Elevated humidity levels in medical, food, and pharmaceuticalproductsmay reduce the products’ shelf life, trigger bacterial growth,and even lead to complete spoilage. In this study, we report a humidityindicator that mechanically bends and rolls itself irreversibly uponexposure to high humidity conditions. The indicator is made of twofood-grade polymer films with distinct ratios of a milk protein, casein,and a plasticizer, glycerol, that are physically attached to eachother. Based on the thermogravimetric analysis and microstructuralcharacterization, we hypothesize that the bending mechanism is a resultof hygroscopic swelling and consequent counter diffusion of waterand glycerol. Guided by this mechanism, we demonstrate that the rollingbehavior, including response time and final curvature, can be tunedby the geometric dimensions of the indicator. As the proposed indicatoris made of food-grade ingredients, it can be placed directly in contactwith perishable products to report exposure to undesirable humidityinside the package, without the risk of contaminating the productor causing oral toxicity in case of accidental digestion, featuresthat commercial inedible electronic and chemo-chromatic sensors cannotprovide presently. Therefore, intelligent tags thatcan dynamically report on the storage environment are considered efficientto address these issues in food safety.The best-by date is mandatory and printedon packages of perishableproducts. Yet, it does not provide dynamic information on productsafety prior to consumption. For instance, several products past thesedates are usually safe to eat depending on consumer handling practices.3 Macroscopic size intelligent tags such as temperature tags9 and gas tags14 have been widely studied and are constantly reported in recent years.In addition to macroscopic size intelligent tags, microparticles15 that can report changes in pH16 or carry encoded information17 and respond to external stimuli18 havealso been reported.Intelligent tags aredesigned to monitor the storage environmentof perishable products in a cost-efficient manner.19 Medicines formulated as tablets and powderscan also disintegrate or cake by absorbing excess moisture.20 Furthermore, medicines can deteriorate or exhibitundesirable chemical reactions, leading to reduced drug potency inthe presence of excess moisture.Detection of humidity is often overlookedin the research fieldof intelligent tags despite its relevance in the storage of perishableproducts. Condensation of moisture on perishables can lead to hasteneddeterioration, bacterial growth, and reduction in their estimatedshelf life. Some common undesirable effects of excess humidity infood include caking, stickiness, lumping, crumbling, and molding.22 while other examples by virtue of geometric deformation have alsobeen proposed.24Research on humidity tags remainssparse to date despite the importanceof humidity control as discussed above. The majority of reported humiditytags are chemo-chromatic; hence, they respond to changes in humidityby changing their color21 proposed a chemo-chromatictag using cobalt chloride to estimate the water activity of driedproducts. The easy hydration and dehydration reaction between CoCl2 and CoCl2·6H2O induces a colorchange, thus indicating a humidity shift. However, direct contactwith consumable products was not advised due to the fact that chronicingestion of cobalt compounds has been widely reported to cause serioushealth problems.25 Therefore, users arerecommended to place this tag on the outside of the packaging, whichintroduces the risk of reading the ambient humidity instead of thetrue condition experienced by the target products. To overcome this,Bridgeman et al.26 proposed a hygroscopiccomposite tag of less toxic chemicals, i.e., sodium borohydride andDenimBlu30 dye. In this tag, DenimBlu30 dye functions as a redox indicatorand evolves from yellow to blue after absorbing water, hence reportingthe humidity increase. However, direct contact is still not recommendeddue to toxicity concerns.Thompson et al.24 presented a tailored shape memorypolymer of which theglass transition temperature can be driven lower to the operationaltemperature by vapor pressure. The absorbed water drives this transitionby increasing the free volume and the mobility of polymer chains.Therefore, the initial rigid polymer develops into a flexible elastomericstate over time under vapor. However, the reversibility of this polymeronly endows an instant humidity value and not the moisture exposurehistory.Abandoning chemical reactions, Snyderet al.31 A noteworthy study by Esteves et al.28 focused on gelatin/IL-based formulations, functioning aselectrical and optical sensors; the formulations can be tailored torespond to humidity and detect volatile organic compounds under dryand humid conditions.Ionic liquids (ILs) have gained attention in recentyears as theycan be immobilized in polymer or metal–organic frameworks,enabling the creation of flexible composites that exhibit high sensitivity,fast response, and wearability as humidity sensors.28 unfortunately, exhibit reversible changes. Sincea perishable product exposed to high humidity conditions still showsdeterioration or bacterial growth even if the humidity is broughtback to the recommended level, it is impractical to interpret spoilageaccurately using reversible indicators. Therefore, a food-grade, edible,nontoxic, and irreversible humidity indicator with a tunable responsetime that matches the spoilage course is desirable.Despite the recent progress in intelligenttags, a biocompatiblehumidity indicator that can be placed in direct contact with foodand medicine without contamination risk has not yet been reportedin the literature to the best of our knowledge. The biocompatibilityof the ionic liquids is not discussed in the above studies; it isquite promising to design fully biocompatible IL humidity sensors.Nevertheless, reading IL-based sensors requires the use of electricaldevices due to the chemisorption of water molecules on the ILs whichenhances its ionic conductivity and capacitance. However, this increasesthe cost of producing humidity tags and has environmental implicationsas the materials used in fabricating these electrical devices aretypically plastics and metals. In addition, the indicators discussedabove,32 Based onthe thermogravimetric analysis, we hypothesize that the bending mechanismis a result of hygroscopic swelling and consequent counter diffusionof glycerol and water. Leveraging our understanding of the bendingmechanism, we demonstrated that EHI’s lag time, response time,and bending curvature can be tuned to expand its application scope.Overall, the developed indicator shows the potential to irreversiblydetect undesired humidity exposure. The humidity indicator’sversatility is attributed to its ability to tune the lag time andresponse time by changing the layer thickness, making it suitablefor applications where humidity changes occur gradually or infrequentlyover an extended period. The demonstrated response time is slow forapplications requiring real-time or near-real time response. The indicator’sdeformation in response to humidity makes it also suitable for useas a humidity-activated actuator, particularly in situations wherelow humidity levels need to be maintained. Moreover, thanks to itsfood-grade hence edible ingredients, the EHI has two distinct advantagescompared to commercial nonedible electronic and chemo-chromatic sensors.First, the EHI can be placed in direct contact with perishable products;hence, it can report on the humidity level inside the packaging withoutany risk of contaminating the product. Second, in case the EHI isaccidentally ingested, for instance, by infants or children, it doesnot pose any risk of oral toxicity.In thisstudy, we report an edible humidity indicator (EHI) madeof food-grade ingredients that responds to undesired humidity exposureby irreversibly deforming and rolling on itself. The indicator ismade of two food-grade polymer films with distinct protein, caseinate,to plasticizer, glycerol, ratios. Caseinate, originating from bovinemilk, has been repeatably reported as a promising biodegradable packagingalternative in recent years.Figures S1 and S2). Thefilms are conditioned by storing them in desired humidity over 24h. The conditioned films are then attached to each other leveragingthe intrinsic sticky nature of films to fabricate bilayer EHIs.The fabrication processconsists of several simple steps 1a and is33 not only due to its attributes such as biocompatibility,biodegradability, and wide availability but also because of its randomcoil structure with the ability to form electrostatic, hydrophobic,and hydrogen bonds. Meanwhile, the casein film on its own is brittleand hence not easy to handle. To improve this, varying amounts offood-grade plasticizer glycerol are added to each layer. To ensurethat EHIs are made of entirely nontoxic ingredients, no glue is adoptedin the stick-together step. Perhaps due to caseinate’sself-healing ability, which rises from its random coil nature andability to form weak intermolecular interactions within polymericnetworks, i.e., electrostatic, hydrophobic, and hydrogen bonds asmentioned above,34 the single caseinatefilms can attach to each other and form a stable bilayer film. A scanningelectron microscope image  showsthat the gap between the two layers is negligible. In general, thiscomposite bilayer contains only edible ingredients, which avoid anysafety concern about direct contact with edible products.To avoid potential oral toxicity issues, EHIs aremade of onlyfood-grade ingredients, namely, caseinate, glycerol, and water. Caseinateis proposed as biodegradable packaging materialR) as illustrated. Prior to humidity exposure, the EHI keepsits flat shape for a relatively long time . We observed two distinctregimes, a Lag stage where the EHI absorbs waterfrom the environment with no significant bending followed by a Response stage where the EHI deforms and gradually rollson itself. After that, the EHI does not revert back to its initialshape when the humidity level is brought down. We refer to this experimentalobservation as the irreversible bending of the EHI, a feature thatis essential for detecting and reporting humidity exposure.The EHI response to accidental humidityexposure is illustrated as bending curvature development 1b. MoreoLag stage as thestage from t0 to troll, where t0 is the moment theEHI is exposed to a high humidity level while troll is the moment the EHI starts to roll  film remains insidethe rolled structure. We refer to this bending direction as rollingonto the less hygroscopic film.The reported bendingand consequent rolling behavior are observedin other systems as a result of a mismatch in layer properties.Figures S1 and S2). If the bending mechanism of EHIs was identicalto the aforementioned hydrogel bilayer structures, bending towardLGF would be expected. As we observe a different bending directionfrom the hydrogels in the literature, the bending mechanism of theEHI can not be solely explained by the distinct water adsorption capacityof two layers attached together, namely, HGF and LGF. A differentrolling mechanism is needed to explain this observation.Surprisingly, such a bendingdirection is not observed in our study.EHI sways back and forth slowly at the early stages of the exposure;it eventually bends toward and rolls onto HGF  regardless of RH value, EHI composition, or geometry. In otherwords, HGF (blue) remains inside the rolled structure and LGF (pink)remains outside 1b. This 42 Improper handlingand storage of these test strips, especially due to humidity exposure,have been reported to give rise to incorrect diagnosis.43 We test whether the EHI can be used to detecturine test strips with compromised functionality due to humidity exposure.For this test, we exposed urine strips with EHIsattached to labhumidity fluctuating between 48–64% throughout the experimentand monitored the bending of the EHI strips 2a, top. ned flat 2c. To suAs different products degrade in distinct time scales,the broaderapplicability of EHI hinges on the tunability of the lag time andresponse time. The bending/rolling mechanism needs to be understoodnot just out of scientific interests but also from an engineeringinterest to benefit EHI design.Figure S3). Hence, we rationalize that a different physicalmechanism should be in play.As HGF (blue) has higher water absorptionability compared to LGF (pink), the EHI should have bent toward LGFif the bending mechanism was based solely on compressive stressesinduced by the layer swelling more. Nevertheless, the observed bendingdirection contradicts this initial expectation. More interestingly,scanning electron microscopy images revealed that the LGF of the EHIbilayer elongated more than the HGF after 2 weeks of storage outsidethe desiccator at ambient humidity conditions (see Table S1).To shed light on the mechanismbehind bending and consequent rolling of the EHI on itself, we conductedthermogravimetric analysis (TGA) on various films: a bilayer EHI(1),a single HGF(2), and a single LGF(3) 3a. PriorTGA profiles show three distinct regimes. In stageI (100 to 200°C), water evaporation induced a similar mass loss trend in allthe samples. Stage III, from around 300 °C, is the decompositionof sodium caseinate, leaving 30% residue at 700 °C. In stageII, where the part of our interest lies, we found that films 1a and1b show an identical trend after the humidity exposure, indicatingthat the compositions of these two films are identical. Therefore,it is rationalized to hypothesize that glycerol diffused from HGFto LGF after humidity exposure, considering that glycerol is the onlychemical that decomposes within stage II.Basedon the TGA observations, we propose a rolling mechanism that involvesthe adsorption of condensed water from the environment and subsequentlythe counter diffusion of glycerol and water across the bilayer. Thehypothesized mechanism 4 is illuStorage stage (at Low RH), LGF and HGF absorb a limited amount of water. Glycerolis constrainedin each layer at this stage since the caseinates are densely packedand the glycerol diffusion is considered extremely slow. Therefore,the EHI is capable of maintaining its flat shape for a relativelylong time under 10% RH. In fact, as already mentioned, the EHI canmaintain its shape for at least 15 days when subjected to low relativehumidity (10% RH) . Thisimplies that the EHI is stable and reliable within our observationperiod and its shape will not be affected until it is exposed to arelatively high level of humidity. Shelf stability should be furtherinvestigated and adjusted based on the shelf life of the product,and EHIs will be implemented.During the t0 when humidity is introduced,both films start to absorb moisture and exhibit hygroscopic swelling.In this stage, which we term as Lag stage, we speculatethat the films absorb more water and exhibit a volumetric change comparedto the initial volume. As previously mentioned, the high concentrationof glycerol in the HGF tends to bond more water, and there is a largervolume change in the HGF (ΔVHGF)compared to that of the LGF (ΔVLGF). On the macroscale, the bilayer first bends toward the LGF withthe HGF as the outer layer and the LGF as the inner layer (Movie S1). However, since the network of thecaseinate film is expanded by the absorbed moisture, the glycerolmolecules from this moment can move from the HGF to LGF due to theconcentration gradient. We hypothesize that moisture uptake and glyceroldiffusion along with water diffusion lead to a competition in volumechange between the LGF and the HGF (ΔVLGF ≈ ΔVHGF). On themacroscale, we hypothesize the lag time observed is due to the timerequired for the condensed water to penetrate the glassy network.At Response stage, the bulk volume of eachlayer further expands; hence, the caseinate proteins are loosely packed,and the size of the pores within the caseinate network further grows.As a result, glycerol in this stage diffuses significantly from theHGF to the LGF. Furthermore, water absorbed by the HGF migrates tothe LGF accompanied by glycerol diffusion. These two factors henceforthhelp the LGF eventually win the competition in the volumetric increase(ΔVLGF ≫ ΔVHGF) and lead the bilayer to roll up with the LGF as theouter layer along with HGF as the inner44 layer on the macroscale.In the Moreover, interface effects suchas the formation of a thick skinlayer due to enhanced evaporation at corners and at the interfacemay alter local microstructure in EHI. The altered crust skin mayslow down the diffusion of water consequently time scale of bending.Response stage, the counter diffusion ofglycerol and water reaches equilibrium, and the two layers becomeidentical in composition as supported by TGA results. The rollingof the EHI on itself stops since there is no concentration gradientthat can drive the dilatation. Similarly, since the bending and rollingare proposed to be driven by glycerol diffusion, the process cannotexhibit reversibility because there is no glycerol concentration differencein the equilibrium bilayer to provide the necessary driving force.After It is crucial to engineering the EHI response,namely, the lag time, response time, and final curvature of rolling,to match the characteristic time scale of decomposition/degradationof the labeled products. In this section, we will demonstrate thatthe EHI response can be tuned by simply adjusting the layer thicknessand aspect ratio.Normalized Area, i.e., Projected area/Initialarea, instead of the curvature, is given to represent the rollingbehavior. The projected area is highlighted by the gray dashed line. Both C1 and C2 showa normalized area increase during the lag stage. This increase inthe area could be attributed to the films swelling when they absorbmoisture, as we already discussed  is 25 min. It exhibits a linear rolling behavior in line withearlier presented results that is completed within 30 min. The rolledstructure is maintained after 150 min. For the thicker combination, the lag time is 60 min;meanwhile, it takes around 90 min to complete rolling. By increasingaround 30% of the HGF thickness, both the lag time and response timeare doubled. We can attribute this longer operation time to two reasons.First, a thicker film spends more time reaching the swollen equilibriumstate. Second, the thicker combination, due to its reduced flexibility,iscussed 4.However, the device is consideredversatile due to the abilityto easily tune its lag time and response time through the layer thickness,making it suitable for various applications where humidity changesslowly or infrequently over a longer period of time, such as monitoringhumidity trends in a climate-controlled environment such as storageroom. The response time varies from 90 to 150 min in the showcasedcombinations. While this showcased response time may not be idealfor applications requiring a real-time response, it is suitable forwarning users of slowly evolving humidity-triggered processes suchas bacterial or mold growth which typically requires multiple hoursto a few days based on environmental conditions and biological species.47 However, contradicting results exist in the literatureregarding the quantitative relationship between the aspect ratio andfinal bending curvature. Stoychev et al.,35 Wang et al.,48 and Abdolahi et al.49 found the bending curvature of the free-standinghydrogel bilayer is independent of the aspect ratio, which agreeswith Timoshenko’s theory.50 However,Kim et al.51 observed a strong dependencebetween aspect ratio and bilayer bending. Moreover, multiple-rollinghas been observed exclusively under the condition of a large aspectratio by Alben et al.45 We already foundthat at 50% RH all our EHIs roll to a radius of around 2.5 mm regardlessof aspect ratio , wherethe rolling degree can be considered uniform within experimental limits.In this study, the relationship between the aspect ratio and the attainedcurvature of the rolled EHI is further investigated for EHIs conditionedat 50% RH and exposed to 80% RH. The results are summarized in The aspect ratio hasalready been consistently reported to influence the bending conformation for other bilayer systems.atio see S6, where51 In this study, we only focus on the final curvature the rolled EHIattains because bending conformation is less critical for humidityinterpretation according to our observations. To eliminate certainconformations, the short edge of the EHI is attached to a solid object.When the short edge is fixed by a clamp, the EHI looks like a cantileverbeam, and the bending occurs along the long edge.As the aspect ratio influences the bending conformation,to bemore specific, bilayers will bend into a shape that minimizes resistanceto bending in an energetically preferred way.R) as illustrated in the inset. We further normalizethe radius with the thickness of the HGF of the EHI to ensure thatwe have a dimensionless graph (R/HGF thickness and theaspect ratio l/w. The coefficientof determination (R2 ≈ 0.97 forC1 and R2 ≈ 0.99 for C2) indicatesa reasonably strong, positive linear correlation between nondimensionalized R and aspect ratio. Since the length and the HGF layer thicknessare fixed, while the width is varied in this set of tests, we canalso put it in a more simple way; i.e., the final rolling radius islarger for smaller widths. It can be seen that C2  overall shows a smaller R, which is within expectation considering that the larger total amountof glycerol transported from the HGF to the LGF can induce more pronouncedswelling in the LGF in C2 compared to that in C1. Moreover, when wecompare the two slopes of C1 and C2, it is evident from the observationthat the aspect ratio exerts a more substantial effect on the finalradius of the rolled bilayer EHI in thinner C1 compared to C2.EHIs of twodistinct thicknesses (both C1 and C2) were preparedin various aspect ratios, conditioned under 50% RH, and exposed to80% RH. The final shape of the rolled EHI is characterized by radius and low glycerol film (LGF), respectively. Anillustration of the film production procedure is in To prepare the film-forming mixtures, ultrapure water  and glycerol  were mixed and magnetically stirred at 600 rpm and60 °C. To this solution, sodium caseinate was gradually added over time intervals of 2–3 min to reachthe desired caseinate/glycerol/water ratio, which is 12:14:100 forthe HGF (dyed blue) and 12:3:100 for the LGF (dyed pink), respectively.The stirring rate was subsequently increased to 850 rpm for 60 min.Afterward, food dyes (Dr. Oetker) were added to distinguish the HGFand the LGF. The final mixtures were centrifuged at 7400 rpm for 12min to remove any bubbles formed during the mixing process.Film-forming mixtures were cast into plastic Petri dishes  and baked in the oven at 40 °C for48 h. After gelation, the films were peeled off and stored in a desiccator(RH 10%). Films are conditioned for 24 h in an environmental chamber under specific RH before the test.Moreover,the thickness of the film was controlled by the amountof the mixture used and measured by a film thickness gauge (Heidenhain).For instance, a 12 mL mixture can obtain an HGF of 0.310 mm thickand an LGF of 0.155 mm thick.53The utility of EHIs is demonstratedwith a urinalysis reagent strip . An EHIstrip (0.155 mm-HGF and 0.155 mm-LGF) of 5 mm by 20 mm was cut andattached to the blank end of the urinalysis strip along the shortside with Norland Optical adhesive that cures using ultraviolet light.All EHIs were conditioned under 10% before usage and then exposedto atmospheric humidity that simulated a consumer leaving the actualstorage bottle open by accident. The test was conducted at a lab temperaturethat fluctuated around 23 °C. After a 48 h atmospheric humidityexposure, we kept the test urine strip back in the desiccator for4 h to examine any reversibility. Following this, a urinalysis testwas conducted on the model strip and compared with an original stripunexposed to atmospheric humidity. The preparation of artificial urineis the same as described in the literature.R) of the rolled EHIswas analyzed using ImageJ. The rolling curvature was calculated as1/R.For visualization, the EHI response was recorded throughoutthe process in the form of a time-lapse with an action camera  to visualize the process. To measure thecurvature evolution, the recorded time-lapse film was extracted percertain frames. The radius  conducted onan SDTQ600 instrument. In each analysis, approximately 8.0 ±0.5 mg of sample was placed in an aluminum oxide crucible and heatedover temperatures ranging from 30 to 750 °C at a rate of 20 °C/min.A continuous nitrogen flow at a rate of 30 mL/min was supplied tothe TGA to maintain an inert atmosphere.Normalized Area to represent the rolling in this test, which was calculated as Projectedarea/Initial area.For the thickness test,two combinations were tested. Combination 1 (C1) was made of a 0.155mm-thick HGF and a 0.155 mm-thick LGF while combination 2 (C2) wasa 0.206 mm-thick HGF and a 0.155 mm-thick LGF. After conditioningseparately under 50% RH for 24 h, the single-layer films were stucktogether and immediately cut into rectangular strips. One end of thesamples was fixed to a glass slide with water-proof tape. The ready-to-testEHIs are 15 mm in length. After that, the samples were transferredinto the environmental chamber at 80% RH and a constant temperatureof 25 °C. The EHI response was recorded in time-lapse from thetop view to visualize the rolling process. Finally, pictures wereextracted, and the projected area of the rolled circle was analyzedusing ImageJ image analysis software. We use R) of the rolls was analyzed using ImageJ as well.For the aspect ratio test, the conditioningand test procedure were the same as the thickness test as describedabove besides that samples were cut into rectangular strips of variouslengths and widths. We only focus on the final geometries. Therefore,images were only captured after the rolling was completed. The radius(


Zygotic genome activation (ZGA) is essential for early embryonic development. However, the regulation of ZGA remains elusive in mammals. Here we report that a maternal factor TDP-43, a nuclear transactive response DNA-binding protein, regulates ZGA through RNA Pol II and is essential for mouse early embryogenesis. Maternal TDP-43 translocates from the cytoplasm into the nucleus at the early two-cell stage when minor to major ZGA transition occurs. Genetic deletion of maternal TDP-43 results in mouse early embryos arrested at the two-cell stage. TDP-43 co-occupies with RNA Pol II as large foci in the nucleus and also at the promoters of ZGA genes at the late two-cell stage. Biochemical evidence indicates that TDP-43 binds Polr2a and Cyclin T1. Depletion of maternal TDP-43 caused the loss of Pol II foci and reduced Pol II binding on chromatin at major ZGA genes, accompanied by defective ZGA. Collectively, our results suggest that maternal TDP-43 is critical for mouse early embryonic development, in part through facilitating the correct RNA Pol II configuration and zygotic genome activation. Zygotic genome activation is crucial for mammalian embryonic development. Here, the authors find that TDP-43 is indispensable for mouse embryonic development and mediates zygotic genome activation through Pol II configuration. The ZGA is the first transcription event, and is essential for embryonic development. The zygotic genome is activated as two waves, namely minor and major ZGA5. Minor ZGA occurs at the end of the one-cell stage with the expression of dozens of genes6, followed by a major ZGA bursting at the two-cell stages with the expression of thousands of genes for early embryogenesis in mice4. During mouse oocyte to embryo transition, the transcription machinery of RNA Pol II undergoes loading, pre-configuration and elongation for gene expression7. Transiently blocking minor ZGA with a transcription elongation inhibitor DRB leads to a majority of mouse embryos arrested development at two-cell stage7. Timely release of DRB inhibition before major ZGA results in abnormal transcription with the reduced enrichment of Pol II at major ZGA and the residual of Pol II binding at one-cell Pol II target genes, accompanied by the globally reduced expression of major ZGA and ectopic expression of one-cell Pol II targets7. These observations suggest Pol II is tightly regulated during the transition from minor to major ZGA in mice.After fertilization, the mammalian embryo reprograms the terminally differentiated gametes to totipotent blastomeres with the potency to generate a whole organism. During oocyte to embryo transition, the embryo undergoes reprogramming and many key events including maternal RNA degradation and zygotic genome activation (ZGA)9. However, genetic depletion of mouse maternal Pou5f1 was shown to give rise to little effect on early embryonic development10. Recently, several factors, such as Dux, Dppa2, Dppa4 and Nelfa, were identified as the important regulators for ZGA genes in two-cell-like embryonic stem cells (ESCs)17. However, genetic disruption experiments showed that most of these factors are dispensable for both the ZGA and early embryonic development in mice20. Therefore, the regulation of Pol II machinery remains poorly understood during the mammalian oocyte to embryo transition.Due to transcription silence before ZGA, maternal effect genes are anticipated to play dominant roles in the regulation of RNA Pol II machinery during the ZGA. Zebrafish transcription factor Pou5f1 primes at the loci of several hundred ZGA genes and is required for the expression of these genes21. TDP-43 includes several domains composed of an N-terminal nuclear localization sequence (NLS), two RNA binding domains (RRM1 and RRM2), a nuclear export signal (NES), and a C-terminal glycine-rich domain22. Pathogenic aggregates/inclusions of TDP-43 in the cytoplasm of nerve cells are major features of a range of neurodegenerative diseases23. A combination of a toxic gain in cytoplasm and a function loss in nucleus of TDP-43 is believed to be the predominant reason for neuronal death in patients suffering from these diseases24. Since its aggregates have been shown to be related to neurodegenerative diseases, great efforts have been made to explore the roles of TDP-43 in different cells and animals26.TDP-43 (Transactive response DNA binding protein 43 kDa), officially named TARDBP (Tar DNA binding protein), is an evolutionarily conserved DNA/RNA binding proteinTdp43/TDP-43/Tardbp universally expresses and encodes a protein sharing high similarity with its human homolog27. Specific deletion of TDP-43 with Stra8-Cre mice results in a severe loss of male germ cells and male infertile28. The intercrosses of heterozygous (Tdp43+/-) mice could not deliver homozygous (Tdp43-/-) offspring and Tdp43-/- embryos die between embryonic day 3.5 (E3.5) and E8.530. TDP-43 null blastocysts were unable to generate mouse embryonic stem cells (ESCs) due to proliferation defects of inner cell mass30. Thus, zygotic TDP-43 is essential for mouse early embryonic development. Although many mouse models have been developed to investigate TDP-43, the current understanding of its functions and mechanisms remains limited. Whether this multifunctional protein is involved in mouse oocyte to embryo transition remains unknown.Mouse 31. Depletion of TDP-43 in mouse oocytes shows that maternal TDP-43 is required for the ZGA and early embryogenesis. Detailed analyses show that TDP-43 interacts with RNA Pol II and Cyclin T1 of the P-TEFb complex, and is required for proper Pol II configuration and the expression of ZGA genes during mouse oocyte to embryo transition.In this work, we find that TDP-43 translocates from the cytoplasm into the nucleus at the mouse early two-cell stage, a simultaneous occurrence with mouse major ZGATdp43 mRNA in mouse oocytes and preimplantation embryos by analyzing the previous data of RNA-seq and Ribo-seq32. The results showed that the levels of Tdp43 expression were dynamic in mouse oocytes and early embryos  mice . Tdp43oKO mice grew normally to their adulthood. The oocytes from Tdp43oKO females underwent efficient elimination of TDP-43 at both the mRNA and protein levels  that normally produced litters (8.8 ± 2.1 pups/litter), Tdp43oKO females did not deliver any pups after mating with fertile wild type (WT) males for over 3 months , E1.5 and E2.5. We isolated a similar number of 1-cell embryos with pronucleus at E0.5 , MII, 1 C (1-cell embryos), E2C and L2C (early and late 2-cell embryos). Our RNA-seq data were well correlated between the replicates   Fig. . Maternarol Fig. . By control Fig. . About 5Tdp43oKO L2C embryos, we knocked down TDP-43 protein in mouse 1-cell embryos using Trim-away with the specific antibody against TDP-43 and cultured these embryos to the L2C stage 35. TDP-43 was efficiently knock-down in the embryos that were injected with Trim21 mRNA and the antibody against TDP-43 (hereafter KD embryos), but not in the Inj-Contr (injection control with Trim21 mRNA and IgG), probably because of TDP-43 localization in the cytoplasm until early 2-cell stage   were also up-regulated in Tdp43oKO embryos  experiments in mouse ESCs with the antibodies against TDP-43 and Polr2a. Our results showed that the TDP-43 antibody specifically precipitated Polr2a Fig. . Convers38 Fig. . Another38 Fig. . The intyos Fig. . Similaryos Fig. . Altoget7. The Stacc-seq data of TDP-43 were highly reproducible between the replicates, and were distinct between FGO and L2C embryos, with only 8.9%  of TDP-43 peaks overlapping  of TDP-43 peaks co-localized with the Pol II peaks in L2C embryos Fig. , while ~c43 Fig. , indicatTdp43oKO embryos prompted us to investigate whether TDP-43 depletion could affect the transition of Pol II occupancy during ZGA. By applying Stacc-seq7, we found that the change of Pol II occupancy at the promoters and gene bodies was correlated with the expression levels of DEGs in Tdp43oKO L2C embryos  and late 2-cell (L2C), as well as the 1 C and L2C specific cluster. The PoI II enrichment of the 1 C specific cluster became gradually weaker, while the enrichment of the L2C specific cluster became gradually stronger in WT embryos 37. Then, we sought to investigate whether TDP-43 regulates Pol II through the components of the P-TEFb complex in mouse ZGA.Our results suggest that TDP-43 was directly involved in the transcription of ZGA genes through Pol II configuration. As mentioned previously, Pol II enters the elongation for transcript production when it is phosphorylated at its CTD Ser2 (P-Ser2) by P-TEFb  family proteins include six members that highly express during mouse oocyte to embryo transition53. Maternal importin α2 (Kpna7) was reported to be required for mouse early embryonic development and fecundity54. Similar to those lacking maternal TDP-43, depletion of maternal importin α7 (Kpna6) resulted in the arrested embryo development at the two-cell stage and the dramatically decreased expression of ZGA genes in these embryos53. Thus, these importin proteins may interact with TDP-43 and regulate its translocation from the cytoplasm into the nucleus during mouse maternal to zygotic transition. Taking together, the regulations of TDP-43 translocation deserve to be further investigated during mouse maternal to zygotic transition. The investigations of TDP-43 translocation may contribute to the treatment of human neurodegenerative diseases, such as amyotrophic lateral sclerosis and Alzheimer’s disease.Since the discovery of TDP-43 aggregations related to neurodegenerative diseases, the translocation of TDP-43 from the cytoplasm into the nucleus has been broadly investigated in the neuronal cells with cytoplasmic TDP-43 aggregations for the treatment of these diseasesAcrv1  gene during mouse spermatogenesis, in which TDP-43 and Pol II binds the promoter of Acrv1 in mouse spermatocytes and round spermatids, but the expression of this gene is exclusive in the post-meiotic round spermatids22, further supporting this hypothesis. Thus, how the cells control TDP-43 for the active transcription in the specific context deserves to be further investigated. In summary, our study provides strong evidence to support that maternal TDP-43 interacts with Pol II and is required for proper Pol II configuration, ZGA, and early embryogenesis.Although TDP-43 co-localizes with Pol II in mouse full-grown oocytes, the absence of TDP-43 barely affects P-ser2 foci formation and Pol II configuration in these cells. Furthermore, the absence of TDP-43 mildly affects the gene expression in mouse full-grown oocytes. These observations suggest that the regulation of TDP-43 on the active transcription through Pol II is a stage- or context-specific function of ZGA during mouse oocyte to embryo transition. A previous report shows that TDP-43 regulates the expression of a testis-specific Tdp43floxp/floxp mice (No. 017591) that possess loxP sites flanking exon 3 of Tdp43 were obtained from Jackson Laboratory and were mated with Zp3-Cre transgenic mice that express Zp3 promoter driving Cre recombinase33. After obtaining Tdp43+/floxp;Zp3-Cre mice, they were intercrossed to produce Tdp43floxp/floxp;Zp3-Cre and other genotype mice. Tdp43flox/flox females were mated with Tdp43flox/flox;Zp3-Cre males to routinely produce Tdp43floxp/floxp  and Tdp-43floxp/floxp;Zp3-Cre mice. Tdp43floxp/floxp;Zp3-Cre females had the oocytes with TDP-43 deletion, thus designed as Tdp43oKO (Tdp43 oocyte-specific knockout) mice. These mice were maintained in a hybrid background of C57BL/6 J and ICR, and were genotyped using tail DNA or toe DNA as PCR templates with the primers of a previous report (PubMed: 20660762)33.Mice and all animal experiments were manipulated under the guidelines of the Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences (IOZ-IACUC-2021-052). Mice were maintained under the controlled environment conditions, including 12-h alternating light/dark cycles, a temperature controlled at 23-25 °C, and a humidity range of 40–65%. They had ad libitum access to food and water. Oocytes and embryos were isolated in M2 medium from 7–8-week-old female mice that were injected with HCG after the injection of PMSG for 46–48 h or mating with males at the following time points: MII oocytes: 13 h after HCG; PN2 1-cell embryos: 24 h after HCG; PN5 1-cell embryos (Late 1-cell): 30 h after HCG; early two-cell embryos: 36 h after HCG; late two-cell embryos: 48 h after HCG. To obtain embryos, females injected with HCG were mated with normal fertile ICR males. Full-grown oocytes were isolated from the ovaries of 7–8-week-old females after only the injection of PMSG for ~48 h and cultured in M2 medium (Sigma 7167). Embryos were cultured in KSOM medium (Millipore MR-106-D).TM mRNA DIRECTTM Micro Kit (Millipore 61021). mRNA was extracted as the instruction of the manufacturer. Reverse transcription (RT) was performed with Takara PrimeScript RT Reagent Kit according to the instruction of the manufacturer. Quantitative RT-PCR was conducted with Abm EvaGreen 2X qPCR MasterMix-No dye kit (Abm MasterMix-S) with specific primers of TDP-43  and Gapdh . The results were repeated with at least three independent samples. Gapdh was used to normalize the gene expression.Oocytes and embryos were transferred to the lysis buffer of Invitrogen DynabeadsOocytes and embryos were fixed with 4% PFA in PBS for 30 min at 37 °C. These samples were washed with PBSA , permeabilized with 0.5% (V/V) Triton X-100 for 30 min at room temperature. After being washed with PBSA, samples were blocked with 1% BSA for 1 h at room temperature. These samples were incubated with primary antibody overnight at 4˚C . After washed with PBST (0.1% triton X-100 in PBS) three times, oocytes and embryos were incubated with specific fluorescence conjugated secondary antibody for 1 h at room temperature . After washed at least three times, the samples were imaged with Zeiss LSM780 or LSM880 or Airyscan880. For TDP-43 staining (Santa Cruz Sc-376532), the zona pellucida of embryos was removed with Tyrode’s solution (Sigma T1788) before fixation.55. The RNA library was constructed with a kit of Vazyme (TD502) and performed as the protocol of manufacture.Oocytes and embryos were treated gently with Tyrode’s solution for removing the zona pellucida. Twenty oocytes or embryos were collected for each group of RNA extraction. These samples were performed for reverse transcription and PCR pre-amplification as the smart-seq2 steps as previously reported56. The gene expression was calculated with gene annotation from Gencode RefSeq database using HTSeq (v2.2.1), StringTie (v2.1.2) and FeatureCounts (v2.0.1)58. DEGs were identified by DESeq2 (v1.24.0)59, and the threshold of adjusted P-value was 0.05 with fold change ≥2. Heatmaps of the genes with expression changes were clustered with k-means clustering analysis and were visualized with the heatmap package in R. 2D. PCA analysis was performed and visualized by using all expressed genes by factoextra (v.1.0.7). Dot analysis was used to compare the up- or down-regulated expressed genes in the samples and the Spearman’s rank correlation coefficient was used to compare the relevance between different samples. GO term analysis for the enriched biological processes was conducted using clusterProfiler (v.4.6.0) and DAVID (v6.8) with the default parameters60.All RNA-seq data were mapped to the mouse mm9 genome by HISAT2 (v2.2.1) with default parameters after removing the low-quality sequencing reads with Cutadapt (v0.3.8)42. Maternal genes were identified as highly expressed in MII oocytes (FPKM > 5), but down-regulated in late 2-cell embryos (at least 3-fold). Genes with FPKM (MII) < 5 and up-regulation  in either 1-cell embryos or early 2-cell embryos were identified as minor ZGA genes, while those genes up-regulated in late 2-cell embryos were identified as major ZGA genes. Other genes with FPKM (MII) > 5 and highly up-regulated at the late 2-cell stage (over 5-fold) were also included in major ZGA genes.Maternal and ZGA genes were defined with the reference RNA-seq data of mouse oocytes and early embryosTrim21, NLS-TDP43 and Ccnt1 were cloned in the vector Pcs2 that contained a fused-expressing mCherry or EGFP. The cloned genes were PCR with the sequence of SP6 promoter and T3 promoter. The cloned genes were transcripted according to the AM1340 manufacturer’s protocol and the products were purified as the mRNA template for the microinjection of mouse 1-cell embryos before PN2 stage. After injected with NLS-TDP43-mCherry and Ccnt1-EGFP, mouse 1-cell embryos were cultured for these gene expression and fixed for the staining of TDP43 and Ccnt1 with specific antibodies. For Trim-away technology, mCherry-Trim21 mRNA and rabbit anti-TDP-43 antibody  or normal rabbit IgG were simultaneously injected into mouse 1-cell embryos before the PN2 stage, then these embryos were cultured in KOSM to develop into late two-cell stage (pHCG 48 h) for the following analysis.Tdp43floxp/floxp and ER-cre mice, and were treated with Tamoxifen for 72 h for the immunoprecipitation experiments (washing with NP-40 containing 500 nM NaCl). 293t cells were maintained in classical DMEM basic supplement with 10% FBS.ESCs were maintained in classical 2i/lif culture. The induced-knock-out(iKO) ESCs were derived from the blastocysts that were isolated from the mating of Vector and transfection reagent PEI (three times of vector molar volume) were dissolved in isopyknic opti-MEM for 5 min and were mixed. Twenty-five minutes later, the mixture of vectors and PEI was added into 293t cells to incubate for 12 h. Then, the culture medium was replaced with the fresh medium. After 36 h of transfection, these cells were collected with the lysis buffer of Beyotime plus protease inhibitor for the immunoprecipitation (IP) experiments.Mouse ESCs or 293t cells were collected in IP lysis buffer of Beyotime with protease inhibitor, incubated on ice for 15 min. After centrifuging (11833 g for 15 min at 4 °C), the supernatant was collected for Co-IP experiments according to the manufacturer’s protocol of Protein A/G Magnetic Beads (Bimake B23202 or MCE HY-K0202). The antibodies included TDP-43 , Polr2a , Flag , and Myc . We also washed the IP mixture of mouse iKO ESCs with NP-40 containing 500 mM NaCl for the Co-IP experiment of TDP-43. The precipitated products were heated for 5 min at 95 °C for Western blotting.Oocytes and embryos were collected in 10 μl 1X protein loading buffer and heated for 5 min at 95 °C. The heated samples were fractionated at 100 V for 2 h using 10–12% NuPAGE Bis-Tris gels and MOPS SDS running buffer, and were transferred to PVDF membranes for 2 h at 200 mA (3.5 h when Polr2a antibody was blotted). Then, the membranes were blocked with 5% skimmed milk in TBST (M/V) for 1 h at room temperature. After washed with TBST, the membranes were incubated with primary antibodies at 4 °C overnight . After washed with TBST, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. After washed with TBST, the membranes were detected for the targeting proteins according to the instruction of SuperSignal West Dura Extended Duration Substrate and were imaged with a LAS-3000 apparatus.Proximity Ligation Assay was performed with Sigma-Aldrich reagents according to the protocol of Sigma Aldrich Duolink® PLA Fluorescence . The primary antibodies of different host species  were used and diluted as immunofluorescence staining. The embryos were collected as above, and their zona pellucida was removed using Tyrode’s solution. These samples were fixed with 4% PFA for 30 min at 37 °C, and permeabilizated with 0.5% Triton X-100 in PBS for 30 min at room temperature. The embryos were blocked with Sigma-Aldrich Duolink® Blocking Solution in a humidity chamber for 60 min at 37 °C. The samples were transferred into the mixed solution with the primary antibodies in Duolink® Antibody Diluent, and were incubated in a humidity chamber at 37 °C for at least 2 h or at 4 °C overnight. The samples were washed with 1X Duolink® washing buffer A, and were transported to the probe mixture prepared as the manufacturer’s protocol, then were incubated in a humidity chamber at 37 °C for 1 h. After washed with Duolink® washing buffer A, the samples were incubated with the ligation mixture that were prepared as the manufacturer’s protocol in a humidity chamber at 37 °C for 30 min. After washed with 1X Duolink® washing buffer A, the samples were incubated with the amplification mixture containing amplification buffer (1:5) diluted with ultrapure water and amplification polymerase (1:80) in a humidity chamber at 37 °C for 100 min. After washed with 1X Duolink® washing buffer B and 0.01X Duolink® washing buffer B, the samples were incubated with 1X DAPI at room temperature for 30 min or at 4 °C for a long time in a dark condition. After washed with PBSA (0.1% BSA in PBS), the samples were imaged by using a LSM880 or Airyscan880 apparatus.7. Briefly, 1 μl fully dissolved 5% digitonin (Sigma D141) was added to Buffer1 , termed DB1. Then, 0.5 μl PA/G-Tn5 (Vazyme Biotech TD902) and 0.5 μg TDP-43 antibody  or Pol II antibody (Active Motif 102660) were also added to DB1 and the mixture was incubated at 4 °C for 30 min. Meanwhile, mouse oocytes and embryos were treated gently with Tyrode’s solution to remove the zona pellucida, then were picked into a 200 μl low-binding tube containing 6 μl of the DB1. After incubated at 4 °C for 10 min, these samples were added with 35 μl of the mixture of DB1, PA/G-Tn5, the antibody and 12.5 μl of TTBL (Vazyme TD502) for cleaving the chromatin around the binding sites and transposing with adapters. Finally, the DNA was purified and PCR was performed as the Stacc-seq steps. We used at least 150 embryos or oocytes for the high quality data of TDP-43 (200) or Pol II (150) in these experiments.Stacc-seq was performed according to our previous report61. For downstream analysis, read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced (RPKM) after removing non-uniquely mapped reads, unmapped reads and PCR duplicates. The RPKM was further normalized with Z-score transformation to minimize the batch effect. UCSC genome browser was used to visualize the signals of Stacc–seq62. Repetitive element enrichment was calculated by comparing annotated repeats overlapped by Stacc-seq peaks and randomly shuffled peaks. All the Stacc-seq peaks were called by MACS (v.1.4.2) with the parameters–nolambda–nomodel63, and were compared by using BEDTools (v2.29.2)64. ChIPseeker (v1.26.2) was used to identify and visualize the genomic features of peaks65. Promoters were defined as ±2,5 kb around the transcription starting sites (TSS). Stacc-seq peaks at least 2.5 kb away from TSS were defined as distal peaks by BEDTools (v2.29.2).All paired-end Stacc-seq reads were mapped to mm9 using Bowtie2 (v.2.2.5) after trimming adapters and low quality sequencing readst-test or two-sided Wilcoxon rank-sum to compare the difference between two groups. P-values < 0.05 were considered as a significance. The immunostaining experiments and Western blots were performed at least three independent repetitions with similar results.Quantitative analyses were expressed as the mean ± SD, based on at least three independent biological samples. Statistical analyses were performed using a two-tailed Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Women typically exhibit weaker attentional control ability than men. Lower resting vagally mediated heart rate variability (vmHRV) is thought to reflect the poorer function of the neurophysiological pathways underlying attentional control and thus, poorer attentional control ability. However, existing findings are inconsistent regarding the relationship between vmHRV and attentional control. Gender may be an important moderator.To examine whether gender moderates the relationship between resting vmHRV and attentional control, and to provide neurophysiological evidence for elucidating gender differences in attentional control ability.Two hundred and twenty college students completed the Attentional Control Scale to evaluate their attentional control ability. Resting vmHRV was assessed during a 5 min baseline period using an electrocardiographic amplifier (ECG100C) of the Biopac MP150 physiological recorder.t = 0.498, p > 0.05), but the scores of the attentional shifting dimension of women were significantly lower than those of men ; (2) Resting vmHRV was significantly negatively correlated with attentional control in women, whereas the correlation was not significant in men; (3) Gender significantly moderated the relationship between resting vmHRV and attentional control ; (4) Among participants with lower resting vmHRV, there was no significant difference in attentional control between men and women , but among participants with higher resting vmHRV, men scored significantly higher than women in attentional control .(1) There was no significant difference in the total scores of the Attentional Control Scale between men and women (Gender moderates the relationship between resting vmHRV and attentional control, with higher resting vmHRV in women reflecting a compensatory response to deficits in attentional control. AttentiAccording to the neurovisceral integration model, attentional control is significantly influenced by prefrontal-subcortical inhibitory pathways . The preHeart rate variability (HRV) is a measure of the variability of the interval between two consecutive heartbeats . VagallyHowever, there are inconsistent findings in existing studies regarding the relationship between resting vmHRV and attentional control ability. Although some studies have shown that resting vmHRV is positively correlated with attentional control , 17, othStudies have shown that there are gender differences in attentional control , 19. WomThe differences in attentional control between men and women have a neurophysiological basis. The brain regions involved in attentional control mainly include structures such as the frontal cortex and cingulate gyrus . NeurostA growing body of research has shown that gender is a significant factor in moderating the relationship between resting vmHRV and mood, such as depression, anxiety and stress perception. For example, there are gender differences in the relationship between resting vmHRV and depression . Higher In summary, gender may be an important factors that moderates the relationship between resting vmHRV and attentional control. However, existing studies have not investigated this issue systematically. The current study aimed to examine how gender moderates the relationship between resting vmHRV and attentional control to shed light on the neurophysiological differences in attentional control between men and women.2.2.1.Participants were recruited through two methods. One group of participants was recruited through campus advertisements and received a cash incentive of 15 RMB for participation. The other group of participants was recruited from students enrolled in public psychology courses and received a course credit award. Inclusion criteria included being right-handed and having normal or corrected-to-normal vision. Exclusion criteria included a history of neurological, psychiatric or cardiovascular disease. A total of 220 participants  were included in this study, with a mean age of 19.31 ± 1.17 years. All participants voluntarily signed the informed consent form before the experiment. As sleep quality, physical activity and caffeine intake are important variables influencing HRV , partici2.2.When participants arrived at the laboratory, they naturally sat on a chair to rest, signed the experimental informed consent form, and filled out a series of self-report questionnaires. After the participants felt completely relaxed, they began to collect resting ECG data for 5 min. During the ECG data acquisition process, the participants sat relaxed in a chair, kept their bodies stable, breathed naturally, and looked at the picture of the neutral cup presented on the computer screen. At the end of experiment, participants scored their emotional arousal on a 9-point scale.2.3.The Attentional Control Scale (ACS) is a self-reported measure of attentional control ability . It is w2.4.To assess the subjective emotional experience during the experiment, we instructed the participants to score their emotional arousal  on a 9-point scale .2.5.Cardiac data were collected using the electrocardiographic amplifier (ECG100C) of the Biopac MP150 physiological recorder. Three silver chloride (Ag-AgCL) disposable electrode pads were attached to the participant’s wrist and two ankles in lead II. The filtering range of the Biopac ECG100C amplifier was 0.5–35 Hz with a sampling rate of 1,000 Hz. The Kubios HRV analysis package  was used2.6.t-tests were conducted to analyze the differences between male and female participants on all variables. Pearson correlation was used to analyze the relationship between vmHRV and other important variables. The SPSS macro PROCESS (Model 1) was used to test the moderating effect of gender  on the relationship between attentional control (independent variable) and resting vmHRV (dependent variable) . All tests were two-tailed and significance levels were evaluated using an alpha of 0.05. Independent samples ariable) . Bootstr3.t = 4.133, p < 0.001). There was no significant difference between male and female participants on resting vmHRV . Women’s attentional control scores were slightly lower than men’s, with the difference not reaching a significant level . However, women scored significantly lower than men on the attentional shifting dimension of attentional control .r = −0.233, p < 0.01).B = −3.088(1.189), 95% boot CI , t = −2.598, p = 0.01). Conditional analysis showed a negative correlation between resting vmHRV and attentional control in women (B = −2.120 (0.779), 95% boot CI , p = 0.007), whereas there was no significant correlation between resting vmHRV and attentional control in men (B = 0.968 (0.898), 95% boot CI , p = 0.282). The moderating effect of gender remained significant when analyzed with age, BMI, respiration and emotional arousal as covariates in the moderation analysis (B = −2.897(1.203), 95% boot CI , t = −2.408 p = 0.017), with a negative correlation between resting vmHRV and attentional control in women (B = −2.072 (0.786), 95% boot CI , p = 0.001) and no significant correlation in men (B = 0.825 (0.912), 95% boot CI , p = 0.367). In other words, among women, the difference in attentional control between participants with lower and higher resting vmHRV was significant; whereas among men, the difference in attentional control between participants with lower and higher resting vmHRV was not significant (see B = 2.284 (1.542), 95% boot CI , p = 0.139), but men scored significantly higher in attentional control than women in participants with higher resting vmHRV (B = −3.377 (1.536), 95% boot CI , p = 0.029).cant see . With ge4.This study aimed to investigate whether gender moderates the relationship between resting vmHRV and attentional control, and to provide neurophysiological evidence for elucidating gender differences in attentional control ability. The results showed that there was no significant difference between men and women in the overall scores of attentional control, but women scored significantly lower than men in attentional shifting. Gender was a significant variable in moderating the relationship between resting vmHRV and attentional control. Resting vmHRV was significantly negatively correlated with attentional control in women, whereas it was not significantly correlated with attentional control in men.Although there was no significant difference in the total scores of attentional control between men and women in this study, women had poorer attentional shifting abilities than men, which confirmed the previous findings. Women had weaker attentional shifting ability than men, as evidenced by a lower efficiency in shifting attention across multiple cognitive processing tasks . AttentiMore importantly, the present study showed that gender moderates the relationship between resting vmHRV and attentional control, with resting vmHRV negatively correlated with attentional control in women, whereas the two were not significantly correlated in men. The conditional analysis revealed that among participants with higher resting vmHRV, men had significantly higher attentional control scores than women. This result explains the mechanism of gender differences in attentional control from the perspective of vagal nerve tone. Women with high resting vmHRV were more likely to have difficulty with attentional control, suggesting differences in the neural mechanisms of attentional control reflected by high resting vmHRV in men and women. Consistent with previous studies, we speculated that higher vmHRV in female participants reflects a compensatory mechanism for deficits in attentional control, as an effort by individuals to compensate for dealing with negative emotional responses , 30. DueSome studies support the idea that higher resting vmHRV in women is a compensatory response to inadequate attentional control. Studies have shown that higher resting vmHRV in women is associated with increased activity in the amygdala (the area associated with threat processing), whereas higher resting vmHRV in men is associated with decreased amygdala activity . This coFrom an evolutionary psychology perspective, the tend-and-befriend theory and the parental investment theory could also provide an explanation for the negative correlation between resting vmHRV and attentional control is limited to women , 42. EvoIn general, the results of this study suggest that women have poorer attentional shifting abilities than men, and that gender moderates the relationship between vmHRV and attentional control. The high resting vmHRV levels in men and women may reflect different emotion regulation mechanisms, with higher vmHRV in women reflecting a compensatory response to deficits in attentional control.5.There are several shortcomings in the present study. First, the present study examined the relationship between resting vmHRV and attentional control in a cross-sectional investigation and was unable to determine a causal relationship between the two variables. Future studies could design longitudinal follow-up experiments to examine the neurophysiological basis of gender differences in attentional control. Second, the present study only used the self-report scale to measure attentional control ability, which may not accurately cover all aspects of attentional control . Future The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Ethics Committee of Nanjing University of Chinese Medicine. The patients/participants provided their written informed consent to participate in this study.XZ contributed to the conception and design of the study. YS checked and corrected the scientific issue of the study. XZ and SW conducted the experiment, analyzed the data, and wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.This research was funded by the Research Project of Jiangsu University Philosophy and Social Science (2021SJA0338).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Herein, we have constructed a magnetic graphene field-effect transistor biosensor (MGFETs) for highly sensitive detection of cardiac troponin I (CTNI). Graphene films transferred to ITO conductive glass as conductive channels. CTNI aptamer was immobilized onto the graphene film via 1-pyrene-butanoic acid succinimidyl ester (PBASE) to capture CTNI. Magnetic nanobeads (MBs) modified with CTNI antibody were added to the reaction chamber to form an aptamer/CTNI/antibody/magnetic nanobeads sandwich-type complex. We found that the magnetic force exerted on the complex leads to an impedance change of the graphene film. The reason for this result is that the magnetic field exerts an influence on the MBs, causing CTNI aptamer strand to bend, resulting in a change in the distance between the double conductive layers of the graphene film surface and the test solution. With periodic sampling integration, different concentrations of CTNI can be detected with high sensitivity. Due to the stringent recognition capability and high affinity between the CTNI aptamer and CTNI, MGFETs have the potential to detect various types of proteins. Furthermore, MGFETs also have the potential to be utilized for the detection of DNA or specific cells in the future. Based on statistical data from the World Health Organization (WHO), cardiovascular diseases have emerged as the primary cause of global mortality, with acute myocardial infarction (AMI) being the deadliest among various cardiovascular conditions , 2. The To address these limitations, various cardiac biomarkers have been developed for the diagnosis of acute myocardial infarction (AMI). These include cardiac troponin I (CTNI), CK-MB, cardiac troponin T (CTNT), and myoglobin , 7. CTNIMainly, CTNI detection methods include enzyme-linked immunosorbent assay , radioimGraphene is a single atomic layer of two-dimensional crystals formed by carbon atoms linked by SP2 hybridization. It has good electron mobility and many oxygen-containing groups that can be used to anchor DNA, proteins, and other biomolecules . Many reIn conventional graphene field-effect transistor biosensors (GFETs), applying a gate electric field results in the formation of double conductive layers between the electrolyte solution and the graphene film. Researchers have constructed a capacitive model of a GFETs . The gatHowever, although these techniques have reached extremely high sensitivity through semiconductor analyzers, they are still costly and complicated to use. Additionally, the application of gate voltage through the insertion of Ag/AgCl electrodes into the solution may affect the concentration of the solution, and the gate voltage may cause structural damage to the biomolecules to be measured –27.Therefore, researchers have been exploring alternative methods to improve the performance of GFET biosensors, such as using non-covalent functionalization with biomolecules, which can improve the sensitivity and prevent damage to the biomolecules –30. OtheHerein, we propose a novel approach for CTNI detection using magnetic graphene field-effect transistor biosensors, which eliminates the need for an Ag/AgCl electrode to provide the gate voltage. Firstly, we transfer graphene films onto glass substrates with two indium tin oxide (ITO) electrodes to establish conductive channels . The graThe conductive glass with ITO electrodes was sourced from Hua Nan Xiang Cheng Ltd. Troponin I and mouse anti-troponin I monoclonal antibodies were obtained from Shanghai Linc-Bio Science CO., LTD. 1-Pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) was acquired from Shanghai Aladdin Biochemical Technology Co., Ltd. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide-hydrochloride (EDC) and N-hydroxy succinimide (NHS) were purchased from Sigma-Aldrich. The troponin I aptamer sequence used, (5-NH2-CGT GCA GTA CGC CAA CCT TTC TCA TGC GCT GCC CCT CTT A-3′-6-FAM), was purchased from Sangon Biotech (Shanghai) Co., Ltd. Carboxylated ferric oxide nanoparticles were obtained from Nanjing XFNANO Materials Tech Co., Ltd. Dimethyl sulfoxide (DMSO) was purchased from Sangon Biotech (Shanghai) Co., Ltd. Sodium dodecylbenzene sulfonate (SDS) and sodium dodecyl sulfate phosphate-buffered saline  were obtained from Sigma-Aldrich . Bovine serum albumin (BSA) was purchased from Shanghai Linc-Bio Science CO., LTD. To evaluate the quality of graphene, a Raman microscopic system  was utilized. High-magnification transmission electron micrographs (TEM) were captured using a JEOL JEM 2100F instrument.To obtain a uniformly dispersed magnetic bead solution, the purchased carboxyl-modified magnetic nanobeads were sonicated for 15 min at room temperature. The carboxyl groups on the magnetic nanobeads were activated by adding 5 µL of 0.2 µM NHS and 5 µL of 0.2 µM EDC to 500 µL of 1 mg/mL magnetic nanobeads. Tchenka et al. , followeIn order to deposit the magnetic nanoparticles at the bottom, a permanent magnet was positioned beneath the magnetic nanoparticle test transistor, and the supernatant was removed using a pipette. The resulting pellet was then resuspended in 1 mL of PBS buffer at pH 7.4.To functionalize the magnetic nanobeads, 10 µL of mouse anti-troponin I monoclonal antibodies with a concentration of 1 mg/mL were added to the activated magnetic nanobead solution prepared with NHS and EDC. The mixture was incubated at room temperature for 2 h. Following this, 10 µL of 10% bovine serum albumin (BSA) was introduced into the suspension and incubated for 1 h to block any potential residual sites on the nanobeads.The fixed magnetic nanobeads were obtained by using a permanent magnet underneath the transistor to hold them in place, followed by aspirating the upper layer of unreacted chemicals using a micropipette. The resulting pellet was resuspended in 1 mL of PBS (pH 7.4) supplemented with 1.0% BSA and stored at 4 °C. .The cleaned graphene film was transferred onto a glass substrate with two ITO electrodes, serving as a conductive bridge between the two ITO electrodes Fig. a. The MGThrough the magnetic force between magnetic beads (MBs) and an externally applied magnetic field, the distance between MBs/aptamer conjugates and the graphene film can be dynamically adjusted. This modulation directly influences the conductivity properties of the graphene film, thereby affecting the impedance of the magnetic field effect transistors (MGFETs) with a dual conductive layer Fig. a. In ourID/IG) and the presence of a small D-peak. Furthermore, the intensity of the 2D-peak of single-layer graphene was more significant than that of the G-peak, while the half-peak width of the 2D-peak gradually increased and shifted to a higher wave number as the number of layers increased. These observations led us to conclude that the transferred graphene films were multilayer films [The transferred graphene was characterized using Raman spectroscopy Fig. . The Ramer films –32.Fig. π–π stacking, while the other end can bind to the 5′-NH2 labeled aptamer. We labeled the 3′ end of the CTNI aptamer with a fluorescent group to verify the firm attachment of the aptamer to the graphene film. The . Alternatively, one end of PBASE can be linked to graphene through The Fig.  showed tCarboxyl-modified magnetic nanobeads were activated by NHS and EDC and utilized as a capture probe for binding to CTNI antibodies. The nanobeads were used to form aptamer/CTNI/antibody/MBs sandwich-type immune complexes. Tubes containing 10 µL of CTNI aptamer at a concentration of 2 µM and 100 µL of 10 µM CTNI were incubated for 2 h at room temperature. A fluorescence detection system detected a fluorescence intensity of about 1400 A.U. The mixture of magnetic nanobeads and CTNI antibody binding, which was completed by the aforementioned experiment, was then taken out (100 µL) and added to the test tube. The solution was incubated for 1 h and then separated using a magnetic precipitation method. A part of the supernatant solution was tested using the fluorescence detection system for detection. The results shown in Fig.  indicateAfter the linkage of the CTNI aptamer to the graphene, different concentrations of CTNI solution were added to the reaction chamber of the magnetically controlled MGFET, followed by the addition of an antibody-modified magnetic bead solution for 30 min. The reaction chamber of the MGFET was then washed with PBS to remove any unbound magnetic nanobead and antibody complexes. A permanent magnet was placed at the back of the MGFET to apply a periodic magnetic field, and the impedance change of the MGFET was detected using a laboratory-made device Fig. a.Fig. 7aIn conventional GFETs, the gate electrode applies voltage to the Ag/AgCl electrode, controlling the directional movement of charge carriers between the conductive solution and the graphene film. In MGFETs, the CTNI aptamer can be considered as an elastic model. By applying a periodic magnetic field, the bending degree of the CTNI aptamer chain can be altered, leading to a change in the distance of the double conductive layer between the graphene film and the electrolyte solution. This results in carrier movement and subsequently alter the conductivity between the drain and the source of the MGFET. The variation in the impedance of the MGFET with magnetic field strength was divided into three components Fig. b. When tThe impedance variation of MGFETs with respect to CTNI concentration at a fixed magnetic field strength was measured to determine the sensitivity and detection limit of the MGFETs Fig. a. The MGWe evaluated the specificity and selectivity of MGFETs by detecting various types of proteins. Cardiac troponin T (CTNT), human serum albumin (HSA), CK-MB, bovine serum albumin (BSA), and glucose were added to test the specificity of MGFETs, and the different types of proteins were tested alongside CTNI under the same experimental conditions , 35. TheTo summarize, a graphene-based MGFETs design has been successfully developed as a biosensor for the detection of CTNI. The structure involves laying ITO films on both ends of a glass sheet to serve as the drain-source of the FETs. Then, by transferring a graphene film in the middle, the graphene film was functionalized with an immobilized CTNI aptamer to capture CTNI in solution. A sandwich-type immune complex was formed with CTNI antibody-modified magnetic nanobeads. Finally, a magnetic field was applied to the GFETs by a permanent magnet immobilized under the ITO conductive glass. To reduce noise interference, we utilized the method of periodic sampling and integration to improve the signal-to-noise ratio, achieving high sensitivity detection of CTNI with a detection limit that can reach 10 pM. Our biosensor provides a promising alternative to conventional Ag/AgCl-electrode-based biosensors. Its use of a magnetic field instead of an electric field to control the GFETs not only enables damage-free detection of the biomolecules but also reduces the cost and complexity of the overall detection system.


Exhaled Drug MONitor – EDMON measures exhaled propofol in ppbv every minute based on multi-capillary column – ion mobility spectrometry (MCC-IMS). The MCC pre-separates gas samples, thereby reducing the influence of the high humidity in human breath. However, preliminary analyses identified substantial measurement deviations between dry and humid calibration standards. We therefore performed an analytical validation of the EDMON to evaluate the influence of humidity on measurement performance. A calibration gas generator was used to generate gaseous propofol standards measured by an EDMON device to assess linearity, precision, carry-over, resolution, and the influence of different levels of humidity at 100% and 1.7%  relative humidity (reference temperature: 37°C). EDMON measurements were roughly half the actual concentration without additional humidity and roughly halved again at 100% relative humidity. Standard concentrations and EDMON values correlated linearly at 100% relative humidity (R²=0.97). The measured values were stable over 100min with a variance ≤ 10% in over 96% of the measurements. Carry-over effects were low with 5% at 100% relative humidity after 5min of equilibration. EDMON measurement resolution at 100% relative humidity was 0.4 and 0.6 ppbv for standard concentrations of 3 ppbv and 41 ppbv. The influence of humidity on measurement performance was best described by a second-order polynomial function (R²≥0.99) with influence reaching a maximum at about 70% relative humidity. We conclude that EDMON measurements are strongly influenced by humidity and should therefore be corrected for sample humidity to obtain accurate estimates of exhaled propofol concentrations.The bedside  Propofol is the most used intravenous anaesthetic for induction and maintenance of general anaesthesia and sedation. Anaesthetic effect can be measured by electroencephalographic systems like the Bispectral Index monitor (BIS). However, the electroencephalogram is influenced by opioids and neuromuscular blocking agents, and is subject to internal and external influences such as eye blinking or sensor movement which add noise to the signal –3. An alInappropriate dosing may cause haemodynamic instability, delayed recovery, or (rarely) intraoperative awareness –12. RealReal-time propofol monitoring from exhaled breath is a rapidly developing field of research. Harrison et al. were the first to perform online measurements of exhaled propofol, reported in 2003 . ExhaledHigh humidity in breath influences the underlying technology of ion mobility spectrometry –28. A muThe EDMON monitor is primarily intended to be used in mechanically ventilated patients. During mechanical ventilation, humidity of breath samples varies depending on various factors including use of heat and moisture exchange filter and fresh gas flow . Under n-1 was freshly prepared by dissolving propofol ≥ 97%  in 1% v/v absolute ethanol  in HPLC-grade water or n-hexane ≥ 99%  for all measurements and stored in a 250 mL glass bottle. To enhance accuracy, the exact propofol concentration of the stock solution was calculated according to the weight of the added propofol determined with an analytical scale .Propofol stock solution of 35.9µg mL-1 N2  . The. The2O)nJ mol-1) 44] hamp hamp2O)nJ mol-1) .ref = 37°C), clinical samples will always contain substantial humidity, but may lay under the 70% threshold. Therefor influences on measured propofol concentrations are prone to differ during changing clinical conditions. Such, the amount of humidity in clinical samples is influenced by patient conditions, heat-and-moisture-exchanging filters, ventilation settings, active humidification, oxygen concentration, fresh mixture volume, inspiratory and expiratory settings, lung-protective ventilation, etc. [Relative humidities between 70% and 100% roughly halved the EDMON measured concentrations compared to measurements without additional humidity. Considering the typical humidity of 27g/m³ in the mouth , which con, etc. –50. EDMOJiang et al.  propose v and 23 ppbv, while imprecision over 100 measurements was ≤ 10%. Resolution is comparable for measurements with and without additional humidity whereas carry-over effects are smaller with additional humidity. The influence of humidity was substantial and results in a sigmoidal decrease in measured propofol concentration by up to 50% at relative humidities exceeding 70%. Quantitative propofol measurements must therefore be adjusted for sample humidity on-line, or at least at the beginning of the measurement, for sample humidity if it can be ensured that the humidity remains stable throughout the procedure, preferably above the 70% threshold. The equations we provide suggest an easy way to adjust for sample humidity.In summary, linearity with and without additional humidity was observed for measured concentrations between 1 ppb


The prognostic role of soluble programmed death ligand 1 (sPD-L1) in digestive system cancers (DSCs) remains inconclusive. This study aimed to explore the predictive value of sPD-L1 expression in DSCs.Comprehensive searches were run on the electronic databases  to identify studies that assessed the prognostic role of sPD-L1 in DSCs. Review Manager software (version 5.3) was used for all analyses. Pooled data for survival outcomes were measured as hazard ratios (HRs), 95% confidence intervals (CIs), and odds ratios and their 95% CIs.The search identified 18 studies involving 2,070 patients with DSCs. The meta-outcome revealed that a high level of sPD-L1 was related to poorer overall survival  and disease-free survival  in DSCs. Individually, the prognostic significance of high level of sPD-L1 expression was the highest in hepatic cell carcinoma  followed by gastric cancer .sPD-L1 may be a prognostic factor in DSCs for overall survival and disease-free survival. Inflammatory cytokines, treatment approaches, and other factors may affect the expression of sPD-L1. Therefore, the prognostic value of sPD-L1 for recurrence and metastasis should be further investigated. sPD-L1 may also predict response to treatment. Well-designed prospective studies with standard assessment methods should be conducted to determine the prognostic value of sPD-L1 in DSCs. DSCs inDespite recent advances, the prognosis of DSCs remains unsatisfactory . GeneralIn the tumor microenvironment, cancer immunity plays a vital role in promoting cancer cell proliferation, survival, and angiogenesis . In the 22.1The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were used in this study . PubMed,2.2The inclusion criteria were as follows: a) patients diagnosed with malignant DSCs  confirmed by pathological analysis; b) the studies were conducted in English; c) human survival  with regard to sPD-L1 levels is provided by hazard ratios (HRs) or survival curves or can be calculated from the text; and d) each study had a sample size of more than 20 cases. The exclusion criteria were as follows: a) letters to the editor, comments, reviews, and animal studies; b) the sample sizes were <20 for each cancer type; and c) survival data were not provided.2.3Data were extracted by two independent reviewers. Information from the included studies was reviewed and extracted. This included the following:1) Authors, publication years, countries, histological types (differentiation), gender, tumor stage, metastases stage, initial treatment methods , study types (retrospective or prospective), sample sizes, ages, the methods for sPD-L1 detection, the cutoff value of sPD-L1, and follow-up time.2) OS and DFS and the predictive value of sPD-L1 for treatment response and metastasis.2.4The Newcastle–Ottawa Quality Assessment Scale (NOS) was used to evaluate study quality, as previously described . The sco2.52 and I2 tests to quantify the heterogeneity  software (Nordic Cochrane Centre) and STATA software (version 12.0) were used for data evaluation . The corogeneity . Heterogectively . If I2 <g’s test .3The selection flowchart is shown in Table 1The information of included studies and quality assessment.3.12 = 71, p<0.001). Furthermore, a high sPD-L1 level was correlated with unfavorable DFS by pooling the data from 10 studies using a random-effects model ; p-value was <0.01 with significant heterogeneity  (As shown in p<0.001) Figure 33.2Six studies focused on sPD-L1 levels and survival outcomes in HCC –57. A st3.3Six studies reported sPD-L1 levels and survival outcomes in gastric cancer patients –51. A pr3.4A study  from Fra3.5A study from China  includin3.6p<0.01).Ha et al.  reported3.7sPD-L1 was measured using ELISA before and after neoadjuvant chemoradiotherapy in 117 patients with rectal cancer in a study from the UK , which i3.8To confirm the stability of the findings, a sensitivity analysis was performed by omitting any single study on OS. The results are reliable, as shown in The results are summarized in Publication bias was evaluated using Begg’s test for OS. The results are shown in 4PD-L1 can be divided into membrane-bound PD-L1 and sPD-L1 . The detThe results of this meta-analysis revealed that high levels of sPD-L1 were associated with unfavorable OS. Several studies have reported that high sPD-L1 expression is associated with poor survival in breast cancer , renal cMonitoring sPD-L1 levels might be helpful for predicting survival in patients with cancer and subsequently improving treatment efficacy . TominagThe potential correlation between sPD-L1 and tissue PD-L1 levels was also investigated. In rectal cancer, PD-L1 expression in biopsy specimens is not significantly different from that in serum PD-L1 . In gastSeveral studies have reported the association of inflammatory cytokines with sPD-L1 level . In esopSeveral studies indicated that sPD-L1 expression was not correlated with age and sex , 52, 61.Inhibition of sPD-L1 can result in a function similar to that of anti-PD-1 or anti-PD-L1 monoclonal antibodies, thereby achieving a checkpoint inhibitory effect . Some stThis study has some limitations. First, some of the studies included in this meta-analysis were retrospective studies, and there might have been selection or publication bias because the positive results were more easily published in the journal, whereas the negative results were not. Second, the cutoff values were not uniform, and heterogeneity might exist. Third, a high heterogeneity was observed in some analyses. The source of heterogeneity may be individual patients with different TNM stages and tumor types, sex, ages, study types, treatment methods, country locations, cutoff values, and follow-up times. To identify the sources of the heterogeneity, subgroup analyses were adopted but failed to determine this.5In conclusion, sPD-L1 can be a prognostic factor for DSCs. High sPD-L1 expression predicted poor OS and DFS. Inflammatory cytokines, treatment approaches, and other factors may affect the expression of sPD-L1. Therefore, the prognostic value of sPD-L1 for recurrence and metastasis should be further investigated. sPD-L1 may be a prognostic factor for treatment response. Well-designed prospective studies with standard assessment methods should be conducted to determine the prognostic value of sPD-L1 in DSCs.Design and manuscript drafting: GL, JR, YQ, and ZZ. Editing and proving: GL, F-MK, and RX. All authors read and approved the final manuscript.