Integrins are a family of transmembrane glycoprotein signaling receptors that can transmit bioinformation bidirectionally across the plasma membrane. Integrin αIIbβ3 is expressed at a high level in platelets and their progenitors, where it plays a central role in platelet functions, hemostasis, and arterial thrombosis. Integrin αIIbβ3 also participates in cancer progression, such as tumor cell proliferation and metastasis. In resting platelets, integrin αIIbβ3 adopts an inactive conformation. Upon agonist stimulation, the transduction of inside-out signals leads integrin αIIbβ3 to switch from a low- to high-affinity state for fibrinogen and other ligands. Ligand binding causes integrin clustering and subsequently promotes outside-in signaling, which initiates and amplifies a range of cellular events to drive essential platelet functions such as spreading, aggregation, clot retraction, and thrombus consolidation. Regulation of the bidirectional signaling of integrin αIIbβ3 requires the involvement of numerous interacting proteins, which associate with the cytoplasmic tails of αIIbβ3 in particular. Integrin αIIbβ3 and its signaling pathways are considered promising targets for antithrombotic therapy. This review describes the bidirectional signal transduction of integrin αIIbβ3 in platelets, as well as the proteins responsible for its regulation and therapeutic agents that target integrin αIIbβ3 and its signaling pathways. Integrins, a family of transmembrane glycoprotein signaling receptors, comprise two distinct, noncovalently associated subunits, α and β. Each subunit consists of a large extracellular domain that contributes to ligand binding, a single-pass transmembrane (TM) domain, and a smaller unstructured cytoplasmic tail of approximately 20~70 amino acids except β4). The cytoplasmic tail provides binding sites for adaptors, signaling proteins, and cytoskeleton-associated proteins, which play an essential role in integrin bidirectional signaling  . In mamm. The cytQuantitative studies using 7E3 mAbs eventually confirmed that each unstimulated platelet presents approximately 50,000–100,000 copies of αIIbβ3 on its surface , and addBidirectional signaling of integrin αIIbβ3 is vital for platelet functions, hemostasis, and arterial thrombosis. Bidirectional signaling of integrin αIIbβ3 also plays an important role in cancer progression through regulating the interaction of integrin αIIbβ3 with the fibrinogen/αvβ3 complex on the surface of tumor cells  and/or rThe inside-out signaling of αIIbβ3 on platelets can be initiated by various soluble agonists, such as epinephrine, ADP, thromboxane A2 (TXA2), or thrombin, which bind to G protein-coupled seven-transmembrane domain receptors (GPCRs). Inside-out signaling can also be initiated by immobilized agonists, such as vWF or collagen, which mainly interact with GPIb-IX-V or GPVI, respectively. Inside-out signaling includes (1) intracellular activators  binding to integrin αIIbβ3 tails, (2) separation of the α and β TM and the cytoplasmic tail, (3) a conformational change of the extracellular domain of αIIbβ3, and (4) increasing ligand binding affinity and avidity. To date, talin, kindlin, and other proteins have been identified as directly or indirectly interacting with integrin cytoplasmic tails to participate in the inside-out signaling of αIIbβ3 .Talin has long been known to play an essential role in integrin activation. As an integrin-actin adaptor protein, it is an autoinhibited dimer with a head-to-tail conformation . It cons744PLY747 motif of the β3 tail is proposed to induce αIIbβ3 activation by disrupting the salt bridge between αIIb and the β3 tail [746A mutation of β3 integrin, which is believed to selectively disrupt the interaction between αIIbβ3 and talin, display impaired inside-out activation of αIIbβ3 [talin-1 in mice showed that integrin αIIbβ3 is unable to activate in response to any tested agonists [Over the past 20 years, studies in cultured cells , mouse m β3 tail . Talin-H β3 tail . Studies β3 tail . Furtherf αIIbβ3 . Conditiagonists , 44. Thiagonists , 44. Thuagonists , 45. Recagonists . Howeveragonists .kindlin-1 gene lead to Kindler syndrome, which is characterized by serious skin blistering, progressive poikiloderma, photosensitivity, and atrophy of the skin [kindlin-3 gene lead to type-III leukocyte adhesion deficiency (LAD-III), as well as recurrent infections, immune deficiencies, and severe bleeding disorders caused by the dysfunction of integrins in leukocytes and platelets; loss of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) in the bone marrow; elevated leukocyte counts; and osteopetrosis [kindlin-2 gene, but kindlin-2 is highly expressed in cancers of the lung, prostate, pancreas, liver, and esophagus [kindlin-2 is embryonically lethal in mice and causes multiple severe abnormalities in zebrafish due to impaired integrin activation [A series of publications have established a requirement for kindlin coordinating with talin for integrin αIIbβ3 inside-out signaling , 52, 53.the skin , 60. Mutpetrosis –64. To dsophagus . Knockoutivation , 66, 67.kindlin-3−/− mice, Moser et al. showed that in platelets lacking kindlin-3, integrin αIIbβ3 could not be activated despite normal talin expression [744PLY747 motif of the integrin β3 tail negatively regulates talin binding [756ITY759 motif also inhibits kindlin-2 binding [Using Chinese hamster ovary (CHO) cells expressing integrin αIIbβ3, the Calderwood group reported that kindlin-1 and talin cooperatively enhance integrin αIIbβ3 activation , 68 and pression . Kindlinpression , and conpression . Howeverpression . Further binding , 71. Sim binding . These o binding . Tyrosin binding . Structu binding . Recent  binding , 76, 77. binding , 78, 79.ILK−/−mice showed increased bleeding time, reduced aggregation, soluble fibrinogen binding, and defects in α-granule secretion [756ITY759 motif of the integrin β3 cytoplasmic tail. β3-Endonexin is present in resting human platelets. Nonetheless, there is little available information about how β3-endonexin regulates integrin αIIbβ3 [PP1cγ−/−) mice, Gushiken et al. showed that PP1cγ mainly participates in thrombin-induced integrin αIIbβ3 inside-out signaling but not ADP or collagen-related integrin αIIbβ3 inside-out signaling. Vinculin, a marker for integrin-mediated focal adhesion complexes, inhibits Rap1-GTP-interacting adaptor molecule (RIAM) binding to talin and plays a role in inside-out signaling of αIIbβ3 [Vcl) showed that tail bleeding times in Vcl−/− mice were prolonged, but platelet functions, including agonist-induced fibrinogen binding to αIIbβ3, spreading, clot retraction, platelet aggregation, and adhesion on immobilized fibrinogen or collagen, were similar to those of wild-type mice [In addition to talin and kindlin, other proteins, such as ILK , β3-endoecretion . These on αIIbβ3 , 92. CIBn αIIbβ3 , 93. Hown αIIbβ3 . ICln bin αIIbβ3 . Using tf αIIbβ3 , 94. Usif αIIbβ3 . Howeverype mice .3-induced thrombus formation between Cib1+/+ and Cib1−/−mice, possibly owing to compensation by CIB2 and CIB3 [Cib1−/−mice showed a rebleeding phenotype and defective thrombosis due to impaired integrin αIIbβ3 outside-in signaling [p. Ter2648SerextTer101) potentiates platelet integrin αIIbβ3 activation by facilitating recruitment of talin to the β3 tail [Several proteins are thought to bind directly to one of the integrin αIIb or β3 cytoplasmic tails to inhibit integrin αIIbβ3 activation. CIB1 plays a role in the possible negative regulation of integrin αIIbβ3 activation by binding directly to the GFFKR sequence of the αIIb cytoplasmic tail , 96, wheand CIB3 . In contignaling . Dok1 isignaling . The intignaling  and impaignaling . Some stignaling , 106. Crignaling . The rol β3 tail . Recent  β3 tail . Due to  β3 tail , 110. α- β3 tail –111, whi1, TP, 5-TH2A, and PAR receptors. PLCβ is downstream of GPCRs, whereas PLCγ is activated by VWF/GPIb-IX-V or collagen/GPVI interactions [2+-independent process [2+ concentration in the platelet cytosol. DAG and Ca2+ activate many isoforms of platelet protein kinase C (PKC) and Ca2+ diacylglycerol guanine-nucleotide-exchange factor I , leading to the conversion of Rap1-GDP to Rap1-GTP and the translocation of Rap1-GTP to the plasma membrane [CalDAG-GEFI−/− mice, induction of inside-out activation of integrin αIIbβ3 by calcium ionophore, collagen, ADP, and a TXA2 analog was strongly inhibited. In contrast, thrombin-induced activation of αIIbβ3 was mildly affected [PKCα−/− mice showed that PKCα was a regulator of inside-out signaling of αIIbβ3 [Rap1b−/− mice demonstrated that ADP- or AYPGKF-induced integrin αIIbβ3 activation was impaired, as was FeCl3-dependent arterial thrombosis [Knowledge of how agonists lead to integrin αIIbβ3 activation by talin and/or kindlin is vital for understanding inside-out signaling of αIIbβ3 Fig. . The iniractions , 119. PI process . Unlike  process , increasmembrane –124. In affected . This fiaffected  in humanaffected . Platelef αIIbβ3  but did rombosis . Interesrombosis , but it rombosis .Fig. 2ScRap1 mediates inside-out activation of integrin αIIbβ3 through another effector, called Rap1-GTP-interacting adaptor molecule (RIAM), on the membrane. RIAM is a member of the Mig-10/RIAM/lamellipodin (MRL) family of adaptor molecules. RIAM recruits talin-1 to integrin αIIbβ3. Knockout of RIAM in megakaryocytes abolishes Rap1-dependent αIIbβ3 activation ; howeverThe outside-in signaling of integrin αIIbβ3 on platelets is triggered by the binding of soluble fibrinogen to activated integrin αIIbβ3 Fig. . Many ofXenopus (CTX) family of the immunoglobulin superfamily. JAM-A likely indirectly associates with integrin αIIbβ3 through CD9 [Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a membrane-spanning immunoglobulin protein that regulates outside-in signaling, but not inside-out signaling, of integrin αIIbβ3 , 137. PEough CD9 . In mousough CD9 , 148.The tetraspanins possess four conserved hydrophobic transmembrane regions: two extracellular loops and two intracellular tails . At least five members of the tetraspanin superfamily, CD151, tumor suppressing subtransferable candidate 6 (TSSC6), CD63, CD9, and CD82, have been reported to be expressed in platelets –153. HowCD151−/− platelets have demonstrated that deletion of CD151 is capable of inhibiting the outside-in signaling properties of αIIbβ3, including reducing agonist-induced platelet aggregation, delaying clot retraction, diminishing platelet spreading on fibrinogen, and reducing formation of filopodia. However, CD151−/− platelets display normal αIIbβ3 inside-out signaling properties, as evidenced by standard agonist-induced binding of soluble fibrinogen or JON/A antibody [12 receptor complex and participates in integrin αIIbβ3 outside-in signaling [12 receptor [Studies using murine antibody , 157. Reantibody . CD151 freceptor . Early sreceptor . Future Gas6−/− mice have shown that Gas6 plays a role in platelet function [Gas6−/− mice have a normal bleeding time but a tendency to repetitively rebleed due to impaired αIIbβ3 outside-in signaling [Gas6−/− mice [Growth arrest-specific protein 6 (Gas6) is a member of the vitamin K-dependent protein family. Recent studies of function –162. Gasignaling . Interes−/− mice . Once Ga−/− mice . There a−/− mice  and the −/− mice , 167, th13 [13 directly binds to the integrin β3 cytoplasmic tail [13 siRNA interference. Gong et al. reported that platelets transfected with Gα13 siRNA spread poorly on immobilized fibrinogen and fail to activate Src. The myr-FEEERA peptide disrupted the Gα13/β3 interaction, thereby hampering Src activation and ultimately inhibiting αIIbβ3 outside-in signaling [SLP-76−/− murine platelets have normal fibrinogen binding but poor spreading. In the absence of SLP-76, collagen-induced platelet aggregation and granule release, as well as the phosphotyrosine of the β3 tail, are markedly impaired [3-induced thrombus formation, are strongly impaired in myosin-deficient mice [Lnk−/−mice exhibit reduced abilities in terms of full spreading on fibrinogen, fibrin clot retraction, platelet aggregation, and stable thrombus formation. Lnk is thought to mediate αIIbβ3-dependent outside-in signaling through facilitating Src phosphorylation of Fyn [WASP−/− mice showed that integrin αIIbβ3 outside-in signaling, such as fibrinogen and JON/A binding under agonist stimulation, is normal, whereas integrin αIIbβ3 outside-in signaling-dependent events, such as spreading on immobilized fibrinogen, fibrin clot retraction, and rebleeding, are impaired [Mucor circinelloides [Some intracellular adaptor molecules, such as the heterotrimeric guanine nucleotide-binding protein (G protein) Gα13 , 169, va13 , the SH213 , myosin 13 , Src hom13 , Grb2 [113 , FcγRIIa13 , lymphoc13 , stress-13 , Disable13 , 179, NL13 , are belmic tail . The sprignaling . VPS33B,ignaling . VPS33B ignaling . SLP-76−impaired , 183. Myimpaired . Outsideent mice . Fibrinoent mice . Platelen of Fyn . Shc andn of Fyn . Disablen of Fyn . The baln of Fyn . NLRP3 rimpaired . Some eximpaired , UV [186elloides , heparinelloides , and hypelloides , also reelloides , reelin elloides , 192, anelloides  were alsThe maintenance of normal platelet integrin αIIbβ3 signal transduction depends on numerous kinases and phosphatases that participate in the cascade of phosphorylation and dephosphorylation. To date, more than 10 enzymes have been reported to be involved in integrin αIIbβ3 outside-in signaling. The earliest phosphorylation event after fibrinogen binding to αIIbβ3 is the activation of Src kinase. Src has been reported to directly and constitutively associate with arginine-glycine-threonine (RGT) residues of the integrin β3 cytoplasmic tail via its SH3 domain , 195. InSyk−/−platelets adhere normally to immobilized fibrinogen [Following platelet activation by agonists, fibrinogen binds to αIIbβ3 and then results in tyr529 of Src being dephosphorylated by protein-tyrosine phosphatase-1B (PTP-1B) . After Sbrinogen  and failbrinogen , 205.PKCδ−/−mice showed that PKCδ is a key negative regulator of filopodial formation, and a lack of PKCδ leads to enhanced platelet aggregation [PKCδ−/− mice compared to that in wild-type mice [PKCθ−/− mice have shown that PKCθ negatively regulates thrombus formation on collagen under flow [PKCι/λ−/− mice show that PKCι/λ is dispensable for αIIbβ3 bidirectional signaling [PI3Kγ−/− platelets have demonstrated a diminished ability to reorganize the cytoskeleton, spread on fibrinogen, and form stable thrombi in vivo using a FeCl3-induced carotid injury model [Pyk2−/− platelets show a significant defect in integrin αIIbβ3 outside-in signaling, similar to the loss of c-Cbl or PI3Kβ activity [2 (cPLA2α) and vimentin, a cPLA2α binding partner, are constitutively associated with αIIbβ3 in mouse and human platelets [2α−/−platelets demonstrated that αIIbβ3 outside-in signaling was impaired and inside-out signaling was partially impaired [Twelve isoforms of the PKC family are involved in most platelet functions required for thrombus formation . Recent regation . Howeverype mice . PKCθ isype mice . PKCθ isype mice , 213, buignaling . Studiesignaling . PI  that acts as an RGD mimetic. The EPIC trial showed a reduced frequency of restenosis in high-risk angioplasty patients who received abciximab infusion [Therapeutic agents targeting integrin αIIbβ3, both approved for clinical use and under development, are shown in Table infusion . Three pinfusion . In receinfusion . Eptifibinfusion . All thrinfusion . Orally infusion . For a minfusion –240. In infusion .Table 2T3-induced occlusive carotid artery thrombosis. Eptifibatide also dramatically prolongs tail bleeding and increases blood loss; however, the myr-FEEERA peptide had no such adverse side effects [Because of the marked inhibition of platelet function, integrin αIIbβ3 antagonists can increase bleeding risk, although many studies suggest that these antagonists do not significantly increase the risk of life-threatening bleeding when compared to standard unfractionated heparin . Severe  effects . Our stu effects , 197. ThThe development of proteomics, biophysics, and gene knockout/knockin technologies has uncovered an increasing number of proteins that participate in the bidirectional signaling of integrin αIIbβ3 and has begun to shed light on their mechanisms and roles in regulating integrin αIIbβ3 signaling. Given the importance of integrin αIIbβ3 bidirectional signaling in maintaining proper platelet function, examining the complex regulatory relationship between these interacting proteins can prove immensely important for understanding the mechanisms of platelet activity, as well as for developing new therapies for cancer and thrombosis based on a deeper knowledge of the underlying physiology. Until now, the complex stoichiometric and spatiotemporal dynamics between integrin αIIbβ3 and its regulatory proteins have remained obscure, but promising new techniques have already presented new opportunities to learn more. Considerable efforts are still needed to fully explore how integrin αIIbβ3 interacts with its regulatory proteins, how its regulatory proteins interact with one another in space and time, and how therapeutic agents targeting integrin αIIbβ3 and its pathways can provide therapeutic benefits while minimizing adverse side effects.


Ciona intestinalis in diverse models of human disease partially alleviates the phenotypic effects of mitochondrial respiratory chain defects. AOX is a non-proton pumping, mitochondrial inner membrane-bound, single-subunit enzyme that can bypass electron transport through the cytochrome segment, providing an additional site for ubiquinone reoxidation and oxygen reduction upon respiratory chain overload. We set out to investigate whether AOX expression in Drosophila could counteract the effects of mitochondrial DNA (mtDNA) replication defects caused by disturbances in the mtDNA helicase or DNA polymerase γ. We observed that the developmental arrest imposed by either the expression of mutant forms of these enzymes or their knockdown was not rescued by AOX. Considering also the inability of AOX to ameliorate the phenotype of tko25t, a fly mutant with mitochondrial translation deficiency, we infer that this alternative enzyme may not be applicable to cases of mitochondrial gene expression defects. Finding the limitations of AOX applicability will help establish the parameters for the future putative use of this enzyme in gene therapies for human mitochondrial diseases.The xenotopic expression of the alternative oxidase AOX from the tunicate  TWNK and POLG, respectively encoding the mitochondrial replicative DNA helicase Twinkle and the catalytic subunit of the mitochondrial replicase pol γ, are associated with diverse human diseases, as well as with aging. In particular, they underlie cases of Progressive External Ophthalmoplegia (PEO) and Alper’s syndrome, for which mitochondrial DNA (mtDNA) depletion, deletions and/or point mutations are frequently observed4. Twinkle and pol γ are essential components of the mtDNA replication machinery. Twinkle hydrolyzes nucleotide tri-phosphate to unwind the parental double-stranded DNA, providing the single-stranded DNA template for pol γ. In concert with the mitochondrial single-stranded DNA-binding protein (mtSSB), Twinkle and pol γ, and possibly replisome proteins that remain to be identified, can promote mtDNA replication and guarantee proper levels of this genome for correct organellar gene expression6. mtDNA carries genes that are essential for ATP production via the process of oxidative phosphorylation (OXPHOS).Mutations in the human genes 7. However, the fact that OXPHOS is often affected in mitochondrial diseases does provide a possible common target for therapeutic interventions: the respiratory chain (RC). Four of the RC complexes transfer electrons to molecular oxygen, a process coupled to energy transduction forming a proton gradient across the mitochondrial inner membrane, whereas a fifth complex uses this gradient to phosphorylate ADP to ATP. Most organisms possess branches of the electron transfer system known as alternative pathways, which are absent in humans, other vertebrates, and insects9. These pathways may represent “safety valves” to alleviate stress arising from RC overload, as is the case in mitochondrial diseases, preventing excess formation of reactive oxygen species (ROS) and allowing reoxidation of primary electron carriers such as NADH. The alternative oxidase (AOX), for example, is a single-subunit enzyme able to transfer electrons from ubiquinol to oxygen, bypassing RC complexes III and IV, without proton pumping. It has been suggested that AOX expression in human mitochondria could be a treatment for patients with mitochondrial diseases, as a bypass therapy10.Mitochondrial diseases exhibit a broad clinical spectrum, often requiring treatments that are specific to each type of disorder, with only a few such treatments shown thus far to be convincingly effectiveCiona intestinalis (Tunicata: Ascidiacea) in cultured human cells, in the mouse, and in the fruitfly Drosophila melanogaster showed several benefits, which are consistent with a role for AOX in buffering OXPHOS stresses, caused by chemical, genetic or physiological damage. It promoted resistance to inhibitors of complexes III and IV14, alleviated the metabolic abnormalities of complex IV-deficient cells derived from patients15 and the deleterious phenotypes of complex IV insufficiency in Drosophila16. AOX function has also been reported to provide some benefit in other models of mitochondrial disorders, such as fly lines with knockdown of pol γ17 or with mutations in dj-1β, the homologue of the human Parkinson’s disease gene DJ112, and in sesB, the homologue of another PEO gene, ANT118. In addition, the rescue by AOX of Drosophila models of Alzheimer’s disease and mid-line closure defects suggests that RC dysfunction and/or excess ROS production are not only involved, but also appear in early stages of these pathogeneses20.The transgenic expression of the AOX gene from Drosophila homologue of Twinkle. Our data shows that AOX does not rescue developmental lethality, nor alter the biochemical and molecular parameters associated with Twinkle and pol γ mutations or knockdown. This restricts the potential therapeutic use of AOX to specific types of mitochondrial dysfunction.We set out to investigate if the beneficial effects of AOX expression in higher metazoans is applicable to fly lines with mtDNA replication defects. Considering the earlier finding that AOX can ameliorate the phenotype of pol γ-depleted flies, which are defective in mtDNA replication, we hypothesized that AOX could have the same effect in flies with defects in the UAS-empty2nd, UAS-mutAOX2nd, UAS-wtAOX8.1 and UAS-wtAOXF6, with the Twinkle variant constructs UAS-Twinkle WT, UAS-Twinkle K388A, and UAS-Twinkle A442P 21. UAS-wtAOX8.1 and UAS-wtAOXF6, when induced, lead to the expression of AOX at low and high levels, respectively21. The Twinkle variant constructs guide the respective overexpression of the wild-type form of the mtDNA helicase (WT), an active-site mutant (K388A), and a PEO mutation-bearing variant (A442P)22. The latter represents a fly mutation homologous to the one found in human PEO families, A475P23.Using standard genetic crosses, we initially created 12 fly lines, in which we combined the AOX variant constructs UAS-AOX; UAS-Twinkle lines with the ubiquitous GAL4 driver daGAL4 . Because the larval body wall consists mostly of muscular and nervous tissues, we tested if tissue-specific expression of the Twinkle mutations using the mhcGAL4 and elavGAL4 drivers, respectively, could reproduce the lethal phenotype observed in the presence or absence of AOX. Consistent with the lack of detectable OXPHOS dysfunction in the larval body wall, flies overexpressing Twinkle K388A or A442P in muscles or neurons completed normal development , so we first verified its effect on the levels of Twinkle mRNA in controls  and induced individuals  to show the efficacy of the construct  knockdown in development (using the ubiquitous Act5CGAL4 driver) and in neurodegeneration models . We therefore created and analyzed eight additional UAS-AOX lines combined with UAS-tamas RNAi or with a polymerase-active-site mutant of the catalytic subunit of pol γ, tamas Q1009A  is(are) operating when AOX is present in the mitochondria of higher animals.It has been shown that the xenotopic expression of tko25t mutation34. This mutant has a defective mitochondrial ribosomal protein S12, impairing organellar protein synthesis and causing developmental delay and neurological problems35. Because another alternative enzyme, the alternative NADH dehydrogenase Ndi1, was also unable to rescue tko25t, the authors concluded that this mutant has a more complex metabolic defect, e.g. affecting multiple RC components, or some other process34. Here, we describe another case of AOX inability to rescue mitochondrial dysfunction in Drosophila. Overexpression of Twinkle mutants or knockdown of the endogenous enzyme caused developmental lethality at different stages with varying levels of mtDNA depletion, which was not alleviated by AOX. AOX was also unable to rescue the lethality caused by expression of a catalytically inactive pol γ or knockdown of its catalytic subunit, nor any of the molecular and biochemical parameters associated with mutant Twinkle expression. At high levels, AOX was in fact more detrimental for flies with Twinkle defects. One possible explanation is that the mutant Twinkle, or the depletion of the wild-type enzyme, in some way affects the organization of the inner mitochondrial membrane, and the further expression of a membrane-targeted protein (active or inactive AOX) synergistically impairs even more larval/pupal viability. Twinkle has been associated with a specific lipid domain of the inner mitochondrial membrane37, consistent with this hypothesis.At the other end of the spectrum of AOX applicability, there is a reported case of a true mitochondrial dysfunction, whose associated phenotypes were not ameliorated by the transgenic expression of this alternative enzyme, namely flies bearing the tko25t in the presence of Ndi1, or Ndi1 plus AOX34. We speculate that in both cases, the metabolic consequences of severely defective mtDNA replication/mitochondrial translation may result in the activation of AOX, leading to an additional decrease in ATP production due to partial diversion of electron flow. Both the multi-subunit mitochondrial ribosomal machinery, of which the tko gene product is an essential part, and the modularly-architectured, multimeric Twinkle enzyme are dependent on the energy of nucleotide tri-phosphate hydrolysis for their proper functions in organellar protein synthesis and genome maintenance, respectively (reviewed in38). For example, the recombinant human wild-type Twinkle not only requires Mg2+ and ATP for dsDNA unwinding and ssDNA translocation, but the enzyme also presents significantly higher stability in vitro in the presence of such compounds39. Therefore, fluctuations in the ATP level in a sensitive mitochondrial genetic condition, such as that found in the tko25t and Twinkle models, may worsen the defects caused by such mutant proteins, which may in turn require even higher than normal nucleotide levels to maintain their fragile stability and/or function.However, similar aggravations of developmental problems were also observed for 29, which in turn impairs mitochondrial RNA and protein synthesis40. tko25t produces a defect directly in the mitochondrial translation apparatus, downstream of Twinkle and pol γ, although its phenotypic consequences appear less severe than those of our mtDNA replication models. Nonetheless, the end result is similar: incorrect or insufficient subunits of all four OXPHOS complexes partly encoded in mtDNA, which AOX cannot circumvent. The fact that the mitochondrial genome encodes seven subunits of OXPHOS complex I and two subunits of complex V  may explain the inability of AOX to rescue mitochondrial gene expression defects, as this enzyme is predicted to counteract complexes III and/or IV deficiencies.One of the primary effects of disturbing Twinkle or pol γ is the decrease of intact copies of mtDNA Drosophila17. We speculate that differences in the genetic background of the fly lines, in dietary conditions, and in experimental design might explain these discrepancies. Even though conditions appear to be homogenous within each study, they are difficult to compare side-by-side. Our implementation of two controls (for promoter dilution and for AOX enzymatic activity), plus two expression levels for AOX, and two defective pol γ models (knockdown and mutant expression), does represent a robust experimental design. On the other hand, the use by Humphrey et al.17 of several neural-specific GAL4 drivers does make their report reliable regarding pol γ depletion in the nervous system. Deficiencies of RC complex III and/or IV in specific neuronal classes are thus implied to underlie neurodegeneration in pol γ disease, in contrast to the broader effects of mtDNA replication defects documented in our study. Another important difference is that the drivers used for ubiquitous knockdown are different: daGAL4, used here, and Act5CGAL4 used by Humphrey et al.17 may differ in their exact strength, tissue and stage-specificity, and response to conditions. Notably, in combination with daGAL4, we found UAS-tamas RNAi to be pupal lethal, whereas in combination with Act5CGAL4, Humphrey et al.17 found it to be only semilethal, with AOX impacting the number of progeny. Clearly, the matter needs further investigation.Although in accord with the above hypothesis, our data appears inconsistent with the previously published findings showing significant improvement in the phenotypes associated with the knockdown of pol γ in diverse cell types of Drosophila: the absence of OXPHOS defects in larval body-wall mitochondria despite severe mtDNA depletion in whole L3 larval homogenates, as well as the absence of developmental lethality in the case of tissue-specific expression of mutant Twinkle in muscle and neurons. Maternal protein, RNA and even mtDNA may supply enough mitochondrial activity in those tissues to complete development. Because the large storage of triglycerides in the L3 larvae could interfere with the respiration measurements, we used a previously validated protocol29 in which the animals were dissected to eliminate all internal organs and the hemolymph, preserving the larval body wall  for these experiments. The results imply either that the larval phenotype described by us and others22 upon mutant Twinkle overexpression is caused by effects in one or more of these internal organs, or else could be due to an additional but unknown biological role of Twinkle, separate from mtDNA maintenance. The findings suggest a crucial role for mitochondria in the brain, fat body or other tissues that generate and respond to signals governing metamorphosis, over which AOX has no influence. Future studies using other tissue-restricted drivers for the larva, including ring gland, fat body, Malpighian tubule, salivary gland, glia, neuroendocrine cells, among others, will help us understand these roles.Our combined biochemical and genetic data also revealed an unexpected phenomenon regarding the overexpression of Twinkle variants in tko25t, Twinkle and pol γ, we would argue that consideration of the possible use of AOX in therapy should exclude deficiencies in mtDNA maintenance or gene expression, unless clear benefits can be robustly demonstrated in a specific, clinically relevant model.As the human diseases associated with Twinkle and pol γ mutations account for a significant portion of all mitochondrial disorders, AOX may have limited applicability in therapy. Indeed, taking into account the combined data on w1118, balancer lines CyO, TM3,Sb and TM6B-Tubby, and driver lines daughterless-GAL4 (daGAL4), myosin heavy chain-GAL4 (mhcGAL4) and embryonic lethal abnormal visionC155-GAL4 (elavGAL4) were obtained from stock centers. The lines UAS-AOXF6, UAS-AOXF24 12, UAS-wtAOX8.1, UAS-wtAOX7.1, UAS-empty2nd, UAS-empty3rd, UAS-mutAOX2nd and UAS-mutAOX3rd 21 were used in genetic crosses with the balancers described above to establish double transgenic lines in combination with UAS-Twinkle WT, UAS-Twinkle K388A, UAS-Twinkle A442P22, UAS-Twinkle RNAi (VDRC ID 108644/KK), UAS-tamas RNAi (VDRC ID 106955/KK) and tamas Q1009A29. Supplementary Table w1118 background for six-to-ten generations, and were maintained in standard diet12 at 18 or 25 °C, with 12 hr light/dark cycles.The standard line w1118) or GAL4-expressing males (daGAL4 or mhcGAL4) per vial, and allowed to lay eggs for 24 hrs at 25 °C in standard diet12, where the offspring were kept until eclosion , denatured at 95 °C for 5 min and resolved by SDS-PAGE on 12% polyacrylamide gels at 120 V for approximately 1.5 h. Proteins were transferred to nitrocellulose membranes using the semi-dry gel transfer system MSD10  for 15 min at room temperature. Membranes were blocked in PBST  containing 5% dried nonfat milk for 2 h at room temperature or overnight at 4 °C. Primary antibodies  were incubated for at least 1 h at room temperature, followed by three washes (10 min each) with PBST. Secondary antibodies [HRP-conjugated goat anti-rabbit and anti-mouse IgGs ] were incubated for at least 2 h at room temperature, and washed as described above. Membranes were incubated in Luminol substrate detection system Immun-Star HRP , and chemiluminescence signals were detected on a ChemiDoc Imaging System .Protein extracts from 10 whole 1-to-3-day-old adult males of the indicated genotype were prepared by gentle grinding of tissues in 200 μl PBS containing 1.5% Triton X-100 on ice. The suspension was centrifuged at 16,000 24, using 5–10 L3 larvae (5 days after egg laying) or adult males (1–3 days after eclosion) per preparation. To estimate relative mtDNA copy number, 15 ng of total DNA were used as template in a 20 μl quantitative PCR reaction, containing 10 μl PowerUp SYBR Green Master MixTM (Thermo Fisher Scientific) and 10 μM of each primer. The primers used were 16S-R (5′-TCGTCCAACCATTCATTCCA-3′) and 16S-L (5′-TGGCCGCAGTATTTTGACTG-3′) to amplify the mitochondrial 16S rRNA gene, and RpL32F (5′-AGGCCCAAGATCGTGAAGAA-3′) and RpL32A (5′-TGTGCACCAGGAACTTCTTGAA-3′) to amplify the single-copy, nuclear RpL32 gene. Reactions were performed in a StepOnePlusTM Real-Time PCR instrument (Applied Biosystems), under the manufacturer’s recommended conditions: 95 °C for 2 min, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. The ΔΔCT values were calculated by comparing the ratios between the mitochondrial and nuclear target genes. The relative mtDNA copy number values were derived by comparing each group of UAS-AOX; UAS-Twinkle lines to the same control sample  that was arbitrarily given the value 1.0.Total DNA was extracted as described previouslymtDNA-helicase (Twinkle) and tamas transcripts were performed essentially as described above, except that 1 μl of a 1:25 dilution of the reverse transcription reaction was used as template, along with 10 μM of the specific primers: TwinkleF (5′CCAAAGCGGTTCTAGTCAGC3′) and TwinkleR (5′GACTGGCATCGTAGTGCAAC3′) for mtDNA-helicase; tamasF (5′AAAATGTTCTCCATCACAAAGG3′) and tamasR (5′TTGGGACGATGAAATACCTC3′) for tamas; and RpL32F and RpL32A (described above) for RpL32. The relative transcript values were derived from the standard curve method, by comparing the ratio between mtDNA helicase and RpL32, or tamas and RpL32 for each UAS-RNAi; UAS-AOX X daGAL4 line to its control sample  that was arbitrarily given the value 1.0.Total RNA was prepared from 5 larvae using the TRIzol® plus PureLink RNA Mini Kit, and was reverse-transcribed using the SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme kit (Thermo Fisher Scientific), according to the manufacturer’s recommendations. The quantitative PCR reactions to estimate the relative amounts of 29 in ice-cold PBS, eliminating the internal organs and the hemolymph, to obtain the larval body wall, which was then weighed in an analytical balance, gently homogenized in isolation buffer , and transferred to the oxygraph O2k chambers . Oxygen consumption was measured at 25 °C in assay buffer  after addition of substrates for complex I , and complex IV  in the presence of 1 mM ADP. Complex I- and IV-driven respiration was corrected for the signal obtained after addition of 50 nM antimycin A and 5 nM rotenone, and 100 nM potassium cyanide, respectively. The antimycin A-resistant complex I-driven oxygen consumption, corrected for the signal obtained after addition of 200 nM propyl-galate and 5 nM rotenone, was attributed to AOX.Five L3 larvae (5 days after egg laying) of the indicated genotype were dissected as previouslypost hoc test, as indicated in the figure legends, except for differences in Twinkle and tamas transcript levels, for which Student t-tests were applied. The analyses were performed using the software SAS University Edition v9.4 (SAS Institute Inc.).Statistical differences among the indicated groups of flies were tested using one-way ANOVA with Tukey Any material or fly line used in this work is available from the corresponding author upon individual requests at any moment.Supplementary Information


Cladonia uncialis, suitable for detection of relatively small fluctuations of usnic acid concentration in response to environmental changes.The purpose of this study was to develop and validate a specific and sensitive liquid chromatography tandem mass-spectrometry method for quantification of usnic acid concentration in the lichen, The resulting method was fully validated according to international guidelines and demonstrated good selectivity and sensitivity with minor levels of a matrix effect and high accuracy. Cladonia, Usnea, Lecanora, Ramalina, Evernia, members of the Parmeliaceae, and other lichen genera [Usnic acid (UA) is one of the most common and most studied lichen-specific secondary metabolites and is taxonomically widely distributed in species of n genera . Isolaten genera  and studn genera , 3–5. Thn genera , and mayn genera , from hen genera , 9, and n genera , 11.Cladonia uncialis (L.) F.H. Wigg. [Under natural conditions, the accumulation of UA in lichens is thought to depend on environmental factors , 12–14 wH. Wigg. –21, fromH. Wigg. –25).All reagents used were of analytical or higher grade and purchased from Sigma Aldrich (Merck KGaA) unless otherwise stated. Usnic acid (UA) standard stock solution was prepared by solubilising 1 mg of UA in 1 mL of 100% acetonitrile.Cladonia uncialis thallus was crushed with mortar and pestle, soaked in 10 mL of 100% acetonitrile, vortexed for 30 s and agitated at 150 rpm on the shaker  for 20 min at room temperature. This extraction was repeated four times with the same material to obtain residual UA. All extracts were pooled into one glass tube, and the combined volume was adjusted to 50 mL with 100% acetonitrile.50 mg of the top 10 mm of dry Cladonia ochrochlora (a non-UA-producing species) was prepared in the same manner to emulate the matrix effect.For the control, an extract from ® Mini-UniPrep® G2, PTFE membrane, pore size 0.2 μm) samples were injected in a volume of 10 μL into a C8 LC column  100 Å, LC Column 100 × 2 mm) and separated by UltiMate™ 3000 RSLCnano System. Chromatographic separation was performed at a flow rate of 0.2 mL/min using a gradient elution program, starting from 80% of eluent A  and gradually changing to 5% A and back over 40 min. Exact gradient parameters: 80% of eluent A/20% B (100% acetonitrile acidified with 0.1% formic acid (v/v)) for 5 min, gradual changing to 5% A/95% B over 23 min, 5% A/95% B for 5 min, return to 80% of eluent A/20% B over 7 min.LTQ XL™ Linear Ion Trap Mass Spectrometer (Thermo Scientific™) was used to conduct mass spectroscopy. Filtered (WhatmanMass spectrometry measurements were performed on an LTQ OrbiTrap XL MS (Thermo Fisher). Samples were introduced to MS via electrospray ionisation using the following conditions: sheath gas flow rate, 30 (arbitrary units); auxiliary gas, 5 (arbitrary units); ESI voltage, 4.0 (kV); capillary voltage, − 35 (V); capillary temperature, 275 (°C); and tube lens voltage, − 110 (V). The collected spectra were scanned over the mass/charge number (m/z) range of 155–2000 atomic mass units . MS spectra were generated by collision-induced dissociation of the metabolite ions at normalized collision energy of 35%.The LC–MS/MS method was validated with respect to the specificity, linearity and sensitivity, precision and accuracy, matrix effects and recovery.C. ochrochlora extracts without UA) with a blank spike (UA in 100% acetonitrile) and a matrix spike (C. ochrochlora extracts spiked with UA).The Specificity test was conducted by comparing chromatograms of 6 matrix blanks (C. ochrochlora extracts) as matrix standards. The final calibration curves included three replicates per calibration point, and linearity was assessed by linear regression.Two types of calibration standards were used for assessment of linearity and sensitivity of the method: different concentrations of UA in a solvent (acetonitrile) only as blank standards, and the same concentrations of UA in a matrix solution  by visual observations of 10 analytical runs. The Limit of Detection (LOD) and Limit of Quantification (LOQ) were calculated using the formulas recommended by the guidelines mentioned above:The intra- and inter-day accuracy and precision measurements were conducted using measurements of three concentrations of UA  dissolved in a matrix (extract) on a single assay, repeated (with triplicates) three times within 1 week.The Matrix effect was determined by comparison of the retention time (Rt) and the level of MS signal of the representative blank matrix spiked with a predetermined amount of UA with those obtained for the corresponding amount of UA in the solvent (100% acetonitrile).C. uncialis and C. ochrochlora, a similar comparison was made using both matrix samples spiked with the same amount of UA. Samples used for measurements contained 20 µL of final extract per mL of acetonitrile.To assure the matrix match between The recovery was determined by comparing MS response level of spiked samples pre- and post-extraction, according to SANCO guide, using the average result of 4 replicates. The recovery percentage was calculated by dividing the value for the MS response of spiked pre-extracted sample by that of post extracted sample.Negative electrospray ionization mass spectrometry has been used for analysis of UA for some time , 27, resOptimisation of the LC–MS/MS protocol was directed toward obtaining distinctive symmetrical peaks combined with good resolution. The C8(2) LC column with 3 µm particle size run at a speed of no more than 0.25 mL/min with acetonitrile buffered with 0.1% of formic acid as a mobile phase demonstrated the best results for LC separation and provided the most favorable conditions for MS/MS measurements.Since some degree of matrix-induced ionisation suppression was previously reported , the modC. uncialis cannot be obtained, another species was chosen as a matching matrix source—Cladonia ochrochlora, a lichen of the same genus and habitat, and known to contain no UA [C. ochrochlora)  were compared using standard addition calibration curves created by spiking those extracts with known concentration of UA  Figs.  and 2a. C. uncialis samples (translated into Y-intercept shift), the calibration curves demonstrated no significant difference in slopes.Although not identical due to presence of UA in the C. ochrochlora extract as a matching matrix for future experiments. The insignificance of the matrix effect on quality of the signal in our experiments in contrast to the matrix effect observed in other laboratories [This supported the use of ratories  could beC. ochrochlora extracts without UA) with samples and spiked blanks (UA added to acetonitrile or matrix solution) demonstrated a high specificity of the method. Neither pure acetonitrile, nor pure matrix chromatograms showed any peaks with the UA fragmentation pattern, while peaks with the UA specific MS fragmentation were observed in samples and spiked blanks at very low (< 2 ng/mL) concentrations.A comparison of blanks  and 32.3 ng/mL (LoQ).While UA indeed could be detected at very low concentrations (< 2 ng/mL), the linearity range was more restrictive: after a series of adjustments the reliable measuring interval fell between 50 and 500 ng/mL. The Limit of Detection (LoD) and Limit of Quantification (LoQ) were calculated using the calibration curve obtained for Cladonia species under natural conditions vary between 0.4 and 3.8 in dry weight percentages, corresponding to 80–760 ng/mL in our experimental conditions [The concentration of the UA sample extracted, using the described procedure with 50 mL final volume of the extract, is expected to be recovered within the quantitative linear range since concentrations of UA in nditions , 30–32. Recovery, a characteristic of extraction efficiency, was measured by comparison of UA-spiked samples where the known concentration of UA  was added after or before the extraction and was 94%, consistent with that of Roach et al. . Since tPrecision was expressed as Coefficient of Variation (CV) and accuracy was expressed as Relative Error (RE), and were evaluated for three concentrations of UA within the linearity range: 50 ng/mL, 200 ng/mL and 500 ng/mL. The resulting fluctuations did not exceed 7% for Intra-assays and 11% for Inter-assays for CV, and 7% for RE calculations in both type of measurements , it is recommended that the calibration samples be included in every sequence in future experiments with re-evaluation of calibration graphs to negate the natural instability of signal in MS.C. ochrochlora) spiked with UA at the concentration of 400 ng/mL, was kept at room temperature in darkness for 1 month and assessed five times during that period. Inter-assay RE was 2.31%, which is an acceptable level of deviation, demonstrating stability of UA under experimental conditions in this study.The matrix solution (extract of Cladonia uncialis under the conditions in this study: sample preparation and storage, maintaining concentration of UA in the extracts between 50 and 500 ng/mL, and regular adjustments of calibration.The method suggested in the present article proves to be suitable for accurate measurements of UA concentration in dried field samples of The method presented in this paper was performed in a single laboratory and the validation was performed on a single species.


Ciona intestinalis in Drosophila perturbs the development of flies cultured under low‐nutrient conditions (media containing only glucose and yeast). Here we tested the effects of a wide range of nutritional supplements on Drosophila development, to gain insight into the physiological mechanism underlying this developmental failure. On low‐nutrient medium, larvae contained decreased amounts of triglycerides, lactate, and pyruvate, irrespective of AOX expression. Complex food supplements, including treacle (molasses), restored normal development to AOX‐expressing flies, but many individual additives did not. Inhibition of AOX by treacle extract was excluded as a mechanism, since the supplement did not alter the enzymatic activity of AOX in vitro. Furthermore, antibiotics did not influence the organismal phenotype, indicating that commensal microbes were not involved. Fractionation of treacle identified a water‐soluble fraction with low solubility in ethanol, rich in lactate and tricarboxylic acid cycle intermediates, which contained the critical activity. We propose that the partial activation of AOX during metamorphosis impairs the efficient use of stored metabolites, resulting in developmental failure.The mitochondrial alternative oxidase, AOX, present in most eukaryotes apart from vertebrates and insects, catalyzes the direct oxidation of ubiquinol by oxygen, by‐passing the terminal proton‐motive steps of the respiratory chain. Its physiological role is not fully understood, but it is proposed to buffer stresses in the respiratory chain similar to those encountered in mitochondrial diseases in humans. Previously, we found that the ubiquitous expression of AOX from  Drosophila expressing the alternative oxidase are unable to complete pupal development if reared on low‐nutrient medium.Additional nutrients are needed, to replace those normally manufactured cataplerotically. Most holometabolous insects, for example, lay their eggs on rich nutrient sources, which sustain rapid growth during larval development. Metabolites that are synthesized and stored during the larval stages are then used to drive the subsequent processes of cellular proliferation, migration, and differentiation. The program of metamorphosis during pupal stage is then executed in an environment where further feeding is not possible.The metabolic processes that characterize the larval and pupal stages may be crudely described as anabolic and catabolic, respectively. Nevertheless, the biosynthesis that occurs during larval development depends on a supply of biological energy, while pupae rely on converting some of their stored resources into new biomolecules, as well as fueling and regulating the morphogenetic program. Importantly, most of the energy generation, as well as the production of relevant metabolites for biosynthesis and signaling, originate within the mitochondria. The tricarboxylic acid (TCA) cycle, in particular, is central to catabolism, anabolism, and signaling, and is in turn reliant on the mitochondrial respiratory chain.Drosophila melanogaster has been extensively studied with regard to the molecular and cellular processes that underpin this developmental program. However, the metabolic events which accompany development have received less attention. D. melanogaster is considered a cosmopolitan species , nor mutations that abolish the synthesis of a vital prosthetic group of the enzyme  larvae reared on the low‐nutrient medium are deficient in specific stored nutrients, regardless of genotype. Together, these results imply that AOX‐expressing flies are unable to make proper use of this depleted metabolic store, so as to complete development. Finally, we identified a specific set of metabolites as the most likely candidates to rectify this deficiency, thus enabling AOX‐expressing flies to fully undergo metamorphosis.22.1Drosophila strains used in the study were the transgenic line UAS‐AOXF6 for C. intestinalis AOX (on chromosome 2), constructed in‐house and described previously , and the standard ubiquitous driver line daGAL4 (insertion on chromosome 3). Both lines were maintained as homozygotes and crossed together as indicated in figures. In addition, RNAi lines 30282  and 65175 , both for ATPCL, were crossed with daGAL4 to test the effects of ATPCL knockdown, using an internal control. Flies were maintained and cultured on standard high‐sugar medium , used at concentrations indicated in the figures. The effects of different media on eclosion were tested by mating batches of 10 virgin females and five males overnight in food vials containing complete medium, then transferring them daily to fresh food vials containing the medium under test, for egg laying. Pupae per vial and the number of eclosed adults were recorded.2.2C. intestinalis AOX and their parental cell‐line were cultured as previously . The medium containing doxycycline was replaced after 48 hr. AOX transgene expression was verified by western blot analysis using a customized antibody, as previously , and extracted by gentle inversion approximately 10 times. After the phases had fully separated they were isolated and each extracted twice more with the opposite solvent. A further 40 ml of water was added to the ether fraction and the two fractions were then left overnight in open beakers for all ether to evaporate. Each fraction was then made up into 40 ml fly food by adding the low‐nutrient medium ingredients, for testing in the eclosion assay, or else analyzed further by mass spectrometry, as described below, or used in respirometry. For further fractionation by ethanol precipitation, 10 ml of the aqueous fraction were decanted into 50 ml centrifuge tubes, and 99% ethanol added to bring the final ethanol concentration to the desired level (between 40% and 75%). After overnight precipitation at −20°C and centrifugation at 14,0002.5l‐Lactate assay kit/Pyruvate assay kit; Sigma). The reactions were performed in a black 96‐well microplate by mixing samples 1:5 with the reagent master mix and incubation at room temperature for 30 min. Fluorescence (excitation 535 nm and emission 590 nm) was measured at 1 s intervals using the same plate reader and normalized for protein content.To assay triglycerides, batches of 10 L3 (wandering‐stage) larvae were homogenized in 100 μl phosphate buffered saline (PBS)‐0.05% Tween , using a disposable plastic pestle. Samples were heated at 70°C for 5 min, cooled to room temperature, and vortexed. Aliquots of the homogenate (5 μl) were added to 100 μl of Triglyceride Reagent (Thermo Fisher Scientific) in transparent 96‐well plates. After incubation at 37°C for 30 min, absorbance at 540 nm was measured using a plate reader  and normalized for protein content based on the Bradford assay . For lactate and pyruvate assays, batches of 10 L3 larvae were homogenized similarly, in 6M guanidine hydrochloride on ice, then incubated at 95°C for 5 min. Supernatants were transferred to fresh vials and stored at −80°C, then thawed on ice and diluted 1:10 with water. Standards and reaction master mix were prepared according to the manufacturer’s protocol  and dried under constant nitrogen flow at room temperature. The resulting residual metabolites were converted into methoxime and trimethylsilyl derivatives by a two‐step procedure. First, dried samples were dissolved in 25 µl of methoxyamine hydrochloride solution , and incubated for 1 hr at 45°C. After the addition of 25 µl of N‐methyl‐N‐(trimethylsilyl)trifluoroacetamide (Sigma‐Aldrich) and a further 1 hr incubation at 45°C, samples were spiked with 25 µl of an alkane‐standard mixture . The analysis was performed using a gas chromatograph  combined with a time‐of‐flight mass spectrometer . Peak identification and data analysis used ChromaTOF software (Leco Corp.), NIST 2014 Mass Spectral Library and open‐source software Guineu v2. See Supporting Information for further details.2.7t test (Microsoft Excel) was used to assess significance when performing pairwise comparisons, implementing the Bonferroni correction where more than two groups were compared. When comparing multiple levels of a single factor to each other, one‐way analysis of variance (ANOVA) followed post hoc by the Tukey honestly significant difference (HSD) test (http://astatsa.com/) was applied. To test which of two factors was a significant determinant of a numerical outcome, two‐way ANOVA (GraphPad Prism) was used. Note that post hoc analysis  is only appropriate in cases where a significant interaction between the factors is detected by ANOVA, or where more than two levels of a given significant factor are compared. χ2 tests to compare observed and expected outcomes were performed online (https://www.graphpad.com/). Details are given in figure legends, as appropriate.Student’s 33.1daGAL4 driver produced a similar number of eggs as control strain matings  which enables AOX‐expressing flies to complete development on an otherwise low‐nutrient medium, we undertook a series of experiments in which we removed individual components from the standard medium, or added individual components to the low‐nutrient medium. Noting the exquisite temperature‐sensitivity of the phenotype citrate Figure a, multiv4 Figure c and eve4 Figure d.3.4Wolbachia  that had a significant, detrimental effect on larval development, but nevertheless allows us to measure relative effects on the completion of metamorphosis, and a much lower dose (15 μg/ml), typically used to eliminate intracellular bacteria such as 3.5Since the addition of specific dietary supplements to low‐nutrient medium failed to rescue the AOX‐associated developmental failure, we attempted to isolate, or at least enrich for, the active material present in the complex food additives. This approach was simplest for treacle, because it contained only very low levels of particulates. The published composition of treacle (Table S1) gives only an approximate description of its biologically active ingredients, insufficient for the present study. Therefore we used simple chemical fractionation to focus attention on the particular, active components. Ether extraction was used to separate treacle into aqueous and nonaqueous fractions which were then tested for their ability to support the development of AOX‐expressing flies. We reproducibly found the complementing activity only in the aqueous fraction Figure a. It was3.6Ciona AOX under the control of a doxycycline‐inducible promoter  and lactate , while AOX expression had no significant effect on these metabolites. Pyruvate, being interconvertible with lactate via LDH was also decreased in larvae grown on low‐nutrient medium , but not by AOX expression. These findings support the hypothesis that the low‐nutrient diet restricts the accumulation of stored nutritional resources and that it is the inability to mobilize these diminished resources that causes developmental failure in AOX‐expressing pupae.The above findings strongly suggest one (or both) of two scenarios, relating nutrient storage and AOX. Either (a) accumulated nutritional reserves are lower in AOX‐expressing larvae cultured on low‐nutrient medium, compared with control larvae or with AOX larvae grown on complete medium; or (b) nutritional reserves accumulated on the low‐nutrient medium are similar, regardless of genotype, but those resources are insufficient to enable AOX pupae to complete development, due to AOX activation during metamorphosis. To test these possibilities, we assayed the relative levels of triglycerides Figure a and lac3.8Drosophila development .Treacle and its various fractions that were tested earlier in the developmental assay were analyzed further by mass spectrometry, alongside standards for 48 common metabolites. Of these, the majority was detectable in treacle, and most of these were present in the aqueous fraction thereof Table . However4In this study, we established that the developmental failure of AOX‐expressing flies on low‐nutrient medium occurs specifically during the pupal stage Figure . Several4.1Drosophila  AOX variant would not suffice, since the transgene was expressed at a much lower level . However, the expression of neither cytosolically expressed GFP  pathway, driven by an insufficient supply of amino acids , full activity was not recovered in either the pellet or supernatant fractions Figure b. Based Importantly, the effect of the missing nutrients need not be solely on the actual synthesis of triglycerides and other stored metabolites for subsequent catabolism. Some of them are known to be involved in the generation of epigenetic signals, which may program metabolism during metamorphosis according to the prevailing conditions. For example, specific catabolic enzymes may be required in higher amounts under conditions of dietary stress, enabling the pupa to survive on a lower repository of stored triglycerides. In combination with partially activated AOX, this could lead to a catastrophic depletion of resources. For example, the list of detected compounds in the active treacle fraction included succinate and several other TCA cycle intermediates that could modulate the activity of alpha‐ketoglutarate‐dependent histone demethylases , AOX expression exacerbated rather than alleviated the phenotype (Dogan et al., Drosophila. Clearly, before AOX is to be developed as a therapeutic, the conditions for, and consequences of, its activation need to be evaluated in detail. However, if judiciously exploited, such properties could also be beneficial, for example in allowing better regulation of the enzyme in a therapeutic setting, or even using AOX to improve adaptation to nutritional stresses.Mice ubiquitously expressing 4.4As discussed in Section 1, the life cycle of holometabolous insects provides a “best of all worlds” scenario, in which maximum advantage can be taken of food resources for growth and dispersal to maintain genetic fitness and adaptability, while minimizing vulnerability to predation.Drosophila researchers culture flies in the laboratory.In an insect species that would be naturally endowed with AOX, there would be strong selective pressure to evolve adjustments to the ways in which the onset of metamorphosis is regulated, so as to take account of potentially decreased bioenergetic efficiency during the pupal stage. This pressure might also favor the evolutionary loss of AOX itself if this inherent vulnerability to starvation outweighs the stress‐resistance AOX may confer. In practice, the natural environment is likely to be much more similar to what we here describe as a low‐nutrient medium than the complete medium on which we and almost all other Since AOX has indeed been lost during insect evolution, our findings could offer one possible explanation.The authors declare that there are no conflict of interests.Supporting informationClick here for additional data file.


While many factors contributed to the successful elimination of polio from India, partnership and coordination mechanisms at multiple levels that have evolved over the years have been an important element. The lessons learned from these partnership and coordination mechanisms among various stakeholders involved in service delivery, surveillance, community mobilization, and governance deserve documentation as a legacy of the program. This article discusses the various processes and techniques adopted to build strong partnerships and coordination mechanisms among stakeholders by optimizing their strengths and using opportunities that lead toward the eradication of polio from India. Secondary data and literature review of relevant reports, papers and documents were adopted as the methodology for developing this research article. The article provides a model conceptual framework for partnerships and applies that framework to the CORE Group Polio Project (CGPP) partnerships in India and the partnerships among stakeholders for polio eradication in India. The learnings and expertise of the CGPP in developing, managing, and nurturing partnerships can be adapted and replicated for elimination or controlling other diseases  and for ending preventable child and maternal deaths. Polio is one of the very few diseases that have the potential to be eradicated because of the following: 1) the poliovirus affects only human beings and there is no animal reservoir, 2) the life span of the virus in the environment is very short, 3) immunity against polio is lifelong, and 4) an effective and low-cost vaccine is available. Nonetheless, the eradication of polio has turned out to be far more difficult than expected, requiring far greater sums of money, much broader stakeholder participation, and a much longer time span.5 At present, wild poliovirus (WPV) transmission is limited to very small geographic areas with small populations within Afghanistan and Pakistan.4 The type 2 WPV was declared eradicated in 2015.3In 1988, the World Health Assembly passed a resolution to eradicate polio. Since then, the Global Polio Eradication Initiative (GPEI), led by the WHO, the United Nations Children’s Fund (UNICEF), the US Centers for Disease Control and Prevention, and Rotary International, has provided support to governments of polio-affected countries. Other partners, including the U.S. Agency for International Development (USAID), the Bill & Melinda Gates Foundation, civil society organizations and non-governmental organizations (NGOs), and networks such as the CORE Group Polio Project (CGPP), have worked together to support the GPEI across the globe. Despite unanticipated challenges and setbacks, the goal of eradication has almost been achieved. The number of polio cases reported each year since 1971 has declined from 49,293 in 1971  to 18 in 2019 as of May 7, 2019 .2,3,5 At1. Strengthening of routine immunization2. Surveillance for acute flaccid paralysis cases3. Supplementary immunization activities†4. “Mop-up campaigns”‡This achievement has been attributed to the success of the polio eradication strategy, which consists of the following four pillars:6 The last case of WPV was recorded in 2011  proved to be the key to India’s success. This required committed leadership, a devoted health workforce, tailor-made strategies, data-driven planning through surveillance and research, meticulous monitoring, targeted communication and social mobilization efforts, strong partnerships, and adequate funding. The remaining polio-endemic countries, Nigeria, Afghanistan, and Pakistan, have now adopted India’s best practices and lessons learned related to polio eradication. India’s polio program demonstrates that ambitious health goals can be reached even in areas with poor health systems, and India’s success now serves as a model for polio eradication and other public health programs around the world.12At the 7th meeting of the South-East Asia Regional Certification Commission for Polio Eradication held in New Delhi on March 27, 2014, the region was declared polio-free, signifying that 80 percent of world’s population now lived in certified polio-free regions.3 and gives a clear road map to all the stakeholders for the last phase of the program. This plan is based on three themes: 1) eradication : stopping transmission; 2) integration: collaboration with immunization and emergency teams to eradicate polio and to protect populations; and 3) certification: certify and containment of all WPV cases and ensure long-term polio security.14 These themes underscore the need for a vital future global health policy and programs.As a longtime partner in the GPEI, the CGPP decided to document the partnership and coordination mechanisms which could be helpful to other public health initiatives. This documentation is also in line with one of the objectives of “Polio Endgame Strategy 2019–2023”The partnership and collaboration model developed by the polio eradication program in India was the result of collective efforts of the government; UN agencies; NGOs at the international, national, and local levels; and other partners who worked tirelessly. The CGPP is one of the valuable partners in the national polio eradication program.§26–27 However, this critical element of polio eradication has not been well documented. In particular, the role of civil society organizations such as the CGPP and the contribution of NGOs have been inadequately documented. One of the leaders of the GPEI has written that “much of the wealth of the GPEI’s lessons will never be captured in textbooks or academic articles because many of the details, especially with regard to what didn’t work, will survive only in the knowledge and experience of thousands of individuals who worked at various levels of this global initiative.”15 With this in mind, the CGPP has decided to document its experiences in India with partnerships as a key element in the success of the GPEI there.Many times it has been said and heard that “partnerships” were key to the success of polio eradication in India as well as globally.1. The CGPP partnership in which international and national NGOs collaborated together at the country level under the leadership of a secretariat to support polio eradication activities.2. The partnership among governments , the WHO, UNICEF, Rotary International, and the CGPP.This article discusses how the partnership for polio eradication evolvedThe authors reviewed reports, articles, working papers, and research articles pertaining to partnerships in health programs in general and to the health partnerships developed for polio eradication in India. We created a general conceptual framework for partnerships and then applied that framework to the CGPP partnership in India and the partnership among stakeholders for polio eradication in India.16 The term “partnership” is generally used interchangeably with various other terms such as collaboration, alliance, coalition, network, interorganizational relationship, joint advocacy campaign, and taskforce.17 A “collaboration continuum” exists which places partnerships along a spectrum ranging from philanthropic, in which a charitable donor and recipient exchange resources focused on specific activities, to integrative, in which “the partners’ missions, people, and activities begin to merge into more collective action and organizational integration.”18Based on a review of the literature, the following definitions and frameworks were found to be the most appropriate for the partnerships formed for polio eradication. A partnership comprises organizations that have common goals and objectives and that combine resources to implement collective activities. Partnerships can prevent duplication of efforts, ensure synergy of resources, and augment the overall leadership within the country.1920A continuum of organizational relationships can be described within a systems science framework, as shown in 20Coalitions have been recognized as particularly effective and efficient vehicles for identifying and prioritizing system-change strategies and for making the changes happen. Partnerships have been identified as playing a central role in enabling community coalitions and community-based health programs achieve system change and address health disparities.21 It achieves this through collaborative NGO action and learning. In 1999, the CORE Group received USAID funding for polio eradication, establishing the Polio Eradication Initiative Project that later became known as the CGPP.The CORE Group is a membership association of more than 100 U.S.-based international NGOs that strengthen local capacity on a global scale to measurably improve the health and well-being of children and women in developing countries.22 The initial paper of this series provides a detailed history of the global CGPP and its secretariat model.15The purpose of the CGPP is to engage NGOs in polio eradication mainly through social mobilization, community-based surveillance for acute flaccid paralysis (AFP), cross-border coordination, and related activities. The CGPP structure includes a secretariat in each country where it operates, CORE Group international NGO members, and their local NGO partners. The secretariat consists of a small team of “neutral” technical advisors . The secretariat team facilitates communication, coordination, and transparent decision-making among all partners—unifying the community-level expertise of international NGOs and local NGOs with the benefit of international knowledge and strategies of the GPEI partners. The CGPP countries have successfully implemented the secretariat model to coordinate and promote civil society engagement in polio eradication, while simultaneously injecting a crucial community-level component through the coordinated activities of thousands of community health workers.15 the CGPP began in 1999 at a critical juncture in the GPEI when it was becoming apparent that small pockets of unvaccinated children in a number of countries were the source of continued transmission of WPV and the traditional campaign approach to immunization, although achieving high levels of coverage, was not effective in wiping out these pockets of persistent transmission.As described elsewhere in this series,15 During this time, the CGPP and UNICEF realigned and redistributed their work at the block (sub-district) level so that there was no duplication of efforts and resources.The CGPP secretariat in India works in close collaboration with the Ministry of Health and Family Welfare, the government of India, the WHO, UNICEF, Rotary International, and USAID. Until 2003, the CGPP and UNICEF had deployed social mobilizers in the areas where the polio program faced major challenges in terms of resistance to polio immunization. Most of the time, both agencies were working in the same geographic areas. After working together for a few years, the CGPP and UNICEF decided to form the Social Mobilization Network (SMNet) to provide more concentrated support for social mobilization in high-risk areas of Uttar Pradesh.The success of the CGPP arises from its ability to manage partnerships with diverse organizations that are working at various levels and that have very different capacities. The CGPP partnership, working since 1999 in India, could be one of the longest surviving international partnerships among public health programs in India. This partnership emerged as the outcome of working together and strong commitment of donors for an important shared goal. Initially, the CGPP did not have any ground rules, bylaws, formal guidelines, or legal standing to bind the partners together. The partnership evolved in the sense that the partners got better and better at working together over time despite the fact that no operational guidelines or terms of reference were available at the beginning. As the CGPP gained experience, a strong and trusting partnership emerged that was also quite flexible in its approaches. The partners of CGPP had different capabilities that were not necessarily related to polio or immunization. Some were good in community mobilization; others had expertise in program management, advocacy, implementation of targeted interventions, monitoring, developing demonstration sites, or implementing programs efficiently. The CGPP collaborated with diverse organizations that were locally recognized in their respective fields. The CGPP strategically positioned them to effectively address the goal of elimination of polio transmission. One size does not fit all, so for each partner, the CGPP secretariat had to use a different approach because their capacity and management structures were different. For instance, some of the local NGOs had simple management structures and was flexible in all forms of implementation. What they required from the CGPP mentoring on the technical aspects of polio eradication. On the other hand, some large organizations had complex, bureaucratic management systems that had to be simplified for speedy implementation in the field. Coordination with the local implementing NGO partners and with government required a different strategy. While working with the government, the CGPP secretariat represented a group of NGOs, with the government in a leadership position. While working with NGO partners, the secretariat provided coordination, technical expertise, and collaboration to build their capacity.23:1. The CGPP/India working with USAID, the CORE Group, and CGPP global headquarters in Washington, DC2. The CGPP/India Secretariat, coordinating the consortium of international, national, and local NGOs and other community-based organizations3. The CGPP/India, working with the SMNet, UNICEF, the WHO National Polio Surveillance Project (NPSP), and Rotary International4. The CGPP/India, working with the government of IndiaSimilarly, to work with donors and partners  required tailored approaches that had their own pace, involvement, and commitments. Fortunately, the CGPP secretariat and its partners learned from experience and started engaging effectively with these different categories of partners to coordinate and to channel resources. Four partnerships evolved over the life of the projectThe CGPP secretariat plays different roles while partnering with different agencies. Its secretariat coordinates with a consortium of international and national NGOs, provides technical support to them, and represents them at various levels. As a partner in the SMNet, the CGPP consortium partners with UNICEF in the field and with the WHO-NPSP, Rotary International, and the government at various levels. 23 The CGPP in India complemented the efforts of the Ministry of Health and Family Welfare, the NPSP, the WHO, and UNICEF in high-risk areas as per their mutually agreed strategy.In 1999, the CGPP established a secretariat in New Delhi to coordinate activities of the CGPP in India as well as in Bangladesh and Nepal. Five CORE Group international NGO members initially agreed to work directly or through their NGO partners: CARE, the Christian Children’s Fund (now Child Fund), Project Concern International (PCI), the Adventist Development and Relief Agency (ADRA), and World Vision. At present, three international NGOs are working with the CGPP in India: PCI, ADRA, and Catholic Relief Services (CRS) India. They and their eight local NGO partners work in 12 districts of Uttar Pradesh, covering a population of approximately 30 million people. Thus, international and national/local NGO partners willingly started working together under the leadership of the CGPP independent secretariat.Soon the CGPP secretariat became the critical nexus between those working in these high-risk areas  and the high-level technical and donor entities .The CGPP secretariat, as well as UNICEF, the NPSP, and Rotary International, transferred the strategies and implementing activities developed at a higher level down to the local field level through their respective organizational channels while at the same time communicating grassroots-level voices to policymakers. The CGPP secretariat also provided technical support to its collaborating NGOs.The CGPP secretariat in India convened quarterly meetings of its implementing partners, including field staff, to review its activities and plans. The secretariat staff made regular field visits, which were useful for supportive supervision and for providing on-the-job training to the field staff. These field visits were also valuable for strengthening coordination with district-level officials of the government and other partners such as the WHO, UNICEF, and Rotary International, all of whom had staff stationed in the area.Every year, the CGPP secretariat in India organized training for all of the NGO staff using master trainers who were handpicked from among the partner NGO staff and then trained by the CGPP. These trainings were effective at building capacity and strengthening coordination.The partnership between various stakeholders was achieved using multiple mechanisms such as the Social Mobilization Working Group (described further in the following paragraphs) and coordination meetings among partners at the sub-national and district levels. The Social Mobilization Working Group consisted of representatives from UNICEF, the WHO-NPSP, the CGPP, Rotary International, and the government of India. This group frequently discussed the communication and social mobilization challenges of the polio eradication program and suggested necessary changes in the strategy.The role of each partner was well defined and distinct to ensure complementarity and to avoid duplication of effort. The government of India provided leadership as well as human, material, and financial resources, whereas the NPSP supervised the surveillance of acute flaccid paralysis, provided technical support for the operation of the polio immunization campaigns , and supported the monitoring of the supplemental immunization activities. Rotary International played a very important role by supporting advocacy and also providing financial resources. UNICEF provided technical support for vaccine procurement and media campaigns at the national and state levels, and social mobilization through the SMNet. The CGPP in India supported social mobilization through the SMNet; represented NGOs at the national and state levels for better coordination, facilitation, and support; and also provided essential technical and supervisory support to the NGOs.UNICEF, the NPSP, Rotary International, and the CGPP formed the SMNet in 2003 in the state of Uttar Pradesh, and their respective roles are outlined in The CGPP and UNICEF developed various materials for training, behavior-change communication, supervision, and human resource management with uniform guidelines. Examples of these tools are provided in 24These materials were used consistently across the SMNet areas. Gradually, the SMNet standardized field staff positions and functions, expanded and refined data collection, and incorporated increasingly focused behavior-change communication techniques. CORE Group Polio Project and UNICEF implemented synchronized social mobilization activities using community-level workers called community mobilization coordinators. The three-tier network of community mobilizers  carries out the main work of the SMNet. Community mobilization coordinators work with underserved communities in planning, implementing, and monitoring social mobilization and other immunization activities in high-risk areas.The purpose of partner meetings was to sort out operational issues and update all the concerned stakeholders about the current field situation. For smooth and effective coordination, mechanisms were created to address the implementing challenges. These structures were made functional through frequent engagements, such as meetings, field visits, and task force meetings held by the CGPP secretariat and other partners.At the national level, representatives of the WHO, the NPSP, UNICEF, the CGPP, and the government of India formed a Social Mobilization Working Group. The India Expert Advisory Group for Polio, represented by independent experts, government functionaries, academicians, and polio partners, met every 6 months to review and guide the program at the national level. State task force meetings were also implemented at the state level in Lucknow, the capital of Uttar Pradesh. For these meetings, government and other polio partners came together to deliberate and decide on program priorities and issues to address.Under these structures, a district task force was formed under the chairmanship of the district collector and magistrate, a government official who is the key decision-maker at the district level. Either the district collector and magistrate  or the district chief medical officer chaired its meetings to review the progress of polio eradication in the district. The meeting was attended by senior officials of all the relevant government departments and representatives of the CGPP, UNICEF, Rotary International, religious leaders, and the NPSP to review the vaccination coverage and identify administrative, operational, and communication barriers encountered during the previous polio vaccination round. As a result of these meetings, it was possible to address gaps and barriers on a timely basis.The district task force meeting was the forum for discussing evidence-based actions and next steps. Accordingly, the district collector and magistrate issued directives. Reaching every child, whether in the house, outside the house, or even out of the village/city at the market place or on the move at migrant/nomadic sites, required support from different departments of the government and from the community. Block task forces were formed at the sub-district level. A block is the smallest administrative unit of the government administrative structure. Block review meetings  were conducted regularly and were attended by district and block level officials of WHO, UNICEF, and CGPP partners.11Village-level Social Mobilization Working Groups and interface meetings produced important functional partnerships and strong coordination among frontline workers . Block-level supervisors from the SMNet and the NPSP also attended these “interface” meetings. These meetings helped to mobilize local resources to address local issues in a timely fashion. Participation of CGPP representatives in these meetings helped to improve networking and coordination with all partners. This led to improved service delivery to the community. One example is the facilitation of health camps/routine immunization drives in selected outreach areas where resistance was high. These were a response to the frequently stated request for better basic health services. These regular meetings and the subsequent health camps led to improved relationships between the health service providers such as immunization workers and the community.Success can only be achieved once challenges have been mitigated. The CGPP also faced several such challenges. Because there were many diverse partners and some were large international organizations (such as the WHO and UNICEF), whereas others were local, small community-based NGOs that did not have much experience with this type of collaboration, there was apprehension and even mistrust between these groups of organizations. The larger partners had doubts about the capacities of NGOs, whereas the NGOs were apprehensive about losing their identity and operational freedom working under a big umbrella. Here, the CGPP secretariat played a facilitating role between them to develop mutual trust and clarify doubts so that good teamwork could be established.Enabling partner NGOs to deliver in an emergency mode as part of a large-scale program while overcoming bureaucratic hurdles was a major challenge. Most of the NGOs did not have the experience of working very rapidly and rigorously, for example. In the first few years of this collaboration, supplemental immunization activity (SIA) rounds were conducted every month. This required a lot of work, and some NGOs were not used to this type of intensive, almost continuous campaign work. The CGPP facilitated and invested resources in building the capacity of these NGOs. Working with UNICEF, the CGPP strengthened the SMNet by, as mentioned earlier, institutionalizing uniform nomenclature and terms of reference for workers/staff and their training. This took a lot of effort, as each NGO had its own organizational hierarchy, salary structure, mandate, and priorities. The changing epidemiology of local poliovirus transmission required considerable operational flexibility and preparedness. The community considered the SMNet staff as the face of all government machinery. The community had apprehensions about the government’s programs and its functionaries. This made working as a bridge between the community and the government a major challenge. Following the strict requirements of the donor to focus all resources exclusively on polio was also challenging because of the great need (and community demand) to expand the focus to other problems beyond polio that were much more important to the community. However, the CGPP and SMNet succeeded in addressing these challenges through constant effort, facilitation, understanding of the complexities at the ground level, and establishment of effective and regular communication between the community, NGOs, government, and donors.25The success of the SMNet in contributing to the elimination of WPV transmission in India is evidence that the secretariat model was effective in enabling international, national, and local NGOs to develop strong long-term partnerships in which mutual respect emerged and in which each organization recognized that all participating organizations were complementing the capacities of the others. Field-based learning and continuous analysis of inputs, processes, and outputs were responsible for the emergence of such strong partnerships. The secretariat model enhanced the partnerships because it facilitated NGO representation in the immunization task force at state and national levels. The secretariat represented all consortium members as a single entity to the government and other international agencies such as the WHO and UNICEF. This also helped the government and polio partners in the sense that instead of dealing with many NGO organizations they had to deal with only one.The CGPP proved to be a good example of a consortium of NGOs coordinated by a secretariat that is not directly involved in implementation. Because of its neutral leadership (in the sense that it was independent of the NGOs themselves), the CGPP secretariat could provide effective coordination with the government at various levels and with UN agencies, and it could monitor program implementation of multiple partners in an unbiased manner. There was ample evidence that the secretariat-led model resulted in clear and concrete benefits to the program and its partners.However, the secretariat model does need some improvement for future replication for purposes beyond polio eradication or for scaling up in other geographic regions. The CGPP secretariat has no formal independent legal identity of its own; neither does it have a memorandum of understanding with the government. The lack of legal status acted as a barrier during negotiations at all levels. A secretariat that is coordinating the efforts of international NGOs, national and state government officials, and multinational organizations needs to have a distinct legal status and authority in financial and human resource matters. In addition, a lack of an operations manual or working guidelines led at times to some confusion in roles and responsibilities.Box 1During the course of implementation over the previous 18 years, various changes in the CGPP partners have taken place. In 2003, the CRS joined the CGPP in new areas. In 2005, there was a reassignment of districts between the CRS and the ADRA. In 2007, World Vision withdrew, and its areas were assigned to the PCI and the local NGO partners of the PCI. All these changes happened at a time when the polio eradication program was facing multiple challenges, such as a large number of families refusing vaccination and an increasing number of polio cases. The WHO, UNICEF, and the government all shared their apprehensions then that these changes might hamper social mobilization in the CGPP areas. But the CGPP secretariat and CGPP partners did manage these transitions very effectively without any discernible effect on program implementation. This was achieved by a very proactive secretariat team at all levels and capacity building of field staff. The result was that an NGO partner was able to join the CGPP at various stages of the CGPP implementation and begin its own involvement at the stage where the CGPP was at the time.As the GPEI nears its goal of polio eradication, now is the right time to obtain legal registration for the CGPP secretariat in India. This would help to sustain the brand value of the CGPP and its relationships with the community and the government. It would also enable the secretariat to address other public health issues in India. A small group of advisors would also help it to further its agenda in the country.There was a need for considerable compromise and adjustment when the CGPP partnership with its consortium members was first established. Problems that arose because of ideological and strategic differences had to be sorted out. Another indicator of success of this partnership model was that changes in leadership and staff turnover did not affect the functioning or the effectiveness of the CGPP. These partnerships and networking have been going on for 20 years now and the SMNet has been functioning smoothly for 16 years. The feasibility of the partnership was facilitated by the fact that the partners were dealing with a global initiative to eradicate polio, which called for united, synergistic action.A large set of diverse partners  all worked together for eradicating the poliovirus from India. This partnership that has been sustained for two decades now proves that simple mechanisms developed by the partners could be major lessons for global public health initiatives that require collaborative functioning.The partners had a common vision, mission, and program goal. This singular focus on polio eradication brought about cohesion of forces despite the diversity of the partners. There was a sense of ownership resulting from involvement of all the partners at every stage of the program. There was minimal conflict of interests because of a clear overarching goal of global importance, a uniform agenda, role clarity, and the operational freedom with which partners had to continue with their other individual agendas apart from polio eradication.A high level of enthusiasm between all the partners could be sustained for a long period of time because of the shared goal of polio eradication. All the partners were willing to seize the opportunity and be part of the global initiative. There was considerable pride in their involvement in the cause of polio eradication. Motivation was sustained because of the measurable reduction in polio cases.26The CGPP secretariat and consortium model ensured transparency regarding the structure of the partnership and the participatory nature of decision-making. The model was also able to establish accountability resulting from participatory monitoring and peer influence. The Polio Eradication Program in India was open to scrutiny by many groups, including the government and international agencies. This was one of the programs that used monitoring data and took observations of the monitors very seriously. Every evening during SIAs, senior functionaries from the government and partners reviewed the performance at all levels and immediate corrective actions were taken by the concerned partners. If there were unresolved communication challenges or if a few families or group of families were refusing to vaccinate their children in a specific area (such as a village or an urban area), then immediately a joint action plan would be made with the CGPP/UNICEF, government, and WHO officials to resolve the issues and vaccinate each and every child. This helped to establish accountability. Characteristics of successful partnerships with communities identified by Shortell et al. and examples of these successful partnership characteristics of the CGPP are summarized in With respect to “patching” and repositioning of assets, it is of particular note that in the beginning, the CGPP was working in many states in India but as program needs changed along with resource constraints, the work areas narrowed to only Uttar Pradesh state. In 2011, a WPV case was identified in Hawrah district in West Bengal. As an emergency response to this, the CGPP established a new network with three local NGO partners and funding support from UNICEF. A team of experienced CGPP staff from Uttar Pradesh were deployed on a rotation basis for field support to local teams.The partnership model developed by the Polio Eradication Program in India was highly functional and played a crucial role in eliminating polio from the country. The CGPP was itself an important partnership that linked NGOs with district- and block-level partners in high-risk areas. The shared goal of polio eradication made it possible for the partnership model to be effective. The success of polio eradication has strengthened the motivation of partners to work together and collaborate for addressing public health issues.27These coordination mechanisms are now being used for strengthening routine immunization and introducing new vaccines to India. Similar coordinating structures such as the Immunization Action Group (IAG) at the national level, and immunization task forces are being formed at state, district, and subdistrict levels for strengthening routine immunization and introducing new vaccines such as rubella, and rotavirus vaccines. Members of the IAG are similar to those of the India Expert Advisory Group (IEAG) for polio, with few additions. Current members and invitees for the IAG are representatives of the Ministry of Health and Family Welfare, the government of India, UNICEF, the WHO, USAID, the Bill & Melinda Gates Foundation, the CORE Group, the Immunization Technical Support Unit, John Snow, Inc., the Indian Council of Medical Research, the United Nations Development Program, and GAVI, the Vaccine Alliance.The state task force on routine immunization now works in a similar fashion as do the state task forces for polio. In Uttar Pradesh state, where polio partners have focused their efforts, they have also continued their contributions to strengthening routine immunization. The state task force on routine immunization continues to meet frequently to discuss progress and challenges. Special routine immunization campaigns such as Mission Indradhanush have adopted a similar approach for coordination that was used for polio immunization campaigns.The CGPP in India has traveled a long journey and encountered multiple challenges, but it found ways of overcoming these challenges. Developing partnerships has proven to be a critical piece of the polio elimination success in India along with social mobilization and communication interventions for polio immunization campaigns, capacity building of functionaries, and monitoring. The government of India and all partners need to continue their efforts to sustain this partnership for addressing other public health priorities.The lessons learned and expertise gained by the CGPP in developing, managing, and nurturing partnerships can be adapted and replicated to control other diseases  and for elimination of preventable child and maternal deaths. Maintaining and further strengthening the partnership model for polio eradication in India and using it to address other public health priorities in India could be the real legacy of the Polio Eradication Program in India.