Drosophila.Existing genetic methods of neuronal targeting do not routinely achieve the resolution required for mapping brain circuits. New approaches are thus necessary. Here, we introduce a method for refined neuronal targeting that can be applied iteratively. Restriction achieved at the first step can be further refined in a second step, if necessary. The method relies on first isolating neurons within a targeted group  according to their developmental lineages, and then intersectionally limiting the number of lineages by selecting only those in which two distinct neuroblast enhancers are active. The neuroblast enhancers drive expression of split Cre recombinase fragments. These are fused to non-interacting pairs of split inteins, which ensure reconstitution of active Cre when all fragments are expressed in the same neuroblast. Active Cre renders all neuroblast-derived cells in a lineage permissive for Gal4 activity. We demonstrate how this system can facilitate neural circuit-mapping in  In humans – as well as flies and most other animals – the brain controls how we move and behave, and regulates heartbeat, breathing and other core processes. To perform these different roles, cells known as neurons form large networks that quickly carry messages around the brain and to other parts of the body. In order to fully understand how the brain works, it is important to first understand how individual neurons connect to each other and operate within these networks.Fruit flies and other animals with small brains are often used as models to study how the brain works. There are several methods currently available that allow researchers to manipulate small groups of fruit fly neurons for study, and in some cases it is even possible to target individual neurons. However, it remains an aspirational goal to be able to target every neuron in the fly brain individually.The Gal4-UAS system is a way of manipulating gene activity widely used to study neurons in fruit flies. The system consists of two parts: a protein that can bind DNA and control the activity of genes ; and a genetic sequence (the UAS) that tells Gal4 where to bind and therefore which genes to activate. Fruit flies can be genetically engineered so that only specific cells make Gal4. This makes it possible, for example, to limit the activity of a gene under the control of the UAS to a specific set of neurons and therefore to identify or target these neurons. Luan et al. developed a new technique named SpaRCLIn that allows the targeting of a subset of neurons within a group already identified with the Gal4-UAS system.During embryonic development, all neurons originate from a small pool of cells called neuroblasts, and it is possible to target the descendants of particular neuroblasts. SpaRCLIn exploits this strategy to limit the activity of Gal4 to smaller and smaller numbers of neuroblast descendants. In this way, Luan et al. found that SpaRCLIn was routinely capable of limiting patterns of Gal4 activity to one, or a few, neurons at a time. Further experiments used SpaRCLIn to identify two pairs of neurons that trigger a well-known feeding behavior in fruit flies. Luan et al. also developed a SpaRCLIn toolkit that will form the basis of a community resource other researchers can use to study neurons in fruit flies. These findings could also benefit researchers developing similar tools in mice and other animals. An essential step in mapping brain circuits is identifying the function of the individual neurons that comprise them. This is commonly achieved by manipulating neuronal function using effectors encoded by transgenes whose expression is targeted to small subsets of cells using the regulatory elements of neurally-expressed genes . While iAn attractive approach to increase the specificity of neuronal targeting is to identify neurons based not only on the genes they express in the terminally differentiated state , but also on their developmental history . Most neRecombinase-based intersectional methods that combine information about lineage and cell type have been developed in both mice and fruit flies and have been shown to substantially restrict targeting to cell groups of interest . HoweverSplit Cre-assisted Restriction of Cell Class-Lineage Intersections,’ or SpaRCLIn) generalizes the capabilities of the CLIn technique introduced by Drosophila lineages while maintaining compatibility with all existing Drosophila Gal4 driver lines. To facilitate SpaRCLIn’s use, we have generated a variety of tools, including two libraries of transgenic fly lines, each of which expresses distinct Split Cre components under the control of 134 different NBEs. We characterize the efficacy of these SpaRCLIn reagents and provide examples of their use in restricted neuronal targeting and circuit-mapping.To achieve such lineage refinement, we introduce here a combinatorial method analogous to the Split Gal4 technique used to restrict neuronal targeting to the intersection of two TEG expression patterns . We restSpaRCLIn was developed to refine the expression pattern of a Gal4 driver using the basic strategy shown in Drosophila, such as MARCM  which together with CreC form the basis of the tripartite Split Cre system , we used them to generate transgenic fly lines in which they were expressed in patterns dictated by individual enhancers that exhibited activity in neuroblasts. Most of the NBEs selected for this purpose were taken from the large collection of enhancer fragments with fully defined sequences created by the Rubin lab  #43. Moss of NBs . Howevers of NBs .A and CreAB fragments under the control of a compound enhancer formed by fusing individual enhancer elements of the NB-specific genes, deadpan (dpn) and nervous fingers-1 . This synthetic dpn-nerfin-1 enhancer (i.e. DNE) combines the complementary temporal characteristics of both component enhancers, maintaining strong, broad, and specific activity throughout embryonic neurogenesis , also generated expression patterns in neuronal lineages of third instar larvae  and compared these patterns with expression observed in all NBE-CreB∩DNE-CreA∩ DNE-CreC >CreTom crosses. The large size of the patterns and the inability to reliably identify identical neurons and lineages across preparations prevented a systematic analysis, but in general the patterns were similar across preparations for any given cross , suggesting that particular enhancers may strongly influence the extent of labeling. Variability of labeling also appeared to be enhancer-dependent in that use of the same enhancer (i.e. R17A10) to drive both Crehis case , their epan-Gal4) that selectively expresses in Bursicon-responsive neurons  that contained actin^Gal80^myr-tdTomato and a 10XUAS-mCD8GFP reporter  intersections, examined using a UAS-GFP reporter, were clearly restricted relative to rkpan-Gal4 expression , a motor pattern normally elicited by gustatory stimuli, but also by the hormone Bursicon in newly eclosed flies . Robust  neurons . Expressn system . In thisreporter . Progenypression , but theC components were combined pairwise with NBE-CreB components made using the same 23 enhancers  screen in which the 23 identified NBE-Crenhancers . The latxtension .pan-Gal4 expression patterns in flies exhibiting this phenotype were substantially reduced for many of the intersections tested and they consistently included particular neurons in the subesophageal zone (SEZ) that were characterized by somata near the saddle, broad arbors along the superior gnathal ganglion (GNG), and axons that extended medially before turning, with one branch coursing down each side of the midline and then turning laterally along the medial-inferior edges of the GNG , all of which exhibited PE induction upon heating. Indeed, all 36 animals from this intersection that tested positive for the PE phenotype and were successfully dissected showed expression in the PErk neurons, while none of the animals (n = 38) that tested negative had such expression (B∩10G07-CreC). All 19 animals that exhibited induced PE in this intersection had expression in the PErk neurons, and in three animals these were the only neurons present. A third intersection that yielded the PE phenotype in all animals likewise showed consistent expression in the PErk neurons, but the correlation between the PE phenotype and expression in these neurons was somewhat less readily established because of expression in other neurons  sites and encodes a Flp recombinase gene that is co-expressed with CreAB (AB gene and thus limit its expression to early (embryonic) neuroblasts  produced progeny with PE phenotypes at frequencies ranging from 9–17%. Although these frequencies were considerably lower than those obtained using DNE-CreAB, the resulting expression patterns were substantially sparser compared with those of progeny from DNE-CreAB crosses (rk neurons (n = 40). In contrast, only one of the animals examined that lacked the phenotype had these neurons (n = 39). A strong correlation between PE and the presence of the PErk neurons was thus observed, again permitting the conclusion that these neurons are substrates for the behavioral phenotype. We conclude that FRTerminator-based Step one screens may serve as a useful shortcut to serial Step one and Step two screens for restricting Gal4 expression and identifying functionally important neuronal subsets.To determine whether the FRTerminator might therefore expedite parsing of Gal4 expression using the SpaRCLIn system, we repeated selected crosses from the rk crosses . All aniThe SpaRCLIn system introduced here permits the refined targeting of neurons within a group of interest based on both their developmental origins and their patterns of gene expression in the terminally differentiated state. By permitting the combinatorial targeting of many, if not most, of the neuroblasts that generate the mature CNS, the SpaRCLIn system provides end-users with a comprehensive, ‘off-the-shelf’ set of reagents for systematically isolating and characterizing the anatomy and function of specific neurons. The reagents that we have created include extensive lineage-selective Split Cre lines for bipartite (Step 1) and tripartite (Step 2) neuronal screens, in addition to a range of tools that facilitate application of the system. Dual effector and reporter constructs reduce the number of transgenes required to implement the system, and a self-terminating Split Cre component (i.e. FRTerminator) can be used to expedite screening in favorable circumstances. The system is compatible with existing Gal4 driver lines and the examples provided here indicate that it is capable of routinely parsing Gal4 expression patterns into subsets of neurons numbering in the single digits.C lines and 70 NBE-CreB∩C Step two crosses—were required to identify two pairs of command-like neurons capable of inducing PE upon activation (i.e. the PErk neurons). Importantly, we discontinued the Step two screen after testing 70 of the 253 possible intersections because of evident redundancy of the command-like neurons. The latter were prominent in the expression patterns of numerous independent Step two intersections and were readily correlated with PE induction in three that produced particularly reduced expression patterns. In the intersection with the sparsest expression, the two pairs of PE-inducing neurons often comprised the entire observable pattern in flies that had the PE phenotype, illustrating the extreme reduction in expression achievable with SpaRCLIn. The demonstration that the PErk neurons can be isolated in single crosses using the FRTerminator indicates that this reduction in expression can be attained without the labor of Step two screening. However, the lower frequency of the PE phenotype in FRTerminator crosses in our example also suggests that FRTerminator-based screens may require testing more animals for each intersection than a standard Step one screen in order to reliably identify positives.Our use of SpaRCLIn to identify the RK-expressing neurons that trigger robust proboscis extension demonstrates SpaRCLIn’s ability to systematically parse a neuronal group and identify the functionally relevant subset. Just over 200 crosses—134 crosses for the Step 1 screen of NBE-Crerk neurons elicits rhythmic proboscis extension, rather than the tonic PE elicited by activation of all rkpan-Gal4 neurons. This suggests that additional RK-expressing neurons—perhaps lacking command capability—modulate the effects of activating the PErk neurons. Based on their induction of rhythmic extension and their apparent lack of a projection to the proboscis muscles, we conjecture that the PErk neurons identified here are not motor neurons, the activation of which results in tonic and often partial PE  for both the rkpan-Gal4 and TH-Gal4 drivers. It is less clear, however, that all members of each lineage are represented as not all NBE’s are active during early NB divisions. This is evident from the restriction in NB expression observed when the FRTerminator construct is used, since this construct acts by eliminating lineages or sublineages in which Cre activity is initiated sometime after neurogenesis has begun. It is also clear that the DNE does not express efficiently in NB lineages in the optic lobe (data not shown). To extend the capability of the system to include these lineages will require either the development of a more general neuroblast-specific enhancer or augmenting the system to include an enhancer that specifically targets optic lobe NBs.The ability of SpaRCLIn to isolate a given set of neurons of interest in a Gal4 pattern depends critically on the extent to which the various Split Cre components are expressed in the neuroblast lineages of the fly. This will be determined both by the breadth of NB expression of the DNE enhancer used here to delimit Cre activity and by the collective coverage of NB expression provided by the NBEs represented in the libraries of CreDrosophila NBs by choosing breakpoints in the Cre sequence preceded by a serine residue—the native condition of the NrdJ-1 and gp41-1 split inteins used here , and all final constructs were verified by sequencing . For routine molecular biology, the following reagents were used according to the manufacturers’ supplied protocols: PCR amplification: Q5 High-Fidelity 2X Master Mix #M0492S ; DNA ligation: Quick Ligation Kit #M2200L ; Cloning: Gateway LR Clonase II Enzyme mix #11791100 , and In-Fusion HD Cloning Plus #638911. gBlocks were used to generate most of the final and intermediate constructs described below, including the DNA fragments encoding the NrdJ-1 and gp41-1 split inteins and the Cre fragments described in the manuscript. DNA sequences of the split inteins were back-translated from the published protein sequences  and all The indicated Cre80Tom constructs were made stepwise using the described procedures.+-p10; Step 1 - Made the intermediate construct ‘M1:’ an NgoMIV-gBlock013-AatII fragment, an AatII-Gal80-SV40-MfeI fragment , and an MfeI-gBlock014-KpnI fragment were placed between the NgoMIV and KpnI restriction sites of pBPLexA::P65. Step 2: PCR amplified a KpnI-IVS-StuI-AgeI-myr-tdTomato fragment  and a p10-XbaI fragment  was inserted into the unique NdeI site between the mini-white gene and the attB sequence of the M1 vector. Step 2: A KpnI-IVS-Syn21-myr-tdTomato-StuI PCR fragment  and a StuI-p10-SpeI PCR fragment  were placed between the KpnI and SpeI restriction sites to replace the LexA::P65 fragment and to produce the intermediate construct ‘M3’ using the In-Fusion HD cloning kit. Step 3: Used Gateway Cloning to add the Actin 5C promoter to produce the Cre80-dTrpA1 and Cre80-Kir2.1.The sequence between the KpnI and NsiI of M3  were replaced with gBlock25 by HD-infusion cloning to make the intermediate construct ‘M4.’ This step removed the tdTomato gene. The BglII-mCD8GFP-XbaI fragment of M4 was replaced with BglII-dTrpA1-XbaI  and BglII-EFGP-Kir2.1-XbaI  PCR fragments and then the actin5C promoter was inserted by Gateway cloning to get B and CreC libraries.All split Cre constructs were made by Gateway cloning (LR reaction). Two sets of destination vectors with split Cre components were made: one for use with entry clones containing promoters, and another for entry clones containing enhancers. The 134 NBE entry clones were combined with the latter to make the expression clones used to generate the CreA (HJP-176) destination vectors for use with promoter entry clones, a KpnI-IVS-NheI fragment made from annealed oligonucleotides, a NheI-gBlock012-AgeI gBlocks fragment and an AgeI-PmeI-WPRE-HindIII PCR fragment (amplified from pBPGAL80Uw-6 using PrimerS472 and PrimerS473) were placed between the NheI and HindIII restriction sites of the pBPGw vector (Addgene Plasmid #17574 A-gp41-1N-AgeI fragment in CreA (HJP-176) with fragments consisting of: NheI-gBlock010-SphI + SphI-gBlock011-AgeI (HJP177), NheI-gBlock008-BsaI+BsaI-gBlock009-AgeI (HJP178), NheI-gBlock007-AgeI (HJP179), or NheI-gBlock010-SphI+SphI-gBlock015-AgeI (HJP180). To create a set of destination vectors for use with enhancer entry clones (‘the U-series’), an FseI-DSCP-KpnI synthetic core promoter  in addition to D. melanogaster. A 607 bp region starting 899 nucleotides 5’ of the transcription start exhibited highly conserved sequence blocks containing transcription factor binding sites, including three CAGCTG E-boxes commonly found in other NB enhancers (nerfin-1 enhancer with two adjacent bp substitutions (G→C and T→C) that were shown to expand the pattern of NB expression  using the pENTR/D-TOPO Cloning Kit).A 2 kb region upstream of the nhancers . A PCR fnhancers , and thepression . The muthttps://www.janelia.org/project-team/flylight). To verify NB expression of the latter NBEs, we applied the same method used to examine Cre activity in the single ventral nerve cord lineage labeled by the R59E09-Gal4 line (C (HJP266) constructs described below with the following genotype: w, DNE-CreB(attP8); UAS-CreC(attP40); CreStop (i.e. actin^STOP^tomato(attP2)), DNE-CreA(VK00027). CNS preparations of the progeny (third instar larvae or adults) were examined for tdTomato expression in NB clones. Selected JFRC Neuroblast active enhancers (NBEs) with ‘sparse’ expression in neuroblasts were amplified by PCR or synthesized when PCR failed  and cloned into either the pCR8-GW-TOPO or pENTR-D-TOPO donor vectors. Primers listed at the Flylight website were used to amplify most JFRC NBEs using genomic DNA from either y; cn bw sp [gift from James A. Kennison] or Canton S wildtype flies as template.Most of the neuroblast-active enhancers used to make the NBE entry clones were from the JFRC Flylight Collection .Candidatal4 line . Flylighstg-14 (otd (The cas-8 and CG7229-5 enhancers  were synstg-14  and otd -14 (otd  enhancerFRT and Syn21-flipase-T2A-CreA-gp41-1N-AgeI-FRT) were synthesized  and put between the AvrII and PmeI restriction sites of DNE-CreA-gp41-1N. The resulting construct can be used in place of DNE-CreA in Step 2 SpaRCLIn screens. It was tested, but its use is not described in this manuscript. This construct was used as an intermediary to make the final FRTerminator construct by inserting gBlock-043 (part of the CreAB sequence and Nrdj-1N) into its SbfI and AgeI restriction sites using the In-Fusion HD cloning technique.This construct (HJP-473) was made as follows: an AvrII and PmeI flanked DNA fragment . The CreStop construct was made using a NgoMIV-loxP-hsp70 terminator-MluI gBlock to replace the loxP-Gal80 in Cre80Tom (HJP223) by In-Fusion HD cloning. UAS-CreC was made by cloning a NotI-NrdJ-1C-CreC-XbaI PCR fragment  between the NotI and XbaI sites of pJFRC1-10XUAS-mCD8::GFP using the In-Fusion HD cloning technique.Two constructs were used to pre-screen candidate enhancers driving Gal4 expression. These included CreStop (HJP225) and UAS-CreEmbryos were prepared and immunostained as described by Flies assayed for proboscis extension were raised at 25°C until the white prepupa stage and then transferred to 18°C until the time of testing. For neuronal activation using dTrpA1, the chambers were placed on the surface of the Echotherm Chilling/Heating Dry Bath IC25  at 31°C. For the Step 1 SpaRCLIn screen, approximately a dozen adult flies (3–10 d old) of each genotype were placed in glass TriKinetics tubes  and videorecorded at 31°C for 3 min using a Sony NEX-VG10 videocamera. Proboscis extension behavior was analyzed from these recordings. If two or more flies exhibited robust, full-length extension, the cross was scored as positive. For the Step two tripartite SpaRCLIn screen, two flies at a time  were videorecorded together in glass minichambers (0.3 cm diameter X 0.7 cm length) for 3 min at 18°C followed by 3 min at 31°C. Flies were subjected to these temperature transitions twice and proboscis extension behavior was analyzed following the recording. The criteria for positive proboscis extension was three or more bouts of full proboscis extension in both tests. For the FRTerminator behavior experiments flies were subjected to only one test. Flies used to make the videos included in the manuscript were back-mounted on a 200 uL pipette tip with 5-Minute-Rapid-Curing, General Purpose Adhesive Epoxy  and placed just above the heating plate, which was adjusted to apply temperature changes. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by K VijayRaghavan as the Senior and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal their identity: Chris Q Doe (Reviewer #1).Thank you for submitting your article "Cre-assisted fine-mapping of neural circuits using orthogonal split inteins" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Drosophila neurobiologists with the bipartite split-GAL4 system, which has become fundamental in targeting genetic manipulations to sparse neuronal subsets. So, it is with great anticipation that one approaches their creative new method for further refining GAL4 expression patterns. Of course, several methods of refinement already exist, such as intersecting split-GAL4 patterns with FLP, GAL80, or killer-Zip constructs. The novelty in the method they report here is in using neuroblast-specific enhancers as the third intersecting component, rather than additional enhancers expressed in differentiated neurons. This idea is not new but has proven difficult to implement because of the lack of well-defined neuroblast enhancers. Luan and colleagues partially solve this problem by making neuroblast enhancers out of bi- and tri-partite intersections of multiple enhancers, one of which is a synthetic pan-neuroblast enhancer. To do this, they exploit the split intein system to reconstruct a functional Cre recombinase, which in turn excises a ubiquitous GAL80 cassette to allow GAL4 expression within the progeny of the targeted lineage. The method is perhaps somewhat cumbersome, given the large number of transgenes involved, but this should be manageable, particularly if one could generate a panel of stocks, each of which containing all of the split-Cre components needed to target a specific lineage.Haojiang Luan, Ben White, and their colleagues gifted The practical utility of this method will depend principally on (a) the coverage and specificity of the collection of neuroblast Cre lines, and (b) the reproducibility of the resulting expression patterns. With respect to coverage and specificity, the collection of lines here is a good starting point, though as the authors acknowledge, it needs some improvement. They envision, not unreasonably, that this will become a community effort over time, as users identify new neuroblast enhancers and gradually improve the collection. The question is whether, as the reagents currently stand, a sufficient utility threshold is reached so that the community will indeed enthusiastically take up this approach and gradually improve the reagents. The first split-GAL4 system was not perfect and few useful lines were initially generated. But it clearly filled a void and was effective enough that it was rapidly embraced and continuously improved. It is hard to see the split-Cre system catching on in a similar way. Partly this is because of the success of split-GAL4, and the existence of alternative methods for further refinement. The need is simply not as great as it was for split-GAL4. But it is also because of the problem of poor reproducibility of the split-Cre system.Because of the variable expression patterns obtained with the split-Cre system, circuit mapping using this system functions similarly to the existing stochastic methods for restricting expression, i.e. large numbers of individuals need to be tested behaviorally and then analysed histologically to determine which cells were targeted, fly-by-fly. This is how the authors use it in the examples they provide here. One would need to consider on a case-by-case basis which is the more suitable approach. Split-Cre likely involves more crosses but fewer flies in order to get a convincing correlation, but may be more prone to failure due to coverage issues or the inability to discriminate between cells in the same lineage. A simple hs-FLP approach would be easier genetically, requiring fewer stocks and crosses, but perhaps require examining more individuals. The best choice will likely depend on the complexity of the initial pattern, but often standard hs-FLP approach is likely to still be the easiest and most reliable.The strengths of the work are also notable. One is the clearly demonstrated ability to narrow a Gal4 pattern by intersecting it with two or three Cre patterns. Another is using the sparse patterns to identify neurons that can induce a specific behavior; leveraging the stochasticity of the expression to correlate expression with behavioral phenotype is really turning lemons (variability) into lemonade (stronger neuron-behavior correlations). Yet another strength is the creation of new transgenics to simplify use of the system . Very thoughtful.Overall, there are some very important concerns that need to be addressed before acceptance.Essential revisions:1) For the above reasons, our enthusiasm for the split-Cre approach is somewhat muted. It is clever and potentially very useful, but we think it still needs further development before it will be enthusiastically taken up by the community. We suggest, reluctantly, that the authors delve further into the reasons for the variability. It might be easier to do this using a simple Cre-dependent reporter (which they have), rather than indirectly by dissecting some GAL4 expression pattern (as they do with TH-GAL4 here). A more robust method, even with only a limited initial set of neuroblast-specific Cre lines, would certainly be a useful tool for the community.2) It is not clear that the NBEs are really neuroblast-specific. Data supporting this conclusion could be readily obtained by staining the NBE-Gal4 UAS-GFP lines with GFP  and Dpn or Wor (for neuroblasts); there should be only one double positive cell in each hemisegment of the VNC or brain lobe.3) It is unclear how reproducible the NBE pattern is when a NBE-Cre(AB) is inserted at different landing sites other than attP2. Are there data on this point?4) How early does the Cre reconstitution start to work? Staining for temporal factor expression may help to reveal the onset time. Summary:Haojiang Luan, Ben White, and their colleagues gifted Drosophila neurobiologists with the bipartite split-GAL4 system, which has become fundamental in targeting genetic manipulations to sparse neuronal subsets. So, it is with great anticipation that one approaches their creative new method for further refining GAL4 expression patterns. Of course, several methods of refinement already exist, such as intersecting split-GAL4 patterns with FLP, GAL80, or killer-Zip constructs. The novelty in the method they report here is in using neuroblast-specific enhancers as the third intersecting component, rather than additional enhancers expressed in differentiated neurons. This idea is not new but has proven difficult to implement because of the lack of well-defined neuroblast enhancers. Luan and colleagues partially solve this problem by making neuroblast enhancers out of bi- and tri-partite intersections of multiple enhancers, one of which is a synthetic pan-neuroblast enhancer. To do this, they exploit the split intein system to reconstruct a functional Cre recombinase, which in turn excises a ubiquitous GAL80 cassette to allow GAL4 expression within the progeny of the targeted lineage. The method is perhaps somewhat cumbersome, given the large number of transgenes involved, but this should be manageable, particularly if one could generate a panel of stocks, each of which containing all of the split-Cre components needed to target a specific lineage.The practical utility of this method will depend principally on (a) the coverage and specificity of the collection of neuroblast Cre lines, and (b) the reproducibility of the resulting expression patterns. With respect to coverage and specificity, the collection of lines here is a good starting point, though as the authors acknowledge, it needs some improvement. They envision, not unreasonably, that this will become a community effort over time, as users identify new neuroblast enhancers and gradually improve the collection. The question is whether, as the reagents currently stand, a sufficient utility threshold is reached so that the community will indeed enthusiastically take up this approach and gradually improve the reagents. The first split-GAL4 system was not perfect and few useful lines were initially generated. But it clearly filled a void and was effective enough that it was rapidly embraced and continuously improved. It is hard to see the split-Cre system catching on in a similar way. Partly this is because of the success of split-GAL4, and the existence of alternative methods for further refinement. The need is simply not as great as it was for split-GAL4. But it is also because of the problem of poor reproducibility of the split-Cre system.Because of the variable expression patterns obtained with the split-Cre system, circuit mapping using this system functions similarly to the existing stochastic methods for restricting expression, i.e. large numbers of individuals need to be tested behaviorally and then analysed histologically to determine which cells were targeted, fly-by-fly. This is how the authors use it in the examples they provide here. One would need to consider on a case-by-case basis which is the more suitable approach. Split-Cre likely involves more crosses but fewer flies in order to get a convincing correlation, but may be more prone to failure due to coverage issues or the inability to discriminate between cells in the same lineage. A simple hs-FLP approach would be easier genetically, requiring fewer stocks and crosses, but perhaps require examining more individuals. The best choice will likely depend on the complexity of the initial pattern, but often standard hs-FLP approach is likely to still be the easiest and most reliable.The strengths of the work are also notable. One is the clearly demonstrated ability to narrow a Gal4 pattern by intersecting it with two or three Cre patterns. Another is using the sparse patterns to identify neurons that can induce a specific behavior; leveraging the stochasticity of the expression to correlate expression with behavioral phenotype is really turning lemons (variability) into lemonade (stronger neuron-behavior correlations). Yet another strength is the creation of new transgenics to simplify use of the system . Very thoughtful.Overall, there are some very important concerns that need to be addressed before acceptance.We thank the reviewers for your thoughtful comments on our submitted manuscript. You raised concerns that we had not previously appreciated and alerted us to unintended omissions. To meet the reviewers’ requests for essential changes, we have made numerous changes, which we believe strengthen the manuscript considerably. These changes and our point-by-point responses are detailed below, but we wished to start by addressing the two overarching reservations raised about the SpaRCLIn technique. The first of these relates to its efficacy, and second to its ease of use relative to existing methods, particularly Split Gal4.With regard to efficacy, major concerns were raised about “(a) the coverage and specificity of the collection of neuroblast Cre lines, and (b) the reproducibility of the resulting expression patterns.” Prompted by these concerns we have introduced new data regarding the spatiotemporal expression of our NB enhancers and its variability. With regard to the neuroblast-specific DNE: we show that DNE-driven Cre activity is evident as early as embryonic stage 8 and is widespread in the developing CNS by stage 13 . As requested by the reviewers, we also demonstrate that the DNE drives Cre fragment expression early enough to label progeny of a defined NB lineage identified by Lacin and Truman, 2016 . Although we cannot rule out the absence of Cre activity in the earliest born neurons, these new data suggest that Gal80 excision occurs relatively early. We anticipate that coverage of adult neurons, most of which are born during larval neurogenesis is much better, except for those in the optic lobe where the DNE is not active as previously noted.The reviewers point out that the best possible coverage is likely afforded by standard hs-Flp methods and that such methods are easier to use. The downside of these methods, however, is their very limited reproducibility. The reproducibility achieved with almost all NBE lines of SpaRCLIn is at least an order of magnitude better than that of Flp-out Gal80 methods . As our dissection of both the TH- and Rk-Gal4 lines shows, the NBE lines with the best reproducibility will express in a given neuron in upwards of 80% of progeny. Although this falls short of complete reproducibility, it does allow an end-user to identify a neuron of interest in a screen and repeatedly return to it for further characterization with some confidence. In contrast, with hs-Flp methods, one is forced to search the entire haystack  again for the desired needle (i.e. neuron) for every new manipulation (i.e. UAS effector) one uses. The utility of hs-Flp methods thus steeply declines if one wants to do more than simply identify a neuron based on one criterion. As the reviewers point out, end users will want to decide which method to choose depending on their needs, but we believe that SpaRCLIn offers sufficient advantages to be an attractive option in several contexts.Drosophila neurobiology and we appreciate their kind comments regarding our contributions to this effort. However, in many cases even the best available tools don’t restrict expression sufficiently to permit assignment of functional identities to particular neurons. This is reflected in the question we most frequently get from potential end-users regarding SpaRCLIn: Does it work with Split Gal4? The coverage of Split Gal4—using the thousands of lines generously generated, characterized, and made available by the Rubin and Dickson labs—is, like that of SpaRCLIn, incomplete , and finding useful combinations – while facilitated by fine registration tools developed by several labs – still generates “no more that 5%” of intersections without ectopic expression . Under these circumstances, end-users will benefit from additional strategies and SpaRCLIn – with its distinct differences from other commonly used strategies – should prove useful.Under what circumstances might an end-user choose to use SpaRCLIn over Split Gal4? We recommend using it when Split Gal4 can’t (or seems unlikely to) deliver the desired refinement in expression. We agree with the reviewers that Split Gal4 and other existing tools have revolutionized Essential revisions:1) For the above reasons, our enthusiasm for the split-Cre approach is somewhat muted. It is clever and potentially very useful, but we think it still needs further development before it will be enthusiastically taken up by the community. We suggest, reluctantly, that the authors delve further into the reasons for the variability. It might be easier to do this using a simple Cre-dependent reporter (which they have), rather than indirectly by dissecting some GAL4 expression pattern (as they do with TH-GAL4 here). A more robust method, even with only a limited initial set of neuroblast-specific Cre lines, would certainly be a useful tool for the community.AB∩NBE-CreC > CreStop crosses . In general, we find that patterns for any given NBE are similar across preparations, with examples presented in a new figure . Although some variation is almost certainly present, the large size of the patterns and our inability to reliably identify identical neurons and lineages across preparations prevented a more detailed analysis. As noted in the revised Discussion , our results suggest that large-scale variability in the activity of individual NBEs is unlikely to explain all of SpaRCLIn’s stochasticity in Step 2 crosses and that further analysis will be required to determine its source. We also now provide a somewhat more extended discussion of the possible sources with addition of the following material: “If a Cre component is only weakly expressed, or expressed late during neurogenesis, a limited amount of active, full-length Cre may be produced and excision of Gal80 may be sporadic in the expressing neuroblasts. […] Finally, because the efficacy of Cre-mediated excision depends on the distance between the loxP sites flanking the excised fragment, it may prove possible to increase the efficacy of excision – and thus reduce stochasticity of expression – using strategies that decrease the distance between the loxP sites flanking the excised fragment.”We share the reviewers’ interest in the cause(s) of variability and have added new information  and a new figure  to the revised manuscript regarding this point. As noted in the Discussion of the original manuscript, erratic expression may derive in part from intrinsic stochasticity of the individual NB enhancers. As the reviewers pointed out, this possibility can be investigated using our Cre-dependent Act5C^Gal80^tdTomato (i.e. CreStop) reporter. We had previously confirmed NBE-driven expression in larval CNS preparations using this reporter , and we have now examined multiple preparations (n= 4 on average) for each of the 134 NBEs to assess their variability in DNE-Cre2) It is not clear that the NBEs are really neuroblast-specific. Data supporting this conclusion could be readily obtained by staining the NBE-Gal4 UAS-GFP lines with GFP  and Dpn or Wor (for neuroblasts); there should be only one double positive cell in each hemisegment of the VNC or brain lobe.active in neuroblasts (in addition to other cell types). This may have been unclear because we described the procedure for selecting and testing previously unpublished NBEs only in the Materials and methods and not in the body of the paper. While the majority of our NBEs were selected from previously published sets , approximately one-quarter (38) were identified by a method similar to that suggested by the reviewers. We used NBE-Gal4 lines from the Janelia FlyLight collection with probable expression in larval NBs to drive a UAS-CreC construct together with CreA and CreB fragments driven by DNE. Expression in NB clones was evaluated using the CreStop reporter. The main text of the revised manuscript now clearly indicates the origin of the NBEs , with all sources cited in a new column in the revised Supplementary file 1.We realized in reading this comment that we had provided too little information about the sources and properties of our NBEs. Few (perhaps none) of them are “neuroblast-specific.” This is why the Split Cre approach was required. Expression of one Cre component had to be driven by a bona fide NB-specific enhancer (viz. the “DNE”) to delimit Cre activity to NBs. Importantly, however, all of our NBEs are 3) It is unclear how reproducible the NBE pattern is when a NBE-Cre(AB) is inserted at different landing sites other than attP2. Are there data on this point?AB construct/line in our paper. The only CreAB line is the one used for Step 1 screening where expression of the CreAB fragment is driven by the DNE. The DNE-CreAB insertion in this case is into the JK22C attP landing site on Chromosome II. This site was chosen because of its previously demonstrated fidelity of expression compared with attP2 .We are somewhat confused by this point. We do not describe an NBE-CreC and NBE-CreB lines. The former (with insertions on Chromosome III: attP VK00033 or VK00027) are used in Step 1 screens to select suitable NBE-CreB lines (with insertions on Chromosome II: attP40) for use in Step 2 screening. Ensuring the similarity of expression between NBE-CreB and NBE-CreC lines made with identical NBEs is thus important. This is an issue that we had investigated, but did not explicitly address in the original manuscript. The revised manuscript, however, incorporates some of our previous findings together with more recent results .We wonder, however, if the reviewer isn’t referring to the reproducibility of expression produced by NBE-CreB lines had been visually inspected by fluorescence microscopy and compared to the corresponding patterns obtained for NBE-CreC lines . The patterns for a given enhancer inserted at the two different attP landing sites were generally similar, although differences in expression levels between the two types of lines made detailed comparison difficult.  Nevertheless, we flagged 24 NBE-CreB lines whose expression pattern differed overtly in one or more respects from the corresponding NBE-CreC lines. We have now tested several of these lines more systematically and provide an example of one such NBE in the revised manuscript in Figure 1—figure supplement 3C, D where it is compared with an example of an NBE that produced similar patterns . The revised manuscript also now alerts readers to the 24 potentially problematic lines, which are marked with asterisks in Supplementary file 1. A footnote in the table of Supplementary file 1 states that users who identify one of these NBE-CreC lines as a “positive hit” in a Step 1 screen may want to continue using this line in the subsequent Step 2 screen (if possible) to preserve its expression pattern.The larval CNS expression patterns produced by all NBE-Cre4) How early does the Cre reconstitution start to work? Staining for temporal factor expression may help to reveal the onset time.As noted above, we now present data in the revised manuscript  that suggests a relatively early onset of Cre reconstitution. Since we assayed Cre-mediated tdTomato expression and because this expression depends on multiple time-dependent steps  our data set an upper bound on the time of Cre reconstitution.


Background and Purpose: Stent-assisted coiling (SAC) of intracranial aneurysms is usually treated with antiplatelet therapy to reduce the risk of postoperative ischemic events. However, using the same antiplatelet therapy for all patients may increase the risk of bleeding in patients with aneurysmal subarachnoid hemorrhage (aSAH). Thromboelastography-platelet mapping (TEG-PM) measures platelet function, which reflects the effect of antiplatelet drugs. This study aimed to evaluate the benefits of individualized antiplatelet regimens based on TEG-PM parameters for patients with aSAH who underwent SAC.Methods: We retrospectively included patients with aSAH who treated with SAC during the period from June 2012 to December 2019. Patients were divided into two groups: patients whose antiplatelet therapy adjusted by TEG-PM parameters after surgery (adjustment group) and patients who were treated with standard dual antiplatelet therapy without TEG-PM test (control group). The occurrence of major/minor bleeding events, major/minor thromboembolic events, and favorable outcome  were compared in both groups during hospitalization.Results: Of 188 aSAH patients considered for this study, 145 met the criteria for inclusion and were included in the analysis (93 patients in the adjustment group and 52 patients in the control group). The risks of minor bleeding events  were significantly lower in patients in the adjustment group. However, there was no significant difference in the rate of major bleeding events at discharge between adjustment and control groups (p = 0.35). The rates of thromboembolic events and favorable outcome were similar in both groups . Furthermore, the minor thromboembolic events rate was significantly lower in the patients treated with treatment plan C . However, there was no significant difference in the rate of other mentioned above complications and favorable outcomes among patients treated with different antiplatelet regimens.Conclusions: Individualized antiplatelet therapy based on TEG-PM parameters might positively impact the bleeding risk of aSAH patients, without increasing the risk for clinically relevant thromboembolic events. Stent treatment technology has emerged as a viable and preferable method for wide-necked, complex-shaped, and dissecting intracranial aneurysms . HoweverThromboelastography-platelet mapping (TEG-PM) is a measure derived from thromboelastography (TEG) for detecting platelet function in the form of platelet inhibition rate, which can indirectly reflect the platelet aggregation function and can benefit to prevent and reduce thromboembolic events after embolization. Therefore, it is widely used in most hospitals around the world to evaluate the efficacy of dual antiplatelet agents before SAC for intracranial aneurysm . CurrentThe purpose of this study is to explore whether the tailor reduction of the dose of antiplatelet drugs based on TEG-PM parameters can reduce the incidence of bleeding events after SAC without increasing the rate of thromboembolic events and whether it will affect the prognosis of patients.This study was a single-institution retrospective study conducted at The First Affiliated Hospital of Chongqing Medical University. The STROBE statement on cohort studies guided study design and manuscript organization. We retrospectively recruited patients with aSAH who underwent SAC between June 2012 and December 2019. Patients who were treated with an antiplatelet regimen based on TEG-PM parameters due to SAC treatment were included into an “adjustment” group. A “control” group consisting of aSAH patients who underwent SAC without TEG-PM test during the period from June 2012 to January 2015 was added to the final analysis . A furthInclusion criteria were as follows: (1) Patients (ages ≥18 years) with aSAH were treated with SAC were included; (2) have complete perioperative-related imaging and laboratory data; and (3) Initial Hunt-Hess grades were grades I–III. Exclusion criteria included the following: (1) treatment with anticoagulants, thrombolytic agents, and other antiplatelet drugs before admission; (2) severe cardiovascular disease or cerebral ischemic stroke; (3) severe hepatic or renal dysfunction, malignant disease, chronic inflammatory disease, significant coagulopathy, or an infectious condition at study entry; (4) combined with other cerebral diseases; and (5) recurrent aneurysm which is treated with coils only or stent.A standardized protocol was strictly followed in all cases. All cases were treated according to the standardized aSAH treatment protocol  consistiPatients in the adjustment group were drawn blood samples for TEG and TEG-PM testing on the 3rd day after surgery draws blood. Blood samples (3–4 ml) were drawn from a single clean puncture of a forearm vein and collected in a 4-ml test tube (Becton-Dickinson) containing lithium heparin.The platelet inhibition was tested using a TEG-PM analyzer . Adenosine diphosphate (ADP) agonists and arachidonic acid (AA) were added to measure the platelet inhibition of the P2Y12 receptor and cyclooxygenase pathways. Inhibition rate (%) is equal to (AA- or ADP-induced clot strength-fibrin clot strength)/(thrombin-induced clot strength-fibrin clot strength) × 100% . The perR, min); the time elapsed from R until 20-mm amplitude is achieved ; the time to reach maximum speed of initial clot formation ; maximum clot strength ; percent of clot lysis 30 min after MA (EPL); and percent of amplitude decay 30 min after MA (LY30).The following TEG parameters were recorded: the time from the start of the sample until amplitude of clot formation reaches 2 mm  for any clinical information about the occurrence of cerebral infarctions and new intracranial bleedings. Minor bleeding was defined as any extracranial bleeding  documented during antiplatelet therapy that does not cause clinical deterioration. Major bleeding was defined as any new hemorrhage that led to clinical deterioration. Accordingly, a minor thromboembolic event was defined as new asymptomatic cerebral infarction in the stent vessel area, which was diagnosed by diffusion-weighted imaging (DWI). Major thromboembolic events included newly developed transient ischemic attack (TIA) or symptomatic ischemic infarctions. We followed up with patients in the adjustment group via hospital medical records or telephone interviews in July 2020, and the median follow-up time was 11  months. Functional outcome was evaluated at discharge and at least 6 months after discharge. Due to the large time span of patients in the control group and the adjustment group, this study only followed up patients in the adjustment group. Favorable functional outcome was defined as mRS <3.t-test for normally distributed and the Mann–Whitney U-test for non-normally distributed data. p < 0.05 was considered statistically significant. The statistical analysis was performed by SPSS 23.0 software .Data are presented as mean SD or expressed in terms of frequency and percentage. Categorical variables were analyzed using the Chi-square test or, if applicable, with the Fisher exact test. Continuous variables were analyzed using the Student's Of 188 aSAH patients enrolled for this study, 145 endovascularly treated individuals (77.5%) met the inclusion criteria and were included in the final analysis see . The meaThere was also no statistical difference in baseline characteristics between patients with different antiplatelet treatment plans in the adjustment group see . Six (6.p = 0.02). But there was no significant difference in the rate of major bleeding events at discharge between adjustment and control groups  –23 have On the other hand, increasing the dose of antiplatelet drugs to reduce the risk of thromboembolic in patients with SAC of intracranial aneurysms needs to be further studied. Although McTaggart et al.  changed Besides, the incidence of TIA in the adjustment group of this study  and the incidence of minor thromboembolic events  were lower than the reported incidence in the literature [1.9%  for TIA,Finally, there is no significant difference between adjustment and control groups with regard to favorable outcomes at discharge. This finding is in line with several previous publications reporting thromboembolic events are not an important risk factor for poor prognosis in aSAH patients treated with SAC of intracranial aneurysms , 33. TheIndividualized antiplatelet therapy based on TEG-PM parameters might positively impact the bleeding risk of aSAH patients, without increasing the risk for clinically relevant thromboembolic events. However, our data underline the need for a prospective randomized trial for a definite safety range of TEG-PM parameters in individuals with aSAH.There are some limitations to our study. First, we included only the patients whose initial Hunt-Hess grades were grades I–III. Therefore, our results and conclusions on the benefits of individualized antiplatelet therapy based on TEG-PM parameters after SAC of intracranial aneurysms cannot be generalized on aSAH patients whose initial Hunt-Hess grades were grades IV–V. Second, the dynamic of ADPi, AAi, and MA-ADP values during follow-up was lacking due to our retrospective design. Moreover, 6.5% of patients in the adjustment group were lost to follow-up, and it cannot be excluded that some of them could have been dead for cerebrovascular events. Last, this was a retrospective study with a small sample size, so further studies are required to support our findings.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the institutional review board of The First Affiliated Hospital of Chongqing Medical University. The ethics committee waived the requirement of written informed consent for participation.YL: conception, collection of data, interpretation of data, and drafting the work. XZ, ZG, JZ, RX, and ZH: collection of data and interpretation of data. XS: conception and revising the work critically for important intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: To describe the favorable clinical outcome in a case with bilateral choroidal neovascular membrane and quiescent Vogt-Koyanagi-Harada (VKH) syndrome by administering bilateral intravitreal aflibercept injections.Case report: A 30-year-old woman was diagnosed with VKH syndrome at another institution and had been in remission with oral mycophenolate mofetil for two years. However, nearly simultaneous right juxtafoveal and left subfoveal type 2 choroidal neovascular membrane was detected two years after the initial diagnosis. The right eye (OD) received three and the left eye (OS) received four aflibercept injections within a time span of eight months. Visual acuity was 20/30 in OD and 20/25 in OS at the last follow-up visit.Conclusion: Although suppression of inflammation is a must in eyes with inflammatory type choroidal neovascular membranes, anti-VEGF  therapy with agents, such as aflibercept in the present case, is a key therapeutic adjunct and may possibly help improve the visual prognosis. Choroidal neovascularization (CNV) may develop in eyes with various posterior uveitic entities during the course of disease. Inflammatory CNVs may develop as the result of inflammation-induced Bruch’s membrane disruption and/or the increase of several inflammatory mediators including VEGF  . CNV canIn a retrospective cohort study including 4,041 eyes of 2,307 patients with posterior uveitis and panuveitis, 81 eyes (2%) had active CNV or sequelae of past CNV at presentation. In this cohort, only one of 320 eyes with VKH syndrome had CNV (0.3%) . HoweverWe hereby report a patient with VKH syndrome in remission with oral mycophenolate mofetil, who subsequently developed bilateral CNV and was treated successfully with intravitreal aflibercept injections.A 30-year-old woman had been diagnosed to have VKH syndrome at another eye center two years prior and had been treated successfully with high-dose systemic steroids and then with oral mycophenolate mofetil (MMF) without any recurrence. She was referred to us due to the development of nearly simultaneous bilateral CNV while she was still on a daily maintenance dose of oral 500 mg MMF. Her color fundus photographs, fluorescein angiogram (FA), and optical coherence tomographic (OCT) examination at the initial diagnosis of VKH syndrome were obtained Figure 1 . Upon ouThe patient was advised to have regular bi-monthly follow-ups unless an unexpected change occurred. Overall she had a follow-up of eight months.There is no clear standard of care for CNV management in inflammatory disorders. Steroids and immunosuppressives are often used in uveitis to reduce the inflammatory stimulus which seems to play a role in CNV formation, and also partly due to their antiangiogenic effect . HoweverCurrently, anti-VEGF injections have replaced photodynamic therapy (PDT) as the main treatment modality for CNVs . There aMansour et al.  evaluateAnti-VEGF treatment protocol is still somewhat uncertain in inflammatory CNVs. A randomized study investigating the efficacy and safety of ranibizumab for the treatment of CNV due to uncommon causes of CNV (i.e. other than age-related macular degeneration and degenerative myopia) revealed that in a group of 178 patients , 18  despite being in a state of remission. We administered three aflibercept injections in OD and four aflibercept injections in OS during an eight-month period as mentioned in the case description above. Concurrently, the patient maintained the ongoing therapy of oral mycophenolate mofetil. Due to a lack of comparative studies, advantage of administering aflibercept over bevacizumab or ranibizumab on inflammatory CNVs is not clear. To our present knowledge, aflibercept administration has not been reported before in CNV related to VKH syndrome. Clinicians should be cautious in differentiating the signs of CNV from the signs of pre-existing uveal inflammation in eyes with various uveitic entities, as the original disease can be active or in remission.Anti-VEGF agents (aflibercept administrations in the present case) appear to be a very valuable adjunct to obtain favorable anatomic and visual outcomes in most of the eyes of patients with VKH syndrome and coexistent CNV.The authors declare that they have no competing interests.Written informed consent was obtained from the patient for the publication of this case report.


An additional PET/CT scan was performed to evaluate the prostate-specific membrane antigen (PSMA) expression, which has recently been reported to be a potential biological marker in a variety of tumors including HCC. High PSMA uptake was recorded in both the metastatic bone lesions and the primary liver lesion/tumor by the 68Ga-PSMA PET/CT.We have reported here the case of a 69-year-old man who presented with spinal cord compression due to bone metastases as the first manifestation of hepatocellular carcinoma (HCC). For the initial staging, the patient underwent


Thyroid cancer is the most common sort of endocrine-related cancer with more prevalent in women and elderly individuals which has quickly widespread expansion in worldwide over the recent decades. Common features of malignant thyroid cells are to have accelerated metabolism and increased glucose uptake to optimize their energy supply which provides a fundamental advantage for growth. In tumor cells the retaining of required energy charge for cell survival is imperative, indeed glucose transporters are enable of promoting of this task. According to this relation it has been reported the upregulation of glucose transporters in various types of cancers. Human studies indicated that poor survival can be occurred following the high levels of GLUT1 expression in tumors. GLUT-1 and GLUT3 are the glucose transporters which seems to be mainly engaged with the oncogenesis of thyroid cancer and their expression in malignant tissues is much more than in the normal one. They are promising targets for the advancement of anticancer strategies. The lack of oncosuppressors have dominant effect on the membrane expression of GLUT1 and glucose uptake. Overexpression of hypoxia inducible factors have been additionally connected with distant metastasis in thyroid cancers which mediates transcriptional regulation of glycolytic genes including GLUT1 and GLUT3. Though the physiological role of the thyroid gland is well illustrated, but the metabolic regulations in thyroid cancer remain evasive. In this study we discuss proliferation pathways of the key regulators and signaling molecules such as PI3K-Akt, HIF-1, MicroRNA, PTEN, AMPK, BRAF, c-Myc, TSH, Iodide and p53 which includes in the regulation of GLUTs in thyroid cancer cells. Incidence of deregulations in cellular energetics and metabolism are the most serious signs of cancers. In conclusion, understanding the mechanisms of glucose transportation in normal and pathologic thyroid tissues is critically important and could provide significant insights in science of diagnosis and treatment of thyroid disease.Video Abstract The up regulation of Glucose transporters (GLUTs) has been reported in thyroid cancer which has been shown to be an index of aggressiveness and loss of tumor differentiationThe identification of different regulators and signaling molecules which involve in the expression and subcellular distribution of several GLUTs can aid in the diagnosis and clinical management of thyroid cancerGLUTs as rate-limiting steps in glucose metabolism of cancer cells and regulators of glucose uptake pathway are promising targets for the development of anticancer strategiesIn glucose metabolism, the translocation of glucose across the plasma membrane known as the rate limiting step happens by carriers belonging to the facilitative glucose transporter (GLUT) and the sodium-coupled glucose co-transporter (SGLT) proteins families. While the SGLTs require energy to the task of glucose transportation, the GLUTs allow the transport of glucose down its concentration gradient without energy dependence . GLUT1 iMost cancer cells alter cellular metabolisms because of achieving high proliferation rates and this can be lead to constitutive stress metabolic phenotype. Tumor cells are capable of switching metabolism from oxidative to the glycolytic phenotype. It is called Warburg effect which is tumor-specific metabolic characteristic and a key metabolic hallmark. Researches around tumor metabolism show that through the alteration of cellular metabolism in which glycolysis and glutaminolysis are up regulated, it is necessary to maintain rapid cell proliferation, tumor progression, and resistance to cell death . GlucoseA defining characteristic of thyroid cancer cells is their strong ability to take up enormous amounts of glucose compared to normal thyroid tissue. Malignant cells rewire their metabolism through enhancement of glucose uptake for promotion of cell growth and survival. Tumor cells enhance glucose uptake across the plasma membrane via induction of a family of facilitative glucose transporter proteins (GLUTs), which classified regarding their tissue-specific distribution and different affinities for glucose and remarkably different transport capacities. In most cases thyroid cancer cells frequently show overexpression of especially the hypoxia-responsive GLUT1 and GLUT3 proteins. Malignant cells characteristically have a reduced ability to use oxidative metabolism, and instead aerobic glycolysis increased rapidly and oxidative phosphorylation remained stable. Increased glycolysis is the main source of energy supply in cancer cells but, due to the lower energy yield of the glycolytic pathway, malignant cells show an increased rate of glucose transport across the plasma membrane to compensate the acquired energy –25.Recently the tumor differentiation relationship with glucose metabolism in the thyroid cancer is evaluated. The glucose metabolic profiles have complex association with tumor differentiation in well differentiated thyroid cancer (Papillary thyroid cancer) and poorly differentiated thyroid cancer (Anaplastic thyroid cancer). According to the Suh H. Y. et al. analysis based on the genetic mutation status, metabolic profiles of thyroid cancer were not easily correlated with tumor differentiation. The cellular expression of GLUT was negatively related to tumor differentiation in thyroid cancers. The enrichment of glycolysis was positively correlated with the well-differentiated thyroid cancer, but it has negative association in the case of poorly differentiated thyroid cancer. In papillary thyroid cancer, the glycolysis signature was positively correlated with tumor differentiation score, while the GLUT signature has negative association with tumor differentiation score. On the other hand, in poorly differentiated thyroid cancer, both GLUT and glycolysis signatures have negative correlation with tumor differentiation score. Their results were agreed with the previous studies because poorly differentiated thyroid cancers need higher glucose uptake by representing high GLUT expression. But, glycolysis signatures of thyroid cancers with poor differentiation have conflicting relation between papillary thyroid cancer and anaplastic thyroid cancer. Overall considering that the association between the differentiation and glycolysis might have ‘U shape’ pattern. The reason of different scores for GLUT and glycolysis in papillary thyroid cancer was different BRAFV600E mutation status. BRAFV600E positive PTC have higher GLUT signature and lower glycolysis signature than BRAFV600E negative PTC .Among the glucose transporters, GLUT1, GLUT3, GLUT4, and GLUT10 are the types that detected to be expressed in all thyroid parenchymal cells regardless of histology. Results have been showed that GLUT1 has significantly higher expression in samples of patients with thyroid cancer in comparison with normal and benign tissue samples from the same patient. It has been reported that other GLUTs were not changed in comparison to GLUT1 in pathologic tissues of same patient. These results indicated that GLUT1 is theoretically responsible for the observed enhanced uptake of glucose during carcinogenesis , 27. TheThere is difference in the expression of hypoxia-related GLUT1 and GLUT3 between benign and malignant neoplasms, as well as non-neoplastic thyroid lesions. The differences in GLUT1 and GLUT3 expression levels are associated with the histological type of thyroid carcinomas. Both hypoxia-related GLUT1 and 3 are involved in the progression of papillary thyroid carcinomas and may be added to a panel of biological thyroid carcinoma markers. There is a close correlation between the overexpression of GLUT1 and GLUT3 proteins and the high levels of GLUT1 and GLUT3 mRNA in the same thyroid cancer specimens. However, in some neoplasm cases the detection of GLUT1 or GLUT3 positive band and the mRNA level was very low. A hypothesis that provides an acceptable explanation for these findings is that the expression of hypoxia-related GLUTs is further influenced by the microenvironment of tumor cells. The deregulation of glucose metabolism in cancer cells is predominantly mediated by oxygen-related transcription factors, such as the hypoxia-inducible factor 1 (HIF-1). HIF-1 induces a number of genes encoding glycolytic enzymes, erythropoietin, the tumor-associated carbonic anhydrases (CAs), CAIX and CAXII, vascular endothelial growth factor (VEGF), as well as the facilitative GLUT isoforms 1 and 3 (GLUT1 and GLUT3). Hypoxia-related GLUTs are characterized by low Km values and high affinity for glucose as compared to other members of the GLUT family .The abnormal surface expression of GLUT proteins and also the activity of GLUT-1 is under the control of multiple signal transduction pathways, including the phosphoinositide 3-kinase (PI3K)/AKT pathway . In thyrThyroidectomy  and radioactive iodine therapy are the routine treatments for patients suffering from thyroid cancer, but it is often not more effective treatment. The recent advancement in molecular-targeted therapies understanding the molecular pathogenesis of thyroid cancer has shown great promise for the development of early diagnosis and appropriate treatment strategies for thyroid cancer. This has basically eventuated from the recognition of molecular alterations . AlthougSince the early observation by Otto Warburg, it has been well known that tumor cells are characterized by an increased requirement for energy metabolism. According to scientific researches approximately 90% of cancers indicates the phenotype of increased glucose metabolism. Additionally, these cells, have a reduced ability to use mitochondrial oxidation and favor the conversion of pyruvate into lactate, despite the availability of oxygen. Because ATP synthesis is not the top priority of the upregulation of glucose transport. Glycolysis is nearly 18 times less efficient compared to oxidative phosphorylation process so cancer cells require an increased rate of glucose uptake into cells for compensation of the low ATP production –37. Alth18F-fluorodeoxyglucose positron emission tomography (FDG-PET) for detection of thyroid cancer patients has come into the spotlight recently. FDG-PET has been identified to display metastasis in 131I scan-negative thyroid cancer patients with a high accuracy, which is thought to be related to increased glucose metabolism in poorly differentiated carcinoma. It has proved that FDG avidity has correlation with tumor size, lymph node metastasis, glucose transporter expression and differentiation [The application of ntiation . Glycolyntiation .FDG, a glucose derivate labelled with F18, showing avidity in cancer types with high tumor related metabolism and upregulation of the glucose transporter system. The role of FDG PET represented by the development of metastatic diseases, which radioiodine therapy was not efficient for them. FDG PET is more sensitive for the patients with an aggressive histological subtype. Highly differentiated thyroid cancer cells still have the sodium-iodide symporter (NIS) expression, which led to iodine uptake in the thyroid. This ability is low for less differentiated cells. On the contrary, FDG is internalised to the cell by a transporter protein (Glut-1), which is overexpressed in malignant cell types. Inverse alterations of either iodine or FDG uptake in metastases are called the flipflop phenomenon .18F-FDG uptake reflects dedifferentiation of thyroid tumors. Radioiodine-negative metastases display increased glucose uptake more frequently than radioiodine-positive neoplasms. In addition, the survival of patients harboring 18F-FDG–positive metastases is significantly shorter than that of cases with 18F-FDG–negative metastases. The molecular mechanisms of glucose metabolism in thyroid cancer are not entirely comprehended. It has been proved that 18F-FDG uptake can be stimulated by TSH in thyroid cancer tissue in vivo. Although, higher sensitivity of 18F-FDG PET for detection of thyroid carcinoma tissue at higher TSH levels has not been proven in clinical experiments. 18F-FDG uptake regulation in the thyroid cancer cells is performing by TSH or cAMP or mediated by GLUT-1. This regulation is correlated with high activity of PI3-kinase caused by the active mutated K-ras oncogene. These results indicate that high 18F-FDG uptake in thyroid cancer cells observed by PET may be the reflection of intracellular signal transduction cascades stimulation by oncogenes.It is commonly accepted that high 18F-FDG within the tumor cell membrane. Although, thyroid cancer cell line ML-1 do not show GLUT-1 expression in their membranes but instead GLUT-3 expression is significantly detected. Ninety-five percent of human follicular carcinomas are deficient for GLUT-1 mRNA. This observation is not completely unexpected and also agrees with results obtained by Ciampi et al., who reported an overexpression of GLUT-3 in other well differentiated follicular thyroid carcinoma cell lines. Phosphorylation is the serious prerequisite for the intracellular trapping of 18F-FDG. This phosphorylation is catalyzed by hexokinase. According to immunohistochemical staining results, the 18F-FDG retention in thyroid carcinoma tissue is shown to have a correlation with hexokinase I expression [It is evident that the specific uptake of this tracer is controlled by the various isoforms of GLUT as well as hexokinase. GLUT-1, GLUT-3, GLUT-4, and GLUT-10 mRNA expression has been detected in normal thyroid tissue. GLUT-1 is the main glucose transporter mediating the transport of pression .18F-FDG PET. Moreover, a positive association was found for cytoplasmic Glut-1 staining. Well differentiated thyroid cancer expressed Glut-1 mostly in the cytoplasm, rather than on the cell membrane. Several studies reported considerable data that Glut-1 expression on the membrane is mainly found around necrotic areas. The yield of 18F-FDG PET positivity seems to be predicted by HK I expression. If affirmed in larger studies, their results may have important clinical implications. Different types of cancer indicate a significant association between 18F-FDG uptake, Glut-1 and HK II expression. Although HK II was expressed in almost all tumors without a significant relation with 18F-FDG uptake. However, correlation of HK I with 18F-FDG was identical with experiment on breast cancer cells. The role of the different isozymes of HKs in thyroid cancer is not clearly understood so extra research is required to explain the function of HKs in thyroid cancer [The first key enzyme involved in glycolysis is a hexokinase, so GLUTs and HKs are important for mediating glucose metabolism in tumorigenesis, particularly GLUT-1 and HK-II. Abnormal expression of GLUT-1 and HK-II in many malignant tumors is associated with invasiveness and metastasis of tumors including head and neck cancers such as thyroid carcinoma. Few studies have focused on HKII in head and neck cancers such as thyroid carcinoma. Several studies have shown that GLUT-1 and HK II expression levels are related to tumor FDG uptake, but there are differences among tumor types. The uptake of FDG by some malignant tumors was related to tumor expression levels of GLUT-1 or HK-II, or both; however, uptake of FDG by other tumors was independent of either GLUT-1 or HK-II expression . As mentd cancer .PTEN (phosphatase and tensin homolog deleted on chromosome 10) is known as a protein and lipid phosphatase which is a tumor suppressor and a negative regulator of cell growth and metabolism. This gene mutated frequently in many advanced human cancers . PTEN exThe PI3k class I (PI3KC1)-AKT pathway and of the AKT downstream effector AS160 (a rab GTPase activator) pathway are involved in the cell surface exposure of GLUT1 in thyroid cancer cells , 59. TheEnvironmental, genetic and hormonal agents are the main sources of increased prevalence of human cancers , which aPTEN and P53 have major duty in driving the plasma membrane localization of GLUT1. They are key regulators of the glucose metabolism and of autophagy, which are the most common deleted or mutated oncosuppressors found in human carcinomas , 66, 67.Point mutations in p53, which occurred in its DNA-binding domain correlated with malignancy and abolished its inhibitory function on transcriptional activity of GLUTs. Among the GLUTs, GLUT1 and GLUT4 gene promoters are the dominant types which affected with P53 mutations in a dose-dependent and cell type-specific manner. It is terminated in enhanced glucose metabolism and cell energy capacity which predicted to simplify tumor cell proliferation. P53 has been indicated a prominent inhibitory effect on transcriptional activity of the GLUT4 compared to GLUT1. This may be arising from the fact that GLUT1 is a general “housekeeping” glucose transporter, whereas GLUT4 is a tissue-specific and insulin-sensitive glucose transporter , 72.MicroRNAs (miRNAs) functions in carcinogenesis are subdivided into oncogenic miRNAs and tumor-suppressive miRNAs. Onco-miRNAs are upregulated in human cancers which promote cell proliferation and inhibit apoptosis, whereas tumor-suppressive miRNAs are down-regulated in human cancer and can prevent cancer development –75. MiRNMiR-125a-5p have emerged as a potent tumor suppressor and regulator of glucose metabolism in several kinds of cancers, mainly in thyroid carcinoma . Since lIodine assumes an effective function in the modulation of thyroid cells activity. The number of glucose carriers in the plasma membrane can be decreased by the autoregulation performance of iodine. In fact, it influences glucose metabolism not only by effecting on the oxidation pathway, but also through an inhibitory effect on the rate-limiting glucose-facilitated transport system. Iodide is able to inhibit TSH induced stimulation of glucose transport. A role for thyroid hormone in iodine autoregulation has also been suggested, but T3 and T4 did not inhibit glucose transport. Iodide inhibits the Vmax of glucose transport without any apparent effect on the Km. This results means that iodine does not affect the affinity of the glucose transport system, but can decrease the number of available carrier sites. Malignant cells and rapidly dividing cells show a high rate of glucose transport activity. Consequently, the inhibitory effect of iodine on glucose transport in thyroid tissue may be relevant both in physiological and in pathological conditions, both at the level of intermediary and nucleic acid metabolism .18FFDG PET/CT) was designated as the flip-flop phenomenon  is a cytoplasmic serine-threonine protein kinase which represent an essential function in thyroid oncogenesis. Detection of BRAF mutation as a common alteration in thyroid cancer is important knowledge. BRAF mutation may be an initiator in transition of papillary thyroid cancer to anaplastic thyroid cancer . The difHypoxia is a critical hallmark of aggressive malignancies that promotes the malignant phenotype and the activation of physiological adaptation of cancer cells. This promotes tumor cell survival and disease progression. Examples of such adaptations for cancer cells include the glucose transporter 1 gene (GLUT1), which increases glucose uptake for glycolysis . AccordiThere is no trace of HIF-1α expression in normal thyroid tissue but on the other hand in the most aggressive dedifferentiated thyroid cancers its expression is high. In PTC, MTC, and FTC, HIF-1 overexpression has been connected with a poor prognosis and distant metastasis , 92. ThyHIF-1 can participate in warburg phenotype through enhancing the glycolysis process. It is done not only by activation of all glycolytic enzymes transcription, but also by increasing their affinity for substrates . In addiThe phosphatidylinositol 3-kinase (PI3K)–Akt pathway is a member of a family of lipid and protein kinases activated by growth factors, which is responsible for regulating growth and survival processes . The assThe effect of oncogenes on cell metabolic shift for maintaining of cell proliferation is the major aspect in thyroid tumors . In manyTSH stands for thyroid stimulating hormone is the major factor for the regulation of function and metabolism of normal thyroid cells. Its stimulation increases glucose metabolism to amplify iodide transport and thyroid hormone (T3 and T4) synthesis . EnhanceIn normal thyroid tissue, glucose uptake can be stimulated by TSH . StimulaTSH exerts its effect on FDG uptake via the TSH receptors in a time- and concentration-dependent manner. In well-differentiated thyroid carcinomas, most of which are still TSH-sensitive, Glucose uptake is increased at high levels of TSH. It has been found that the expression level of the TSH receptor messenger RNA in thyroid tumors is related to the level of differentiation and that poorly differentiated thyroid carcinomas may lack TSH receptors completely , 112. FuThe c-MYC proto-oncogene is an obvious cause of cancers. Glucose metabolism genes were demonstrated to be directly regulated by Myc. Chief among these are the glucose transporter GLUT1, hexokinase 2 (HK2), phosphofructokinase (PFKM), and enolase 1 –115. GluReactive oxygen species (ROS) as upstream signals of AMP kinase (AMPK) protein can change cell metabolism and increase Warburg effect by upregulation of AMPK. AMPK is a metabolic stress-sensing cytosolic enzyme that activated in shortage of energy for regulation of metabolism and cell growth . In contCancer historically definition described it as an abnormal cell proliferation but recent evidences proved that cancer is indeed a kind of metabolic disorder. It seems that in the near future, metabolomics studies, besides conventional methods will be used for diagnosis and differentiation of different types of thyroid cancers and will most likely introduce altered metabolic pathways as therapeutic targets . The fasAs glucose uptake into cancer cells is the rate-limiting step in glycolysis, targeting GLUTs by blocking their glucose transport channel with small molecules should be a viable mechanism of nutrient deprivation in tumors . The pri


N2 fluorination of aliphatic compounds promoted by ionic liquids is first discussed, focusing on the mechanistic features for reaction using alkali metal fluorides. The influence of the interplay of ionic liquid cation, anion, nucleophile and counter-cation is treated in detail. The role of ionic liquid as bifunctional (both electrophilic and nucleophilic) activator is envisaged. We also review the SNAr fluorination of diaryliodonium salts from the same perspective. Nucleophilic fluorination of guanidine-containing of diaryliodonium salts, which are capable of forming hydrogen bonds with the nucleophile, is exemplified as an excellent case where ionic interactions and hydrogen bonding significantly affect the efficiency of reaction. The origin of experimental observation for the strong dependence of fluorination yields on the positions of -Boc protection is understood in terms of the location of the nucleophile with respect to the reaction center, being either close to far from it. Recent advances in the synthesis of [18F]F-dopa are also cited in relation to SNAr fluorination of diaryliodonium salts. Discussions are made with a focus on tailor-making promoters and solvent engineering based on ionic interactions and hydrogen bonding.We review recent works for nucleophilic fluorination of organic compounds in which the Coulombic interactions between ionic species and/or hydrogen bonding affect the outcome of the reaction. S This feature is especially important when the radioisotopic fluorine-18 [18F] is to be incorporated into an organic substance for clinical applications to positron emission tomography (PET) [18F]F-labeled molecules can generally be produced in higher radiochemical yield and higher activity than the electrophilic substitution using carrier added [18F]F2 sources by several orders of magnitude. Third, the reaction yields from nucleophilic fluorination may improve tremendously (up to > 90%) in reasonable reaction time (<2 h) by employing a variety of promoter/catalyst with a minimum amount of by-products. Recent use of alkali metal fluoride in ionic liquids (ILs) have clearly opened this possibility by designing and applying various ‘task-specific’ ILs for nucleophilic fluorination. This novel capability of ILs as promoter/catalyst is really the results of the fact that ILs comprise the ionic species (cation and anion), but the detailed mechanism has been seldom understood. Here we review recent advances in the catalysis/promotion of SN2 fluorination by ILs, focusing on the mechanistic features of the process. We show that the rates and yields of nucleophilic fluorination may be improved by monitoring and controlling the Coulombic forces and weak interactions  between IL cation, anion and substrates .Nucleophilic fluorination ,2,3,4 ushy (PET) ,8,9. SecNAr fluorination of diaryliodonium salts that are gaining much importance as a useful path to incorporating 18F and 19F to aromatic compounds. Since diaryliodonium salts consist of diaryliodonium cation and the counter-anion such as Br−, OTf− etc., Coulombic interactions with the alkali metal counter-cation and the nucleophile F− would be critical to determine the efficiency of fluorination. When the substrate contains functional groups such as hydroxyl (-OH), amino  that are amenable to hydrogen bonding, the situation becomes too complicated to scrutinize by intuition only. In the second part of this brief review, we show that nucleophilic fluorinations of diaryliodonium salts using alkali metal fluoride are indeed significantly affected by these electrostatic interactions. In some cases, the position of the nucleophile F− relative to those of the electropositive C atom and the leaving group may be determined by these ionic interactions and hydrogen bonding. Either the nucleophile F− is positioned close to the electropositive C atom for efficient fluorination, or F− may be located far from the center of reaction as the results of intricate interplay of the electrostatic interactions. In the latter case, of course, fluorination will not proceed at all. In order to scrutinize the configuration of the reacting system  in pre-reaction complex and transition state to analyze the experimentally observed efficiency of reaction, quantum chemical calculations are indispensable, and we discuss the relevant recent works.These interactions also determine the efficiency of SN2 [X] resulted in excellent yields (>90%) in reasonable reaction time (<2 h) with a minimal amount of by-products. Use of cosolvent such as acetonitrile did not affect or improve the reactivity of fluorination. They also showed that fluorination in [bmim][X] did not require anhydrous condition, demonstrating the extreme flexibility of ionic liquids for reaction conditions. However, less than stoichiometric amounts of IL (0.5 equiv) as catalyst/promoter showed rather poor performance for the nucleophilic fluorination.The first demonstration  of promo+ or Cs+, thereby reducing its retarding Coulombic influence on the nucleophile F−. That is, the counter-cation becomes a ‘ghost-like’ agent by the action of the negative charge of IL anion. The intricate interplay (Coulombic forces) of the counter-cation, the nucleophile, IL cation and anion helps to form a pre-reaction complex and transition state that is optimal for SN2 fluorination, prohibiting the formation of by-products  that were proved to act as catalyst/promoter in nucleophilic fluorination, but were probably better because of the explicit negative charge of IL anions. The mechanism of these very interesting observations was elucidated by Lee and co-workers  in quantproducts . The rol+ bridges the nucleophile F− and the leaving group -OTs, helping the formation of compact pre-reaction complex with optimal configuration for SN2 fluorination. For 2 eq. of [bmim][F] used iodonium tosylate for 18F-incorporation on electron-rich aryl rings  by harnessing these interactions? Among the numerous studies of SNAr fluorination, diaryliodonium salts containing side chains that are amenable to form hydrogen bonds seem to be the best example to address this question.One such strategy results from the question: Could we monitor and control the position of F18F-labeling of guanidine-containing radiopharmaceutical F-dopa. There have been considerable amount of efforts [18F]F-dopa by SNAr scheme have also experienced severe problems of poor yields along with many undesirable by-products. Besides these problems, the number of steps in the synthesis of this elusive radiotracer is of profound importance in order to be used for clinical purposes. Wirth and co-workers’ recent work [NAr fluorination of diaryl iodonium salt based on DiMagno and co-workers procedure [19F) and “hot” ([18F]) fluorination. The origin of the observed poor yields was also studied by quantum chemical calculations F-dopa, further complication would result from the use of base such as K2CO3 employed to extract 18F−. The alkali metal cation K+ may certainly be unfavorable due to its interactions with the nucleophile 18F−, and using Cs+ instead may be better. More recent experimental work by Maisonial-Besset et al. [18F] labelling of L-dopa  and counter-cation (to the nucleophile) and the base may lead to robust synthetic route to [18F]F-dopa with excellent yield and in short reaction time sufficiently satisfactory for clinical usage.Another  efforts ,74,75,76ent work  seems torocedure ) despitef L-dopa  without 18F]Fluorination, is usually very difficult due to unfavorable solvent effects on the small-sized F fluoride when the reaction is attempted in organic solvent. Careful choice of efficient promoter/catalyst is usually required for this process. Use of alkali metal fluoride in Lewis base solvent/promoter such as bulky alcohols and oligoethylene glycols proved to be such a breakthrough for efficient nucleophilic fluorination. Employing ILs for that purpose is another example, in which the IL anion acts as Lewis base due to its ionic nature. We discussed that systematic design of the structure of ILs allowed very efficient SN2 fluorination with a minimum amount of by-products in reasonable reaction time. The harnessing of ionic interactions and hydrogen bonding also proved critical in controlling the reaction yield and rates. For SNAr fluorination of diaryliodonium salts, we presented a number of examples where quantum chemical analysis allowed the elucidation of underlying mechanism and design of experiments. We presented the synthesis of [18F]F-dopa by SNAr fluorination of diaryliodonium salts as a very recent example. We believe that our present review would help to advance further developments concerning the tailor-making of promoters/catalysts and solvent engineering based on ionic interactions and hydrogen bonding in the field of organic synthesis, medicinal chemistry, and environmental chemistry.Nucleophilic fluorination, and especially [