-human integrin β4 , rabbit anti-human integrin β5 , and rabbit anti-human Glypican 1 .LUC2 siRNA or Survivin siRNA (10 μg) were mixed in 100 μL of PBS with 10 μL of Exo-Fect Exosome transfection solution (System Biosciences), followed by a heat-shock protocol to complete the RNA nanoparticle loading. Cholesterol-modified 3WJ/FA or cholesterol-modified 3WJ/EGFR (or 3WJ) nanoparticles were incubated with RNA-loaded exosomes for decoration at an average ratio of 5,000:1 (RNA:exosome) at 37°C for 45 min and then left on ice for 1 h to prepare the FA/EV and EGFR/EV. To purify RNA-decorated EVs, 400 μL of RNA-decorated EVs was washed with 5 mL of PBS in a SW-55 tube that contained 20 μL of 60% iodixanol cushion and spun at 100,000 × g for 70 min at 4°C. All the pellets in the cushion were collected and suspended in 400 μL of sterile PBS for further use.Exosomes (EVs)  and RNA nanoparticles harboring LUC2-siRNA/Cy5, 3WJ/EV/LUC2-siRNA/Cy5, 3WJ/FA/LUC2-siRNA/Cy5, and 3WJ/LUC2-siRNA/Cy5 at 37°C for 1 h before flow cytometry. Samples were incubated at a concentration of 100 nM of the Cy5 RNA strand.For cell binding assays, HT29 cells were incubated with FA/EV/LUC2-siRNA/Cy5 as well as control groups including 3WJ/EV/LUC2-siRNA/Cy5 and EV/LUC2-siRNA/Cy5 were incubated with HT29 cells at 37°C for 1 h. Samples were incubated at a concentration of 100 nM of the Cy5 RNA strand. After washing with PBS, the cells were fixed by 4% paraformaldehyde and stained by Alexa Fluor 488 phalloidin (Invitrogen) according to manufacturer’s instructions and DAPI for cell nucleus staining and mounted with ProLong Gold Antifade Reagent . The cells were then analyzed for binding and cell entry by an Olympus FV3000 Confocal System microscope.To study internalization, FA/EV/LUC2-siRNA-tru beacon, 3WJ/LUC2-siRNA beacon, 3WJ/LUC2-siRNA Cy5) were loaded to exosomes, respectively, further decorated by FA as described above. After incubation with HT29 cells for 0.5, 2, and 4 h, cells were fixed and stained as described above. Samples were incubated at a concentration of 100 nM of the Cy5 RNA strand. Cy5 signal was monitored for each group at different time points by confocal microscopy under the same parameters.To monitor intracellular Dicer processing, RNA nanoparticles (3WJ/