Electroporation (EP) is one of the successful physical methods for intracellular drug delivery, which temporarily permeabilizes plasma membrane by exposing cells to electric pulses. Orientation of cells in electric field is important for electroporation and, consequently, for transport of molecules through permeabilized plasma membrane. Uptake of molecules after electroporation are the greatest at poles of cells facing electrodes and is often asymmetrical. However, asymmetry reported was inconsistent and inconclusive—in different reports it was either preferentially anodal or cathodal. We investigated the asymmetry of polar uptake of calcium ions after electroporation with electric pulses of different durations, as the orientation of elongated cells affects electroporation to a different extent when using electric pulses of different durations in the range of 100 ns to 100 µs. The results show that with 1, 10, and 100 µs pulses, the uptake of calcium ions is greater at the pole closer to the cathode than at the pole closer to the anode. With shorter 100 ns pulses, the asymmetry is not observed. A different extent of electroporation at different parts of elongated cells, such as muscle or cardiac cells, may have an impact on electroporation-based treatments such as drug delivery, pulse-field ablation, and gene electrotransfection. Exposure of cells to electric pulses of adequate amplitude and durations leads to transient increase in their plasma membrane permeability and the phenomenon is termed electroporation (EP). Increased plasma membrane permeability allows transmembrane transport of otherwise impermeant molecules into cells . With moElectroporation can also be used for intracellular drug delivery. Since it is a physical method, it bypasses the potential safety issue of viral vectors . As a drThe transport of molecules through permeabilized plasma membrane occurs presumably through aqueous pores that form in the process of electroporation . The tra2+) is a very convenient way of evaluating the extent of EP, because calcium concentration in cells is kept very low (around 100 nM) due to its signaling role [2+ ions compared to other EP detection dyes such as propidium iodide (PI) or YO-PRO-1, this method is considered very sensitive and can detect smaller pores, such as those of nanometer size that emerge in EP with ns pulses, as well as lower levels of membrane electroporation [2+ can also be transported through voltage-gated calcium channels (VGCC) or other channels [2+ concentration  can be a result of its release from internal stores [2+ pathways, such as calcium-induced calcium release [2+ comes to the cell interior from the extracellular medium through EP pores [The influx of ions through a permeabilized plasma membrane can also be an indicator of EP . Monitoring role . Due to poration ,21,22. Hchannels ,24,25. Ml stores ,27,28 an release ,30 and s release . NeverthEP pores ,27,32 anWhen a spherical cell is exposed to a homogeneous electric field, an induced transmembrane voltage (ITV) which varies with position on the membrane is observed: it reaches its peak values at the poles of the cell facing the two electrodes ,34. ConsOrientation of cells in electric field is important for electroporation and, consequently, for transport of molecules through permeabilized plasma membrane. We have previously shown that elongated cells oriented parallel and perpendicular to electric field are not uniformly electroporated when exposed to electric pulses of different durations . Therefo2+) in cells was monitored with a fluorescent calcium indicator Fura-2. The concentration of Ca2+ in intact cells is maintained very low (around 100 nM). After electric pulse exposure, intracellular concentration of Ca2+ was increased due to Ca2+ uptake from external medium  and/or the release of Ca2+ from internal stores  which resulted in Fura-2 response (increased Fura ratio 340/380). To obtain a comparable intracellular Ca2+ concentration, as indicated by Fura-2 response, a much higher electric field had to be applied when cells were exposed to shorter pulses than when longer pulses were used [H9c2 cells were exposed to pulses of different pulse duration: single pulses of 100 ns (40 kV/cm), 1 μs (2500 V/cm), 10 μs (1000 V/cm), and 100 μs (400 V/cm)  takes several minutes [Relative Ca minutes , we comp minutes . We perf2+ concentration is increased in all cells. Relative Ca2+ concentration in cells gradually rises through the first few seconds after the pulse and reaches its plateau. However, the relative Ca2+ concentration in cells exposed to 100 ns does not reach the plateau during 10 s of image acquisition but continues to rise with a slower pace. Relative Ca2+ concentration in cells exposed to 1 μs, 2500 V/cm pulses should be higher with a higher electric field applied, however, the applied electric field was maximal with our exposure setup. Relative intracellular Ca2+ concentration increases with increasing pulse amplitude  and cathode (−) (2+ concentration in poles facing cathode (−) and anode (+) in parallel and perpendicular cells, where these regions can be easily defined. Relative Ca2+ concentration (expressed in Fura 340/380 ratio) in anodic (+) and cathodic (−) poles of parallel and perpendicular cells together were not significantly different, except immediately after pulse (at 0.25 s) (2+ concentration (expressed in Fura 340/380 ratio) after 100 ns, 40 kV/cm thus shows no apparent calcium progression in the direction of the electric field neither in parallel nor in perpendicular cells (2 incubation (in contrast with experiments with microsecond pulses that were done at 37 °C and CO2 incubation). Therefore, effects and contributions of different conditions on results are possible.Microscopic images revealed that after 100 ns, 40 kV/cm pulse exposure no difference in Cahode (−) A–D occur 0.25 s) A. The di 0.25 s) B which par cells A,B. Expe2+ uptake was slightly higher at the pole facing cathode  and anode (+) is very small, yet significant.After exposing cells to 1 μs, 2500 V/cm pulses, the relative Ca cathode C,D. Due 2+ uptake was significantly greater at the pole of the cell facing cathode (−) than at the anode (+). In cells of both orientations , during the first few seconds after 10 and 100 μs pulse application, relative Ca2+ concentration increased more at the pole facing cathode than at the anode. In cells exposed to a 10 μs pulse, Ca2+ concentration (expressed in Fura 340/380 ratio) was significantly higher at the pole facing cathode than at the pole facing anode from 0.25–2.25 s after pulse . During the following seconds, the difference in uptake at the cell poles diminishes, Ca2+ diffuses throughout the cell and relative Ca2+ concentration at poles becomes of similar value (5.25 s after pulse application the difference between cathode and anode (Fura ratio (cathode)—Fura ratio (anode)) reached a median of −0.322, with Q1 and Q3 −2.907 and 4.808, respectively). When cells were exposed to 10 μs, 1000 V/cm E,F or 102+ concentration was significantly higher 0.75–1 s and 1.75–2 s after the pulse at the pole close to the cathode than at the pole close to the anode . In cells pulsed with 100 μs pulse, relative Ca2+ concentration (expressed in Fura ratio) from a representative parallel cell after 100 μs pulse exposure , the source of Ca2+ ions relative Ca2+ concentration increase in cells after electroporation was determined  in cells increased, although not to the same extent as in conditions with external Ca2+ (in DMEM culture medium). This means that the release of Ca2+ from internal stores  occurred due to 100 ns, 40 kV/cm pulse application. In conditions with external Ca2+, we can however observe, in addition to Ca2+ released from internal stores, an uptake of external Ca2+ from extracellular medium. With the use of SMEM medium without Catermined . When cetermined A in cond2+ increase, i.e., Ca2+ is not released from internal stores , suggesting all of the Ca2+ ions that contributed to the increased relative Ca2+ concentration in DMEM culture medium (with Ca2+) are taken up from the poration medium, i.e., from the extracellular medium.On the contrary, when cells were exposed to a single pulse of 1 μs B, 10 μs 2+ ions if the release from internal stores is eliminated. For this purpose, we incubated H9c2 cells with thapsigargin, a well-known blocker of sarco-endoplasmic reticulum Ca2+-ATPases (SERCA) that passively depletes the ER [2+ concentration was not observed  showed high basal internal Ca2+ concentration (baseline not subtracted) that cannot be restored in these conditions for 30 min (2+ entry from the outside medium through Ca2+ channels of different pathways of store-operated (capacitive) Ca2+ entry [2+ concentration  cannot be neglected. On the other hand, cells treated with 100 nM thapsigargin that were later incubated in DMEM (containing Car 30 min A. The re2+ entry . The expntration A which p2+ concentration in at the poles facing anode (+) and cathode (−) in thapsigargin treated cells  after electroporation with a single 100 ns, 40 kV/cm in medium with Ca2+ ions (DMEM) (2+ concentration differences between cathode (−) and anode (+) were still not significantly different, and the values are around zero. However, for final confirming asymmetrical Ca2+ uptake after 100 ns pulse exposure, plasma membrane channel inhibitors should be used. We analyzed relative Cas (DMEM) B. The rep < 0.05).When results were analyzed separately for parallel and perpendicular cells , cells pIn a theoretical paper from Saulis, electroporation with pulses with low amplitude (just above the threshold for electroporation) could lead to predominantly anodal (+) EP . We thus2+ uptake (2+ uptake was still slightly but significantly more prominent on the cathode (−) side of cells leading to positive values of difference between cathode and anode (Fura ratio (cathode)—Fura ratio (anode)). When cells were pulsed with a regular, 400 V/cm pulse of reversed polarity, the direction of Ca2+ uptake was reversed (see When cells were exposed to 100 μs pulses of low electric field strength (200 V/cm), we did not observe any change of the direction of Ca+ uptake A,B. Ca2+rsed see C,D, the rsed see D.It was described previously that orientation plays a significant role in electroporation extent when cells are exposed to electric pulses of different durations in the range from 100 ns to 100 μs . We ther2+ after exposing cells to a single 1–100 µs pulse is polar and asymmetrical: it is higher at the pole facing the cathode (−) than at the pole facing the anode (+). 100 ns pulse, on the other hand, causes symmetrical Ca2+ uptake, with no apparent preference of poles facing to either electrode, except at first time point (250 ms after the pulse). Interestingly, Gabriel and Teissié [The results of our study show that the uptake of Ca Teissié  reported Teissié ,45. It w Teissié .Several other studies reported asymmetric transport at the poles of cells facing the electrodes after micro and millisecond pulse exposure. However, the preferences of the side of the cell reported in studies are different: the uptake of molecules was preferentially on the negative  side, which is in agreement with our results ,40,56,61+-free medium where the resting potential was lower [The anodal (+) preference was explained as a consequence of superposition of induced transmembrane voltage (ITV) and resting potential: mostly, resting potential of the cells is negative inside and it adds to the transmembrane potential at the anode (+) and subtracts transmembrane potential at the cathode (−) side ,43,47. Tas lower , howeveras lower ,51,62 orThe cathodal (−) preference was also explained with the superposition of ITV and resting potential. Poration that starts on the anodal side of the cell produces current flowing through the porated area, which reduces the transmembrane potential on the anode (+) side while increasing the induced transmembrane potential on the cathodal (−) side. Permeabilization on the cathode (−) side exceeds that on the anode (+) side ,39,54,562+ and ethidium bromide) appear to enter predominately through the anodic side, and the larger probes (such as ethidium homodimer and propidium iodide) enter through the cathodic side [According to theoretical predictions by Saulis , both stdic side . Howeverdic side . 2+ uptake and thus failed to confirm his predictions.Saulis also predicted that if the pulse length is short enough, the pores on the cathodic (−) side do not have time to appear and can lead to predominantly anodal (+) EP, particularly with electric pulses just above the threshold for electroporation . We thus2+ can also be dragged in the direction towards the negative electrode by electrophoresis during longer pulses [2+ drag in the direction towards the negative electrode would lead to preferentially anodal (+) uptake. The unidirectional flow through electropores could also be caused by electroosmosis during the pulse, when ionic aqueous medium causes a net hydrodynamic flow toward the electrode with the polarity opposite to the dominant charge in the medium [Asymmetry was also shown to depend on the probe charge that is affected by electrochemical potential ,55, esper pulses ,63. Elecr pulses ,51. It ir pulses . Electroe medium ,62.Asymmetric polar transport may depend on asymmetric membrane structures that can behave in a different way on different sides ,39,45, a2+ transport when the poration medium was changed from low salt medium to high salt medium. This reversal was explained by rapid resealing of membrane pores in high ionic strength medium. Consequently, the smaller sized pores at the anode-facing side would close rapidly, and thus the probes can enter only through the cathodal (−) pole [2+ in high salt medium. On the other hand, Semenov et al. exposed embryonic rat cardiac myocytes to a single 4 ms pulse in a physiological saline that was also high salt medium, and reported preferentially anodal (+) uptake of Ca2+ [Poration medium composition was also suggested to affect polar asymmetry in transport of molecules. Tekle et al. observed a reversal of polar preference of ethidium bromide and Ca(−) pole . Djuzeno(−) pole . Our exp(−) pole  that rep of Ca2+ . The rea2 incubation , this may have affected our results. When H9c2 cells were exposed to a single 100 ns pulse, an asymmetry of polar transport was not observed, except at the first time point 250 ms after the pulse. In other studies, similar to those that investigated EP with µs and ms pulses, all possible outcomes were reported: symmetrical ,39,49, p2+. nsEP caused more pronounced asymmetry in uptake of Ca2+ than Ba2+, which may be caused by local amplification of Ca2+ by store disruption, CICR, store-operated Ca2+ entry, or some other process or attenuation at the opposite pole by sequestration of Ca2+ into the ER [Preferentially anodal asymmetry with ns pulses was explained again with superposition of ITV and resting potential, the differences in pore density and size on poles, cylindrical nanopores with nonuniform cross section, and also by field-driven translocation of phospholipids . Anionico the ER .2+ is released from internal stores, as seen by an increase of relative internal Ca2+ concentration after 100 ns pulse exposure to cells in the medium without Ca2+. This means that the release of Ca2+ from the internal stores can be the reason behind the absence of asymmetrical transport. Sun et al. also speculated that intracellular disturbances accounted for observed symmetrical Ca2+ uptake in spite of preferentially anodal (+) PS translocation [2+ from internal stores, mainly endoplasmic reticulum (ER) [2+ cycling system [2+ transport was also observed in cardiac cells after ns pulse exposure where internal Ca2+ stores were depleted [2+ from internal stores was not the reason for symmetry. As we can see in location . It is klum (ER) ,27,28. Mg system . Symmetrdepleted , so at l2+ can be further amplified with store-operated (capacitive) Ca2+ entry (SOCE) or Ca-induced Ca2+ release (CICR) from internal stores which may also account for the absence of asymmetrical transport. It was suggested that nsEP cause a depletion of Ca2+ stores in ER , which in turn activates plasma membrane calcium influx through channels linked to SOCE [2+ concentration, which led to inositol trisphosphate receptor (IP3R) -dependent release from ER [2+ channels in plasma membrane), caffeine (for depleting Ca2+ from the ER by stimulating ryanodine receptors), and cyclopiazonic acid (for blocking re-uptake of Ca2+ to the ER) in the paper from Semenov [The increase of internal Ca to SOCE . However to SOCE . The sec from ER ,30. Thes Semenov , CICR is Semenov ,23,71, a2+ after nsEP in both normal cells and cells with depleted internal stores of Ca2+. We performed these experiments with the use of thapsigargin, a known inhibitor of SERCA pumps that enables a depletion of internal Ca2+ stores [2+ from the internal stores (ER) may interfere with asymmetrical Ca2+ uptake after 100 ns pulse exposure but does not solely account for a symmetrical increase of Ca2+ throughout the whole cell. The thapsigargin treatment also triggers additional Ca2+ uptake from outside medium through store-dependent and store-independent channels in plasma membrane contributing to different pathways of store-operated Ca2+ entry (SOCE) [2+ through permeabilized membrane. This was also the case in our experiments, since the base level of internal Ca2+ remained high  the polar uptake of Ca+ stores ,59. In ty (SOCE) , which aned high A. Theref2+ ions after EP is important due to their signaling role, regulating many cellular functions [2+ can function as a stressor in cell damage, death, and survival [2+ acts locally [2+ concentration in cells after electroporation. This can be very important in tissues with elongated cells since it was shown that orientation of these cells in electric field affects extent of electroporation [The uptake of Caunctions . Moreovesurvival . Calciumsurvival ,74. Ca2+ locally ,72, therporation . A diffe2+ into cells, however, in the case of 100 ns pulses, Ca2+ release from internal stores (ER) or other more complex pathways of Ca2+ control  may play an important role in asymmetrical transport detection. Moreover, we did not confirm the hypothesis that, with the use of low amplitude 100 µs pulses, we can achieve the reversal of polar asymmetric Ca2+ uptake [To summarize, we confirmed that pulse duration affects polar uptake of Ca+ uptake .2 atmosphere on a 12 mm diameter coverglass in 24-well plates one or two days before the experiment at 5 × 104 and 3 × 104 cells per well, respectively. For microsecond pulse exposures, H9c2 cells were seeded in their culture medium in two well Lab-Tek chambered coverglass  one or two days before the experiments at 8 × 104 and 5 × 104 cells per well, respectively.H9c2 rat cardiac myoblast cell line (European Collection of Authenticated Cell Cultures ECACC 88092904) was used in experiments. They were selected because we already studied the effect of orientation on electroporation on this cell line , and areRelative intracellular calcium concentration was detected using the fluorescent calcium indicator Fura-2 acetoxymethyl (AM) ester . When calcium concentration is elevated, the Fura-2 dye changes the fluorescence spectrum, which can be detected with ratiometric measurements ,21.2 chamber was used to enable consecutive pulse application to the same cells during a long period of time (up to 30 min) under controlled conditions. For nanosecond pulse exposure, the microscope stage heating and CO2 chamber were not used in order to prevent the drying of the sample (cells on coverglass on top of microelectrodes). Cells were illuminated with a monochromator  with Xe light at two excitation wavelengths (340 nm and 380 nm) and an exposure time of 100 ms for both wavelengths, using appropriate filter set , therefore, the emission wavelength was detected at 510 nm. Images were acquired with the software VisiView (Visitron) in a time-lapse acquisition mode . Pulses were delivered during the first two seconds of image acquisition and were later adjusted to the same pulse delivery time . The results from 100 µs pulse application were compared to slow-acquisition mode  as described previously [H9c2 cells were stained with 2 µM Fura-2 AM in their culture media  was subtracted from the images. The ratio images were obtained by the following procedure: fluorescence intensity of each pixel in images excited at 340 nm was divided by that of images excited at 380 nm. Fura-2 340/380 ratio in all pixels was multiplied by a VisiView preset factor of 100 to add numerical precision to 16-bit images with integer pixel values. Increased internal calcium concentration resulted in increased Fura-2 340/380 ratio. Cells were exposed to electric pulses using two different electrode configurations, gold microelectrodes for delivering a single, 100 ns, 40 kV/cm pulse, and Pt/Ir wire electrodes for delivering single pulses of 1 μs, 2500 V/cm, 10 μs, 1000 V/cm, and 100 μs, 400 V/cm  to reachFor 100 ns pulses, Fura-2 labeled cells on round coverglass with cells facing down were placed on 40 µL drop of DMEM medium on top of gold microelectrodes, 2.1 µm thick, mounted onto a cover glass holder, with a gap of 100 µm between the electrodes . The covFor electroporation with 1, 10, and 100 µs pulses, two parallel Pt/Ir wire electrodes, with 0.8 mm diameter and 4 mm distance between inner edges, were placed to the bottom of the Lab-Tek chamber. Pulses were delivered by a laboratory prototype pulse generator (University of Ljubljana) based on H-bridge digital amplifier with 1 kV RF MOSFETs  ,50,77 fo2+  and supplemented with 5 μM calcium chelator EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetracetic acid). After 12 min, the medium was changed to DMEM medium (contains 1.8 mM Ca2+), and 13–15 min after the first pulse, cells were exposed to the same pulse again. However, for 100 ns, 40 kV/cm pulses, changing the medium was not possible due to the setup configuration (cells on coverglass on top of the electrodes). Therefore, we exposed the cells to 100 ns electric pulses only in SMEM without Ca2+ and supplemented with EGTA and compared these results to the ones performed in DMEM medium [2+ stores (ER), cells were incubated with 100 nM thapsigargin during Fura-2 labeling (for 30 min in DMEM medium).To identify the source of calcium ions for elevated internal calcium concentration, we performed the following experiments. Cells dyed with Fura-2 were exposed to a single electric pulse of the same duration and electric field strength , but in calcium-depleted medium—SMEM medium—without CaM medium . For depImage analysis was done using an open-source image-processing program ImageJ  and Excel . Cells in ratio images were manually encircled and mean fluorescence intensity ratio Fura-2 340/380 was determined for each cell. Time of pulsing was determined from graphs as the last time point where fluorescence is at the baseline. Since pulses were applied manually, the separate experiments were adjusted to the same pulsing time when averaged (number of experiments: 3–9) or compared to slow-acquisition mode. The baseline of each cell (fluorescence at time 250 ms before pulse exposure—the last time point before pulsing) was subtracted from each time point.For determining the direction of calcium with respect to the position of electrodes, cells from separate experiments were pooled from the experiments and analyzed. The fluorescence intensity ratio Fura-2 340/380 was determined in manually encircled regions facing the anode (+) or the cathode (−) only in parallel and perpendicular cells since the regions can be best determined in these cells. The orientation of cells in electric field—parallel and perpendicular—in all experiments including controls was determined with ImageJ, as described previously . In briePlot profiles for p < 0.05) in fluorescence intensity ratio Fura-2 340/380 between poles facing anode (+) and cathode (−) were determined by Wilcoxon signed rank test .Statistical analysis was performed using Excel and SigmaPlot 11.0 . The results in figures and the text are expressed as means ± SD. Significant differences , or cathodal (−), but the phenomenon needs further research for optimization of drug delivery techniques.The orientation of cells exposed to electric field is important, especially in elongated cells. The uptake of Ca


Changes in the function or abundance of the SYT1 and SYT7 isoforms alter the number and route by which SVs fuse at nerve terminals. Several SYT family members also regulate trafficking of other subcellular organelles at synapses, including dense core vesicles (DCV), exosomes, and postsynaptic vesicles. Although SYTs are linked to trafficking of multiple classes of synaptic membrane compartments, how and when they interact with lipids, the SNARE machinery and other release effectors are still being elucidated. Given mutations in the SYT family cause disorders in both the central and peripheral nervous system in humans, ongoing efforts are defining how these proteins regulate vesicle trafficking within distinct neuronal compartments. Here, we review the Drosophila SYT family and examine their role in synaptic communication. Studies in this invertebrate model have revealed key similarities and several differences with the predicted activity of their mammalian counterparts. In addition, we highlight the remaining areas of uncertainty in the field and describe outstanding questions on how the SYT family regulates membrane trafficking at nerve terminals.The Synaptotagmin (SYT) family of proteins play key roles in regulating membrane trafficking at neuronal synapses. Using both Ca SV rtentials . This quTrichoplax adhaerens genome, suggesting a non-neuronal origin of SYTs during a period when multicellular communication was emerging [SYTs are conserved family of membrane-trafficking proteins containing a single transmembrane domain and two paired cytosolic C2 domains Figs. b, 3. Theemerging , 97. Theemerging . Many SY2+ based on conservation of negatively charged aspartate residues within their C2 domains that mediate this interaction [2+ , while two lack conserved Ca2+ binding aspartate residues  [2+ sensors driving fast synchronous SV fusion in specific neuronal populations [Syt1 null mutants generally die as embryos or during early larval development [Syt4 and Syt7 are viable and fertile as adults [2+ sensors in Drosophila [SYTs can be subdivided into whether or not they are likely to bind Caeraction . For the, SYT14) , 100. Thulations , 63, 101elopment , though elopment . These selopment , 82, 84.elopment –107. Muts adults , 105, ths adults . These iosophila –110. Theosophila –114, homosophila , 116, asosophila  and Granosophila . In contosophila –122, but2+-dependent lipid binding modules. They represent one of several Ca2+ binding domains found among proteins, with EF hands representing another prominent motif that translates intracellular Ca2+ rises into downstream effector responses [2+ elevation through its lipid-binding properties. Such an evolutionarily conserved role can be postulated for some SYT family members, which are tethered to intracellular membrane organelles like SVs via their transmembrane domains. This would allow the C2 domains of SYTs to bridge distinct membrane compartments and bring two lipid bilayers in close proximity for potential fusion or lipid mixing. Although C2 domains are largely considered Ca2+-dependent lipid binding modules, they also mediate a host of Ca2+-independent interactions. As noted above, multiple SYT family members lack the required aspartate residues that coordinate Ca2+ binding, suggesting these isoforms use the C2 domain as a protein–protein interaction module instead. This is also the case for SYTs that display Ca2+-dependent lipid binding, as other regions of the C2 domain interact with multiple effector proteins, including the SNARE complex [2+-independent manner to facilitate vesicle docking [The domain structure of the SYT family is highly conserved across all isoforms, with a single-pass transmembrane domain, a variable linker and two tandem cytosolic C2 domains, termed C2A and C2B , 124. C2esponses –128. Wit complex , 129. In docking , 130–1322+ sensors for DCV fusion that mediates release of neuropeptides and neuromodulators [2+ regulation of SV and DCV release by SYTs in presynaptic terminals represent the most-well characterized role, there is also evidence they function in postsynaptic vesicle trafficking [2+ influx is required to regulate membrane trafficking to support retrograde signaling and postsynaptic neurotransmitter receptor cycling. Family members of the SYT4/SYT11 and SYT3 subgroups, along with SYT1 and SYT7, have been implicated in these postsynaptic processes. Finally, the group of SYT proteins with degenerate Ca2+ binding sites within their C2 domains are likely to participate in distinct Ca2+-independent membrane trafficking steps [Beyond the role of specific isoforms in SV fusion, SYT family members also function as Cadulators , 133–142fficking , 143–148ng steps –100. In 2+ sensor for activating fast synchronous fusion in all species examined to date. In mammals, SYT1 is the most highly expressed member of the SYT subgroup found on SVs and serves as the sole Ca2+ sensor for synchronous fusion for most neurons in the CNS. SYT2 and SYT9 share largely redundant roles with SYT1 in regulating SV fusion for a smaller population of CNS neurons and most PNS neurons [2+ sensor for fusion is widely accepted, the protein also functions in additional steps of the SV cycle, including docking, priming, and endocytosis [SYT1 is the best characterized member of the SYT family. It is found on SVs and functions as the primary Ca neurons , 101. In neurons , 149–156ocytosis , 157. Weocytosis , 158–1632+ binding mutants that act in a dominant-negative manner [Syt1 mutants has been the predominant use of the 3rd instar NMJ preparation for analysis. Although this synaptic connection is excellent for physiological studies, it requires animals to progress through earlier larval stages that last for days where compensation through homeostatic mechanisms could mask some phenotypes. In addition, reduced activity in Syt1 mutants may have consequences on synaptic development. A few studies have used the more difficult embryonic NMJ preparation where developmental defects and compensation are less likely to occur [The multi-functional nature of SYT1 has made it challenging to separate its role for specific steps in SV trafficking, as null mutations disrupt all its properties. This has been partially addressed in Drosophila using point mutants altering a single amino acid, or by rescuing null mutants with SYT1 transgenes containing mutations in one or several residues , 164–168e manner , 75, 156to occur , 75, 151to occur , 169.Syt1 mutants revealed multiple defects in SV release at larval NMJs. The first key observation was that evoked release was dramatically reduced while spontaneous mini frequency was elevated  were mutated to neutralize the negative charge and prevent Ca2+ binding. The most common approach was to generate D to N substitutions to create D1,2N and D3,4N mutants in either or both C2 domains and use these transgenes to rescue Syt1 null mutants. This approach indicated the C2B domain had a critical role in evoked release, with a near complete loss of synchronous SV fusion in C2B D1,2N or C2B D3,4N mutants [2+ binding to the C2B domain as the critical trigger for exocytosis, while C2A Ca2+ binding appeared less important. However, these results have become more difficult to interpret with the observation that mutations in the C2B Ca2+ binding pocket cause dominant-negative phenotypes in Drosophila, mice, and humans [Syt1 null mutants rescued with C2B D1,2N or C2B D3,4N display less SV fusion that the null mutant itself [2+-independent membrane interactions via the loops that potentially block fusion sites. As such, the preferential role of Ca2+ binding to C2B as the sole trigger for SV fusion requires re-evaluation. More recent mutagenesis work using different amino acid substitutions within the C2 domain loops that alter hydrophobicity of residues rather than charge indicate C2A Ca2+ binding also has an important role in SYT1 [2+ binding to both C2 domains is important for the full activity of SYT1 in triggering synchronous SV fusion. Mutations disrupting Ca2+ binding to both C2 domains of SYT1 still support SV docking and endocytosis, indicating these functions of the protein are largely Ca2+-independent [2+ binding may enhance endocytosis rates [2+ binding-defective SYT1 animals have a far greater increase in spontaneous release compared to null mutants . To date, increases in SV release probability have not been reported in mammals. Both proteins also negatively regulate spontaneous release, as mini frequency is reduced when SYT7 is overexpressed in either mice or Drosophila [Syt1/Syt7 double mutants in both species [Syt7 mutants have a larger pool of releasable SVs and a faster refilling rate after stimulation  in the C2A domain of SYT4 and SYT11. The mammalian Syt4 gene was independently identified as an activity-regulated intermediate early gene induced by seizure activity [2+-insensitive SYT isoform might have a neuroprotective role [2+, SYT4 was hypothesized to reduce SV release following excessive neuronal hyperactivity. This model assumed SYT4 was a resident SV protein like SYT1.The mammalian SYT4 protein was identified in searches for novel SYT genes prior to the advent of genome sequencing , 293. Eaind Ca2+ . The faiactivity . These tive role . By olig2+ unlike its mammalian orthologs [2+-regulated membrane trafficking is known to occur within postsynaptic compartments and can use similar vesicular trafficking components to those found in presynaptic terminals [Syt4 null mutants were generated in Drosophila [2+ sensor for retrograde signaling at NMJs [Drosophila encodes a single SYT4 family member and initial studies suggested SYT4 overexpression could reduce SV release , consistrthologs , 298. Caerminals , 299–302erminals , 303–308osophila . Animals at NMJs , 305.Syt4 mutants. Drosophila NMJs display a unique form of presynaptic plasticity following high frequency stimulation. Within a minute after strong stimulation of the motor nerve, a robust increase in the frequency of spontaneous release is observed  to the postsynaptic membrane. Syx4 mutants displayed reduced synaptic growth and a loss of activity-induced enhancement of spontaneous release [Syt4 mutants [2+-regulated manner that initiates a presynaptic response. Similar to SYT1, SYT4 interacts with known regulators of endocytosis like AP-2 [2+ channel abundance and Ca2+ release cooperativity at presynaptic AZs [Syt4 mutants, indicating the t-SNARE SYX4 supports additional postsynaptic trafficking steps independent of SYT4 in Drosophila.To identify components of the SYT4 retrograde signaling pathway that function in the postsynaptic compartment, rather than in presynaptic exosome delivery, a UAS-SYT4-pHlourin (SYT4-pH) construct was generated and expressed directly in postsynaptic cells with a muscle GAL4 driver to visualize its trafficking to the plasma membrane . A targe release , similar mutants , 305. Thike AP-2 , 292. Alptic AZs . These pDrosophila homolog of the mammalian SYX3 and SYX4 postsynaptic t-SNARE subfamily [2+ in mammals, with postsynaptic SNAREs and SYTs controlling long-term potentiation (LTP) and activity-dependent AMPA receptor trafficking [SYX4 is the sole ubfamily , 316. Pofficking , 317–326fficking , 328. Thfficking .2+ binding. Unlike the lack of Ca2+ binding by mammalian SYT4 and SYT11, Drosophila SYT4 binds Ca2+ and its C2 domain Ca2+ binding properties are essential for retrograde signaling [2+. Mutation of this residue back to an aspartate in the mammalian homologs restore Ca2+ binding, indicating the on/off nature of this interaction is tied specifically to this residue [2+ binding aspartate residues in C2A and C2B are conserved across evolution in the SYT4 family, unlike the higher divergence seen in other SYT family members that act through Ca2+ independent mechanisms. Finally, membrane organelles containing mammalian SYT4 or SYT11 can undergo Ca2+-regulated fusion [2+ that is not captured in in vitro studies. One possibility is that the serine residue in the Ca2+ binding pocket mediates lower affinity Ca2+ interactions that are difficult to detect in vitro. Another option is the serine residue is a site of phosphorylation that would reintroduce stronger negative charge in the Ca2+ binding pocket, allowing SYT4 to function in a phosphorylation-controlled manner in vivo. Mutagenesis of this serine in Drosophila has demonstrated the residue is critical for SYT4 function in vivo [2+ binding in vivo.Going forward, many open questions remain for how SYT4 regulates trafficking of membrane organelles at synapses. One major question surrounds the role of Caignaling , 298. Th residue . In addid fusion , 323. Th in vivo . The spe2+ and regulate trafficking. How SYT4-containing exosomes transit the synaptic cleft and are taken up by the postsynaptic cell is unknown. Once inside the muscle, how do SYT4 proteins transit to a specific postsynaptic compartment to regulate synaptic plasticity? What potential retrograde signaling molecules are present in these compartments? Alternatively, does SYT4 act primarily at the postsynaptic membrane to control endocytosis of trans-synaptic signaling components? Future work on this family should provide important insights into similarities between Ca2+-regulated trafficking steps in pre- versus post-synaptic compartments.Additional questions on SYT4 function revolve around the discovery that the protein is found on exosomes in Drosophila. There are no current studies indicating the mammalian homologs are present on exosomes. SYT4 positive compartments co-localize with DCV markers and BDNF in mammals , 330, whSyt genes are expressed at lower levels or in specific neuronal subpopulations in Drosophila [2+ binding residues that indicate a role as Ca2+ sensors. Antisera raised against the two proteins demonstrate they are expressed in multiple neuropeptide releasing neurons in both the CNS and PNS [2+-dependent release of neuropeptides, though genetic analysis will be required to define their function. It is unclear if the more abundant SYT isoforms  also serve as Ca2+-sensors for DCV fusion in Drosophila, given their established role for this step in many mammalian neurons. With the limited expression of Syt-α and Syt-β in specific neuronal subpopulations, and the presence of DCVs in most Drosophila neurons, it is likely the more abundant SYT isoforms also participate in DCV trafficking and fusion.Beyond the abundant SYT family members , four additional osophila , 100. Sy and PNS , 334–3362+ binding residues and are unlikely to function as Ca2+ sensors. RNA profiling and in situ analysis indicate these genes are expressed at very low levels  , 341. LEfunction . SYT2 mufunction . This grachinery , 340. Inachinery . In thisout mice .2+ binding site of human SYT1 have been identified, resulting in a severe neurodevelopmental disorder with infant hypotonia, hyperkinetic movement disorder, profound developmental delay, cognitive dysfunction, and failure to develop language [2+-binding pocket, the location of all mutations identified to date, is a privileged site for alleles that can dominantly disrupt SV release.Recently, similar heterozygote dominant mutations in the C2B Calanguage , 345. Th2+ binding residue, cause lethality when overexpressed in Drosophila [Syt1 null phenotypes, indicating the amino acid change completely abolished the ability of the protein to function. How do these mutant SYT1 and SYT2 proteins dominantly interfere with neurotransmitter release? The studies in Drosophila suggest mutant SYT alleles are likely to multimerize with endogenous SYT1 or SYT2 and disrupt the normal function of a SYT oligomer in a dominant fashion. Another model based on studies in mice suggests loss of Ca2+-dependent lipid binding is the primary mechanism [2+ binding mutants was performed in Drosophila that identified interactions required for the pathogenic mechanism [2+-independent lipid interactions through the C2B polybasic region, to dominantly poison the endogenous SV fusion machinery. These interactions likely position mutant SYT proteins near release sites where they dominantly disrupt fusion even in the presence of a normal copy of the protein. Two candidate mechanisms for the dominant effect seem most likely. In one model, a defect in the ability of the C2B domain loops to penetrate lipid bilayers upon Ca2+ influx would poison the fusion machinery by oligomerizing with the wildtype copy of SYT1. Another possibility is that the disease alleles result in neutralization of negative charge or increased hydrophobicity of the C2B loops, resulting in spurious activation of Ca2+-independent lipid interactions by the mutant protein. This could bring a SYT oligomer to membranes at inappropriate times, or act to clog release sites as a monomer only that blocks the activity of the normal allele. With genome sequencing becoming more common, it is likely more cases of SYT-mediated neurological disorders will be found given a disruptive amino acid change in the C2B Ca2+-binding pocket in a single copy can cause disease.Drosophila contains a single ortholog of the human SYT1 and SYT2 proteins, and mutations corresponding to several of the dominant-negative disease alleles have been modeled , 346. Giosophila . This muechanism . How thiechanism . This stSyt7 mice displaying manic and depression like behaviors [Syt7 mRNA, and several SNPs in the Syt7 gene have been identified in BD individuals [Beyond neurological disorders caused by SYT1 and SYT2 mutations, the remaining links to disease are still somewhat speculative and relatively understudied. Disruptions of SYT7 have been suggested as a risk factor for bipolar disorder (BD) in humans, with mutant ehaviors , 348. A ividuals , 348. SYividuals  and a roividuals , 262. A ividuals –353. SYTividuals , 354, buividuals –367. Ove


A writing system is a graphic code, i.e., a system of standardized pairings between symbols and meanings in which symbols take the form of images that can endure. The visual character of writing implies that written characters have to fit constraints of the human visual system. One aspect of this optimization lays in the graphic complexity of the characters used by scripts. Scripts are sets of graphic characters used for the written form of one language or more. Using computational methods over a large and diverse dataset , we answer three central questions about the visual complexity of written characters and the evolution of writing: (1) What determines character complexity? (2) Can we find traces of evolutionary change in character complexity? (3) Is complexity distributed in a way that makes character recognition easier? Our study suggests that (1) character complexity depends primarily on which linguistic unit the characters encode, and that (2) there is little evidence of evolutionary change in character complexity. Additionally (3) for an individual character, the half which is encountered first while reading tends to be more complex than that which is encountered last. It is aa in the Latin script is defined by a basic shape which can be used in different languages to encode different sounds, depending on the context  in which it is used. Conversely, a writing system can be written using different scripts, e.g., Serbo-Croatian.Writing systems are based on characters organized into sets, here called scripts. Characters, in our definition, are the basic symbols used to visualize (write or print) a language. A script as we define it is a set of graphic characters used for the written form of one or more languages. A script is also sometimes one of several that are used for a language's writing system. Scripts, writing systems, and spoken languages rarely overlap perfectly. For instance, the Latin script is used by a variety of writing systems, to write a diversity of languages. Scripts do not determine what writing encodes, but they determine what writing looks like. For instance, the character Written languages contrast with spoken languages. Writing is a relatively recent innovation: it can be traced back to a few invention events that occurred no more than a few thousand years ago. Writing requires, to a much larger degree than speaking, an explicit and deliberate effort in learning and transmitting it. While spoken languages extensively recruit auditory perception, written languages rely on the visual modality. This implies that written characters have to fit constraints of the human visual system . At leasVisual complexity influences performance in reading and visual discrimination tasks . Lower v1.1Script: A set of graphic characters used for the written form of one or more languages, or the “graphic form of the units of a writing system”  type: A way to categorize scripts based on the semiotics of the writing system they are used for, i.e., the linguistic unit their characters encode. Well known types include alphabets, syllabaries, logographies, etc. Here we often write “type” as short-hand for “writing system type”.Symbol/character: We use the words “symbol” and “character” interchangeably. They refer to basic symbols (shapes) used to visualize (write or print) a language.Font: A set of graphical representations of the shapes in a script. For instance, the font for the letters of the Latin script. This paper is not concerned with typographic variants, such as italic vs. regular type, or serif vs. sans-serif variants of a given script. We focus on the variation between scripts, which is considerable, rather than on variation inside scripts.1.2Two main drivers of character complexity have been hypothesized: its graph inventory size, and the type of the writing system it is associated to . The siz1.2.1Studies investigating the relation between complexity and graph inventory size have yielded conflicting results. One limitation of both studies is that they treat scripts as independent datapoints, even though distinct scripts are not independent. Scripts are related to other scripts. Many alphabetic writing systems originate from the Greek alphabet in more or less direct ways, for instance. Not accounting for common ancestry can be particularly problematic for cross-cultural data, as some of the characteristics observed in a population may be due to their common ancestry : it is necessary to account for the fact that some observations are not independent from one another . Here, w1.2.2Several arguments suggest that most of the variance in character complexity should be captured by knowing which script a character is from. First, inclusion in a given script captures many important sources of variance in character complexity that do not vary at the level of individual characters. This includes the material that the script is usually written on; the shape of the basic strokes making up the script; or general stylistic influences. Second, something like the principle of uniform information density , which g1.3Graphic complexity increases the cognitive cost of processing signals . GraphicHigher visual complexity makes visual stimuli, and thus characters, harder to memorize and to recognize  – see SeComplex drawings and scribbles are known to simplify in experimental settings, because simpler shapes take less effort both to remember and to produce. Drawings, in particular, have been among the first type of stimuli used in transmission chain experiments . TransmiIt has been suggested that changes in writing systems over time are relatively directed and that relatively iconographic or figurative variants (think Egyptian hieroglyphs [Egyp]) give rise to more abstract and simpler characters . Iconic 1.3.1If pressures for simplification drive the evolution of scripts, we can expect that idiosyncratic scripts, i.e., scripts invented de novo by illiterate inventors in the recent past, with no overwhelming influence from one single ancestor, would have had less exposure to such pressures. In turn, this predicts that the characters of recently created scripts would be more complex than those of scripts that were exposed to evolutionary pressures for a longer period of time.1.3.2Branching-out events occur whenever a script differentiates from its parent script: a large share of scripts were formed by branching out from other scripts. They did so either as independent offshoots of continuing scripts (Thaana [Thaa] from Arabic [Arab]), or as continuations of extinct scripts (Tifinagh [Tfng] or Greek [Grek] from Phoenician [Phnx]). Such branching-out events provide the opportunity to increase a script's efficiency, by simplifying its characters. If branching-out events favored an increase in efficiency, this would predict that the characters of the “parent” script would, on average, be more complex than their offspring's characters.1.4Studies of the Latin script suggest that visual complexity is not homogeneous within its letters. Latin letters tend to be “right-facing” : most ofRecent work argues that the distribution of information inside Latin letters is biased towards the left half—the first half in reading and writing order. In line with this, The contradictory nature of claims and evidence on the distribution of visual information among letter halves is partly due to the lack of a clear and robust metric for visual information. Graphic complexity, as measured here, can (in our view) be used as a proxy for visual information , providiWith rare exceptions (e.g. early Sumerian writing) reading and writing directions tend to coincide, and the direction of inscription tends to be the same between letters and inside letters: if letters are written from left to right, then the strokes inside these letters also tend to be written from left to right. There is good evidence that the strokes written first are more important in identifying a letter than those written last, at least for Chinese characters  and LatiWe expected to find that letters in scripts written and read from left to right (like Latin) are “left-heavy”: their left half would be more complex than their right half. Conversely, letters in scripts written from right to left should be found to be “right-heavy”. When testing this prediction, we must control for a possible tendency for visual information to concentrate on the left or right side of characters, independently of reading direction. Visual perception has inbuilt asymmetries between the left and right visual hemifields, at least for some domains such as face perception e.g. . WhetherWe pre-registered two predictions based on the hypothesis that visual information clusters on the side of characters that the reading eye encounters first. Visual complexity should be higher in character halves that come first in reading order: left for Left-right scripts (i.e. scripts customarily written and read from left to right), right for Right-left scripts. Our second prediction was that complexity differentials between halves are specifically predicted by order: left halves are not more complex than right halves when they come last in reading order, and vice-versa.22.1https://osf.io/9dnj3/) where all analyses carried out were pre-registered and described. Pre-registration consists in describing both the research design and analysis plan as independently as possible from data collection. Data and R scripts used to produce the results and figures can be found at https://osf.io/9dnj3/.We kept a complete research diary on the Open Science Framework website , and enriched with official proposals to encode new scripts not included in the standard, but under consideration at data collection time (November–December 2018). This study excluded the following scripts. Secondary scripts, defined by https://osf.io/9dnj3/ for an exhaustive list of exclusions and the justification for each.The inventory of scripts included in our study was compiled from the Unicode 10.0, updated according to the Unicode 11.0 . See 2.3s = 0.87, p < 0.001 (as measured on pictures of full characters).Following previous studies in cultural evolution , two mea2.3.1Perimetric complexity is defined as the ratio of inked surface to the perimeter of this inked surface . It is oThe measure was implemented in Wolfram (Mathematica) . Perimet2.3.2Algorithmic complexity measures are obtained by compressing the image file corresponding to a character . Algorithmic complexity is then the size in bytes of the compressed file: it offers an estimation of the length of the shortest computer program that produces the picture of the character without loss of information. This measure correlates well with perimetric complexity .2.4Our analyses required a standardized collection of pictures, in which the amount of variation due to the use of different fonts would be minimized, while the variation due to actual character shapes would be preserved. Among other things, fonts vary on two properties that can affect the measures of character complexity: size and line thickness.2.4.1A picture of each character was generated using a range of Unicode identifiers  and a font . The script generating the pictures (written in bash) fixed the size of the picture at 500 by 500 pixels, and an initial font size for drawing the symbols at 60. Whenever a script presented characters that would be to too big to fully fit within the 500 by 500 pixels canvas, it was rerun at a smaller point size. In such cases, we decreased point size 5 by 5, until reaching a size at which all characters would fit inside the canvas. This was necessary for only four scripts .2.4.2within each script.In order to standardize our pictures for size across scripts, we adaptively resized them. We first trimmed all the pictures. We then selected, for each script, the character with the largest picture . From this picture, we derived a ratio specifying how much it had to be resized for its largest dimension to fit a 490 by 490 pixels square (maintaining the aspect ratio and thus avoiding distortions). Finally, we used this ratio for resizing all pictures from the same script, and placed the resulting pictures back on a 500 by 500 pixels white canvas—see 2.4.3In order to obtain a collection of characters with the same constant line thickness, we used a combination of functions in Mathematica: first thinning, then pruning, and finally, dilation See .. The Th2.4.4Algorithmic complexity metrics were computed on pictures having a black foreground (black character) over a white background. Each character's picture also went through the Potrace algorithm , to elim2.4.5Each character picture in our dataset was automatically split vertically, i.e., it was cut into two halves (right and left) separated by a vertical line.To do so, we used the pictures exactly as they had been processed as described in the preceding steps. We split them in two equal-size halves, and re-padded them to an equal, constant size see . Padding2.5The variables included in our analyses were the graph inventory size of each script (the number of characters that it includes), its family (a category based on each script's geography and ancestry), its type , and whether or not the script was idiosyncratic. We classify scripts created by identifiable creators in the last two centuries, with no predominant influence from any single existing script, as “idiosyncratic”. For analyses using character-level measures, an additional grouping variable script refers to the specific script to which they belong. Whenever applicable, ancestor, i.e., which other script is considered an ancestor of the script, was also used.2.5.1Our starting point was the dataset published in 2.5.2Graph inventory size was measured as the number of unique characters included in our sample for each script. When a character exists in several possible versions depending on its position , we counted each version as one distinct character, following the Unicode Standard.2.5.3-Middle Eastern family: direct descendants of the main scripts of the Middle East .-Phoenician family .Our script family variable mostly followed the classification established by This family variable is not strictly phylogenetic: in addition to phylogenetic information in the form of ancestry , family also includes geographic information. This classification adheres to and reflects previously established conventions in reference documents seminal to the study of writing systems . A stricAs for all other variables, each script's last common ancestor was determined by pooling together information from all our sources. When sources were consistent with one another but differed in their specificity, the most specific source (citing the ancestor that was closest in time to its descendant) was chosen.2.5.4-alphabet: “the characters denote consonants and vowels”.-abjad  as well as particular syllables”. Our dataset included four such scripts: Egyptian hieroglyphs [Egyp], Chinese characters [Hani], Anatolian hieroglyphs [Hluw] and Tangut [Tang].-featural: “the shapes of the characters correlate with distinctive features of the segments of the language”. The only such script in our sample is Hangul [Hang].Based on definitions from 2.5.5Scripts were considered idiosyncratic if they fulfilled the following criteria. (1) precise information is known about their inventors . (2) There is no scholarly consensus that they derive their shape from the influence of one single identified ancestor. Most resemble no known script; others fuse many influences together so that no single dominant influence is discernible. (3) The script was invented after 1800. This definition excludes invented scripts such as Cherokee [Cher], which was invented de novo by an identifiable inventor, but nonetheless bears the dominant influence of one script .2.5.6n = 109) we used Morin's information after double-checking it against Omniglot. Scripts that were not referenced on Omniglot were double-checked with the ScriptSource website. For the remaining 24 scripts, a consensus method was applied.Direction of writing was obtained from four sources — two websites: Wikipedia and Omniglot, and two reference books . We deciFor six scripts, we found discrepancies between Morin's coding and Omniglot (or ScriptSource if the script wasn't referenced on Omniglot). In two cases we established that Morin was mistaken (Meroïtic hieroglyphs [Mero] and Meroïtic cursive [Merc]). In four other cases, disagreements between sources were important enough to conclude that the script was either not well documented, or extremely variable, with regard to direction of writing. These scripts were thus excluded: the Batak script (for Karo) [Batk], the Japanese syllabaries [Hrkt], the Linear B script [Linb], and the Old North Arabian script [Narb].33.1Size has an impact on character complexity: the more characters in a script, the more complex the characters. However, this effect depended on whether large scripts (with graph inventory size >200), including logosyllabaries (or morphosyllabaries), were included or not see .Fig. 5ScThe size variable (number of characters in a script) was used as predictor in a nested regression analysis. The scripts were grouped by family to account for shared cultural influences between distinct scripts. These families were used as the grouping variable in linear mixed models with random intercept, using the lmer function of the lme4 package for R . A null N = 47,880 characters from 133 scripts), the best null model for characters' perimetric complexity included both type and script (the latter variable nested by family) as random effects. A model adding size as a fixed effect shows larger scripts to be more complex than smaller ones . Models were refitted using maximum likelihood for comparison purposes, showing that the test model was more informative  .On the full dataset , similar to t = 1.086, p = 0.28) nor for algorithmic complexity .Using a subset comprising exclusively scripts with less than 200 characters (Most of the effect of graph inventory size seems to depend on the inclusion of a few very large systems (mostly East Asian) which also tended to have very complex characters. We thus replicated the results from both 3.2We predicted that the script to which a character belongs would account for over half of the variance in character complexity. Contrary to our prediction, the script variable accounted for less than half of the variance, and in fact type captured more of the variance in character complexity than either script or family. This was true for both perimetric and algorithmic complexity, see The intraclass correlation (ICC) was calculated on raw values for perimetric complexity measures and on log-transformed values for algorithmic complexity (in order to avoid convergence issues), using the ICC1.lme function in the “psychometric” R package . 38.57% 3.3t = 0.226, p = 0.822 for perimetric complexity; β = −0.003, 95%CI, df = 125.05, t = −0.108, p = 0.914 for algorithmic complexity), when compared to the best null model for characters' complexity . Additionally, idiosyncratic scripts were neither more nor less complex than the other scripts from the Recent Inventions family that were not idiosyncratic .Our hypothesis predicted that characters from idiosyncratic scripts would be more complex than characters from non-idiosyncratic scripts. The null model for this hypothesis thus did not include family as a random effect: random effects only included type and script. Contrary to our predictions, adding the Idiosyncratic variable did not improve the model fit, for both perimetric and algorithmic complexity measures. The test model failed to show any effect of idiosyncratic  was subtracted from the descendant's average complexity (as pre-registered). Our dataset included information on 102 branching-out events, from 29 different ancestor scripts. The most frequent parent script was Brahmi [Brah], with 25 offspring scripts. A parent script had, on average, 3.55 descendants (SD = 4.98).t = 0.563, p = 0.577). Perimetric complexity tended to increase (not decrease) with branching-out events, but this trend failed to reach significance , see When controlling for ancestry , algorithmic complexity did not seem subject to any systematic effect: no significant increase or decrease in complexity occurred with branching-out events  and algorithmic complexity (BF = 5.06) – see A Bayesian one-sample 3.4.1df = 140.67, t = 4.444, p < 0.001). Adding the script's average character complexity to our model did not make it more informative , suggesting the effect is evenly spread between high-complexity and low-complexity scripts. Visual search for potential outliers did not suggest the effect to be driven by outlier scripts. Adding script directionality to the model improved model informativeness (ΔAIC = 4), but the effect of directionality was small and non-significant , and not indicative of marked differences between Left-right scripts and Right-left scripts. First halves are more algorithmically complex than last halves, regardless of the script's average complexity and directionality.We calculated, for each character and for both measures of complexity, the first-half / last-half differential, i.e., the complexity of the first half, minus that of the last half. The first half was the half coming first in writing order, i.e., left in a script written from left to right, right in a script written from right to left. Data points were individual characters, nested by script and by family. A significantly positive intercept, meaning that first halves are, on average, more complex than last halves, controlling for family or writing direction, would confirm our prediction. This proved true for measures of algorithmic complexity . However, script directionality strongly influenced the first-half / last-half complexity differential. Our model was made more informative by the inclusion of script directionality (ΔAIC = 5), showing a higher first-half / last-half differential for Right-left scripts . Visual inspection of the data confirmed this: the first halves of characters are perimetrically more complex in Right-left scripts, but not in Left-right scripts . The order model included a clear negative effect for order , consistent with our previous results on this measure. For perimetric complexity, however, the informativeness advantage in favor of order was not large enough to be interpreted (ΔAIC = 1.3).We tested this prediction by comparing two models. Each model used character halves as data points, nested according to the character from which they were taken, the script of that character, and the family of that script. We added each half's position in reading order (first or last) as a fixed effect to the first nesting model (order model). In the second nesting model (side model), however, we included each half's laterality (left or right) as a fixed effect. For algorithmic complexity, the prediction was confirmed: the order model was more informative than the side model , this should lead it to overestimate complexity (compared to our other measure). This is a reason to doubt its reliability in the case at hand.df = 0.0004, t = 11, p < 0.0001), indicating that perimetric complexity is more likely to be higher than algorithmic complexity on right-side letter halves, controlling for order (not controlling for order does not change this result).There is also reason to believe that our measurements disagree specifically in ways that confound our predicted effect. Perimetric complexity, compared to our other measure, specifically overestimates the complexity of right-side letter halves (independently of whether they come first or last in writing order). We showed this by considering how the discrepancy between our two measures varies. The discrepancy between perimetric complexity and algorithmic complexity was calculated by normalizing each complexity measure (z-score) for each character half, then subtracting normalized algorithmic complexity from normalized perimetric complexity. Normalization is necessary to ensure that bring our two heterogeneous measures to the same scale so they can be compared. The resulting discrepancy score then served as the dependent variable in a nested regression model. The data points were discrepancy scores for letter halves, nested by letter, script, and writing system type. To these nesting variables we added two fixed effects, one for side , and one for order . The model found a clear effect of side on the discrepancy score . Both final models showed a negative and significant estimate for the effect of directionality . The complexity differential in favor of the left half is diminished in scripts written from right to left, i.e., when the right half comes first in reading order. This effect is at least as clear for perimetric complexity as it is for algorithmic complexity. Controlling for each script's overall complexity by adding it as a fixed effect did not change this result, nor did it make either model more informative .For each character , we calculated the Left-right differential, i.e., the complexity of the Left half minus the complexity of the Right half. We then built a null model that predicts this Left half / Right half differential using script and family as a nesting variable. We then included the script's directionality (Right-left as opposed to Left-right) as a fixed variable. Doing so made the model more informative for both complexity measures .Contrary to our predictions, the script that a character belonged to did not account for as much variance in character complexity as writing system type did. In previous studies, causality was usually assumed to flow from type to graph inventory size to character complexity. This assumption underpins 4.2not more complex than scripts that were exposed to evolutionary pressures for several centuries. Overall, character complexity did not decrease when parent scripts branched out into descendant scripts. We provide and discuss two possible interpretations of these results: (1) differences in the use of scripts may generate statistical noise, or (2) the decrease in character complexity occurs early and rapidly in a script's history.Overall, there was little evidence of a decrease in complexity over the long-term history of scripts. We put forward two hypotheses derived from our assumption that scripts should manifest a historical decrease in character complexity. Neither were supported. Idiosyncratic scripts were Social and cultural factors varying from context to context could have impacted the complexity of scripts' characters. Distinct scripts were used for distinct purposes, and by distinct populations. It is unclear how variation in function would have impacted complexity directly, but it implied different populations of users, ranging from trained scribes to nearly everyone in the population. Higher complexity might have been maintained more easily when scripts were used only by a specialized fraction of the population. Finally, in some cases, writing, and especially handwriting, is made to reflect social belonging, through unnecessary sophistication . NeverthAnother possibility would be that compression processes were not captured in the data we gathered and analyzed. This could be the case if the graphic simplification of characters occurred early and rapidly in the history of scripts. This is also in line with the fact that in experimental settings (e.g. ), such e4.3For algorithmic complexity, we found, as predicted, that the first halves of letters were more complex than their last halves, regardless of the direction in which the script is written (left-to-right or right-to-left). For perimetric complexity, this first-half advantage was not significant, contrary to what we had predicted. However, we had reasons to believe perimetric complexity to be biased at the level of character halves. Cutting character pictures in half creates new delineations absent from the complete picture, and perimetric complexity is by definition highly sensitive to contour lengths. Furthermore, we showed that our two measures differed in the amount of complexity they measure on the right-side and left-side of letters, quite independently of writing direction. This laterality bias confounds the original tests of our hypothesis. We avoided this bias by considering the influence of direction of reading upon the left-half / right-half complexity differential. We found a clear effect consistent with our prediction for both complexity measures. The direction of reading and writing modulates the distribution of visual complexity within characters, in the predicted direction. The complexity differential between left halves and right halves is increased in Left-right scripts, and decreased in Right-left scripts, clearly and significantly for both complexity measures.Previous studies had claimed that visual information was unequally distributed within letters, but important divergences exist between authors. The “left-heaviness” of Latin letters has often been noticed , but canInsofar as visual complexity can be interpreted as a proxy for visual information, our study confirms, on a large observational dataset, the intuition that the majority of experimental studies had gotten from studying a few scripts. The direction of reading and writing drives the distribution of complexity inside letters: left halves are more likely to be more complex when they come first.From a cultural evolution point of view, this result supports the burgeoning body of work studying the ways in which letter shapes fit subtle cognitive and perceptual biases. Interestingly, here, the cognitive bias in question is not derived from the structure of natural scenes as in , and nei4.4The current study has several limitations. First, the images we used to analyze scripts were idealizations (as any representation of a script must be). They abstract away a great deal of internal variation due to time, space, and differences between writers, etc. There is also a need to study writing systems on their own, through their own chronological trajectory, which this study was not meant to fulfil , or as the inventory of patterns or features are re-used by different characters in the same script . This main use differs from the complexity measured on their characters independently from one another. When used alongside simpler characters, higher complexity characters can benefit from a processing advantage, for instance during visual search (Third, character complexity also depends on the way characters are combined and occur in real world settings. The visual complexity of scripts l search . More coIn this study, we focused on the visual aspect of characters, and how they are perceived, i.e., how they are recognized as characters by a reader. Nevertheless, characters also have to be produced. For most of the history of writing, characters were hand-written. Although more complex characters would, overall, be more effortful to produce than simpler ones, some shapes may be easier to produce than others. Changes related to motor production may also have an impact on the graphic complexity of characters see  could ofThe complexity of written characters is a fundamental aspect of their legibility, determining their recognizability and the cognitive effort required to process them . Underst


The recognition of microbe-derived molecules activates Ca2+ channels or Ca2+ pumps that trigger a transient increase in Ca2+ in the cytoplasm. The Ca2+ binding proteins , known as Ca2+ sensors, relay the Ca2+ signal into down-stream signaling events, e.g., activating transcription factors in the nucleus. For example, CaM and CML decode the Ca2+ signals to the CaM/CML-binding protein, especially CaM-binding transcription factors (AtSRs/CAMTAs), to induce the expressions of immune-related genes. In this review, we discuss the recent breakthroughs in down-stream Ca2+ signaling as a dynamic process, subjected to continuous variation and gradual change. AtSR1/CAMTA3 is a CaM-mediated transcription factor that represses plant immunity in non-stressful environments. Stress-triggered Ca2+ spikes impact the Ca2+-CaM-AtSR1 complex to control plant immune response. We also discuss other regulatory mechanisms in which Ca2+ signaling activates CPKs and MAPKs cascades followed by regulating the function of AtSR1 by changing its stability, phosphorylation status, and subcellular localization during plant defense.Calcium (Ca This letrations , 2021. C toolkit  e.g., Cars (TFs) .2+ concentrations are an early event during immune responses, where the Ca2+ channels and Ca2+ pumps are activated to form specific Ca2+ signatures to each stimulus , e.g., botrytis-induced kinase 1 (BIK1), activate the Ca2+ channel through the phosphorylation of CNGC4, but not CNGC2. In addition, BIK1 also phosphorylates CaM7 that, in turn, binds to the IQ motif in CNGC2 and CNGC4 to suppress the activity of the heteromeric Ca2+ channel , which functions as an extracellular H2O2 receptor and is required for H2O2-induced Ca2+ rise (2+-permeable channel to trigger the programmed cell death (PCD)  or Arabidopsis thaliana signal responsives (AtSRs) could be a sensor of the Ca2+ burst  . Another not Cl– . These cated PCD . Most st2+ burst  as descr2+ changes, and plays a suppressor role in the plant immune system  and reactive oxygen species (ROS) concentrations, and enhanced resistance to bacterial and fungal pathogens , non-race-specific disease resistance1 (NDR1), and non-expresser of PR genes1 (NPR1) to modulate their expression in SA-mediated plant immunity (isochorismate synthase 1 (ICS1) to suppress plant immunity at both 20oC and 28oC , and the CaMBD binds to Ca2+-bound CaM . CaM binding to AtSR1 is essential for the function of AtSR1 . Yuan etand NDR1 . Given tound CaM , AtSR1 aresponse .camta1/2/3 and the transcriptional expressions of agd2-like defense response protein 1 (ALD1) and flavin-dependent monooxygenase 1 (FMO1), which both encode two pip biosynthesis enzymes, were greatly induced in camta1/2/3. Moreover, CBP60g and SARD1 were identified to regulate Pip production by ALD1 and FMO1; also, AtSR1 protein interacted with the CGCG box in the promoter of calmodulin binding protein 60g (CBP60g), but not that of SAR deficient 1 (SARD1), to repress the CBP60g expression  crosstalk to mediate plant immune response. This idea is supported by the observation that the application of Pip to plant leaf promoted NPR1 stability -mediated plant immune response . The biopression . In addi as well . These otability . How AtScis-regulatory element was highly enriched in the eATP-responsive promoters (RNA-dependent RNA polymerase 6 (RDR6) and bifunctional nuclease-2 (BN2) to induce the transcriptional expression of RDR6 and BN2, respectively. RDR6 is known to convert single-stranded (ss) RNA into double-stranded (ds) RNA to induce RNA silencing  employs an effector protein, V2, to impair the interaction between CaM3 and AtSR1 to suppress plant defense  . The flgvia the truncated NLR TN2, where CPK5 is required for this TN-mediated immunity (camta3 number 1 (DSC1) and DSC2 directly interacts with AtSR1, where DSC1 and DSC2 guard the guardee AtSR1. Upon infection of avirulent Pst strains carrying avrRps4 and avrRpt2, a rapid degradation of AtSR1 is induced probably following phosphorylation and/or ubiquitination of AtSR1. The degradation of AtSR1 contributes to the activation of downstream immune responses by de-repression of defense-related genes, e.g., EDS1 and NDR1  is partially suppressed in the cdk8 null mutant. Further study revealed that CDK8 regulates SA biosynthesis genes, such as ICS1 and EDS5, probably via AtSRs-mediated transcriptional reprogramming, although a detailed mechanism of how CDK8 regulates AtSRs remains to be studied  is required for most transcriptions of general protein-coding genes and several non-coding RNA (ncRNAs) genes . The med studied . Another studied . It is iIAA1 and IAA19 to regulate their expressions, and regulates DWF4 expression through binding to its promoter  of AtSR1 suppresses its negative function in plant immunity, however, the molecular mechanism of recovery of AtSR1 after successful prevention of pathogen infection needs to be addressed. There are several major questions: (1) The role of AtSR1 in plant growth remains unclear  How AtSR1 crosstalks with hormonal pathways, such as gibberellins (GAs), brassinosteroids (BRs), ethylene (ET) and auxin is not understood.In recent years, a great deal of progress has been made in understanding Ca unclear . (2) Fur2+/CaM-mediated signaling plays a unique role in both pathogenic  and symbiotic interactions [Ca2 +/CaM-dependent protein kinase (CCaMK)] in plants. It is well recognized that CCaMK, a Ca2+/CaM-binding protein (Since the focus of this special issue is on signaling in plant biotic interactions, it is appropriate to point out that Ca protein , plays a protein . HoweverPY, KT, and BP were involved in writing this review. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Upon stimulation with extracellular ATP, these cells displayed a concentration-dependent increase in intracellular Ca2+, which was mediated by release from intracellular stores. SiNPs inhibited the Ca2+ responses to ATP within minutes of application and at low micromolar concentrations, which are significantly faster and more potent than those previously reported for the induction of cellular toxicity and pro-inflammatory responses. SiNPs-induced inhibition is independent from the increase in intracellular Ca2+ they produce, is largely irreversible and occurs via a non-competitive mechanism. These findings suggest that SiNPs reduce the ability of airway epithelial cells to mount ATP-dependent protective responses.Because of their low cost and easy production, silica nanoparticles (SiNPs) are widely used in multiple manufacturing applications as anti-caking, densifying and hydrophobic agents. However, this has increased the exposure levels of the general population and has raised concerns about the toxicity of this nanomaterial. SiNPs affect the function of the airway epithelium, but the biochemical pathways targeted by these particles remain largely unknown. Here we investigated the effects of SiNPs on the responses of 16HBE14o- cultured human bronchial epithelial (16HBE) cells to the damage-associated molecular pattern ATP, using fluorometric measurements of intracellular Ca Nanoparticles (NPs) designate small-sized natural or engineered particulate matter with dimensions of less than 100 nm in at least one dimension and physical and chemical properties that differ from those of the bulk material. They are composed of a wide range of materials, such as metals, inorganic carbon, and organic compounds. In the last decades, NPs have gained popularity in many industries, as their unique properties make them suitable for use in cosmetics, biotechnology, food, pharmaceutical and chemical industries ,4,5,6,7.Human exposure to NPs has increased considerably since the nano-technological revolution. Their small dimensions and unique physico-chemical properties facilitate the ability of nanoparticles to cross different barriers and to reach distal organs ,10,11,12Besides its role in gas exchange, the airway epithelium protects the body from foreign substances. The cells lining the respiratory tract have key roles in mucociliary clearance and contribute to the initiation of airway inflammation ,26,27,282+ homeostasis, disrupt the plasma membrane upon uptake and alter membrane fluidity and integrity . In contrast, activation of P2Y receptors activates inositol triphosphate (IP3)-mediated signaling, which opens IP3 receptor Ca2+ channels in the membrane of endoplasmic reticulum (ER), leading to Ca2+ release from these stores  recordings in all series of experiments, indicating that no major changes in cell functionality leading to compromised plasma membrane integrity underlie the observed acute inhibitory effect of SiNPs on ATP response.Using this experimental paradigm, we found that SiNPs reduce the response to ATP in a concentration-dependent manner. This inhibitory effect was not only observed in the presence of the SiNPs, but also found to persist and to be actually stronger up to 35 min after the removal of SiNPs from the extracellular solution . Future 2+ signals detected with Fura2. However, this mechanism can be discarded, as it is expected to operate for all intracellular Ca2+ signals, and we have previously demonstrated that the SiNPs actually enhance the response of TRPV1 to the agonist capsaicin , respectively.Alternatively, it could be envisaged that SiNPs inhibit the Caembranes , and thiembranes . This maHowever, the hypothesis considering that SiNPs directly target components of the ATP signaling pathway has a limitation. The cellular concentration of SiNPs and thereby any putative direct effect on a signaling component would be expected to decay, not to increase, upon washout. Thus, it remains difficult to explain why the concentration dependencies of the SiNPs action show a more potent effect on the ATP responses during washout than during the application of the particles.2+ emerged as a candidate factor to be involved, as we observed that SiNPs induced such effect in a rather irreversible manner, in agreement with our previous report [2+] induced by the particles. Furthermore, the experiments in which we used ionomycin revealed that cells undergoing intracellular Ca2+ overload were able to respond, albeit more slowly, to a first application of ATP. Taken together, our data strongly indicate that the increase in Ca2+ induced by the SiNPs is not the primary factor underlying the decrease in the responses to ATP.A more plausible explanation is that the SiNPs trigger an inhibitory mechanism that outlives and is further enhanced after the ceasing of the accumulation of the nanoparticles in the cellular compartments. In this direction, the increase in intracellular Cas report . However2+ response, but do not act on any factors determining the sensitivity to ATP. For instance, SiNPs may decrease the number of ATP receptors available for activation at the plasma membrane and/or their maximal level of activation, but do not decrease their affinity for ATP. The latter is informative, as it indicates that SiNPs do not interfere with the binding and unbinding of ATP to and from P2Y receptors. Furthermore, because we assessed the responses to ATP from the amplitude of the Ca2+ increase, none of the events that determine the sensitivity to ATP may be affected. Future experiments, complemented by mathematical modeling, may help in discerning what elements of the pathway may be affected or not by the SiNPs.Although our present experiments did not allow pinpointing a precise target of SiNPs action, they did let us conclude that SiNPs act via a non-competitive inhibitory mechanism. Thus, these particles may affect any of the elements of ATP signaling cascade that determine the maximal intracellular CaAnother curious result was that the inhibitory effect of SiNPs appeared to reach a plateau at concentrations above around 10 μg/mL, indicating that they are unable to inhibit the responses to ATP completely when applied at concentrations up to 100 μg/mL. Interestingly, we previously found a qualitatively similar effect for the inhibition of TRPV4 in 16HBE cells, but the plateau for maximal inhibition was reached at concentrations higher than around 300 μg/mL  was calculated from the ratio of fluorescence measured upon alternating illumination at 340 and 380 nm. Experiments were performed using an inverted microscope with MT-10 illumination system and the xCellence Pro software of the microscope . All measurements were performed at 35 °C. Fluorescence intensities were corrected for background signal, and intracellular Ca2+ concentrations were calculated as described previously [Caeviously . Data we2+] prior to the application from the peak value reached during challenging . The baseline was calculated by determining the mean [Ca2+] during the last 20 s before the application of the compound. Data are given as ± standard error of the mean.To determine the amplitude of the response induced by the compounds of interest, we subtracted the baseline [Ca2ATP stock solution to the corresponding extracellular solution (standard Krebs or Ca2+-free Krebs). Ionomycin, an ionophore that triggers Ca2+ influx, was used to show that the effect observed during the application of SiNPs is not calcium-mediated and thus is a result of the interaction of SiNPs with subcellular structures. Ionomycin at 1 μM concentration was obtained by dilution of 2 mM stock in Krebs. Solutions used in measurements performed in the absence of extracellular Ca2+ were prepared with Krebs titrated to pH 7.4 with NaOH . The solutions containing ATP were prepared by adding the appropriate amounts of a 50 mM Naith NaOH .


Biomaterial-associated infections and reactions between implants with human cells have become a standard clinical concern. In this study, a green synthesized βW, synthesized from rice husk ash and a calcined limestone precursor, was incorporated with mullite, maghemite, and silver to produce β wollastonite composite (βWMAF) to enhance the tensile strength and antibacterial properties. The addition of mullite to the βWMAF increased the tensile strength compared to βW. In vitro bioactivity, antibacterial efficacy, and physicochemical properties of the β-wollastonite and βWMAF were characterized. βW and βWMAF samples formed apatite spherules when immersed in simulated body fluid (SBF) for 1 day. In conclusion, βWMAF, according to the tensile strength, bioactivity, and antibacterial activity, was observed in this research and appropriate for the reconstruction of cancellous bone defects. However, their widespread application is limited because they do not meet several of the regenerative needs of bone tissue and practical needs related to handling and surgery. There are potential and growing applications of bioactive calcium silicate for commercial bone replacement, such as in dentistry , load-be2) , and tit2) [2) . The jus2) [2) . The use2) [2)  and tita2) [2) . Using m3O4) and maghemite (γ-Fe2O3), the two typical forms of magnetic nanoparticles, are most suitable in biomedical science and engineering due to their non-toxic property, biocompatibility, high surface to volume ratio, ability to excellently carry a broad range of biological ligands, and existence of natural routes for its degradation [Iron compound nanoparticles or magnetic nanoparticles have an essential aspect in biomedical engineering and biology. Magnetite  and Staphylococcus aureus (S. aureus) microbial growth inhibition, has been widely studied [Currently, antimicrobial activities of the silver ion have been reported against both Gram-positive and Gram-negative bacteria, protozoa, fungi, and certain viruses . Silver  studied ,31,32. M studied ,34 and c studied ,36 doped studied . Another studied .This research aims to create a green synthesis of β-wollastonite obtained from rice husk ash and calcined limestone incorporated with mullite, maghemite, and silver nanoparticles to test its tensile strength, magnetic properties, bioactivity, and antibacterial potential.2) source from fired rice husk was obtained from a rice mill industry in Penang, Malaysia. Meanwhile, calcium oxide (CaO) was collected from a nearby limestone quarry in Perak, Malaysia. The β-wollastonite composite (βWMAF) consists of 20 wt.% of mullite, 5 wt.% of maghemite (γ-Fe2O3), and 5 wt.% silver (Ag). Mullite was obtained from the calcination of andalusite for 8 h at 1450 °C and collected from a mining quarry in Terengganu, Malaysia. Mullite selection of 20 wt.% refers to a previous study by Farah et al. [Staphylococcus aureus (S. aureus) ATCC11632 and Gram-negative Escherichia coli (E. coli) ATCC10536 were used for antibacterial activity evaluation in the antibacterial study. A culture agar  and a culture broth  were used as a media cultivation nutrient. The maghemite nanoparticle was self-synthesized using the co-precipitation technique, and the raw materials were used as anhydrous iron (II) chloride  and anhydrous iron (III) chloride , supplied by Sigma-Aldrich  was synthesized from agricultural and natural rice husk ash and limestone sources. The silica ; the CaO:SiOus study ,41.To produce the βWMAF sample, the mullite powder, maghemite, and silver were milled using ball milling at 250 rpm for 15 min to obtain the homogenous mixture. The βW as a control sample and βWMAF mixture were pressed under 2.5 tons to get a disc (12.0 ± 0.1 mm diameter × 2.0 ± 0.1 mm thickness).The phase identification of βW, mullite, maghemite, silver, and βWMAF was detected via X-ray diffraction  using Cu Kα radiation (λ = 0.15406 nm) with a step size of 0.025°/step, a counting time of 0.1 s, and the Bragg’s angle ranging from 20–70°. The morphology and elemental analyses were examined using a field emission scanning electron microscope  and an energy dispersive  spectrometer, and the samples were platinum sputter-coated before analysis. The microscope was operated at a voltage of 3.0 kV.The diametral compression or Brazilian disc test is an excellent way to measure the tensile strength of brittle materials, such as ceramic and biomaterials . Theory t = 2P/πDT, where P is the peak load (N), D is the diameter (mm), and T is the sample thickness (mm).In this experiment, the diametral tensile strength of the βW and βWMAF was evaluated using the Brazilian test, referring to the ASTM C496 standard . The len3 and 1.4 × 103 Oe with a 0.1 emu sensitivity.Magnetic properties were evaluated using a vibrating sample magnetometer  βW and βWMAF powders. The external magnetic field vibration could be applied both in the transverse and longitudinal directions of the vibration. Each sample’s magnetic moment was measured over a range of magnetic fields applied between −1.4 × 10The ability to form apatite on the surface of the βW and βWMAF discs was tested using an SBF solution whose ionic composition is similar to that of human physiological body fluid. Generally, βW and βWMAF discs were soaked in 30 mL of SBF solution for 1, 7, and 14 days in a polyethylene bottle and retained in an incubator at 37 °C, referring to the procedure by Kokubo and Takadama . Every tTo evaluate the ion dissolution from βW and βWMAF discs, the SBF was prepared according to Kokubo’s method . The βW Escherichia coli ATCC10536 and Staphylococcus aureus ATCC11632, the antimicrobial activity was carried out using a disc diffusion method using Mueller Hinton Agar (MHA). All the specimens and apparatus were sterilized in an autoclave at 121 °C for 15 min), and all the tasks were inside the laminar airflow chamber. The bacteria cultures were prepared in nutrient broth  at a concentration of~1.5 × 108 CFU/mL corresponding to the density of 0.5 MacFarland. Next, under sterilized conditions, E. coli and S. aureus cultures were swabbed on the agar petri dish using a sterile cotton swab. The βWMAF disc was placed on the agar, followed by the βW disc as negative control and Ag disk as a positive control. These petri discs were incubated at 37 °C for 24 h. Subsequently, the zone of inhibition (ZOI) was calculated in millimeters (mm) for duplicate specimens for each sample.Using The bioactivity test, magnetization test, antibacterial test, and five samples for diametral tensile strength were duplicated. For n = 2 and 5, all data were expressed as means and standard deviations.2O3), (b) mullite, (c) silver, (d) βW, and (e) βWMAF powders. The prominent peak of each raw material was an exhibit at βWMAF (2O3) phase was observed at theta 35°, for mullite at 26.1°, silver (Ag) clearly at 38.2°, and most of the peak of βWMAF was dominant by β-wollastonite phase (β-CaSiO3) at 30°, 50.2°, 53.3°, and 39.1°. Referring to previous reports, amorphous CaSiO3 has a faster apatite-formation capacity owing to the accelerated release of Ca ions relative to alpha-wollastonite (α-CaSiO3) and beta-wollastonite (β-CaSiO3) [at βWMAF e. The pr-CaSiO3) . ConsequThe micrograph of the βW and βWMAF discs are presented in The addition of mullite to the βWMAF increased the DTS compared to βW. The maximum strength of the βW sample in the absence of reinforcement was 0.69 ± 0.13 MPa. The addition of 20 wt.% mullite as reinforcement to the βWMAF increased the strength to 2.39 ± 0.25 MPa, and it was appropriate for cancellous bone implant or scaffold with a compressive strength between 2–12 MPa . Farah e2O3), β-wollastonite (βW), and βWMAF powders at room temperature are shown in −1 at 25 kOe, because the combined Fe ions formed a magnetite structure. Similarly, almost no hysteresis loops were found on the curves of the βWMAF, indicating superparamagnetic behavior. Further research needs to investigate βWMAF properties due to the heating ability, specifically, using βWMAF as thermoseeds for hyperthermia treatment of solid cancer tumors under an alternating magnetic field.Magnetization curves of the maghemite (γ-Fe3) phase (ICDD no. 00-043-1460), as shown in Meanwhile, for βWMAF, some phases were detected, such as β-wollastonite, mullite, maghemite, and silver, before soaking in the SBF for 14 days b. The β-2+) and silicon (Si4+) ions released by the samples entered the SBF solution as they were immersed in it. The H+ ions replaced the Ca2+ ions in the samples in the SBF solution as the samples were immersed in the solution, resulting in the formation of silanol (Si–OH) on the surface and an increase in the pH of the SBF solution [43−) ions reacted with Ca2+ ions in the solution to form the HA phase on the sample [10(PO4)6(OH)2):The calcium  absorption of βW and βWMAF samples were responsible for the 1419.23 and 1420.03 cm−1 bands. Previous research has shown that the bioactive process began between the 1008.50 and 930.76 cm−1 bands, as evidenced by the presence of non-bridging oxygen-stretching modes [−1 for the βWMAF sample, higher than the βW sample, which also occurred in stoichiometric apatite [ samples ,50. Befoples [−1 ,52. β-wong modes . These b apatite . This fi4+, Ca2+, Fe3+, Ag+, H+, OH−, and HPO42−. A day after soaking, the pre-nucleation aggregates in the arrangement’s spherule shape were in balance with the ions in the SBF solution, as revealed in 3+, Fe3+, and Ag+ in βWMAF. The factor may cause the crystal growth on the spherules surface Ca/P ratio, which was 2.25 (βW) and 1.76 (βWMAF) upon 14 days of immersing , the disc diffusion method was applied by using bacteria . The inh E. coli .However, no inhibition zones were found on βW, implying that βW lacked bactericidal or bacteriostatic activity. The most common cause of implantation infections that resulted in implant failure was bacterial adhesion to the implant material. Several studies on metal ion-based modification by loading antibacterial agents such as Cu, Ag, and Zn were published to improve silicate bioceramic antimicrobial activity . This ioE. coli and S. aureus. In vitro bioactivity findings revealed that apatite crystals were close-packed and fragile, with a quicker growth rate on βWMAF than on βW. The most crucial antimicrobial analysis indicated that βWMAF samples were antibacterial against E. coli and S. aureus. Furthermore, cytotoxic assays of the βW and βWMAF must be critically characterized. Another issue is creating a βWMAF bioceramic composite with strong antibacterial characteristics and low cytotoxic risk. As a result, further changes to the fabrication to improve crystallinity and reduce the dissolution of Fe and Ag during the early stage may render the bioceramics composite non-toxic to human cells. As a result, the current study findings provide new insights into the first step of future complex work to fabricate magnetic antibacterial β-wollastonite bioceramic composite scaffolds for bone regeneration. Development of calcium and silica-based material biowaste further improves the significant advantages in biomaterial applications.The magnetism, bioactivity, and antibacterial activity of βW and βWMAF synthesized from locally available materials, such as rice husk ash (RHA) and limestone ash, were investigated. The inclusion of 20 wt.% mullite into the βW increased the compressive strength up to 200%, which is appropriate for the cancellous bone application; moreover, adding 5 wt.% Ag into the βW produced an excellent antibacterial property against