This review reports the progress of the recent development of graphene-based microfluidic sensors. The introduction of microfluidics technology provides an important possibility for the advance of graphene biosensor devices for a broad series of applications including clinical diagnosis, biological detection, health, and environment monitoring. Compared with traditional  sensing systems, the combination of graphene and microfluidics produces many advantages, such as achieving miniaturization, decreasing the response time and consumption of chemicals, improving the reproducibility and sensitivity of devices. This article reviews the latest research progress of graphene microfluidic sensors in the fields of electrochemistry, optics, and biology. Here, the latest development trends of graphene-based microfluidic sensors as a new generation of detection tools in material preparation, device assembly, and chip materials are summarized. Special emphasis is placed on the working principles and applications of graphene-based microfluidic biosensors, especially in the detection of nucleic acid molecules, protein molecules, and bacterial cells. This article also discusses the challenges and prospects of graphene microfluidic biosensors. The rapid development of Microfluidics Technology in recent years has caused a revolutionary impact in the fields of physics ,2, mater2) [Due to the reactivity, surface and interface effects, quantum size effect, Raman spectrum effect, catalytic efficiency, electrical conductivity, and other characteristics of nanomaterials, nanomaterials have excellent applications in the field of microfluidics technology ,22. Grap2) ,28. The 2) ,30,31. T2) ,33,34. T2) ,36,37. B2) . Besides2) ,40.The combination of graphene and microfluidic sensors can amplify their respective strengths to enable even more valuable and potential applications. Santangelo et al.  developeThe purpose of this article is to provide a comprehensive overview from production to the application of graphene microfluidics devices and the latest progress of graphene microfluidics sensors in recent years. In Fabrication methods of graphene may be divided into top-down and bottom-up two main categories.The top-down method is mainly exfoliation , and also includes the reduction of GO, sonication, etc. Micromechanical exfoliation is the first method used to obtain graphene with different layers from graphite . Recentl12CH4 or 13CH4, which rapidly diffuses into most metals. Then, when the carbon in the metal reaches supersaturation at a certain temperature, due to carbon segregation, balanced graphene will form on the surface. Moreover, the carbon concentration and the cooling rate directly determine the number of layers of graphene on the metal surface. Besides the metal substrates, the synthesis of graphene on the insulating substrates has also been studied to be feasible. Tai et al. [4 partial pressure or H2/CH4 ratio, were also experimentally investigated. In short, it is possible to deposit multiple layers of graphene on an insulating substrate without catalysis. Furthermore, to manufacture high-quality graphene, a higher decomposition temperature is required on an insulating substrate than on a metal substrate including Cu or Ni.The bottom-up approach is to synthesize graphene using small carbon-containing molecules as raw materials, which mainly includes chemical vapor deposition (CVD), pyrolysis of SiC, and crystal epitaxy. The high purity and crystalline graphene can be obtained by all of these techniques. Nowadays, the CVD method has proven to be the most widely used fabrication technique for obtaining high quality and large area graphene due to process tunability and mass production. The growth of graphene by CVD can in principle be divided into two types, that is, the carburizing mechanism and the surface growth mechanism. The difference between the two mechanisms lies in the carbon content of the metal substrate. CVD methods use carbon compounds such as methane as carbon sources to grow graphene by decomposition at high temperature on the substrate. The growth of graphene on Cu metal surfaces by CVD processes, which reduce the decomposition temperature and graphitisation temperature of carbon precursors, is now being extensively studied. The growth of graphene on Cu, Ni and Cu/Ni surfaces was investigated by Li’s group  using cai et al.  conducteAlthough graphene exhibits great application potential in many fields due to its excellent physical and chemical properties, it is worth mentioning that the strong vdW force between graphene sheets is far greater than the interaction with solvents, making it difficult to dissolve in water and common organic solvents, which also limits its wide application in microfluidic platforms. This is also the biggest obstacle to the application of graphene as a sensing medium layer to sensor devices. Hence, it has greatly significant to modify the characteristics of graphene and expand the application of it in the function of nanoelectronic devices especially in the microfluidics field via functionalization. Although graphene has a stable hexagonal structure and chemical inertness, the actual graphene produced has some defects and its edges are very active, which provides the possibility to realize the functionalization of graphene. One of the most effective methods to functionalize graphene is to reduce or peel the graphite with an alkaline metal in an appropriate solvent and then the intermediate graphene formation is quenched with an electrophilic body. Besides, the functionalization of graphene can avoid agglomeration by producing the strong polar-polar interaction of hydrophilic. The functionalisation of graphene covers many areas of research, including the chemical modification of its surface, reactions with various molecules, and covalent and non-covalent interactions ,61,62. T2 carbon atoms of graphene to form covalent bonds. The common free radicals are aryl diazonium salts and benzoyl peroxide, which are synthesized by Bergman cyclization and Kolbe electrosynthesis. In addition to free radicals, dienophiles also can react with the sp2 of graphene. For example, one of the most common dienophiles Azomethine ylide, it has been successfully used to functionalize carbon nanostructures  via 1, 3 dipole ring addition reaction. The dihydroxyl phenyl group was decorated on a graphene sheet with pyrrolidine rings, which was formed perpendicular to the graphene surface by the addition of azomethine ylide precursors  can also be used to modify molecules to functionalize the graphene surface and form a stable dispersion system. It is essential to increase the solubility and avoid agglomeration of graphene by the non-covalent functionalization with different organic compounds. Moreover, as a hydrophobic material, graphene must be non-covalently functionalized to make it soluble in polar solvents. To achieve the non-covalent bonding, there may have multiple routes including π-interaction, electrostatic, hydrophobic, vdW interactions. Among them, it must be said that π-interaction is an attractive method for graphene functionalization because it can connect functional groups to graphene without disturbing the electronic network of graphene.As a π-system, non-covalent molecular interactions involving graphene are essential for stabilizing proteins, nucleic acid molecules, inorganic molecules and functional nanomaterials . Due to cation-π interactions, so that various receptors with strong binding energies and high selectivity for metal cations can be explored. The πcation-π interactions also exhibit different advantages to metal cation-M interactions, such as a stronger total binding energy.The H-π interaction, one of the hydrogen bonds, plays an important role in adjusting the geometry and the nature of the complex. Additionally, the substantial contribution provides by dispersion can further stabilize the H-π complexes. At present, the π-π interaction is the most attractive non-covalent interactions, that is, the negatively charged and diffused electron cloud of the π-system exhibits attractive interactions. For example, the dispersion interactions also take hold when two π-system possess the same electron densities in noncovalent π-π interactions . HoweverFor the microfluidics field, one particularly attractive idea is to develop integrated “lab on a Chip (LOC)” systems that can reproduce laboratory-scale processes in a simplified way that costs less, takes less time, and takes up less space than traditional equivalents. Besides, graphene and its derivatives  are very suitable for the basic technical functions of microfluidics, so they have been widely used in LOC devices.As a typical representative technology of a LOC, microfluidic technology has developed rapidly. The microfluidic chip is the main platform for the realization of microfluidic technology. The main feature of the device is that the effective structures  containing fluids are at least micron-sized. Besides, in the microfluidic-based LOC devices, fluid transport is carried through laminar co-flow or immiscible flow in a segmented flow within a small channel. Therefore, the fabricating process of the microfluidic chip is particularly important. It has been reported that many microfluidic fabricating processes have been proposed for various materials and applications. Under the major fabricating processes, many sub-processes including etching, lithography, thermoforming, hot embossing, polymer casting, and bonding are used individually or collectively to implement the final form of LOC devices. Among them, hot embossing and bonding are the most widely used methods in microfluidic chip processing. Glass microfluidic chip prepared by hot embossing have great potential in the fields of medical detection, drug analysis, air monitoring and optical sensors. Jiang et al.  fabricat2 silicon channels with vertical walls and flat bottom surface in cross section by optimizing the power and pulse frequency of a nanosecond laser, which is a method for fabricating microfluidic channels in silicon using a laser system. The design of the microfluidic chip includes a resonance channel branched into a three-pronged outlet, and a glass wafer is used to seal the microfluidic channel using an adhesive. Due to its good electroosmosis, light transmission, surface biocompatibility, and processing technology similar to silicon materials, glass [There are a wide variety of materials used to make microfluidics device, mainly includes silicon, metals, semiconductors and other inorganic materials, as well as polymers, hydrogels, paper and other organic materials . The difs, glass  is widels, glass  develope2 flow, which is perfectly suited to the practical needs of microfluidic chips. Olmos et al. [Although glass has many advantages compared with other materials, the manufacturing cost of glass-based microfluidic chips is high and time-consuming and laborious, which limits its use. Polydimethylsiloxane (PDMS) is currently the most widely used polymer material in the field of microfluidic chips, and it is often combined with glass to make microfluidic chips. PDMS can be applied to biomedicine in the field of microfluidics, including on-chip devices and rapid real-time monitoring. Due to the enhanced signal response and ease of manufacturing, many microfluidic preparation processes use PDMS as basic material. Surface treatment, especially functionalization, has changed the physical and chemical properties, so it is aimed at a very wide range of sensing applications for PDMS-based microfluidic systems. The most important surface modification techniques commonly used in microfluidics are plasma and UV. Liu et al.  reporteds et al.  introducGraphene, as a two-dimension material, has a large surface and high surface-area-to-volume ratio. On account of the special 2D structure, each carbon atom is exposed on graphene’s surface. Graphene is sensitive to changes in the environment caused by the adsorption of the analyte to its surface, in particular to the binding of organic or inorganic molecules on the surface . Hence, 2+ increases, but the Fermi energy level decreases. In the electrochemical microfluidic sensor, the sensing platform achieves significant sensitivity and low detection limits, although the area of graphene in contact with the solution is small. This work fully proves that the sensing platform can continuously monitor toxic heavy metals in real-time, and is convenient for direct sample research, which can be applied to daily life and industrial manufacturing. At present, it also shows some shortcomings, such as difficulty in determining the concentration of a single heavy metal in the presence of other heavy metals [Currently, based on the development trend of electrode surface functionalization and device module miniaturization based on electrochemical sensors, a new generation of nano-modified and integrated microfluidic technology on-chip detection system has been developed. For example, graphene is widely used as a working electrode in electrochemical detection platforms for the detection of heavy metals. The electrochemical analysis method is one of the most commonly used methods, because of the advantages of low power consumption, high sensitivity, short analysis time, and easy direct measurement . In 2019y metals , defects2 and Si are used as the gate dielectric and back gate, respectively. The Cr/Au, which is patterned by photolithography, is located on both sides of the graphene surface and acts as an electrode. The design of the graphene channel FET sensor with the graphene/Au/Cys-PP structure is then completed by a sequence of operations involving the evaporation of gold nanoparticles (NPs), immersion in a cysteamine (Cys) solution and immobilisation of the protoporphyrin IX (PP). 2+ aqueous solution is dropped into the chamber, the Cd2+ ions are combined with the doped graphene on the channel surface. After that, in the process of dropping the mixed solution that produces •OH, the Ids monitored by the graphene/Au/Cys-PP-Cd2+ sensing layer shows a rapid response when corresponding to 1 × 10−4 M •OH within 2 s, as shown in 2+ structure enables real-time label-free detection of •OH and its concentration. Because of its label-free, high sensitivity, selective detection function, and miniaturization function, it has important value in human health and environmental monitoring.FETs are also one of the most widely used electrochemical microfluidic devices. Graphene has a suitable condition it was generally used as a channel material for FETs with applications in sensors and biosensors. Compared with other FETs devices made of classic semiconductor materials , GFETs show the development of flexible, stable, and biocompatible merits . Many meIn recent years, graphene-based FETs have attracted a lot of attention in various electrochemical and biosensor applications. Hence, a GFET is promising in the detection of various molecules, such as exosomes, bisphenol A (BPA), etc. For example, Yu et al.  have devWith the continuous improvement of microfluidic technology and the continuous penetration and integration with other disciplines, several research hotspots have emerged in recent years, of which microfluidic optical devices are typical representatives. A new cutting-edge cross-discipline formed by the combination of optoelectronics and microfluidics technology combines microfabrication technology with physics, chemistry, biology, etc., and realizes the special functions of optical or optoelectronic devices and systems through precise control of microfluidics. With the birth of the new discipline of microfluidic optics and the development of new technologies, microfluidic optics will play a more important role in the future of optical technology. In contrast to traditional optical systems with large volume, high cost, and poor adjustability, the fusion of microfluidic technology and optical devices provides the possibility of miniaturization, arraying, low-cost and high-precision control of microfluidic optical devices.3, and the diameter of the two ports connecting the channel is about 10 μm. Based on the theoretical analysis of the reflection of the coupling structure, the strong interaction between a part of the incident energy and rGO is very sensitive to the RI change of the low refractive index medium. Therefore, when the pressure of the aqueous solution in the microfluid changes, its RI will change accordingly, which is measured by the photodetector. In their work, a fixed frequency (1 MHz) weak ultrasonic wave was used to provide a pressure of 1kpa to the water, and the RI change of 1.35 × 10−7 was obtained by using the formula (dn) and ultrasonic pressure (dP) in the water. The RI of water changes periodically with the ultrasonic frequency. Integrated optics can accommodate the compact arrangement of microfluidic channels and optical devices, which has broad application prospects for the integration of fluid optical sensors with high sensitivity and high throughput. Various optical methods have been applied to microfluidic sensings, such as light absorption, Raman scattering or surface plasmon resonance, and other measurement methods. Besides, graphene possesses excellent optical properties, especially under the total internal reflection structure, the enhanced interaction between graphene and light and its polarization-dependent properties, making graphene combined with microfluidics technology demonstrate potential applications in optical sensing. Wu et al.  developeD) and sensitivity (S) for graphene-based microfluidic optical sensors are determined by the methods described above. The relationship between them and RI changes is as follows:−8 and a sensitivity of 3.5 × 109 mV/RIU for this microfluidic optical sensor. To facilitate the reliability of the experiment and optimise the signal-noise ratio (SNR), the high RI changes in the aqueous solution caused by high-pressure ultrasound were also monitored using the microfluidic optical sensor  has become a vital nanomaterial in the field of biosensors due to its unique optoelectronic properties, especially in the manufacture of low-cost optoelectronic devices. The channel size and flow characteristics of microfluidic devices have also become favorable conditions for the study of biosensors and biological systems. In the field of sensing and biosensing, the research goal of combining microfluidic technology is to develop integrated miniaturized equipment that can achieve high sensitivity and selectivity, fast response, small throughput, and automated testing.d shows an upward trend in the range of 55 to 62 μA. Besides, even at concentrations as low as 1 μg/mL, a significant current response to BPA injection can be observed. Due to BPA molecules in a high-concentration solution can form covalent compounds with DNA molecules, which destroy the DNA structure and chemical bonds, resulting in ssDNA molecules to fall off the graphene surface, as shown in 2) group of an antibody (cMAb). After injection into the microfluidic channel, cMb molecules are directly trapped by cMAb on the channel surface (see The combination of microfluidic technology and biosensing provides the advantages of high-throughput analysis and portability, thereby realizing intelligent real-time detection. Due to the different sizes of biomolecules, graphene nanomaterials can be used to functionalize and detect different biological analytes , such asface see f, leadin1 CFU/mL to 107 CFU/mL, and reaches saturation at 107 CFU/mL. Compared with GO-based immunochips, the use of GO nanosheets to wrap cMWCNT exhibits a synergistic effect, which improves the sensing properties of Salmonella typhimurium cells and has better biological detection properties, such as sensitivity and detection limit. Singh et al.  preparedHere, more work is still needed to demonstrate the selectivity or specificity of the graphene microfluidic biosensor to the analyte. Ideally, it is more hope that the sensor can have a large response to the target detection object, but almost no response to any interference. In this regard, it is necessary to improve the functional layer, or more methods to capture the target.In this review article, we discussed the latest work on graphene and microfluidic technology, focusing on microfluidic sensors based on graphene nanomaterials for the detection of chemical substances and biomolecules. The combination of graphene and microfluidics can take advantage of the unique physical and chemical properties and analytical characteristics of graphene, such as real-time rapid detection, high sensitivity, low consumption, and easy operation. In recent years, more and more devices based on graphene integrate microfluidic have been developed and widely used in various fields. This article first introduces the preparation methods of graphene and its derivatives and the functionalization of graphene. Next, briefly introduce the development of microfluidic technology, and analyze the preparation of microfluidics and material research. It mainly discusses the manufacturing process of different types of microfluidic platforms and microfluidic devices. The main applications of graphene microfluidic devices are divided into three parts: electrochemistry, optics, and biology. It ranges from the detection of chemical molecules to the electrical signals of nucleic acid and protein molecules and even the detection of bacterial cells.Although graphene microfluidics devices have made remarkable achievements in many fields such as biomedicine and health detection, research on graphene-based microfluidic sensors is still developing and improving, and there is much work to be done. In order to better use the graphene sensor, it can be converted into a sensing system that can be used in many daily life environments, such as drinking water quality monitoring, blood glucose level detection in the body, or as a urine analyzer. The main challenges for researchers in the microfluidics biosensors, in especial, using the graphene and turn them into production . However


C. elegans is an excellent in vivo model to study the regulation of cell invasion during development. Here, we have examined the function of egl-43, the homolog of the human Evi1 proto-oncogene , in the invading AC. egl-43 plays a dual role in this process, firstly by imposing a G1 cell cycle arrest to prevent AC proliferation, and secondly, by activating pro-invasive gene expression. We have identified the AP-1 transcription factor fos-1 and the Notch homolog lin-12 as critical egl-43 targets. A positive feedback loop between fos-1 and egl-43 induces pro-invasive gene expression in the AC, while repression of lin-12 Notch expression by egl-43 ensures the G1 cell cycle arrest necessary for invasion. Reducing lin-12 levels in egl-43 depleted animals restored the G1 arrest, while hyperactivation of lin-12 signaling in the differentiated AC was sufficient to induce proliferation. Taken together, our data have identified egl-43 Evi1 as an important factor coordinating cell invasion with cell cycle arrest.Cell invasion allows cells to migrate across compartment boundaries formed by basement membranes. Aberrant cell invasion is a first step during the formation of metastases by malignant cancer cells. Anchor cell (AC) invasion in  C. elegans, the so-called anchor cell, can invade into the adjacent epithelium during normal development. Our work has identified an oncogenic transcription factor that controls the expression of specific target genes necessary for cell invasion, and at the same time inhibits the proliferation of the invading anchor cell. These findings shed light on the mechanisms, by which cells decide whether to proliferate or invade.Cells invasion is a fundamental biological process that allows cells to cross compartment boundaries and migrate to new locations. Aberrant cell invasion is a first step during the formation of metastases by malignant cancer cells. We have investigated how a specialized cell in the Nematode  C. elegans is a genetically amenable and tractable model that has provided important insights into the molecular pathways regulating cell invasion and uncovered the molecular similarities between tumor cell and developmental cell invasion  transgene as a marker.AC invasion was scored in mid-L3 larvae after the VPC had divided twice (Pn.pxx stage) as described . 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CopenhaverEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: This article by Deng and coworkers is a study of EGL-43, the C. elegans ortholog of the human Evi1 proto-oncogene, and how it regulates the fate and function of the invading anchor cell (AC). The authors show that egl-43 inhibits AC proliferation by repressing lin-12 expression.  Furthermore the authors show that a positive feed-back loop exists between egl-43 and fos-1 in the AC, maintaining high expression of both for the induction of genes required for AC invasion.I have no major criticisms of this work. It appears solid and complete, and the text is well-written and clear. The only thing I felt was lacking was more discussion of the role of egl-43 in the fate decision of the AC as compared to other studies on this subject, in particular the recent work from the Greenwald lab . How does egl-43 function fit in with the HLH-2 expression clock described in this paper?Reviewer #2: Summary:The research described in this manuscript examined the function of egl-43 in regulating C. elegans anchor cell (AC) invasion. Using CRISPR/Cas9 mediated genome editing, the authors have engineered several new egl-43 alleles that provide valuable insight into egl-43 function. Specifically, they identify the long isoform (egl-43L) as the predominant isoform functioning during AC invasion as well as regulation of egl-43 by fos-1 via a cis-regulatory FOS-Responsive Element. Using multiple cell cycle reporters, the authors also show that egl-43 is required for the AC to undergo G1 cell cycle arrest, and that egl-43 depletion results in proliferative ACs. The authors claim that this cell cycle dependent function of egl-43 is independent of the nhr-67 / CKI-1 pathway known to be involved in AC invasion, although I feel that this is not well-supported by the evidence currently provided in the manuscript. Opportunities to include more convincing controls and to quantify data were sometimes missed by the authors, though should be easily remedied by either better quantification of existing data or new experiments. Suggested revisions to the manuscript include validation of RNAi reagents, explicit indication of sample sizes, and additional experiments (outlined below). Despite the fact that some of this data  has been recently shown in a bioRxviv preprint ), I feel that the mechanistic insights gained through the authors’ careful dissection of the egl-43 locus is complementary and I am enthusiastic about seeing this work published. Furthermore, the novel finding that ectopic expression of Notch intracellular domain is sufficient to induce proliferation in a normally post-mitotic differentiated cell is a very exciting finding and would be of interest to a broad readership.Comments:General comments:Assuming PLoS Genetics allows for citations of preprints, given the nature of overlap between this work and that of a recently updated bioRxviv preprint ), I think it would be useful for the field if the authors discussed their results in the context of data showing that egl-43 regulates both hlh-2 and nhr-67 in a cell-cycle dependent manner as well as feedback between EGL-43 and FOS-1. The main discrepancy between this work and Medwig-Kinney et al. (2019) is whether or not egl-43 and nhr-67 function independently of each other in mediating G1/G0 arrest in the AC. See below for specific experimental suggestions that might clear this discrepancy up.I would recommend for showing single channel fluorescence images to use grey scale, which the human eye can see subtle differences in easier than false colored images, and only use false colored images for overlays.In the results and brief Discussion section the authors miss the chance to put their data using endogenously-tagged alleles in the context of what has been shown by their and other labs previously using transgenes - for example, autoregulation of egl-43 has been shown multiple times based on transgenes and the first potential explanation for this is that levels of egl-43 are extremely important - see Wang et al. 2014 (doi: 10.1016/j.bbrc.2014.08.049) - where they show that egl-43 functions through an incoherent feed forward circuit with negative feedback in regulating MIG-10 levels in the AC.For the most part, the authors represent fluorescence quantification data through box plots, which depict median values. However, given the wide spread of some of this data , median may not be the best statistic to show. I would recommend using an alternative method of data visualization, such as violin plots including mean values and standard deviation.Introduction:Potential typos (minor):“selected” → “select” (paragraph 1)“trackable” → “tractable” (paragraph 1)“EGl-43” → “EGL-43” (paragraph 3)“the VU cell undergoes three rounds of cell divisions” - This is not 100% accurate, as the ρ cells undergo an extra round of division. See Newman, White, & Sternberg (1996).Results:“FRT” should be defined upon 1st use of the acronym. The FRT experiments are really elegant - I’m wondering, is the reduced penetrance in these lines as compared to RNAi due to produrance of the protein during the length of time it takes for the flipase to remove the genomic region flanked by FRT sites? I couldn’t tell from the images - it looks like there is no expression, but it would be useful to quantify this.“We found no obvious difference in the expression pattern of the two egl-43 reporters, suggesting that the long egl-43L isoform accounts for most of the expression observed.” - This claim can be supported by evidence showing quantitative comparison of expression levels in both reporters (not directly provided).How was the egl-43Si RNAi construct validated? The targeting sequence is presumably much smaller  than typical RNAi constructs, so the efficiency may be significantly lower. Also, how did the authors determine the 5’ UTR sequence, as I could not find it annotated on WormBase? The authors also may want to consider that there is evidence  that RNAi can act on pre-mRNA, which would indicate that this construct may recognize the introns of pre-spliced egl-43L transcripts.Figure 1B-C: It would be helpful to see quantification of this data presented as well.How was the sample size for the egl-43L RNAi vs. egl-43 RNAi experiment (Results paragraph #2) determined? Typically a minimum sample size of 28 is required to perform a significance test at ɑ = 0.05.Figure 2: The number of animals observed with the representative phenotype shown, with respect to the total number of animals observed, should be indicated in Figures 2A,C-D. The n indicated in the bar graph in panel B is difficult to read due to the small font size .The source of the RNR-1::GFP strain/construct  should be cited in addition to the WormBook chapter.Is the characterization of the endogenously-tagged MCM-7::GFP described elsewhere? I know that the transgene has been used as a reporter for actively cycling cells by the van den Heuvel lab . If this is the first description of the endogenous MCM complex as a reporter for S-phase onset/cycling cells it would be worth characterizing it first and then using it as a reporter. I believe the data, I just think it would be nice to highlight that it’s a GFP-knock in - you could cross the allele into the MCM-4::mCherry transgene from the van den Heuvel lab and just demonstrate that they show the same exact pattern of localization in a cell cycle-dependent way as a supplemental figure?The original CDK biosensor citation should be included as well from Spencer et al. 2011 when citing its use as it was co-opted from mammalian cell culture.Image quantification: The Materials and Methods section is specifically lacking a description of how the CDK sensor was quantified, and in general more information is needed in reference to image quantification for all of the data in the manuscript - “built-in measurement tools” in Fiji/ImageJ could mean many different things - how did the authors correct for background/camera noise? Were measurements made from single confocal z-planes? Are the authors’ reported mean grey values or integrated density . Did the authors use thresholding and the wand tool to select the region of interest, or did they hand draw regions of interest?In the updated Medwig-Kinney et al. pre-print, it is shown that the regulatory relationships between egl-43 and nhr-67 do not exist until post-AC-specification. I recognize that this data was not available at the time of submission. However, this could explain why the authors do not see a significant change in nhr-67::GFP expression in the AC following egl-43(RNAi). More importantly, however, I would suggest that the authors examine mitotic ACs for regulation of gene expression rather than looking at earlier stages, as it is impossible to know whether or not an AC is out of cycle (beyond using a second set of reporters for cell cycle state) so you can not assess whether the single AC you are measuring is going to invade or not. As to the authors’ statement that the proliferation of the AC results in dilution of protein expression - this data exists. I would point the authors to data using the transgene containing the full (~5kb) cdh-3 promoter fused to GFP. In Matus et al. (2015), I found that this promoter was expressed at ~97% of wild-type levels in proliferating ACs , while other reporters are clearly down-regulated, suggesting that GFP is not simply diluted as ACs become mitotic and proliferate but that the actual transcriptional program is changing due to inappropriate cell cycle entry.This also brings up an important point on the use of the cdh-3 promoter for driving constructs of interest in the AC. We have found that the smaller ACEL used to drive cdh-3 (~1.5kb) for many of the transgenes from the Sherwood lab, also used in this paper (qyIs23 and qyIs50), is regulated by nhr-67 and egl-43, which is why we used the full cdh-3 promoter (~5kb) to generate new AC reporters for studying nhr-67 loss of function in Matus et al. 2015. I bring this up because if anything, the use of the smaller cdh-3>constructs could under-report the number of ACs due to depletion of the promoter. It took a little digging for me to figure out that the new constructs were designed with the full cdh-3 promoter, so it would be helpful to distinguish this in the text/methods. We used cdh-31.5 vs cdh-3 in our original paper if that nomenclature is helpful.Figure 3G,I: The overexpression of CKI-1 in a lineage should cause G1/G0 arrest. It would appear that the authors are making the claim that egl-43 mediates cell cycle arrest independent of CKI-1, but a more parsimonious explanation would be that depletion of egl-43 results in downregulation of the cdh-3 promoter driving CKI-1 expression, and in cases where you see multiple ACs, those ACs do not have a critical threshold of CKI-1 activity to prevent cell cycle entry. One suggestion would be to quantify levels of CKI-1 in all of the animals and see if there is a statistical correlation between CKI-1 levels and number of ACs observed. While, the 2 AC phenotype could be the result of perturbing AC/VU specification, 3+ ACs shouldn’t be observed if the cdh-3>CKI-1 is functioning 100% of the time.Figure 3G-I: The key says “control siblings” - does this mean that these are the progeny resulting from a cross? I assume not, but this terminology may be misleading and whether animals of homozygous or heterozygous is an important distinction.Figure 4A: This data should be quantified. Also how was expression of lin-12 determined? How were the boundaries of ACs versus VUs determined in adjacent cells? The endogenously- tagged lin-12::GFP reporter from Attner et al. (2019) has both membrane bound and nuclear localization, making it easier to distinguish which cell has active Notch signaling - this strain might be easier to use and would better make this really stunning point that active Notch signaling post-AC/VU decision can force the differentiated AC into the cell cycle and inhibit invasion.Figures 4A-C: When was lin-12 RNAi treatment administered? Knockdown of lin-12 prior to AC/VU specification may confound the number of ACs observed. It would be worthwhile to try an L2 plating of lin-12(RNAi) and see if, at some penetrance, you can repeat your experimental results.Figure 4D: What percentage of animals are the phenotypes shown indicative of?Figure 5B: It would be helpful to show GFP::EGL-43L expression without fos-1(ar105) in the background here.The authors postulate that egl-43 has cell cycle dependent (lin-12) and independent (fos-1) roles, but this is not supported by the data showing that lin-12(RNAi) rescues AC invasion in egl-43(RNAi) animals.The authors should mention the potential effects that the endogenous transcriptional reporters (with pre-floxed SEC) have on protein function.The introduction and discussion need elaboration, specifically with regard to links between Notch signaling and cell cycle regulation, in C. elegans and other model systems.The authors argue that egl-43 and nhr-67 control cell cycle arrest in distinct pathways. To show this convincingly, they should perform the lin-12/Notch experiments with nhr-67 RNAi perturbation experiments, the expectation would be that nhr-67(RNAi) does not induce lin-12 expression if the two pathways are independent. Alternatively, as we believe that egl-43 does regulate nhr-67 activity, it would be interesting if this was still the case - that nhr-67(RNAi) does not regulate lin-12, as we have recently shown that endogenous lin-12::GFP is strongly down-regulated pre-AC/VU decision in our bioRxiv preprint. If you find that nhr-67(RNAi) doesn’t turn on lin-12::GFP, it could also suggest that egl-43 has nhr-67-dependent and nhr-67-independent roles in maintaining the AC in a post-mitotic state, and provide an explanation why nhr-67(RNAi) on an nhr-67(pf88) hypomorphic allele doesn’t significantly increase the AC invasion/proliferation defect .Supplementary tables:Tables S2 and S3 is missing the plasmids and primers used to generate the egl-43Si and egl-43Li RNAi constructs. Information regarding the targeting sequences used would also be helpful to include.Table S2 contains primers whose sequences are not provided in Table S3. Namely oTD140-143.Table S3 contains sequences of primers that are not defined in Table S2 or elsewhere. Namely the OEL316-319.Reviewer #3: The C. elegans AC-VU cell fate decision is a classic example of Notch-based lateral inhibition, and the two fate outcomes differ in proliferation and invasive (basement membrane breaching) activity. In this manuscript, the authors follow up on prior studies from >10 years ago that showed that EGL-43, an Evi1-related transcription factor, is required for both the VU cell fate and for AC invasion. They focus particularly on EGL-43 function during AC invasion.Key findings:-The long isoform of egl-43 is required for AC invasion despite unimportance of several specific protein domains unique to this isoform .-EGL-43 inhibits AC proliferation by promoting G1 arrest, and does so by (directly or indirectly) repressing LIN-12/Notch, which promotes proliferation and inhibits invasion . This is the most impactful result in the paper.-EGL-43 does not affect expression of two other factors involved in AC invasion (NHR-67 and HDA-1), or vice versa, but EGL-43 and FOS-1 mutually upregulate each others’ expressionOverview:The paper is generally well written, though data presentation needs clarification in several places. The presented results seem solid and the paper definitely moves the field forward, but it stops short of a really impactful, mechanistic understanding. Therefore, my enthusiasm for the current version is modest.Specific points:1. There are some “rigor and reproducibility” issues in the data presentation that need to be addressed:Fig 1B quantification: how many animals were examined, how many showed these expression patterns, and how many had multiple ACs and/or no invasion?Fig 2B: statistics? Are the changes over time significant?Fig 2C and 2D quantification: how many animals were examined, how many showed these expression patterns?Figure 3H and 3I: What % of animals had multiple ACs? How does this relate to the % that had invasion defects?Figure 4F and 4G: Are the %s in G based on pooling the 3 lines of each genotype, or are they based on one specific line? What % of the animals scored in G had multiple ACs and is there an absolute correlation between the two types of defects?Addressing any of the below would add more meat and impact to this paper, but addressing points 2,3,4 would be especially relevant:2. Circumstantial evidence suggests that the egl-43 long isoform might be required because it is the predominant one transcribed in the AC, but the authors can't exclude that there is something unique about the protein made by this isoform. Transgenic rescue experiments with each isoform could address this question more definitively.3. The authors present more evidence for the previously observed correlation between cell cycle arrest and invasive activity, but the basis for this correlation remains unclear. On p. 10 the authors speculate that "EGL-43 might perform two functions in the AC; first to repress AC proliferation and second to activate pro-invasive gene expression". But data in Figure 4 suggest that EGL-43 might do one thing (inhibit lin-12) to accomplish both results. What is the explanation for why lin-12(gf) suppresses proliferation and invasion defects , but CKI expression mostly does not ? At what step of the cell cycle is lin-12 acting? Does lin-12 affect expression of the mentioned "pro-invasive" genes?4. Is the cross-regulation of egl-43 and fos-1 functionally important? Can cdh-3>fos-1 rescue egl-43 defects or vice versa? Or are they both needed in parallel?5. The paper does not show whether lin-12, fos-1, or any of the other downstream effectors of EGL-43 are direct targets, so it does not provide insights into EGL-43 binding properties or mechanisms of transcriptional regulation (e.g. is EGL-43 both a tx repressor and activator?).6. Given that egl-43 is expressed in both AC and VU, and seems to regulate different target genes in each, what is the basis for target specificity?**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: No: We cannot find information pertaining to the sequence of the egl-43Si 5'UTR used in the generation of the RNAi targeting clone to the short isoform of egl-43. Also, I could not find the data supporting the claim that the different GFP-tagged alleles have the same expression levels in the AC - Fig. S2 should use the unmodified allele to normalize data to.Reviewer #3: No: numerical data for bar graphs has not been provided**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  and your responses to reviewer comments. If eligible, we will contact you to opt in or out. *Dear Dr. HajnalThank you very much for submitting your Research Article entitled 'The C. elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.After carefully reading through Reviewer#1's comments (self identified as David Matus), I think he brings up a reasonable editorial suggestion in improving the manuscript--more strongly suggesting an alternative model that EGL-43 might also function to repress the cell cycle in part by promoting cki-1 expression.  I don't think you need to bring up the ChIP data, but I leave that up to you.  If you can make this change and outline how you have done so in your letter, I will make sure this elegant work is accepted quickly--it will not be sent out for re-review.In addition we ask that you:archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. 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CopenhaverEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #2: Summary:I commend the authors on the extensive revisions they performed to address many of the comments provided by myself and other reviewers previously. Both the text and graphs are much clearer now. The only result that we’d like to bring attention to is still the discrepancy in data between this report and recent work from our laboratory. We are not asking for additional experiments at this time, but would just like to offer some caveats that might change the interpretation of the data, regarding the role of EGL-43 in maintaining the post-mitotic state of the AC independent of the activity of CKI-1.In 2015 we reported that an integrated transgene (cdh-3>CKI-1::GFP) completely rescued the AC proliferation and invasion defects in the hypomorph, nhr-67(pf88). In our 2020 paper we demonstrated this same transgene also completely rescued invasion and proliferation in nhr-67(RNAi) using the enhanced RNAi vector in the T444T backbone. In Figure 3F and 3G it appears that your extrachromosomal cdh-3>CKI-1::SL2::mNG transgene only partially rescues nhr-67(RNAi) . Both the use of extrachromosomal arrays, which can vary in expression levels and the construction of your transgene using an SL2::mNG may explain the difference between our results and your results presented in Figure 3. Specifically, Ahier and Jarriault  argue for using 2A viral peptides rather than SL2 sequences in generating fusion proteins where stoichiometry between the protein of interest and the fluorescent protein are important to maintain. Here’s the relevant text from their discussion section:“This is in contrast to the SL2 approach as operon sequences may be under the influence of cis-regulating elements (enhancers or silencers) that are difficult to predict . Moreover, when using an SL2-based approach, several additional parameters can impact on the expression levels of protein products, which could vary widely. For example, after trans-splicing, each subsequent mRNA molecule may have its own specific stability, and each transcript is read individually by different ribosomes, permitting variable translation.”In our integrated transgene, cdh-3>CKI-1::GFP, we avoid this by generating the fusion between CKI-1 and GFP.*If* the extrachromosomal lines more robustly rescued nhr-67(RNAi), I would be inclined to believe that egl-43 could be functioning independently of CKI-1 through LIN-12, but the more parsimonious explanation is that you’re not generating enough CKI-1 to restore the AC to a G0 cell cycle arrested state. I think this only affects the summary model - that the repression of LIN-12 is required with NHR-67 to maintain G0 arrest is an amazing result. I think that we don’t know (yet) whether LIN-12 functions to either activate cyclins and CDK complexes or repress CKI-1. I think this is made even more complex because of the likely feedback between CKIs and CDK/Cyclin complexes. I would be fine with showing dotted arrows with a question mark between LIN-12 and the cell cycle machinery, to show that the current data can’t resolve this relationship.Finally, I also noticed that cki-1 might actually be a direct target of egl-43, from your L3 ChIP data presented in the excel file. This is exciting, but obviously potentially counter to your proposed model. It suggests that nhr-67 and egl-43 might co-regulate CKI-1 activity in the AC to maintain it in a post-mitotic G0 arrested state, though we still don’t know if nhr-67 directly regulates CKI-1.chrII:7810217-7810482 cki-1; cki-1 356.7129 15.22800064chrII:7809138-7809378 cki-1 170.3209 6.874500275Reviewer #3: The authors have responded satisfactorily to my prior comments, and the revisions have significantly improved the paper.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #2: YesReviewer #3: Yes**********what does this mean?). 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PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via  12 Mar 2020PGENETICS-D-19-01671R2 Caenorhabditis elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion The Dear Dr Hajnal, Caenorhabditis elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.We are pleased to inform you that your manuscript entitled "The The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. 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In obsessive–compulsive disorder (OCD), functional behaviors such as checking that a door is locked become dysfunctional, maladaptive, and debilitating. However, it is currently unknown how aversive and appetitive motivations interact to produce functional and dysfunctional behavior in OCD. Here we show a double dissociation in the effects of anxiogenic cues and sensitivity to rewarding stimuli on the propensity to develop functional and dysfunctional checking behavior in a rodent analog of OCD, the observing response task (ORT). While anxiogenic manipulations of perceived threat  and actual threat (punishment of incorrect responding on the ORT) enhanced functional checking, dysfunctional checking was unaffected. In contrast, rats that had previously been identified as “sign-trackers” on an autoshaping task—and therefore were highly sensitive to the incentive salience of appetitive environmental cues—selectively showed elevated levels of dysfunctional checking under a range of conditions, but particularly so under conditions of uncertainty. These data indicate that functional and dysfunctional checking are dissociable and supported by aversive and appetitive motivational processes, respectively. While functional checking is modulated by perceived and actual threat, dysfunctional checking recruits appetitive motivational processes, possibly akin to the “incentive habits” that contribute to drug-seeking in addiction. Obsessive–compulsive disorder (OCD) is a common and highly debilitating mental health disorder with an estimated lifetime prevalence of 2.3% . It has The mechanisms underlying excessive checking are a matter of debate . A promiIt is important to note that checking behavior itself is not necessarily maladaptive and constitutes a normal part of the behavioral repertoire of healthy people. However, the compulsive checking observed in OCD patients is clearly dysfunctional and maladaptive. We have previously argued  for the To probe the psychological and neurobiological bases of checking, we previously developed a fully translational, rodent-to-human analog of OCD-like checking in rats that allows functional and dysfunctional checking to be assessed independently—the observing-response task ORT; . To brieConsidering the adaptive and maladaptive nature of functional and dysfunctional checking, respectively, we hypothesized that these two behaviors may be supported by different psychological mechanisms. Increases in checking correlate with uncertainty and anxiety as described above, consistent with dysfunction of the security motivation system, but we propose that the security motivation system specifically contributes to functional checking behavior. In contrast, the compulsive and dysfunctional checking that is specific to OCD, and not generalized anxiety or depression, we hypothesize to reflect uncontrolled habitual responding supported by the appetitive motivational system . This hyWe tested the relationship between the security motivation system and checking using three different approaches. First, we manipulated the level of uncertainty in task reinforcement, as in our previous research  or the cue itself (“sign-tracking”). These different behavioral responses to a Pavlovian cue are considered to reflect the extent to which the cue acts as an incentive stimulus , and areThus, the present study investigated the psychological mechanisms underlying functional and dysfunctional checking behavior in a fully translational, rodent-to-human analog of checking in OCD. We hypothesized that functional and dysfunctional checking behaviors are supported, respectively, by aversive (security motivation) and appetitive representations within the motivational system. We specifically predicted that: (i) presentation of anxiogenic cues, whether uncertainty, perceived threat  or actual threat (punishment of incorrect responding), would increase functional, but not dysfunctional checking responses; and (ii) that sign-trackers would be selectively more sensitive to appetitive Pavlovian cues, manifest as increased dysfunctional checking independent of all experimental manipulations.Supplemental Fig. 1A; Group: F = 17.3, P < 0.0001, η2 = 0.51; Group × Session: F = 2.31, P = 0.047, η2 = 0.12; Šidák-corrected pairwise comparisons showed that goal-trackers made more magazine entries than controls in all sessions, all P’s < 0.02]. Sign-trackers approached the lever more than the other experimental groups throughout training .Animals were trained to associate the to-be-observing lever with sucrose pellet delivery in a food magazine located on the opposite side of the conditioning chamber through a Pavlovian conditioned approach (autoshaping) procedure. Thirty-seven animals underwent Pavlovian conditioning, while 10 animals served as unpaired controls that received the same number and frequency of lever presentations but received all sucrose pellets at the start of the session. All animals that underwent Pavlovian conditioning were subsequently classified as sign-trackers, goal-trackers, or intermediate phenotypes depending on whether approach was toward the lever, the magazine or both, respectively. Following classification , the cohort consisted of 11 sign-trackers, 11 intermediates, and 15 goal-trackers. Intermediate animals were not included in subsequent analyses. Consistent with these group assignments, goal-trackers made greater numbers of magazine entries during the autoshaping sessions . All animals also made greater numbers of dysfunctional extra observing lever presses . Testing our a priori hypothesis that sign-trackers would show more dysfunctional checking, we found that sign-trackers made more eOLPs than both goal-trackers  and controls . Thus, uncertainty increased both functional and dysfunctional checking in all animals, but consistent with findings in OCD patients  and once responding was stable, their performance under conditions of increased uncertainty was assessed. Consistent with our predictions, all animals increased the numbers of functional observing lever presses (OLPs) made under conditions of uncertainty, regardless of phenotype . Uncertainty did not impair the ability of the animals to use the cue light to guide responding  but it did make it harder for all animals to discriminate between the active and incorrect levers while the cue light was off . Thus, as expected, the introduction of uncertainty to the ORT led to a greater reliance on checking behavior and the consequent cue light to guide responding, as compared to the more predictable schedule in the “Baseline” sessions.Uncertainty affected other measures of responding in the ORT as expected, based on our previous data . Under cF = 40.0, P < 0.0001, η2 = 0.55; Stimulus × Group: F < 1; Group: F = 2.62, P = 0.09]. However, despite producing equally strong fear memories, presentation of threat-associated contextual cues did not affect responding on the ORT in the same way for goal-trackers and sign-trackers. Only goal-trackers increased functional OLPs in the presence of threat-associated contextual cues . In contrast, threat-associated contextual cues did not alter levels of dysfunctional eOLPs , although sign-trackers continued to make greater numbers of eOLPs throughout testing as compared to controls . Therefore, threat-associated contextual cues did increase checking behavior, but only functional  checking, and only in goal-trackers.To assess the impact of perceived threat on checking behavior, animals underwent Pavlovian fear conditioning to associate an electric footshock with a discrete auditory stimulus and portable contextual cues, in different chambers to those used for the ORT. All animals acquired the Pavlovian fear memory equally well, showing greater fear to the discrete tone cue than to the contextual cues  though the low rate of incorrect lever pressing was unaffected . The ability of animals to use the light CS to guide lever choice was also reduced in the presence of threat-associated contextual cues , though lever choice with the light off was not altered in either the threat-associated and neutral contexts .Consistent with a generalized suppression of behavior, threat-associated contextual cues produced a slight trend toward a reduced rate of active lever pressing  and those classified as sign-trackers made more approaches toward the lever . This cohort consisted of six sign-trackers, 15 intermediates, and 17 goal-trackers.Animals that had undergone autoshaping were classified on their Pavlovian conditioned approach behavior as for Experiment 1. As expected, animals classified as goal-trackers made more magazine entries . However, when incorrect responding was no longer punished, the functional OLP checking responses of sign-trackers were impervious to the removal of the shock  and did not return to baseline like goal-trackers and controls . Thus, while functional checking increased in all groups in an adaptive manner in response to the introduction of punishment to the task, sign-trackers were unable to adaptively reduce their functional checking when punishment was no longer applied.The introduction of punishment to the ORT increased functional checking in all animals in a shock intensity-dependent fashion . However, with the introduction of punishment, all groups increased their dysfunctional checking in a shock intensity-dependent manner . Dysfunctional checking returned to baseline levels when incorrect responding was no longer punished in all groups , though sign-trackers continued to show greater levels of dysfunctional checking than the other groups. Therefore, as for Experiment 1, sign-trackers showed higher levels of dysfunctional checking overall, but punishment led to increases in dysfunctional checking in all animals.Dysfunctional checking eOLPs; B was alsF = 14.0, P < 0.001, η2 = 0.32; Group: F < 1; Group × Intensity: F = 1.56, P = 0.16; post-hoc tests revealed that the rate of active lever pressing was decreased at 0.4 mA, P < 0.0001, d = 0.58, and 0.5 mA, P < 0.0001, d = 0.97]. Once shocks were no longer delivered in the ORT sessions, no suppression was observed and the rates of active lever presses returned to baseline levels . As expected, punishment of incorrect responding also decreased the rate of incorrect lever pressing, and at lower shock intensities than for active lever pressing . This reduction in incorrect lever pressing also affected discrimination measures, as animals better discriminated between the two levers in the shocked sessions when the light was off , though lever choice when the cue light was on did not differ between the shocked and nonshocked sessions . This suggests, expectedly, that rats avoided pressing the incorrect lever after this response was punished.As for the presentation of anxiogenic stimuli in Experiment 1, presentation of aversive stimuli—in this case, punishment of incorrect responding—suppressed overall rates of responding in all animals, in a shock intensity-dependent manner .Following completion of testing on the aORT, we sought to determine whether prior experience of punishment would affect the capacity of anxiogenic stimuli to alter functional and dysfunctional checking in sign-trackers and goal-trackers. Specifically, we wanted to test whether subsequent shock exposure in the contextual fear conditioning (CFC) procedure would reinstate the levels of functional checking previously observed during the aORT sessions. As for Experiment 1, rats underwent Pavlovian CFC in a separate chamber, containing stimuli that could be transferred to the ORT chambers. To determine whether the footshock delivered during CFC would reinstate the responding observed on the aORT, animals were first tested in the normal ORT chambers 24 h after CFC in a “reinstatement” test, and 48 h after conditioning, they were tested in the ORT chambers in the presence of the threat-associated contextual cues. All of the animals showed similar levels of conditioned fear when tested in the fear conditioning context and presented with the discrete auditory cue associated with shock, with fear to the cue being higher than to the context in all animals . Furthermore, the delivery of footshock in the fear conditioning session did not reinstate the responding observed previous on the aORT, with no observed differences in functional checking in the “Reinstatement” test . The patterns of dysfunctional checking  = 7.67, P = 0.002, η2 = 0.34; post-hoc tests showed that sign-trackers made more eOLPs than both controls at P = 0.007, d = 1.01, and goal-trackers at P = 0.002, d = 1.06], though in contrast to Experiment 1, these decreased in the presence of threat-associated contextual cues . Dysfunctional checking in the “Reinstatement” test was no different than previous baseline measurements  indicating that the experience of shock in CFC did not reinstate levels of responding to those observed on the shocked aORT sessions.Prior experience of the aORT did not alter the effects on checking of threat-associated contextual cues that were previously observed in Experiment 1. Again, only goal-trackers elevated their functional checking when tested in the presence of threat-associated contextual cues , while the rate of incorrect lever pressing remained unaffected . This time, discrimination between the active and incorrect levers was not affected by the presence of threat-associated contextual cues, either when the cue light was on  or off .Further replicating the findings of Experiment 1, threat-associated contextual cues suppressed the rate of active lever pressing [Overall, Experiment 2 showed that actual threat (punishment of incorrect responses with an electric footshock) increased functional checking in all animals. Dysfunctional checking was also increased in all animals, though it remained elevated in sign-trackers compared to goal-trackers. Importantly, while goal-trackers were able to adaptively reduce their levels of checking when incorrect responses were no longer punished, sign-trackers continued to show elevated responding, indicating that they were insensitive to the changes in reinforcement contingencies.Excessive checking behavior is common in OCD, but checking itself is a normal part of the behavioral repertoire. We have previously argued  that theConsistent with our observations in a separate study , we found that rats classified as sign-trackers following autoshaping training showed higher levels of dysfunctional checking than goal-trackers or non-autoshaped controls, and that this was particularly so under conditions of uncertainty. A similar finding, that sign-tracking itself increases when reward is uncertain, has recently been reported . IncreasHowever, sign-trackers were not more sensitive to aversive Pavlovian cues; the levels of dysfunctional checking were similar in both the neutral and aversive threat-associated contexts, although it should be noted that sign-trackers showed elevated levels of dysfunctional checking in the neutral context, perhaps masking any further elevation of dysfunctional checking by the presentation of threat-associated contextual cues. In contrast, goal-trackers showed an increase in their functional checking in the presence of these aversive cues, supporting the hypothesis that functional checking depends upon aversive motivational systems, but dysfunctional checking does not. Notably, these data could not be attributed to any differences in shock sensitivity or the strength of fear conditioning, as sign-trackers, goal-trackers, and controls all showed equivalent fear memory when explicitly tested on contextual and discrete Pavlovian fear conditioning. Furthermore, when incorrect responses were explicitly punished in the aversive ORT (aORT) all groups increased their functional checking behavior, as predicted. However, when incorrect lever presses were no longer punished, only sign-trackers continued to check at high levels; other groups’ checking responses returned to baseline levels. These data support the view that functional and dysfunctional checking depend differentially on the aversive and appetitive motivational systems, respectively.While sign-trackers’ functional checking responses showed enduring changes in response to punishment of incorrect responses with shock, it was the functional checking responses of goal-trackers that were sensitive to the presence of threat-associated contextual cues. Previous studies have shown that goal-trackers freeze more to contextual cues and sign-trackers to discrete cues following Pavlovian fear conditioning ; howeverWhile all groups responded similarly in the aORT to the escalating intensity of punished incorrect responses by increasing their functional OLPs, once the shocks were no longer delivered, goal-trackers and control rats rapidly returned to baseline levels of functional checking. Sign-trackers, in contrast, appeared impervious to the absence of shock, continuing to check at high levels. There are at least two potential explanations for this checking behavior: one based on the informativeness of the cue produced by the checking response and the other on its reinforcing properties. The presentation of the cue produced by functional checking provides information to the animal, by indicating which lever is currently reinforced, thereby reducing uncertainty. The “uncertainty reduction hypothesis”  posits tAn alternative view to account for the persistent functional checking in sign-trackers is the “conditioned reinforcement hypothesis” . This viRegardless of whether functional checking is supported by the informational or reinforcing value of the cue, the high levels of dysfunctional checking in sign-trackers cannot be supported by the cue itself because extra observing lever presses (eOLPs) do not result in illumination of the cue light. These responses may have been due to continued sign-tracking to the lever, presentation of which had been reinforced during autoshaping training. However, this seems unlikely, given that these animals increased their eOLPs as training in the ORT progressed, during which time the association between the observing lever presentation and reward delivery was no longer reinforced. To fully discount this possibility, future studies will classify animals as sign-trackers and goal-trackers following autoshaping in an entirely distinct apparatus, such as a touchscreen chamber.A second, and we suggest more likely, possibility is that the operant response of checking itself became imbued with motivational significance—the incentive motivational properties once directed toward the Pavlovian cue was now directed to the observing response, akin to the “incentive habits” view of addiction . This woOverall, these data support the view that functional and dysfunctional checking are dissociable and supported by aversive and appetitive motivational processes, respectively. Functional checking behavior can be modulated by perceived threat and uncertainty, and by the capacity of discriminative environmental cues to act as conditioned reinforcers. However, dysfunctional checking appears to recruit appetitive motivational processes, likely akin to the “incentive habits” that contribute to drug-seeking behavior in drug addiction. This view of dysfunctional checking is consistent with patient data reporting increased habit learning in OCD . IndividSubjects were 96 male Lister Hooded rats  with weights at the start of procedures of 156–222 g for Experiment 1 (mean: 196 g) and 274–352 g for Experiment 2 (mean: 306 g). Animals were housed in groups of 4 with a cardboard tube as enrichment under a reversed light–dark cycle (lights on at 19:00). Testing took place 5 d a week, typically between 08:00 and 13:00. Before any testing began animals were food restricted and fed 10–20 g of standard laboratory chow (SDS) at the end of each day's testing, in addition to any food earned during operant sessions. One rat was excluded after being observed having multiple seizures; data from this animal have been removed from all analyses. Experiments were run as two separate squads of 48 rats. This research has been regulated under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review and approval by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB) and was conducted under PPL 70/7548.Rats were trained in 18 operant-conditioning chambers (Med-Associates) equipped with three levers, two of which flanked a food receptacle where 45 mg sucrose pellets  were delivered. Above of each of these levers was a stimulus light. A third lever was located on the opposite wall, above which was a white house light that remained illuminated throughout the session.In brief, animals were trained on the ORT and underwent separate autoshaping training sessions to enable classification into goal-tracking and sign-tracking phenotypes. Animals were then tested in the ORT under conditions of uncertainty, where it was more difficult for animals to discriminate between active and inactive/incorrect levers without the use of the discriminative stimulus, which could be illuminated by responding on the observing lever. In the “standard” version of the ORT used in Experiment 1, the incorrect lever was not reinforced and can therefore be considered equivalent to “inactive” lever pressing in our previous work. However, in the aversive ORT used in Experiment 2, pressing the incorrect lever was punished and it is therefore inappropriate to refer to these as “inactive” lever presses. For consistency, we have referred to these as “incorrect” lever presses in both experiments reported here.Following this, the effects of threat-associated stimulus were assessed by fear conditioning animals to portable contextual and discrete stimuli. The effect of presenting these threat-associated stimuli on performance in the ORT was subsequently examined, in addition to the effect of contextual and discrete shock-paired stimuli on conditioned freezing behavior.Animals were trained on the ORT as previously described . AnimalsFollowing reliable discrimination of the active and incorrect levers, and completion of autoshaping training, animals were trained in the full version of the ORT. The observing lever was now presented; responses on this third lever illuminated the cue lights, which were otherwise turned off. Responses on the observing lever resulted in illumination of the cue light above the active lever for 30 sec for the first four sessions, and 15 sec thereafter. The observing lever and surrounding areas were baited with powdered reward pellets for the first two sessions of ORT training to encourage exploration and engagement. Unless stated otherwise, active lever presses continued to be reinforced on a VR15 schedule and the identity of active and incorrect levers changed every 90 sec.bserving lever presses (OLPs), which were functional observing presses that resulted in the illumination of the light about the currently active lever; (ii) extra observing lever presses (eOLPs), which were dysfunctional presses on the observing lever, performed while the stimulus light was already illuminated. These responses did not prolong the duration of stimulus light illumination; (iii) active lever presses (rate per minute); (iv) incorrect lever presses (rate per minute); (v) discrimination (light on); and (vi) discrimination (light off), which were measures of the accuracy of lever pressing with the light on and off, respectively. This was calculated asResponses on the ORT were collected automatically by a computer running the Whisker Control server . Six depSupplemental Data Files (https://doi.org/10.17863/CAM.41573).During autoshaping sessions, the lever that would subsequently be used as the observing lever was presented 30 times. Ten seconds after its presentation it was retracted, followed by delivery of a single sucrose pellet. Ten animals were assigned to a control group in which lever retraction was not paired with pellet delivery; instead, these animals received 30 pellets at the start of each session, before the house light was illuminated. Animals received a total of 17 autoshaping sessions. Animals were classified as goal-trackers, sign-trackers, or intermediates based on behavior during these sessions (see below). Intermediates were excluded from statistical analysis but continued to be tested with the other subjects. The data from these animals can be found in the After 10 sessions of training in the ORT animals underwent uncertainty training. During these sessions, the active and incorrect levers changed on a variable time (VT) 70 sec schedule (20–120 sec). Active lever presses were now reinforced on a variable (VI) 15 sec schedule (10–20 sec).After testing under uncertainty animals underwent CFC. Procedures were adapted from Animals were first habituated to these modified boxes for a 5-min session, where the house light was illuminated but no shocks or tones were delivered. The following day, animals were placed in the chambers for 3-min fear conditioning session. After 120 sec of habituation, a tone was presented for 30 sec which coterminated with delivery of a footshock . The house light remained illuminated for a further 30 sec and the animal was removed. Boxes were cleaned with 70% ethanol between fear conditioning sessions.Testing took place the following day. The effect of threat-associated contextual cues on responding in the ORT was assessed first. These sessions took place in the chambers in which animals had been trained for the ORT, now modified to include the contextual cues present for the fear conditioning session. Later, on the same day, animals underwent a contextual fear memory retention test, during which they were placed in the fear conditioning chambers for 3 min. Twenty-four hours after this test, fear to the tone was assessed by presenting four 30 sec tones, each separated by 120 sec. These sessions took place in the chambers used for fear conditioning but were modified to resemble the chambers used for ORT training .Fear conditioning training and testing sessions were video recorded and manually scored offline by an experimenter blind to group assignment (ALM), using a custom script written for PsychToolbox for MATLAB, produced by GHV. Freezing, defined as a cessation of all movement other than those required for respiration, was scored continuously, with the values being expressed as a percentage of the total time that could be spent freezing.Animals in this experiment underwent pretraining and autoshaping sessions identical to those in Experiment 1. However, instead of the introduction of uncertainty to increase checking, animals underwent testing in the aORT, where incorrect lever presses were punished with a mild footshock, of increasing intensity. The shocks were then disabled for six sessions before animals underwent CFC, as described for Experiment 1.In the aversive version of the ORT shocks were delivered contingently upon incorrect lever presses, on the same schedule that the active lever delivered pellets . Animals underwent testing at progressively increased intensities of footshock . The maximum number of shocks an animal could receive in a single session was 30; sessions continued in the absence of any further shocks if this limit was reached. The average of both sessions at each intensity was used for analysis. Following completion of testing under the five different intensities in the aORT animals continued testing in the ORT in the absence of shock for a further 4 d.Following aORT training, animals underwent CFC as in the previous experiment. However, in order to dissociate the possible reinstating ability of experiencing shock in a novel context  animals lever presses:magazine entries during lever presentation during the last 2 d of autoshaping training and was conducted by an experimenter blind to performance in the ORT (DME). Attempts were made to determine subgroups based on clear splits on the distribution of animals’ responding. Although more standardized protocols have been proposed for the classification of goal and sign-trackers (https://doi.org/10.17863/CAM.41573).Grouping of animals to autoshaping and control groups was conducted based on total rate of active lever pressing and discrimination ratios; efforts were made to ensure that no pre-existing differences existed between groups before autoshaping training began. Grouping of ST, GT, and INT animals was based on the ratio of trackers , we founhttps://doi.org/10.17863/CAM.41573). Baseline performance for use in the aORT was obtained from the two sessions before aORT testing began. The values for each shock intensity are an average of the 2 d testing.Baseline performance in the ORT in Experiment 1 was based on the last five sessions of task performance. The average of two five-session blocks of uncertainty sessions were used for analysis . Data we2) was calculated in SPSS . Cohen's d was calculated for all significant post-hoc tests. In cases where post-hoc tests report the results of within-subject comparisons dz values are reported,  In cases of significant main effects, but no significant interaction, effect sizes were calculated for the averaged of values across the range of values of the second factor.Experiments were analyzed with mixed 2 × 2 ANOVAs. The degrees of freedom for all analyses with repeated measures factors with more than two levels were adjusted with the Greenhouse-Geisser correction where Mauchly's test indicated that sphericity had been violated. Significant main effects and interactions were followed up with simple effects analysis, where the baseline sessions were compared against all other sessions, with the Šidák correction for multiple comparisons applied. Statistical analysis was conducted with GraphPad Prism (ver. 6.07) for Windows. Partial eta squared (η


Involvement of metals or silica in the pathogenesis of sarcoidosis has been suggested by several case reports and specific epidemiological studies. However, the combination of occupational exposure and an immunological reaction has not been studied before in a group of sarcoidosis patients and non-sarcoidosis controls.In 256 sarcoidosis patients and 73 control patients with obstructive sleep apnea, exposure to metal and silica was assessed using a questionnaire consisting of a complete occupational history subsequently linked to job-exposure matrices. Next, immunoreactivity to aluminium, beryllium, zirconium and silica was determined in 33 sarcoidosis and 19 control patients using a lymphocyte proliferation test.p = 0.21). A significantly higher percentage of the sarcoidosis patients tested showed immunoreactivity to metals or silica compared to the control group .In sarcoidosis, 83 out 256 patients (32.4%) had occupational exposure to metals or silica, compared to 24.7% in the control group (Immunoreactivity to silica and metals was only found in sarcoidosis patients, supporting the hypothesis that these antigens may be involved in the pathogenesis of a distinct subgroup of sarcoidosis patients. This indicates that when searching for causative agents in sarcoidosis patients, besides beryllium, also zirconium, aluminium and silica deserve clinical investigation. Sarcoidosis is a systemic disease characterized by the formation of noncaseating granuloma, mostly affecting the lungs, skin, eyes and lymph nodes . The aetAlthough involvement of metals and silica in sarcoidosis pathogenesis has been suggested by several case reports and epidemiological studies, it has not been systematically studied in a larger group of sarcoidosis patients. The aim of this study was to characterize both occupational exposure and immunoreactivity to metals and silica in a group of sarcoidosis patients and controls. Since sarcoidosis is a very heterogeneous disease, we hypothesize that metals or silica are involved in the pathogenesis of the disease in only a subgroup of patients. Therefore, we have investigated whether occupational history linked to a job exposure matrix (JEM) can be an appropriate tool to identify which sarcoidosis patients have to be further tested for metal and silica immunoreactivity.Sarcoidosis patients visiting the Interstitial Lung Disease (ILD) outpatient clinic of St. Antonius Hospital  received an invitation letter to participate in the study. The diagnosis of sarcoidosis had been established according to the criteria of the American Thoracic Society/European Respiratory Society . The chaSarcoidosis and control patients received an online questionnaire asking for a complete occupational history. The patients’ job histories were coded using the International Standard Classification of Occupations 1968 (ISCO-68) . This clA random selection of 45 sarcoidosis patients with occupational exposure to silica or metals were asked to donate a blood sample for a LPT on beryllium, aluminium, zirconium and silica. From the group of sarcoidosis patients without exposure, 10 patients who had an appointment in the near future were asked for the LPT as well. Those patients were selected in such way that age and sex did not differ from the exposed sarcoidosis patients invited for the LPT. The first 20 control patients included were also asked to donate a blood sample.6 cells were incubated for 5 days in HEPES-buffered media containing at least two consecutive dilutions of an antigen: aluminium nitrate, beryllium sulphate, silicon dioxide or zirconium dioxide. After cultivating for 5 days, proliferation was measured by incorporation of H3-labelled thymidine. Radioactivity was measured with a scintillation counter . Stimulation indexes (SI) were calculated by dividing the antigen specific counts per minute by the mean counts per minute of the basal controls. SI of ≥3.0 were considered as a positive test results, based on the MELISA® guidelines [Blood was drawn in citrate tubes and sent within 24 h to Prohealth Medical, Nederweert, The Netherlands, where the memory lymphocyte immunostimulation assay (MELISA®) LPT was performed. MELISA® is a clinically validated blood test that detects type-IV allergy (sensitization) to multiple metals , 17. Furidelines .5 PBMCs/200 μL were incubated for 6 days in medium (RPMI-1640 GlutaMax supplemented with 5% autologous plasma and penicillin/streptomycin) containing concentrations of 10− 4 M till 10− 10 M BeSO4. Cell proliferation was measured by incorporation of H3-labelled thymidine. An SI of ≥3.0 was considered positive. The highest SI measured for each sample was used in the analysis. For calculating the correlation between the SI for beryllium obtained by MELISA® and obtained in Leuven, one additional patient, who had already performed both test for clinical evaluation, was included.Patients with an SI of ≥3.0 for beryllium were invited to donate a second blood sample, which was sent to the department of Laboratory Medicine, University Hospitals Leuven, Belgium, where a second beryllium LPT was performed according to the protocol described in the ATS official statement on diagnosis and management of beryllium sensitivity and chronic beryllium disease . BrieflyP-values < 0.05 were considered significant.Study data were collected and managed using REDCap electronic data capture tools hosted at St. Antonius hospital. Data were analysed using IBM SPSS statistics version 24. An unpaired T-test was used to compare numerical data between the sarcoidosis and control group. Non-parametric tests were used for non-normally distributed data (Mann-Whitney U test). Categorical data were compared between the sarcoidosis and control group using the Chi-squared test. If expected cell frequencies were below 5, Fisher’s exact test was used for categorical data up to two categories. Odds ratios were estimated for sarcoidosis and occupational metal and silica exposure. Correlation between the MELISA® LPT and validated beryllium LPT was determined with Spearman’s rho correlation. A total of 590 patients were invited to participate in the study, of whom 314 responded and were willing to fill out the questionnaire. Finally, 256 sarcoidosis patients completed the occupational history and were included in the study. Seventy-three patients with OSA were included to serve as a control group. Demographics of all sarcoidosis and control patients are presented in Table The distribution of sarcoidosis patients and controls over the major occupational groups is shown in Table Percentages of sarcoidosis patients and controls are based on all jobs performed during working life until the diagnosis of sarcoidosis was made (sarcoidosis group) or until the moment of study enrolment (control group). If participants had more than one job within the same group, only one was counted. Minor occupation groups without sarcoidosis patients and controls are not shown.p = 0.21) . No significant differences were observed in occupational metal or silica exposure between the sarcoidosis and control group . Hence we compared the distribution of sarcoidosis and controls patients also over the minor groups belonging to major group 7/8/9 Table . No signup Table .Table 3P < 0.01) . One sarcoidosis patient had a positive LPT for multiple antigens, namely zirconium and beryllium. In total, 3 sarcoidosis patients had an SI of ≥3.0 for beryllium and were invited for a second beryllium LPT using the ATS protocol. The highest SI values obtained by MELISA® for those three patients and the additional clinical patient were 3.96, 4.94, 9.05 and 24.48. The highest SI values of the second beryllium LPT following the ATS protocol for these patients were 1.27, 3.42, 5.93 and 379, respectively. When the results of those 4 patients were combined, a strong correlation of 1.00 (P < 0.01) was found between the beryllium SI of the MELISA® and LPT using the ATS protocol.Immunoreactivity was only observed within the sarcoidosis group. Seven 21.2%) sarcoidosis patients had a positive LPT for at least one of the antigens tested, compared to none of the controls .Occupational exposure as assigned by JEMs was compared with LPT results. Four of the 26 sarcoidosis patients 15.4%) with metal or silica exposure showed immunoreactivity to at least one of the antigens tested. Among the 7 sarcoidosis patients without JEM assigned exposure, 3 (42.9%) had a positive LPT for the antigens tested  and will, by definition, not capture individuals experiencing coincidental high risk exposure while carrying out otherwise low-risk jobs . It is i2+ cation induces a conformational change in the HLA-DP2/peptide complex. Immunoreactivity develops when T lymphocytes regard the displaced peptide as foreign (non-self) and become activated [A limitation of the study is that the group for whom a LPT has been performed was quite small. The LPT could not be performed on blood samples of some sarcoidosis patients, due to insufficient lymphocyte counts. Lymphocytopenia is a common observation in sarcoidosis patients , not relctivated . PossiblAnother study limitation may be the sensitivity of the assays performed. For beryllium LPTs, about 1% false positive and 30% false negative results are observed. The specificity of this assay is quite high (0.969) although the sensitivity is determined to be lower (0.686) . Data onIn conclusion, immunoreactivity to silica and metals found in sarcoidosis patients only, supports the hypothesis that these antigens may be involved in the disease pathogenesis of a distinct subgroup of sarcoidosis patients. Since job history with assigned exposure did not differ between sarcoidosis patients and controls, our data indicate that even very low incidence or occasional exposures could be relevant in sarcoidosis patients. Future studies may consider testing all sarcoidosis patients for immunoreactivity without screening them on exposure beforehand. Finally, the data presented in this paper suggests that besides beryllium, also zirconium, aluminium and silica can be considered as possible antigens in this heterogeneous disease. Future studies should however be initiated to clarify underlying immunological pathways and mechanisms in order to establish a more definitive role for these antigens in sarcoidosis.Additional file 1 Table S1. Estimated odds ratios for sarcoidosis and JEM assigned exposure