Caenorhabditis elegans is suitable for gene discovery approaches that may help identify molecular mechanisms of gravity sensing. It is unknown whether C. elegans can sense the direction of gravity.Gravity plays an important role in most life forms on Earth. Yet, a complete molecular understanding of sensing and responding to gravity is lacking. While there are anatomical differences among animals, there is a remarkable conservation across phylogeny at the molecular level. C. elegans nematodes align their swimming direction with the gravity vector direction while immobile worms do not. The worms orient downward regardless of whether they are suspended in a solution less dense (downward sedimentation) or denser (upward sedimentation) than themselves. Gravitaxis is minimally affected by the animals’ gait but requires sensory cilia and dopamine neurotransmission, as well as motility; it does not require genes that function in the body touch response.In aqueous solutions, motile C. elegans provides a genetically tractable system to study molecular and neural mechanisms of gravity sensing.Gravitaxis is not mediated by passive forces such as non-uniform mass distribution or hydrodynamic effects. Rather, it is mediated by active neural processes that involve sensory cilia and dopamine. The online version contains supplementary material available at 10.1186/s12915-021-01119-9. Caenorhabditis elegans (C. elegans), a model organism that has proven powerful for the molecular genetic dissection of other sensory modalities including olfaction, gustation, and mechanosensation,Gravity plays an important role in most life forms on Earth, ranging from single cells to plants  and animC. elegans senses and responds to gravity was triggered by observations that C. elegans swims in water-filled conduits. Since C. elegans is heavier than water, one would expect it to sink to the conduit’s bottom unless it were able to sense the direction of gravity and adjust its swimming direction to negate gravitational settling. Careful observations [C. elegans can sense and respond to gravity has remained unanswered.Our interest in whether rvations  have revC. elegans is heavier than the buffers typically used in laboratory experiments, it settles to the bottom of the vessel when suspended in such solutions [C. elegans offers important experimental advantages to unravel the molecular mechanisms of gravity sensing, including a small and simple nervous system, accessibility to rapid genetic manipulation, and ease of cultivation. What we learn in such studies is likely to be of significance to other animals, including humans.Since olutions . Our obsθ) and azimuthal (φ) angles as functions of time and position beneath the liquid surface  are heaθ ~ 5°. As time elapsed, the polar angle θ gradually increased. In the last frame (J), the animal was ~ 11.5 mm beneath the water surface and its polar angle was θ~142°. Thus, in a 10-s period, the worm changed its swimming direction from nearly upwards to nearly downwards.As time went by, the wild-type worms varied their swimming direction to align with the direction of the gravity vector. Figure This behavior is exhibited more clearly in panel K, wherein we colored the skeletons to correspond with their angle of decent and translated the skeletons of the animal to align their centroids. The animal rotated to align its direction of swimming with the direction of gravity. We observed this same behavior numerous times in two different labs   [pdf on a sphere [θ = 180o)   of the aa sphere  centeredconcentration parameter λ  is analogous to the inverse of the variance in a normal distribution. λ➔0 corresponds to a uniform distribution. When λ ≥ 1 and λ ≥ 3, over 73% and 95% of the animals are facing downwards (θ > 90°), respectively.where the f(θ) is nearly symmetric about the horizontal direction (θ = 90°), indicating equal probability towards upward and downward swimming. As time increases, the KDE skews in the direction of increasing polar angles, the average orientation angle increases, and the scatter about the average polar angle decreases. As the worms descend, they rotate to increase their polar angle and align their direction of swimming with the direction of gravity.Close to the liquid’s surface (shortly after release), λ for our data by fitting the cumulative distribution function (cdf) associated with Eq. cdf. When the wild-type animals were at depth d = 4 mm beneath the surface, λ ~ 0.18, reflecting a nearly uniform distribution. As the animals’ depth increased, the animals had more time to align with the direction of gravity, and the skewness of the KDE and the magnitude of λ increased.We computed the concentration parameter λ∞ ~ 6.3 and β ~ 0.02 mm−1  of young adult wild-type worms’ centroid in the direction of swimming depended nearly linearly  on (− cos θ)  Fig. .3\documUs as the animal’s swimming velocity and Ug as its sedimentation velocity in the gravitational field. The term (−Ug cos θ) is the projection of the sedimentation velocity on the animal’s swimming direction. In contrast to rigid cylindrical rods [Ug depended only weakly on the orientation (θ). This is perhaps because the worm is neither straight nor rigid. We estimate 28°/s. In summary, wild-type animals align their swimming direction with the gravity vector. Is this a passive or active response to gravity?We interpret cal rods  whose seunc-54 [unc-54 mutants (about 33% of the population). We expect paralyzed animals to have a mass distribution like that of wild-type motile control animals. This is most likely to be true in the case of the wild-type animals paralyzed with brief exposures to sodium azide because this paralysis is fully reversible after washing out the azide.To address this question, we monitored the orientation of sedimenting animals with severe movement impairment. We achieved muscle paralysis  by three different methods: (A) exposing wild-type animals to an elevated temperature (heat shock), (B) immersing wild-type worms in sodium azide solution, and (C) testing animals that carry a mutation in the major muscle myosin gene unc-54 . We consd < 100 mm, and Fig. d < 200 mm; sodium azide-paralyzed animals: Fig. Sd = 100 mm and d = 200 mm, and unc-54: Fig. Sd = 40 mm). In all cases, the descent angle θ is nearly symmetric with respect to the horizontal plane (θ ~ 90o) and the orientation distribution does not vary with depth/residence time  of the paralyzed worms (heat-shocked and unc-54) favorably agrees with the estimated contribution of gravitational settling to the velocity of the WT animals . As time passed, the peak of the KDE shifted to larger values of the polar angle θ and the magnitude of the concentration factor λ increased from near 0 to 2.7, consistent with the animals adjusting their orientation to align with the direction of gravity.At short times, the KDE Fig.  resembleIn summary, animals suspended in a liquid that is either lighter Fig.  or heaviunc-29 mutants  in body movements? The angle of descent of starved (> 1 h from the last feeding) wild-type animals Fig. S and unc-ts Fig. S, which aly Fig. S.Importantly, even animals with reduced swimming vigor, given enough time, orient to align with the gravity vector. Positive gravitaxis behavior does not solely rely on vigorous muscle movements.C. elegans are mediated by neurons that extend cilia to the nose of the animal. Is gravity sensation mediated by ciliated sensory neurons? To test this hypothesis, we analyzed the angle of descent of animals mutant for the gene osm-6 or for the gene che-2, which encode, respectively, the intraflagellar proteins 52 (IFT52) and 80 (IFT80) and in which sensory cilia are globally disrupted [We reasoned that if the worm uses its nervous system to sense gravity and orient in the downward direction, we should be able to impair this behavior by disrupting sensation while minimally impairing movement. Many sensory functions of isrupted .λ of the mutants che-2  and osm-6  did not vary as they descended. Their angles of descent were uniformly distributed with nearly equal probability for upward and downward swimming  mutants that carry an early stop mutation in the cat-2 gene were severely defective in orientating with the direction of gravity , we tested an independent cat-2 allele, n4547, which contains a 1007-bp deletion and is presumably a null mutant [n4547 mutants also showed defective orientation with the gravity vector ,” we cultivated cat-2 mutants for 65 h prior to our experiment on an agar plate doped with dopamine and then performed the experiment using regular M9 buffer. In the second set of experiments, dubbed “M9 buffer doped with dopamine (M9DD),” we cultivated cat-2 mutants prior to the experiment on regular NGM agar plates and then performed the experiment in M9 buffer doped with dopamine. In the third set of experiments, dubbed “Agar and M9 Doped with Dopamine (AM9DD),” we cultivated cat-2 mutants on a dopamine-doped agar and carried out the experiment in an M9 solution doped with dopamine. M9DD animals exhibited nearly uniform kernel density distribution, failed to gravitax, and descended with an average inclination angle of 88°±56° (N = 84) . The contrast between the skewed kernel density function of the ADD animals . Therefore, pharmaceutical treatment with dopamine rescued cat-2 mutant ability to react to gravitational forces. While the ADD experiment could be consistent with either functional or developmental roles for dopamine in gravitaxis, the AM9DD experiment, which showed the best rescue of the cat-2 defect, supports the former.If the 4) Fig. S. In contls Fig. S and the ls Fig. S is strikosm-6, cat-2, and dopamine-treated cat-2  exhibited similar orientational distributions as their well-fed counterparts. Our experiments suggest that worms on agar do not exhibit gravitaxis.Furthermore, we monitored the orientation of both animal types as functions of time. Both the wild-type control animals and C. elegans and various mutant animals to Earth’s gravity under various conditions  or genetic impairment (unc-54), animals with globally disrupted cilia (osm-6 and che-2), and animals defective in dopamine biosynthesis or signaling  did not align with the direction of gravity when suspended in liquid. Furthermore, Bristol N2 wild-type worms and mec-3 mutants did not align with the direction of gravity when placed in a vertical agar plate.We monitored the responses of wild-type We determined that the probability distribution functions of gravitaxis-capable, suspended animals and gravitaxis-deficient animals are statistically distinct with the Kolmogorov-Smirnov (KS) two-sample test Table S. The hypC. elegans such as gait synchronization [In prior experimental and theoretical studies, we found that various behavioral propensities of nization , swimminnization , 18, andC. elegans body length is not known. The animals store fat primarily in the intestine [unc-54 mutants differs from that of wild-type C. elegans, such a deviation is far less likely in the azide-paralyzed animals since the azide paralysis is rapidly and fully reversible. Lack of gravitaxis in the dopamine-deficient mutant cat-2 , slender objects with fore–aft symmetry sediment at the angle at which they had been released [Re > 0.Indeed, top heaviness does not necessarily imply alignment with the direction of gravity. Hydrodynamics characterizes the behavior of moving objects based on the ratio between inertial and viscous forces—the Reynolds number released . ObjectsRe > 0, cylindrical objects with uniform mass distribution rotate to attain a horizontal  posture and settle horizontally [U of C. elegans is ~ 0.5 mm/s  as a function of Wo at the greatest depth (residence time) for which we have experimental data. Figure λ as a function of depth. We find no correlation between gravitaxis and motility. Mutant worms that are either less motile or more motile than wild-type worms fail to gravitax. Among animals with the same motility, some gravitax while others do not. For example, the swimming gaits of the gravitaxis-competent WT and the gravitaxis-incompetent osm-6 are similar, with approximately the same frequency, the same amplitude, and the same Wo number  would align in the upward direction. Our experiments using the LUDOX solution that is slightly denser than the animals Fig.  indicateOur findings indicate that downward swimming is caused neither by animal’s mass distribution nor by hydrodynamics and therefore is most likely mediated by the animal’s nervous system. Could the observed downward swimming behavior be caused by the nervous system sensing physical factors other than gravity?C. elegans prefers low oxygen tensions [2 consumption and C02 release by the worms themselves, which is likely negligible on the time scale of our experiments. Finally, by experimenting with the Australia-derived wild-type isolate AB1 and with well-fed and starved worms, we largely exclude the possibility that gravitaxis is explained by the previously described magnetotaxis behavior [Our experiments took place in a vessel at equilibrium with the ambient and subjected to uniform room light and temperature. In the absence of gradients of light intensity or temperature, phototaxis or thermotaxis is unlikely. While tensions , 24, ourbehavior .Although we cannot exclude with absolute certainty that yet-unappreciated factors other than gravity affect the behavior of the animals in our experiments, our collective observations indicate with a high likelihood that the animals respond to gravity.If neural circuits are responsible for sensing and responding to gravity, we should be able to render the animal gravitaxis-incompetent by switching off one or more genes associated with such neuronal circuits. Here, we do so by experimenting with mutant animals that are defective in various neural functions.C. elegans such as olfaction, gustation, thermosensation, nose-touch, and electrosensation are mediated by neurons that extend cilia to the nose of the animal. Animals mutant for the genes osm-6 or che-2, in which sensory cilia are globally disrupted [mec-4 and mec-3, which are involved in the transduction of mechanical stimuli to the body, did gravitax. These observations suggest that the neural mechanism of gravitaxis requires the function of one or more sensory ciliated neurons.Many sensory functions of isrupted , did notC. elegans, including the sensation of bacterial particles [To begin identifying the relevant neurotransmitter systems, we examined the role of dopamine neurotransmission. Dopamine is required for the function of certain mechanosensory behaviors in articles  and habiarticles , 26. In articles —that sigarticles , chemicaarticles , and motarticles . All doparticles , 31.cat-2 alleles and of pharmacological restoration of cat-2 gravitaxis by exogenous dopamine demonstrate an essential role for dopamine in this behavior.Our experiments with mutants harboring a defective dopamine biosynthetic enzyme indicate that dopamine is required for gravitaxis. Our observations of defective gravitaxis in two cat-2 mutants was restored by cultivation during larval development with exogenous dopamine. In contrast, gravitaxis behavior was not restored by the presence of exogenous dopamine during only the time of the assay. While these observations might suggest a developmental role for dopamine, the observation of strong rescue of gravitaxis when we combined exposure to dopamine larval development with exposure to dopamine during the experiment suggests at least some active role for dopamine during gravitaxis behavior.Dopamine could be actively required at the time of liquid suspension for the animal to successfully gravitax. Alternatively, dopamine could be required for the development of the competency for gravitaxis. Our experiments do not clearly distinguish between these two roles. Gravitaxis behavior in C. elegans, dopamine activates the mammalian D1-like dopamine receptors DOP-1 and DOP-4 and the mammalian D2-like receptors DOP-2 and DOP-3 [In nd DOP-3 . DOP-1 and DOP-3 . Our expnd DOP-3 , an impoC. elegans is elusive. A few invertebrates use their head as the test mass (statolith) to detect the direction of gravity [The organ responsible for gravity sensing in  gravity . We do nIf the head does, indeed, serve as the test mass, how is information on head position transmitted to the nervous system? The RIA interneurons encode head orientation  and coulC. elegans, if any, is not known. Gravitaxis may be an ancestral trait. C. elegans has other traits such as electrotaxis—the role of which in the animals’ natural ecology is also unknown.The ethological significance of gravitactic behavior in C. elegans mutants and genetic tools to silence or activate neurons, this assay could identify the molecular pathways required for gravity sensing and behavioral response.Our method of monitoring the swimming orientation of downward sedimenting animals provides a means to identify genes required for gravity perception. In combination with the rich library of Our experimental assay in which we suspended animals in a medium slightly denser than the animals could potentially serve as a high-throughput, forward genetic screening platform to sort out active animals that are defective in gravitaxis and identify the genes that are required for gravitaxis. Following mutagenesis, active animals can be suspended in a solution slightly denser than themselves, gravitaxis-competent animals would swim downward while gravitaxis-defective animals would float to the top. The latter can be collected and subjected to molecular genetic studies. We have previously used a somewhat similar approach to identify genes responsible to sleepiness .Such assays can be used to decipher the neural circuits responsible for gravity sensation and identify which sensory neurons encode gravitational stimuli, which ones decode this information, and what molecular pathways facilitate signaling. While there are significant anatomical differences among animals, there is a remarkable conservation across phylogeny at the molecular level. Knowledge gained in such studies may be beneficial to human health.C. elegans responds to gravity and that specific neural circuits are required for responding to gravitational clues. Our work suggests the possibility of leveraging the powerful genetic and physiological toolkit of C. elegans to elucidate the molecular and circuit mechanisms of gravity sensing.Sensing the direction of the Earth’s gravitational field is essential for the spatial orientation and navigation of animals, including humans. The molecular and circuit mechanisms of gravity sensing and responding to gravity are elusive. We have shown that On the day prior to the experiment, well-fed fourth larval stage hermaphrodites were placed on an agar plate containing a bacterial lawn of OP50. Day 1 adult worms were harvested from the agar plate by floating the worms in M9 buffer and then transferring the worm suspension into a 1.5-mL conical tube. Following centrifugation  for a few seconds to sediment the worms, the supernatant was decanted. The worms were then washed three times with 1 mL M9 buffer by repeating the centrifugation/decanting steps. We experimented mostly with “recently-fed” worms—the time elapsed from floating the worms off their cultivation plate to the completion of the experiment was < 30 min. A few of the experiments were intentionally carried out with “starved” worms—the time elapsed from floating the worms off their cultivation plate to the start of the experiment was ~ 1 h.To paralyze wild-type worms by heat shock, we suspended the worms in 1 mL M9 buffer in a 1.5-mL conical tube and placed them for 1 h in a water bath at 40 °C. The experiment was then performed at room temperature (21~22 °C) within 30 min from the removal of the animals from the water bath. Observations of these worms showed that they were fully paralyzed for over 30 min after the heat shock.To transiently paralyze wild-type worms chemically, we suspended the worms in 10 mM sodium azide in M9 buffer for 5 min. We then performed our experiments with the worms suspended in the same 10 mM sodium azide solution.C. elegans strains used in our study were N2 , AB1 , CB190 (unc-54(e190)), CB1072 (unc-29(e1072)), CB1033 (che-2(e1033)), PR811 (osm-6(p811)), CB1138 mec-3(e1338), TU253 mec-4(u253), CB1112 cat-2(e1112), MT15620 cat-2(n4547), LX636 dop-1(vs101), LX702 dop-2(vs105), and LX703 dop-3(vs106). The strains were obtained from the Caenorhabditis Genetics Center (CGC). Worms were cultivated in a 20 °C incubator on the surface of NGM agar plate  seeded with Escherichia coli OP50 as the food source. All experiments were performed with hermaphrodites.To achieve a buffer density greater than that of the worms, we mixed a suspension of colloidal silica particles in water  with M9Animals were exposed to dopamine (DA) by cultivating them on DA-doped agar and/or by adding dopamine to the M9 solution in the cuvette during the experiment. Agar doping: 400 μl of 50 mM DA hydrochloride (Sigma) dissolved in M9 buffer was added to an agar plate seeded with bacteria. Plates were dried for 1 h . We cultCuboid polystyrene cuvettes with a square cross-section 12 mm × 12 mm and heights ranging from 45 to 200 mm filled with M9 buffer at room temperature (21~22 °C) were used in our settling experiments. To verify that the cuvette material does not affect animal behavior, experiments were repeated in glass tubes with similar results. Twenty microliters of a worm suspension at a concentration of about 1.5 worms per microliter was extracted with a plastic tip pipette and transferred into the cuvette by slowly expelling the worms into the cuvette solution either above or below the liquid surface.X-Z plane and the other on the Y-Z plane at the cuvette’s center. Each image size was 640 × 480 pixels, which results in an aspect ratio of 4:3. As it settled, a worm stayed within the field of view of the two cameras for about 10 s. Images were processed with a Matlab R2018b graphical user interface (GUI), following the scheme described in [unc-29 (17%). When studying unc-54, we were interested only in fully paralyzed worms (about 33% of the population) and we censored animals that showed any feeble swimming movements.The worms were monitored with two cameras (IMAGING SOURCE DMK 33GP031 with a 25-mm lens and IMAGING SOURCE DMK 22BUC03 with a 12-mm lens) acquiring images at 30 frames per second from two orthogonal planes  suspension was added to the whole surface of the agar and dried. Adult worms were harvested from their cultivation agar plates, washed with NGM buffer three times, and pipetted as a suspension onto the agar plate. After removing excess liquid, the number of worms in each quadrant of the plate was counted. Then, the agar plate was positioned in the vertical plane with one of the cross legs parallel to the direction of gravity and monitored with a camera. Images were collected at the rate of one image every 20 min for 12 h. At the end of the experiment, the agar plate was inserted on the stage of a stereo microscope and the number of worms in each quarter was counted within less than a minute.Two percent agarose  was dissolved in a NGM solution and poured into a round dish plate (diameter = 3.5 cm) marked with a cross on its bottom to demarcate four quadrants. After drying the agar, bacteria , following the image processing scheme described in  and outlX-Z plane as  and in the Y-Z plane as , where n is the frame number. The cameras’ pixel size was correlated with the physical length by placing a ruler in front of the camera at the same distance from the camera lens as the cuvette’s mid-plane. The pixel size P in the X-Z plane was Y plane was Rx, Ry, and Rz between the animal’s head (H) and tail (T) along the x, y, and z coordinates are, respectively, We denote the coordinates Fig.  in the XThe distance between the animal’s head and tailThe inclination (polar) angle with respect to the vertical axisθ = 0° corresponds to the upward direction. The azimuthal angleThe angle The algorithm was verified by reproducing the dimensions and inclination angle of a 3D-printed calibration jig Fig. S.To monitor the worms’ orientation as a function of residence time in solution, images of worms were recorded and processed at various positions beneath the liquid surface Fig. . The fieThe position of the worm’s centroid (geometric center)The components of the worm’s centroid displacement between subsequent frames are given by The velocity of the worm iswhere the factor 30 is the video frame rate. The polar angular velocityThe grayscale threshold for detecting worms was manually adjusted using the ImageJ program to transform the captured grayscale images into a binary scale: zero for black (outside the worm) and one for white (inside the worm). The grayscale ranges from 0 to 255. The threshold for detecting a worm varied among experiments and ranged from 32 to 85.n = 0) of each video, we manually defined an imaging region. Pixels in the region outside this imaging region were assigned a zero (black) value. The most frequently occurring binary value (mode) within our imaging region, excluding the worm, was subtracted from all subsequent frames in the video.Background subtraction. In the first frame (n + 1) an extended bounding rectangle with 10 pixels added to the width and length of the bounding rectangle of frame (n) to form a search-region for the worm following its displacement in the time span between frame n and frame (n + 1).A tight bounding rectangle, containing the worm, was manually defined in the first frame. The positions of the tips of worm’s head and tail were manually marked. We then defined in frame .Immobile (non-swimming) worms were excluded from analysis except for experiments purposely carried out with paralyzed worms  and motion-impaired worms , images in which worm paths overlapped, as assessed by visual inspection, were censored. We estimate that less than 15 frames of all frames analyzed were censored.The raw data from our experiments is provided in an accompanying excel file  based on experimental data. To construct the KDE and the cumulative distribution function (CDF) from our data, we used MATLABTM function “ksdensity.” Figure SStatisticians use the kernel distribution estimate (KDE) to estimate the probability distribution function . θ = 0 and θ = π are, respectively, the upward direction and the downward direction. Since our data suggests  and there is an equal probability of finding animals swimming in and against the direction of gravity   Fig. S13\documλ by fitting equation (S10) to our experimental CDF. We consider cases with λ > 1 as exhibiting gravitaxis.We obtained the experimental estimate of TM function kstsest2). We tested two null hypotheses to examine whether two samples come from the same distribution. The first null hypothesis To infer whether the orientational behavior of any strain differs from either that of the wild type (N2) or the paralyzed animals, we carried out the Kolmogorov-Smirnov (KS) test for two samples (MATLABAdditional file 1: Supplemental Information. Figures S1-S30. Tables S1-S3.Additional file 2: Raw Data.


In this article, we study the coupling of a collection of molecular oscillators, called repressilators, interacting indirectly through enzymatic saturation. We extended a measure of autocorrelation to identify the period of the whole system and to detect coupling behaviors. We explored the parameter space of concentrations of molecular species in each oscillator versus enzymatic saturation, and observed regions of uncoupled, partially, or fully coupled systems. In particular, we found a region that provided a sharp transition between no coupling, two coupled oscillators, and full coupling. In practical applications, signals from the environment can directly affect parameters such as local enzymatic saturation, and thus switch the system from a coupled to an uncoupled regime and vice-versa. Our parameter exploration can be used to guide the design of complex molecular systems, such as active materials or molecular robot controllers. In the past two decades, advances in synthetic biology have provided researchers with the ability to implement complex dynamical systems both in vivo ,2 and inAmong those dynamical systems, particular focus has been given to oscillators, both for their complex behaviors ,7 and thThe effect of direct coupling between repressilators through communication modules has been studied previously , and canAs such, we chose to use a scanning strategy to explore the impact of indirect saturation on a system of three indirectly coupled repressilators . We exteAdditionally, such coupling can be tuned over time, as it will depend on the saturation level and activity of the enzymes, and thus it can be used as a control mechanism for the system . As periIn the original implementation of the repressilator, the genes lacI, tetR, and cI express their respective protein and inhibit the transcription of the gene tetR, cI, and lacI, respectively. As such, when lacI is being expressed, tetR is being inhibited, meaning that its protein is being degraded over time, thus freeing cI from inhibition. Once that inhibition is low enough, cI will be expressed, thus eventually inhibiting lacI. In turn, tetR will eventually stop being inhibited and start inhibiting cI, completing the cycle.The original repressilator mechanism can be modeled as:n is the cooperativity of repression [jth mRNA and protein of repressilator i, respectively. In this model, the degradation term for k is the maximum rate of degradation. k for the degradation of mRNAs and k for the degradation of proteins have the same value. That assumption is based on the fact that their respective value are close enough in the original model [Here, pression . α0 and cay rate . Next, wominator . k is thal model , throughWe set ures see . The iniN is the number of samples. To measure a period of any of the oscillator, we look for the time lag that provides a strong autocorrelation (0.7 or above). Specifically, period We used the autocorrelation function in order to calculate the overall period of the system. The normalized autocorrelation function with time lag iven by: (3)RmijmWe then calculated the difference of periods We measured the synchronization of the system over the parameter range of e system . As expeIn order to further analyze the system, we rely on time-series data , bottom.nization .We can note that the shape of the oscillations is greatly affected by the enzymatic coupling. Even at low coupling levels c,f, the Finally, we performed a spectral analysis of the system see  which hiIn this paper, we showed that indirect interaction through competition for enzymes between independent molecular oscillator could lead to their synchronization, as long as their impact on enzymes are similar. An interesting phenomenon arises when the activation mechanism of a weaker oscillator is prevented by the saturation of enzymes by the stronger ones, leading to a sharp transition to an almost doubled period. Those results can be contrasted with directly coupled repressilators, in which the parameter plane of production versus coupling shows a transition between synchronization and chaotic behaviors .Molecular implementation of the system can be done directly with the PEN DNA toolbox framework ,6, as itFinally, indirect coupling in molecular robotics, is usually seen as having a negative impact on the system as those are more non-linear and thus harder to control. However, our results show that, in this case, it provides a complex behavioral space allowing us to sharply transition between different coupling modes through small variations in enzymatic saturation d–f. In pSystems made of a single molecular oscillator have been used to perform computation , control


According to our thermogravimetric—differential thermal analysis and X-ray diffraction results, the calcination temperature was 650 °C. The microstructure, crystal structure, and chemical composition of the nanofibers were observed using field-emission scanning electron, X-ray diffraction, and energy-dispersive X-ray spectroscopy. Commercial particle samples and samples containing 10 wt% and 20 wt% nanofibers were fabricated, and the electromagnetic properties were analyzed with a vector network analyzer and a 7.00 mm coaxial waveguide. Regardless of the nanofiber content, Ni0.5Zn0.5Fe2O4 was dominantly affected by the magnetic loss mechanism. Calculation of the return loss based on the transmission line theory confirmed that the electromagnetic wave return loss was improved up to −59.66 dB at 2.75 GHz as the nanofiber content increased. The absorber of mixed compositions with Ni0.5Zn0.5Fe2O4 nanofibers showed better microwave absorption performance. It will be able to enhance the performance of commercial electromagnetic wave absorbers of various types such as paints and panels.Ni With the development of wireless communication technology, the use of portable electronic devices has rapidly increased. At the same time, there are many concerns about the harmful effects of electromagnetic (EM) waves on the human body ,2,3, andAt the same time, the morphological character of the absorbent particles has a great influence on the EM wave absorbing performance and the selection of frequency. Even if absorbers have the same chemical composition, they need to be controlled the microstructure. Many studies have been carried out to modify or optimize the absorber by applying various microstructures: mesoporous structures ,28, coreAmong them, electrospinning is a method to fabricate nanofibers by applying a high voltage to two spaced electrodes and jet spraying and stretching a precursor solution using the generated electrostatic force. Due to stretching, the solvent is volatilized with a rapid increase of surface area and the solute remains in the form of very thin fibers. As a result, a one-dimensional structure with a high aspect ratio and a nanoscale diameter can be obtained, and it can be transformed to ceramic nanofibers via calcination. Since it is a continuous process that can obtain very long and uniform nanofibers, it is being applied in various fields such as biomedical , photovo0.5Zn0.5Fe2O4 (NZF), which is often discussed as an EM wave absorber [In this study, we fabricated Niabsorber  as a nan3)2·6H2O , Zn(NO3)2·6H2O , Fe(NO3)2·9H2O , Polyvinyl pyrrolidone , and N,N-dimethylmethanamide , commercial NZF nanoparticles , epoxy binder .The reagents used to prepare the precursor solutions were as follows: Ni(NO3)2·9H2O, 10 mmol of Ni(NO3)2·6H2O, and 10 mmol of Zn(NO3)2·6H2O were added to 30.0 g of DMF and mixed for 2 h. Then, the second solution was added to the first solution and stirred for 6 h. The prepared precursor solution was loaded into a 12 mL polypropylene syringe with an inner diameter of 15.56 mm. The syringe was connected with a stainless-steel nozzle adapter and a 23 gage capillary using polypropylene tubing. The distance between the tip of the capillary and the grounded collector was set up to 20 cm. Then the adapter and collector were connected to the power supply and a high voltage of 23 kV was applied to start the electrospinning process. A flow rate of 0.2 mL per hour was constantly applied to the syringe using a syringe pump. Room conditions were temperature of 24 °C and humidity less than 40%. As spun nanofibers were physically separated from the collector using a Teflon tweezer and then dried at 70 °C for 6 h in a dry oven. After that, dried nanofibers were calcined using a box furnace. The calcination conditions were as follows: stabilized at 30 °C, ramping to 200 °C, hold 2 h at 200 °C, ramping to 650 °C and hold 2 h. The temperature ramping speed was 5 °C per minute. The schematic diagram of the fabrication process is shown in To fabricate NZF nanofibers via electrospinning, we prepared a precursor solution. First, 10.0 g of PVP was mixed with 60.0 g of DMF for 6 h using a magnetic stirrer. In another beaker, 40 mmol of Fe under the pressure of 250 MPa and then were cured at 200 °C for 30 min.Since calcined nanofibers are obtained as scaffolds, a dispersing process was needed. Nanofibers were added to 75% ethanol in a 50 mL vial and sonicated for 2 h and dried at 70 °C in a dry oven. The calcined nanofibers/commercial particle NZF mixture was mixed for each other content; 0 wt%, 10 wt%, and 20 wt% of nanofibers/nanoparticles powder of 0.45 g and epoxy binder of 0.05 g were mixed using a mortar. The prepared powders were uniaxially pressed to fabricate toroidal samples  of the as-spun nanofibers was carried out using a Thermogravimetric Analyzer . The crystal structure of the calcined nanofibers calcined at each different temperature was analyzed by an X-ray diffractometer . The morphology and microstructure of the NZF nanofibers and commercial particles were analyzed by a field emission scanning electron microscope  and energy-dispersive X-ray spectroscopy (EDS). To obtain the complex permeability and permittivity, an electromagnetic property test of the prepared toroidal samples was carried out using a vector network analyzer .The TGA-DTA was carried out to determine the calcination temperature of the as-spun nanofibers and the result is presented in An initial mass decrease of about 6% occurred in the temperature range of 60 °C to 140 °C, and it was likely due to the volatilization of adsorbed moisture and residual solvent. The DTA peaks are 100 °C and 135 °C, respectively, and these reactions are endothermic near the boiling points of water and DMF. Endothermic DTA peaks are observed at 100 °C, 135 °C and it is near the boiling point of water and DMF. The mass loss of 16%, starting at around 180 °C, lasts up to 200 °C. The mass-loss rate decreased from 200 °C to 250 °C and then increased. This result explained how the endothermic reaction of glass transition suppressed the exothermic of PVP decomposition. After that, a strong exothermic reaction and mass loss were observed near 260 °C, which was likely rapid PVP decomposition by ignition and combustion. Then there was a continuous exothermic reaction and mass loss by the decomposition of residual carbon black and crystallization of metal ion.0.5Zn0.5Fe2O4 spinel structure was confirmed by comparing it with the Crystallography Open Database ID 96-900-9921 [α1 = 1.78897 Å).900-9921 , and theAs expected from the pattern, the average crystallite size gradually increased according to calcination temperature, and rapid growth was observed at 750 °C. Since this study focused on the geometrical advantages of electrospun nanofibers as an EM wave absorber, the calcination temperature was determined as 650 °C, which has the most similar crystallite size to commercial nanoparticles. Considering that the shape factor compared to nanoparticles is reduced due to the shape anisotropy of the 1-D nanostructure, 650 °C was selected not 550 °C.r = ε′ − jε″ and µr = µ′ − jµ″. The real parts are related to the storage of electromagnetic energy and the imaginary parts show the dissipation of energy. Each real and imaginary part of the complex permittivity and permeability of the NZF nanofibers is shown in The electromagnetic properties could be explained using permittivity and permeability. These material constants were expressed as a complex form, namely, εThese constants were obtained using VNA and a 7.00 mm coaxial waveguide. Samples A, B, and C contained 0 wt%, 10 wt%, and 20 wt% of NZF nanofibers, respectively. The composition of each sample is shown in δε = ε″/ε′, tan δµ = µ″/µ′, respectively. In general, it is expected that the larger the loss tangent and imaginary part of the absorber, the better the EM wave absorbing performance. The dielectric and magnetic loss tangents are shown in (c) and (d) of μ′ and μ″ than sample A due to the high shape magnetic anisotropy of the nanofiber structure. Multiple magnetic resonance loss was not observed, and the magnetic wall resonance in the low-frequency range is dominant. High shape magnetic anisotropy of nanofibers makes it difficult to change the magnetic domain direction and increase the demagnetizing energy, so hysteresis loss which is mainly contributed by changing the domain direction is increased. Since the microstructures containing nanofibers have higher porosity than those without, samples B and C are expected to have a larger surface area than sample A. Therefore, it can be expected that increasing magnetic loss via eddy current. Since eddy current loss is defined by permeability, thickness, and electrical conductivity, the better magnetic loss can be expected from Sample C, which has the largest imaginary part of complex permeability. The eddy current loss factor can be evaluated according to the following Equation (1) [There was a tendency for the coefficient to according to the content of nanofibers. The permittivity of the samples was almost independent of frequency. The real parts of the permittivity were measured from 4.01 to 4.91 and imaginary parts were almost near 0, which means that the permittivity of the NZF absorbent has almost no influence on a change in the EM wave absorbing performance. At the same time, real parts of permeability showed a decreasing tendency from 2.72 to 0.85 at the ~3.56 GHz frequency. The peaks of imaginary parts were observed to be near 1 at 0.79 GHz. All the permeability coefficients decreased with an increasing frequency, which was likely due to magnetization relaxation. The ratio of the imaginary part related to energy dissipation and the real part showed energy storage called electric and magnetic loss tangent, which are written as tan tion (1) ,45.(1)Cf is the frequency, d is thickness, and d is thickness of the absorber layer, and c is the velocity of the EM wave in free space. By substituting the obtained variables into (2), the incident impedance inZ of the absorber is calculated, and the return loss can be obtained by substituting inZ and the free space impedance Z0 into (4).In this formula, In this study, we prepared NZF nanofibers using electrospinning and calcination. The crystal structure and 1-D nanostructure were well-formed, and the average diameter was measured to be 133.56 ± 12.73 nm. We observed that the NZF absorber performance was dominantly affected by magnetic loss regardless of the nanofiber content. We measured the EM properties of samples of pure commercial nanoparticles and samples containing nanofibers of 10 wt% and 20 wt%. We found that as the nanofiber content increased, the EM wave absorption performance improved. In all the samples, the frequency at which the minimum RL value was confirmed was near 2.8 GHz and the thickness was near 12 mm. The increasing of absorbing performance due to nanofiber content is consistent with the results of previous studies that demonstrated that the mixed structure of acicular and particles increased the absorber performance. Our study confirmed that the nanofiber structure is a superior structure for improving the performance of the EM wave absorber and is expected to be effective for any type of magnetic absorber, such as panels, films, and paints.


The PAN nanofibers were pre-treated with NaOH, and further coated with zinc hydroxide, which was subsequently converted into ZIF-8 by the addition of 2-methyl imidazolate. In the resulting flexible ZIF-8/PAN composite nanofibers, ZIF-8 loadings of up to 57 wt% were achieved. Scanning electron microscopy and energy-dispersive X-ray spectroscopy (EDS) showed the formation of evenly distributed submicron-sized ZIF-8 crystals on the surface of the PAN nanofibers with sizes between 20 and 75 nm. X-ray photoelectron spectroscopy (XPS) and carbon-13 nuclear magnetic resonance (13C NMR) investigations indicated electrostatic interactions and hydrogen bonds between the ZIF-8 structure and the PAN nanofiber. The ZIF-8/composite nanofibers showed a high BET surface area of 887 m2 g−1. CO2 adsorption isotherms of the ZIF-8/PAN composites revealed gravimetric CO2 uptake capacities of 130 mg g−1 (at 298 K and 40 bar) of the ZIF-8/PAN nanofiber and stable cyclic adsorption performance.Polyacrylonitrile (PAN) nanofibers were prepared by electrospinning and coated with zeolitic imidazolate framework-8 (ZIF-8) by a phase conversion growth method and investigated for CO 2 capture.Poly acryl nitrile (PAN) nanofibers were prepared by electrospinning and coated with zeolitic imidazolate framework-8 (ZIF-8) by a phase conversion growth method and investigated for CO A part of the activities to achieve these goals is more efficient gas separation, capture and storage technologies for carbon capture and storage (CCS). Adsorption-based methods, such as pressure swing adsorption (PSA)2 or temperature swing adsorption (TSA)3 technologies offer opportunities for tailor-made gas separation of CO2 from gas mixtures4 and subsequent storage, for example in biogas upgrading5 and storage as biomethane (CH4).6 CH4 produced from sustainable sources such as biogas, can help to reduce anthropogenic CO2 emissions. One key point to improve adsorption-based technologies is exploring and improving advanced microporous materials (sorbents),7 such as zeolites,8 carbon molecular sieves9 and active carbon.10 It has been shown that by heteroatom-doping the CO2 adsorption can be significantly affected (e.g. in activated carbons) by creating highly polar adsorption sites for the CO2 molecule and Lewis acid–base interactions. This usually results in improved selectivities in CO2 separation.11–13 Another promising new class of materials are metal–organic frameworks (MOFs), which are assembled from metal ions/clusters and organic linkers.14 These materials can be prepared from a variety of chemical compositions, resulting in structures with ultrahigh surface area of several thousand m2 g−1, large pore volumes with tailorable pore size and specific chemical sites for capturing guest molecules by adsorption. In these materials, the extremely large internal surface area and pore channels can be modified to introduce specific surface charges, hydrophilicity/hydrophobicity or uniformity of pore channels. Zeolitic Imidazolate Frameworks (ZIFs), a sub-class of MOFs, have been proposed for gas separation and storage applications.15 These materials have first been synthesized by Yaghi's group.16 The structure consists of zinc(ii) cations and 2-methylimidazole anions (2-mim). Within the class of MOF materials, ZIFs have an interesting framework structure with high thermal and chemical stability. According to a report from Park et al.,15 ZIF-8 has a large sodalite (SOD) zeolite-like cavity (11.6 Å) and smaller apertures (3.4 Å). The size of these pores, the high achievable specific surface area of more than 1000 m2 g−1, and large micropore volumes indicate that this material has promising potential for CO2 capture and separation.Recently, the NET-ZERO 2050 report was published by the European Climate Foundation,30 recently, new approaches have been proposed to structure adsorbents  into nanofibers to enhance gas transport,17 adsorption capacity18 and adsorption kinetics.19 For example, zeolite nanofibers of ZSM-5 have been fabricated by electrospinning, post shaping and carbonization of polyvinylpyrrolidone (PVP)20 the performance has been evaluated for CO2 separation from CO2/CH4 mixtures in synthetic biogas mixtures.Beneath shaping of microporous materials into useful structures, such as pellets, granules or extrudates,et al.,21 including in situ growth of MOF nanocrystals on the surface of polymer nanofibers. The preparation of MOF-polymer nanofibers through electrospinning is an elegant way to shape MOF materials into hierarchical porous nanofibrous sorbent materials at mild conditions with large surface-to-volume ratio, tailored pore size, and high permeability for gas separation processes. In this study we propose a phase inversion method to grow ZIF-8 on the surface of electrospun polyacrylonitrile (PAN) nanofibers for CO2 capture. We investigated the influence of the growth of the ZIF-8 nanoparticles on PAN nanofibers as a function of reaction time to maximize the CO2 gas uptake, which was expected to increase with the amount of the microporous ZIF-8 sorbent loaded on the inactive PAN nanofiber as backbone.Due to the temperature instability of MOF materials, this approach to fabricate MOF-nanofiber composite materials is not feasible for the majority of MOF-based nanofibers. However, different approaches for the structuring of MOF-nanofiber materials for energy and environmental applications have been recently reviewed by Dou The resulting ZIF-8/PAN composite nanofibers have been characterized by scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-EDS), X-ray diffraction pattern (XRD), surface area and pore size distribution analysis and by more advanced characterization techniques, such as X-ray photoelectron spectroscopy (XPS) and nuclear magnetic resonance spectroscopy (NMR).2 uptake of the ZIF-8/PAN composite nanofibers have been tested at pressures up to 40 bars and the cyclic adsorption–desorption properties of the composites have been investigated.Finally, the CO2.2.1in situ growth of ZIF-8, the raw chemicals zinc nitrate hexahydrate (Zn(NO3)2·6H2O), sodium hydroxide (NaOH) and 2-methylimidazole (2-mim) with purity over 99.0% were purchased from Sigma-Aldrich. For the electrospinning (ES), polyacrylonitrile (PAN) with an average molecular weight of 150 000 was chosen as polymer with a purity of 99.0%. The PAN was purchased from Parchem fine & specialty Co., Ltd N,N-Dimethylformamide (DMF) with purity of 99.0% was purchased from Sigma-Aldrich. Besides, the solvent of methanol and ethanol with purity of 99.9% were purchased from Sigma-Aldrich. The deionized water (D.I.W) was produced in the laboratory by an ultrapure system with a resistivity of 18.0 mΩ cm−1. All chemicals used in this work have not been further treated or purified. High purity gases of CO2 (99.99 mol%), CH4 (99.99 mol%), He (99.99 mol%) and N2 (99.99 mol%)  were used for gas adsorption experiments.For the 2.2st step, the PAN solution was electrospun into PAN nanofibers. In the 2nd step, the PAN nanofibers were immerged into a heated sodium hydroxide solution (75 °C) to activate the surface. In the 3rd step, the metal-contained (Zn2+) precursors were attached on the active PAN surface. In the last step, the in situ growth process of ZIF-8 was initiated by the addition of the complementary precursor (2-mim solution).The preparation of the ZIF-8/PAN composite nanofibers can be divided into four steps: in the 1For the preparation of the PAN nanofibers, 1 g PAN and 10 g DMF were mixed and sealed into a 50 mL plastic bottle with cylindrical zirconia balls to ball-mill by regular ball milling at 50 rpm for 24 hours. Then the dispersion solution was electrospun by needle-based electrospinning equipment (Linari s.r.l) with the voltage of 30 kV and a distance of 140 mm between the electrospinning needle and receiving substrate to prepare PAN nanofibers.For the activation of the PAN nanofiber surface with hydroxide groups, 0.5 g of PAN nanofibers were immerged into the 50 mL NaOH-D.I.W solution (60 g NaOH dissolved into 500 mL D.I.W) and heated at 75 °C for 20 min to activate the fiber surface. The treated PAN nanofibers were 3 times washed with ethanol and dried afterwards in air at room temperature.3)2 solution for 20 min to form Zn(OH)2 on the surface of PAN nanofibers. The applied Zn(NO3)2 solution consisted of 6.29 g Zn(NO3)2·6H2O in 180 mL of a 1 : 1 volumetric solvent mixture of methanol and ethanol.For the application of the metal precursor, the surface-treated PAN nanofibers were immersed into 100 mL of a 120 millimole per liter (mM) Zn2 solution directly, where the Zn(OH)2-coated PAN fiber had been immerged already to complete the in situ growth process. Every 30 minutes, the samples were taken and dried after ethanol washing to identify the optimum loading and structure of ZIF-8 in the composite. The applied 2-mim solution consisted of 7.49 g 2-methylimidazole in 190 mL of a 1 : 1 volumetric solvent mixture of methanol and ethanol. According to reaction time the samples were labelled ZIF-8/PAN-X 2 solution and 50 mL 480 mL 2-mim solution were prepared (as described in Section 2.2) and mixed by vigorously magnetic stirring for 1 hour at room temperature, then the suspension solution was centrifuged to obtain the ZIF-8 colloids. Followed, the ZIF-8 colloids were washed with ethanol for 3 times. Finally, the ZIF-8 nanocrystals were dried at 100 °C to remove the residual ethanol, methanol and moisture.For the synthesis of ZIF-8 nanocrystals in powder form, 50 mL 120 mM Zn(NO2.4Scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-EDS) were performed by a Zeisis Merlin FEGSEM, the samples were sputter-coated with the thin layer of the gold (around 12–16 nm).−1. All of the samples were attached on a silicon-substrate sample holder. The miller indexes were identified by Match! 3 software.X-ray diffraction (XRD) measurements were performed by a Rigaku Smartlab X-ray diffractometer, which was operated at 40 kV and 30 mA using Cu Kα radiation with step speed of 5° minX-ray photoelectron spectroscopy (XPS) was performed in the range of 200 to 1200 eV with ZIF-8 nanocrystals, pure PAN fiber and ZIF-8/PAN composite nanofibers in a angle resolved XPS chamber for depth profiling of surfaces (equipment from Thermo Scientific Nexsa).13C nuclear magnetic resonance spectroscopy (NMR) was performed on a Bruker Avence III 300 Wide Bore solid state NMR spectrometer, using a 4 mm probe. The frequency of 13C was 75 MHz with the pulse program of CP-Toss, using 4 mm rotors at a MAS spinning frequency of 6500 Hz. The 90° 13C pulse-length was 4 μs, and pulse length in decoupling sequence is 7. Recycle delay is 5 s, while the sweep wide is 30 241.94 Hz. The carbon resonance line of adamantane was used as an external chemical shift standard and was assigned a value of 38.48 ppm. The scans for CP-Toss experiments was 1k. Besides, the samples were frozen and milled into powder with help of liquid nitrogen before it was filled into NMR tube.2 uptake measurements were also carried out in this equipment. All samples were degassed to below 10−6 bar and 383 K for 8 hours prior to the measurement.The surface area as well as pore-related information  determination of ZIF-8/PAN composite materials and ZIF-8 powders were measured by nitrogen adsorption isotherms at −196 °C . The CO2 adsorption performance was measured by an IsoSORP adsorption analyzer , on samples with a weight of about 0.5 g. All samples were degassed at 110 °C under high vacuum for 4 h, following a buoyancy test with helium at 22 °C to determine the mass and volume of each sample. Then, the CO2 adsorption–desorption isotherms of the obtained ZIF-8/PAN composite were measured with CO2 dosing from high vacuum to 40 bar, then to high vacuum with an equilibrium point every 10 bar. The equilibrium for each step was set until the deviation of mass was less than 0.1 mg per 10 min.The high-pressure CO3.in situ growth of the ZIF-8 on the surface of electrospun PAN nanofibers resulted in flexible, paper-like ZIF-8/PAN composite nanofiber mats with light yellow colour, whereas the solvothermally synthesized ZIF-8 powder showed white nanocrystals.The 3.1in situ growth durations of 30 min  can be observed for the ZIF-8/PAN composites samples with decreasing reaction time, et al.26The pattern-ray diffractogram of the synthesized ZIF-8/PAN composite nanofibers matched closely with the simulated X-ray diffractogram of ZIF-8 powder, which can be assigned to cubic 2/PAN composite nanofibers (zinc hydroxide attached to the PAN nanofiber before the in situ growth of the MOF) and of the ZIF-8/PAN composite-90 nanofiber structure. The larger size and broader distribution of the Zinc in the initial Zn(OH)2/PAN composite  was carried out. The scan of a XPS spectrum of ZIF-8/PAN-90 and ZIF-8 powder exhibit peaks of Zn 2pectively .27 for the ZIF-8/PAN-90 composite nanofiber, the ZIF-8 powder and pure PAN nanofibers, respectively . These peaks have no major chemical shift. PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>CH–) when comparing the spectra for the ZIF-8 nanopowder with the ZIF-8 crystallites attached to the ZIF-8/PAN-90 composite nanofiber. Similar chemical shift of the C2 peak in ZIF-8 has been observed when organic molecules, such as caffeine, were encapsulated in the pores of ZIF-8 to form van der Waals bonds.28 However, more detailed analysis supported by DFT calculations29 would be required to further proof electrostatic interactions and hydrogen bonding between the ZIF-8 and C–N groups of the PAN nanofiber  of the sample obtained after short reaction time (30 minutes), indicate that the ZIF-8 has not been fully crystallized or has major surface defects. The broad and slightly displaced XRD peaks for this sample in 3 g−1 and the surface area to 888 m2 g−1 for the sample ZIF-8/PAN-90. Considering a maximum ZIF-8 loading of 57.19 wt% in ZIF-8/PAN-90 achieved in this work, the surface area of the pure ZIF-8 crystallites in the composite can be calculated to 1552 m2 g−1, assuming negligible surface area of the PAN nanofibers. Using similar estimates (43 wt% PAN in the composite), the actual micropore and total pore volumes of the ZIF-8 crystallites in the ZIF-8/PAN-90 composite should be about 0.54 and 0.88 cm3 g−1, respectively. This indicates that by the in situ growth method proposed in this work, ZIF-8/PAN nanofiber composites with high surface area, pore volume and ZIF-8 particle loadings have been achieved compared to ZIF-8-nanofibers in other studies. 2 uptake performance of the ZIF-8 nanofiber composite materials in this study compared to other studies.A large peak centered around a pore radius of about 11 Å can be assigned to micropores, whereas two smaller and broader peaks between 20 to 70 A are related to micro and mesopores. 2 uptake and re-generation of the ZIF-8/PAN-90 at 25 °C for 1 bar and for 40 bars for four cycles in 2 uptake of ZIF-8/PAN-90 nanofibers at 1 bar and 40 bars is 7 cm3 g−1 and 130 mg g−1.2 separation with fast adsorption–desorption kinetics. Reasonable high CO2 uptake of ZIF-8/PAN-90 nanofiber composites have been achieved compared to similar studies on ZIF-8,36 suggesting that these materials are interesting for CO2 separation from gas streams.These initial results demonstrate good initial cyclic stability for CO4.13C NMR analysis indicate electrostatic interaction and hydrogen bonds between the PAN matrix and the ZIF-8 nanocrystals confined to the PAN nanofiber surface, resulting in good attachment of the ZIF-8 nanoparticles and relatively high CO2 uptake. We believe that this method to grow MOF structures into polymer nanofiber structures offers further opportunities to produce nanofibrous adsorbent materials for other applications, such as air or liquid filtration or in medical applications.In summary, we successfully demonstrated a phase inversion method to grow ZIF-8 crystals on the surface of a PAN polymer nanofiber matrix without sacrificing the surface properties of the material. XPS and There are no conflicts to declare.


Penaeus monodon and P. vannamei) that is identified by the presence of floating white fecal strings on pond water in grow-out ponds. Although the clinical manifestations of WFS are well defined, the underling etiology remains obscure. WFS has been associated with several enteric pathogens, including Enterocytozoon hepatopenaei (EHP). The association is based on studies that found areas where WFS has been reported, the prevalence and severity of EHP infection are high. In this study, we describe an experimental reproduction of WFS in P. vannamei pre-infected with EHP and challenged with a unique isolate of Vibrio parahaemolyticus isolated from the gastrointestinal tract of a shrimp displaying WFS. Upon laboratory challenge, shrimp displaying white fecal strings and white discoloration of the gastrointestinal tract were analyzed by histopathology, in-situ hybridization and quantitative PCR. Histological analysis confirmed the lesions of EHP and septic hepatopancreatic necrosis in the hepatopancreas of shrimp exposed to both pathogens. Quantitative PCR showed shrimp infected with both EHP and V. parahaemolyticus had a significantly higher load of EHP compared to shrimp infected with EHP alone. This is the first demonstration of experimental reproduction of WFS under laboratory conditions when animals are infected with EHP and V. parahaemolyticus concurrently. The data revealed a synergistic relation between EHP and V. parahaemolyticus isolate that led to the manifestation of WFS. We propose the gross signs of WFS can be used as an indicator of the presence of EHP infection in association with a particular strain of an enteric Vibrio spp. in countries where EHP is endemic.White Feces Syndrome (WFS) is an emergent disease of penaeid shrimp ( Enterocytozoon hepatopenaei (EHP), and White Feces Syndrome (WFS) whose causative agent or agents remain unidentified.The shrimp gut microbiota is fundamental to the host’s nutrition, growth, pathogen resistance, and maintenance of the internal homeostasis , 2. FeedP. monodon [P. vannamei [P. stylirostris [HPM is a disease of the hepatopancreas in penaeid shrimp caused by several species of microsporidians including EHP. This microsporidian species has been found in several Asian countries such as China, Vietnam, Malaysia, Indonesia, Thailand, and India –6. Recen monodon , P. vannvannamei , and P. irostris , 13. EHPirostris , 10.Vibrio spp., that cause septic hepatopancreatic necrosis (SHPN) [At a farm level, WFS manifests as white fecal strings floating along the surface water in grow-out ponds. Shrimp in these ponds display unusual clinical signs such as poor growth rate, size disparity, gastrointestinal tract with yellowish to whitish discoloration, softshell, and chronic mortalities , 8, 14. s (SHPN) , 6, 10.Vibrio parahaemolyticus as the missing link between EHP and WFS. Co-infecting EHP-infected shrimp by an immersion challenge with a unique strain of V. parahaemolyticus that was isolated from gut of a shrimp (P. vannamei) displaying WFS allowed us to provide the first experimental evidence of WFS reproduction. To our knowledge, this is the first demonstration of the reproduction of WFS under controlled laboratory conditions using two unique pathogens.In this study, we isolated and identified a strain of P. vannamei were obtained from the Oceanic Institute . For at least two consecutive years, this population has tested negative by PCR for all OIE-listed pathogens as well as OIE non-listed pathogens including EHP. The bioassay was carried out in the Aquaculture Pathology Laboratory of the University of Arizona. All procedures for the sampling and euthanasia of shrimp were performed following the guidelines established by the American Veterinary Medical Association (AVMA) [Specific pathogen free (SPF) n (AVMA) .P. vannamei shrimp displaying WFS in a grow out pond was dissected, and a small section of the GI tract was directly plated on a Thiosulfate Citrate Bile Salts Sucrose (TCBS) (DifcoTM) plate and incubated at 30 0C for 24 hr. After an overnight growth, only sucrose negative Colony Forming Units (CFU) were present in the plate. A CFU was picked and plated on a Tryptic Soy Agar (Sigma Aldrich) plate containing 2.5% NaCl (TSA+) for further maintenance of the culture. The bacterial identification was carried out by PCR amplification and sequencing of the 16S rRNA region [ToxR gene and PirABvp genes were performed following previously published protocols [TM) supplemented with 2% NaCl (TSB+) was used for culturing the isolate at 30°C overnight with gentle shaking (100 rpm) to reach 1.5 × 109 CFU/mL.The gastrointestinal (GI) tract of a juvenile A region . In addirotocols , 19. To P. vannamei infected with EHP (Thailand isolate). In order to produce EHP-infected shrimp, a co-habitation challenge was conducted by rearing fifty SPF P. vannamei (average weight 2.5–3.5 g) with fifty confirmed EHP-positive shrimp in a 1000 L tank for 60 days. The hepatopancreas of the experimentally challenged shrimp were dissected and preserved in Davidson’s fixative and 95% ethanol. EHP infection in the SPF shrimp was confirmed by PCR and histopathology at the end of the cohabitation challenge.The EHP isolate used in this study was obtained from a candidate SPF population of V. parahaemolyticus (WFS isolate) as an inoculum and both SPF and EHP-infected population of P. vannamei. Two independent trials were conducted . In each trial, four 90-L tanks were filled with artificial seawater adjusted to 30 ppt , the temperature was kept at 26°C (±0.6) and the pH ranged from 7.5–8.0. For trial 1, seven SPF and eight EHP-infected P. vannamei (average weight 6.0. ±1.1 g) were used whereas in trial 2, ten SPF and eight EHP-infected P. vannamei (average weight: 7.1±2.0 g) were used for the experimental infection. The setup of the tanks for the trials 1 and 2 is summarized in To reproduce WFS in a controlled laboratory setting, an experimental bacterial immersion challenge was conducted by using the V. parahaemolyticus challenge was conducted via an immersion challenge by adding an overnight culture into the aquaria to obtain a final bacterial concentration of 1.0 × 106 CFU/mL. The bacterial inoculation was conducted four times throughout the challenge on 0, 1, 4, and 18 days post-infection (dpi). The EHP positive control group was not inoculated with V. parahaemolyticus, while the SPF group was not infected with EHP nor V. parahaemolyticus. Mortality, presence of white fecal strings and white color in the gastrointestinal tract (GI-Tract) were recorded daily throughout the experiment. Moribund as well as surviving shrimp at the end of the experiment were fixed in Davidson’s AFA fixative [For both trials, fixative . At the fixative  and the The Davidson’s alcohol-formalin-acetic acid (AFA)-fixed samples were processed, embedded in paraffin, and sectioned (5 μm thick) per standard methods , 21. Aft5’-GCC TGA GAG ATG GCT CCC ACG T-3’) and EHP 510 R (5’-GCG TAC TAT CCC CAG AGC CCG A-3’) primers [in situ hybridization (ISH). These primers were tailed at 3′-end with digoxigenin-11-dUTP . The Davidson’s fixed shrimp were processed, and tissue sections were made (5 μm thick) (Lightner 1996a). After deparaffinization, hydration, proteinase K digestion, and pre-hybridization, sections were overlaid with 100 μL hybridization solution containing DIG-labeled primers (100 fmol). The slides were placed on a heated surface at 90°C for 10 min and hybridized overnight at 50°C. Final detection was performed with an anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche) that was visualized using nitro blue tetrazolium and 5-bromo-4- chloro-3-indolyl phosphate [The EHP 510 F  were used for amplification using the following cycling parameters: initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30s, 63°C for 30s, and 72°C for 30s, and a final extension at 72°C for 5 min. Following amplification, the PCR products were electrophoresed in 1.5% agarose gels containing 1X GelRed and visualized under ultraviolet light and digitally photographed by the Gel documentation system from Bio-Rad ®. EHP detection and quantification was carried out by quantitative PCR (qPCR) based on the EHP SSU rDNA following a published method [5′-AGT AAA CTA TGC CGA CAA-3′) and primer R:157 (5′-T TAA GCA GCA CAA TCC-3′), and a TaqMan probe (5-FAM-TCC TGG TAG TGT CCT TCC GT-TAMRA-3′) were used. DNA concentration was normalized at 20 ng ul-1. The amplification reactions were conducted as follows: 0.5 μM of each primer, 0.1 μM of TaqMan probe, 1× TaqMan Fast Virus 1-Step Master Mix (Life Technologies), 50 ng of DNA and HPLC water in a reaction volume of 10 μl. The qPCR profile consisted of 20s at 95°C followed by 40 cycles of 1s at 95°C and 20s at 60°C. Amplification detection and data analysis for qPCR assays were carried out with the StepOnePlus real-time PCR system (Life Technologies).Detection of ublished . A 368-bd method . Primer Statistical analyses were conducted using GraphPad Prism Version 6.0. One-way ANOVA (α = 0.05) was used to determine statistical variances between groups. Tukey’s multiple comparisons post-hoc was used to analyze statistical difference between the EHP load among shrimp sampled from different treatments after logarithmic transformation of data.P. vannamei shrimp displaying clinical manifestation of WFS was plated on a TCBS plate. Presence of only CFU Sucrose negative on TCBS plate (green colonies)  was successfully amplified from an isolated CFU and the amplicon sequenced using forward and the reverse primers used for the PCR amplification. The consensus sequence showed highest identity with several strains of V. parahaemolyticus (>99% identity) in NCBI database . The V. parahaemolyticus 16SrRNA sequence was deposited in GenBank with the accession numbers MW526256. Interestingly, NCBI entries showing highest identity were also isolated from shrimp farms as well. To further confirm the identity of Vibrio isolate, a 368 bp DNA representing the toxR gene was amplified by PCR (vp gene could not be amplified from the same isolate (V. parahaemolyticus.A loopful of the GI tract of a olonies)  indicated by PCR . However isolate  indicatiV. parahaemolyticus via immersion. In both trials 1 and 2, the white discoloration of the GI tract in shrimp was observed initially at 8 dpi and only in the tank with shrimp pre-infected with EHP. Subsequently, white fecal strings adhering to the anal opening and fecal string floating on tank water were observed for this treatment  as well as in EHP + V. parahaemolyticus challenged group. For confirmation of EHP infection, samples from trial 1 were further analyzed by in-situ hybridization using EHP-specific probe. The presence of plasmodium and spore stages typical of EHP infection are shown in In situ hybridization using EHP-specific DIG-labeled 18S rRNA probe reacted intensely to the inclusion bodies within the cytoplasm of the infected cells, thus confirming the presence of EHP. The severity of infection was graded G1 to G4  and challenged with a particular Vibrio parahaemolyticus (secondary pathogen) isolated from the GI tract of a shrimp displaying WFS. The data presented here provide empirical support of a strong association of EHP with WFS as recorded in different regions of the world [Here we describe for the first time the experimental reproduction of WFS in shrimp, he world , 8, 10. Vibrio cholerae as the etiologic agent of WFS [Vibrio spp. that cause SHPN [Candidatus Bacilloplasma and Phascolarctobacterium and decreases in Paracoccus and Lactococcus may contribute to WFS in shrimp [White Feces Syndrome has proven to have one of the most elusive etiology of any of the shrimp disease that have been characterized to date. It has been more than a decade since the first formal report of EHP infected shrimp showing WFS was published , althougt of WFS  althought of WFS . WFS appuse SHPN , 10. In use SHPN . Researcn shrimp . Heavy gn shrimp , 27.While studies to reproduces WFS in a laboratory setting have been attempted by several authors few have been successful in consistently reproducing this syndrome and determining the etiology. Huang et al. (2020)  recentlyVibrio parahaemolyticus isolate. This demonstrates that, in order to induce clinical manifestations of WFS, both EHP and this particular Vibrio parahaemolyticus isolate are required. Shrimp exposed only to EHP or Vibrio parahaemolyticus did not develop WFS. Therefore, WFS manifestation involves a unique combination of two pathogens that had not been observed previously for any infectious disease of shrimp known to date. Typically, in a shrimp grow out pond, the cultured population starts showing the presence of EHP first. Later on, during the grow-out cycle, shrimp start developing white discoloration of the GI tract, and floating white fecal strings start appearing in the pond. By H&E histology, we observed a similar sequential order in cellular pathology during the infection process. A primary pathogen, in this case EHP, caused the typical histological lesions and later on affected shrimp display SHPN caused by opportunistic V. parahaemolyticus that was used to challenge the EHP-infected shrimp. The histopathology image in Vibrio sp. resulting WFS.Under laboratory challenge experiments, we were able to confirm the presence of white fecal strings only in shrimp that were pre-infected with EHP and subsequently exposed to the V. parahaemolyticus in both trials, displayed a high size disparity which is higher or comparable to shrimp infected with EHP or V. parahaemolyticus alone  mentioned is required to observe high size variation. In previous studies, shrimp displaying WFS show a copy number in the order of ~ 106 copies ng-1 of extracted HP DNA [Shrimp in the treatment group EHP+us alone . A simild HP DNA .V. parahaemolyticus is very likely due to the synergistic actions of the microsporidian and a particular bacterial strain on hepatopancreatic tubules. As mentioned previously, Huang and colleagues suggested that WFS is caused by intestinal microbiota dysbiosis [The reproduction of WFS in shrimp infected with EHP and challenged with ysbiosis  and thisysbiosis . In EHP-Cryptosporidium spp. [Alterations involving the gastrointestinal track, where a sign of the disease are whitish stools, are common to all animals including humans. In humans, whitish stools are caused by changes in the gut microbiome  or by paium spp. . This miVibrio spp. where the intestine in shrimp, with and without WFS, were analyzed. An increase of intestinal Vibrio population from a normal shrimp with 1.8x107 CFU/g population versus WFS-shrimp with 6.1x107 CFU/g. Some of these Vibrio. spp found in those samples were V. parahaemolyticus, V. vulnificus, and V. damsela. [V. parahaemolyticus as the unidentified pathogen co-infecting with EHP on shrimp manifesting WFS. However, it is possible that other Vibrio species in addition to V. parahaemolyticus could induce WFS.Our study provides conclusive evidence that EHP is not the only pathogen associated with the presence of WFS. In Thailand, some studies have shown the association of WFS in shrimp population with an increase of damsela. . This coVibrio parahaemolyticus leads to the development of WFS. These findings also corroborate and provide an explanation as to why WFS is manifested in areas where EHP and V. parahaemolyticus are also endemic.EHP as a primary enteric pathogen of penaeid shrimp. Infection with EHP in association with a particular strain of S1 Raw images(ZIP)Click here for additional data file.S2 Raw images(TIF)Click here for additional data file.S3 Raw images(ZIP)Click here for additional data file.S4 Raw images(ZIP)Click here for additional data file.S5 Raw images(ZIP)Click here for additional data file.S1 VideoVibrio parahaemolyticus. The white fecal strings still attached to the anal cavity is observed.This specimen was pre-infected with EHP and then challenge with a strain of (MOV)Click here for additional data file.


Deep learning has brought a rapid development in the aspect of molecular representation for various tasks, such as molecular property prediction. The prediction of molecular properties is a crucial task in the field of drug discovery for finding specific drugs with good pharmacological activity and pharmacokinetic properties. SMILES string is always used as a kind of character approach in deep neural network models, inspired by natural language processing techniques. However, the deep learning models are hindered by the nonunique nature of the SMILES string. To efficiently learn molecular features along all message paths, in this paper we encode multiple SMILES for every molecule as an automated data augmentation for the prediction of molecular properties, which alleviates the overfitting problem caused by the small amount of data in the datasets of molecular property prediction. As a result, by using the multiple SMILES-based augmentation, we obtained better molecular representation and showed superior performance in the tasks of predicting molecular properties. Traditionally, drug discovery is time-consuming and very expensive. For understanding the properties of a compound, many results of the simulations can be obtained via the experience of a chemist or pharmacist. The overall process is significantly complex, long, and always inefficient. Deep learning has brought a rapid development in the field of drug discovery and is expected to accelerate the process of drug discovery –8. NeverInspired by natural language processing techniques, many deep learning models use the simplified molecular input line entry system (SMILES)  as a linA related method to this paper is the SMILES enumeration . SMILES The key idea is that we focus on a multiple-SMILES representation learning as data augmentation for various downstream tasks. As we all know, the deep learning model must be fed with a large amount of data. Through learning a large amount of data, the model can find the law and obtain the potential knowledge of the data. Despite the presence of a large number of molecules, labeled datasets are scarce. In the task of molecular property prediction, the number of molecules in some datasets of property prediction is also very small. It leads to the problem of unstable prediction performance using the deep learning models due to overfitting and underfitting.Inspired by the SMILES enumeration  and all In the following, we will describe technical details. We first give the mathematical definition of the problem  and then𝒞𝒩𝒩, where kernel is the convolution kernel, channel denotes the convolution channel, and padding represents the type of padding. A recurrent neural network can be defined as ℛ𝒩𝒩gate, where gate denotes the type of gate, such as LSTM and GRU. Let Xos be the original SMILES strings that have been cleaned and are valid molecules. Let mapping function of multiple SMILES be fms. Xms is the multiple SMILES sequences. We define the vectorization function as fvect. The problem is to learn the function fres that maps the multiple SMILES vectors to molecular representation Xmol. The mapping relations are represented as follows:We define a feed-forward convolutional neural network as fms, fvect, and fres. The final molecular representation is what we want to get and can be further used in specific tasks such as molecular property prediction.The whole process includes three mapping functions, namely, fms can be implemented using the method of renumbering atoms in RDKit [The SMILES  is a popin RDKit  after pefvect can be implemented using language translation technology in the field of natural language processing, which is an effective method for learning from text data. We need to construct a character set for all SMILES sequences in some datasets, which is similar to the corpus in natural language processing. Then randomization and vectorization to convert the SMILES array to a one-hot vector are performed. The mapping function fres for obtaining molecular representation can be learned using stacked CNN and RNN mixed architecture. The RNN consists of an input layer, a hidden layer, and an output layer. X is an input vector, H indicates the hidden vector of the hidden layer, O represents the output vector. Wxh,  Who, and Wh denote the weight matrix from input layer to hidden layer from hidden layer to output layer, and hidden layer, respectively.The last mapping function Ot of RNN at time t is related not only to the input xt at time t but also to the hidden layer value ht−1 at time t − 1. It is shown that RNN can better deal with sequence information, that is, the previous input is related to the subsequent input. SMILES string is precisely this sequence structure, which can extract features with designed RNN architecture.t and time t − 1. The result of the output layer at time t must be based on the input at time t and the result vector of the hidden layer at time t − 1. The process can be summarized in the form of matrix as follows:𝒫 and 𝒬 indicate some kind of neural network. Finally, the mapping function fres for obtaining molecular representation can be represented using CNN and RNN as follows:f denotes the mapping of full-connection layer, pooling layer, and gather layer.The message passing process for stacked CNN and RNN mixed architecture can be found in Extensive experiments have been implemented to evaluate the performance of molecular representation using the multiple SMILES-based augmentation for the tasks of molecular property prediction. We will describe the datasets, baselines, and experimental results.We use five molecular property datasets recommended by MoleculeNet  for the ESOL: ESOL  containsLipophilicity: lipophilicity  includesFreeSolv: FreeSolv  providesHIV: HIV  is used BACE: BACE  is used The details of used datasets are shown as follows:The datasets must be cleaned before being input into the neural network. The cleaning and preprocessing process are shown in CheMixNet: CheMixNet  was propSmi2Vec-BiGRU: Smi2Vec + BiGRU  was propXGBoost: XGBoost  is an enMultitask: Multitask network  was propMPNN: Message Passing Neural Network (MPNN) is a generalized graph-based architecture , includiGC: GC  is a staWeave: Weave  implemenPretraining GNN: pretraining GNN  proposedDrug3D-Net: Drug3D-Net  is a griMultiple SMILES (RNN (one layer), RNN (two layers), CNN_RNN): this is the method presented in this article. The neural network architecture includes one layer RNN, two layers RNN, and the mixed networks of CNN and RNN.We compared our method with the following models:In this experiment, we use root mean squared error (RMSE) and mean absolute error (MAE) to evaluate the performance of regression tasks. Similarly, we use the loss function of “binary_crossentropy” for classification datasets. We use the average area under the receiver operating characteristics curve (AUROC) and the area under the precision-recall curve (AUPRC) predicted from the test set to evaluate the performance of the model for classification tasks. Our experiment was trained based on the Keras framework and TensorFlow . We usedSolubility, lipophilicity, and free energy are very important physical chemistry properties, which are essential properties to understand molecular interaction with solvents. β-secretase 1 (BACE). The larger the AUROC and AUPRC score, the better for HIV and BACE. Our method based on mixed CNN and RNN architecture achieved the best performance on AUROC and AUPRC scores in the test set for HIV and BACE datasets. In HIV, we obtain 0.9767 AUROC and 0.9798 AUPRC scores compared with the 0.9621AUROC and 0.9617 AUPRC of the 3D-based method Drug3D-Net, although the Drug3D-Net considered the information of molecular geometry. In addition, the performance of our method exceeds that of the pretrained model pretraining GNN with a large margin.In summary, our method shows superior performance in both regression datasets and classification datasets, which implies the good molecular representation ability of our proposed method.For different neural architectures of our multiple SMILES methods (shown in Tables In this study, we make full use of the nonunique nature of the SMILES string to perform randomization of a SMILES string multiple times for efficiently learning molecular features along all message paths. By encoding multiple SMILES for every molecule as an automated data augmentation, we obtain better molecular representation and the proposed method shows superior performance in the tasks of predicting molecular properties, which alleviates the overfitting problem caused by the small amount of data in the datasets of molecular property prediction.