Vascular smooth muscle cell (VSMC) proliferation plays a critical role in atherosclerosis. At the beginning of the pathologic process of atherosclerosis, irregular VSMC proliferation promotes plaque formation, but in advanced plaques VSMCs are beneficial, promoting the stability and preventing rupture of the fibrous cap. Recent studies have demonstrated that microRNAs (miRNAs) expressed in the vascular system are involved in the control of VSMC proliferation. This review summarizes recent findings on the miRNAs in the regulation of VSMC proliferation, including miRNAs that exhibit the inhibition or promotion of VSMC proliferation, and their targets mediating the regulation of VSMC proliferation. Up to now, most of the studies were performed only in cultured VSMC. While the modulation of miRNAs is emerging as a promising strategy for the regulation of VSMC proliferation, most of the effects of miRNAs and their targets in vivo require further investigation. Atherosclerosis is the main cause of cardiovascular diseases (CVDs), which are a leading cause of death worldwide ,2. AtherAberrant VSMC proliferation represents a significant problem not only during initial plaque formation but also after surgical interventions like percutaneous transluminal coronary angioplasty or bypass surgery contributing to pathological re-narrowing (restenosis) of the affected blood vessel . RestenoRecent studies have demonstrated that microRNAs (miRNAs) are expressed in the vascular system and are involved in the control of VSMC proliferation ,16. EachSome miRNAs have been implicated in regulating important VSMC nodal regulators such as cytokines/growth factors and their receptors , insulin-like growth factor-1 (IGF-1)), regulators of cell cycle progression , a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1)), signaling cascades -mediated VSMC signaling pathways, extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinases (MAPK), TGF-β signaling cascades (Smad), signal transducer and activator of transcription (STAT)), transcription factors , Krüppel-like factor-4 (KLF4)), nuclear receptors , neuron-derived orphan receptor 1 (NOR1)), and others , mammalian target of rapamycin (mTOR)). This minireview briefly summarizes recent findings on the regulatory effects of miRNAs on VSMC proliferation, including both miRNAs that are associated with inhibition or promotion of VSMC proliferation, see IGF-1 gene [It was shown that the increase of miRNA-9 inhibited the serum-induced proliferation of VSMCs by directly targeting the PDGF receptor (PDGFR) disrupting downstream signaling cascades . FurtherF-1 gene . It was F-1 gene . In addi1/S cell cycle transition by binding to the 3′-UTR of ADAMTS1 and decreasing the levels of its mRNA and protein expression [It was reported that miRNA-22 inhibited VSMC proliferation and migration through repressing ecotropic virus integration site 1 protein homolog (EVI-1) gene expression , which ipression . Furtherpression . In addipression . Increaspression . There apression . The celpression . The deg1 stage by directly decreasing AKT2 (AKT serine/threonine kinase 2) protein expression [There are some miRNAs regulating signaling cascades in VSMCs. It was shown that the overexpression of miRNA-15b/16 promoted SMC contractile gene expression while attenuating VSMC proliferation by repressing the potent oncoprotein Yes-associated protein (YAP). Knockdown of endogenous miRNA-15b/16 in VSMCs attenuated VSMC-specific gene expression and promoted VSMC proliferation and migration . A diffepression . The ovepression . The trapression . Anotherpression . The sigpression . The infSp-1) gene and then repression of transcription factor Sp-1 expression [Some miRNAs have been implicated in the regulation of transcription factors. It was demonstrated that miRNA-15a, which is upregulated in VSMCs treated with a transcription factor KLF4, can strongly inhibit the proliferation of VSMCs . Some stpression . MiRNA-1pression . Anotherpression . Additiopression . miRNA-1pression . Furtherpression . Some stpression ,42. Defipression ,91. Adenpression , inhibitpression . It has pression . Furtherpression . Transcrpression . The miRSome miRNAs have been implicated in regulating nuclear receptors in VSMCs. It was reported that the overexpression of miRNA-152 decreased cell proliferation in LPS-treated VSMCs by downregulating DNA methyltransferase 1 (DNMT1) and subsequently decreasing the methylation of a nuclear receptor ERα gene promoter region . AdditioSome observations suggest that the induction of miRNA-1 by myocardin led to an inhibition of VSMC proliferation by downregulation of Pim-1, which is also named as serine/threonine-protein kinase Pim-1 ,24. It wIt has been demonstrated that miRNA-26a promoted VSMC proliferation by directly targeting Smad1 and Smad4, which are two TGF-β- and BMP-related pro-differentiation factors . VSMCdef1/S transition, and increased levels of proliferating cell nuclear antigen and E2F1 by directly targeting the retinoblastoma (RB) protein mRNA-3′-UTR and then suppressed the expression of RB [It was reported that miRNA-17 stimulated the proliferation of VSMCs, enhanced cell cycle Gon of RB . In addion of RB . It was on of RB . Transfeon of RB . The hypon of RB , and groon of RB . One stuon of RB . Expresson of RB . The proon of RB . Reduction of RB . MiRNA-2on of RB . Furtheron of RB . It is son of RB . So far,It has been shown that overexpression of miRNA-133 inhibited VSMC proliferation in vitro through an ERK1/2 kinase-dependent pathway and increased expression of Sp-1 . FurtherUbc9 and KLF4), which further repressed miRNA-200c levels and accelerated VSMC proliferation [A study suggested that the increased amount of miRNA-29a enhanced VSMC proliferation and promoted atherogenesis, probably through downregulation of Fbw7 (F-box and WD repeat domain-containing 7)/CDC4 (cell division control protein 4) expression by targeting the 3’-UTR of Fbw7/CDC4, subsequently increasing a transcription factor KLF5 stability by reducing the Fbw7/CDC4-dependent ubiquitination of KLF5 . In addiferation . All theZDHHC14 (Zinc Finger DHHC-Type Containing 14) gene [In VSMCs cultured in the presence of the proliferation stimulator PDGF-BB, miRNA-29b was upregulated significantly . miRNA-214) gene . Overexp14) gene . The tarSome studies suggest that the function of miRNA-21 is likely to be complex and highly context-dependent. In addition to promoting contractile gene expression, miRNA-21 has been found to promote VSMC proliferation . MiRNA-2ERα gene, Cx43, NCKAP1, ADAMTS1, cyclin D1, IGF-1, AKT2, Notch1 gene, PDGFR downstream signaling cascades, Ang-II-mediated VSMC signaling pathways, TGF-β signaling cascade , ERK1/2, p38MAPK, Wnt4/Dvl-1/β-catenin signaling pathway, Syk/ STAT3-5 axis, DNMT1, MST2, Raf-1, MEK, eNOS, Cdc42, cyclin D1, FGF1, INSR, NOR1, Jun B/myosin light chain 9, mTOR, KRAS, LOX-1 and MEKK1/ERK/KLF4 signaling. The targets modulated by miRNAs implied in increased proliferation of VSMC include the RB protein, NF-κB p65, CDK6, TGF-β- and BMP-related pro-differentiation factors (Smad1 and Smad4), Fbw7/CDC4, KLF5, SIRT1, CREG, CDKN1A, GAX, Sp-1, ERK1/2 kinase-dependent pathway, SIRT1, KLF4, p21, Ubc9, CKI p27Kip1, p57Kip2, and c-kit, ZDHHC14, PTEN, Bcl-2, and PDCD4. The majority of the studies up to now were performed only in cultured VSMC. The effects of these miRNAs and their targets in vascular muscle tissue in vivo need to be further investigated. Furthermore, there is one open question on the specificity of miRNA action for VSMCs. To this end, there are some tools (such as TissueAtlas [https://ccb-web.cs.uni-saarland.de/imota/)) that have been generated and can be used to check whether the miRNAs and their target genes are expressed in specific tissues [Extensive research during the last decade investigated the association of miRNAs with VSMC proliferation. In this minireview, we briefly summarize the miRNAs that regulate VSMC proliferation. These miRNAs exert their regulation of VSMC proliferation by regulating a number of target proteins and signaling cascades. The targets modulated by miRNA implied in the inhibition of VSMC proliferation include PDGFR, KLF4, YAP, EVI-1, HMGB1, Cyclin D1, p21, Sp-1, S100A4, SRF, LRP6, PAPP-A, myocardin, ELK-1, sueAtlas  and IMOT tissues . Additio


Emerging applications demand that bolometers work at room temperature, while maintaining high speed and sensitivity, properties which are inherently limited by the heat capacity of the detector. To this end, graphene has generated interest, because it has the lowest mass per unit area of any material, while also possessing extreme thermal stability and an unmatched spectral absorbance. Yet, due to its weakly temperature-dependent electrical resistivity, graphene has failed to challenge the state-of-the-art at room temperature. Here, in a departure from conventional bolometry, we use a graphene nanoelectromechanical system to detect light via resonant sensing. In our approach, absorbed light heats and thermally tensions a suspended graphene resonator, thereby shifting its resonant frequency. Using the resonant frequency as a readout for photodetection, we achieve a room-temperature noise-equivalent power (2 pW Hz Bolometers are highly sensitive instruments that can detect radiant energy. Here, authors report micro-bolometers based on suspended graphene nano-electromechanical membranes that can detect light at room-temperature with a NEP coefficient of 2 pW/Hz^1/2 and bandwidth up to 1.3 MHz. Emerging applications3 in scientific and thermal imaging, remote environmental monitoring, THz communication, solar probes, and terahertz communication coupled with the need for increased portability demand that future bolometers work at room temperature and push the limits of speed  and sensitivity , while also maintaining a large spectral BW. A common method to modify the speed and sensitivity is to change the thermal resistance between the bolometer and its environment1. However, both the speed and sensitivity are inversely proportional to the thermal resistance, so a sensitive bolometer is often slow.The bolometer is an essential tool used to detect massive energetic particles and electromagnetic radiation. A primary benefit of the bolometer is its ability to detect light deep into the infrared4, because it has the lowest possible heat capacity per unit area of any material. Moreover, graphene possesses an ultra-broadband spectral absorbance6 and is thermally stable up to at least 2600 K (ref. 7), so a graphene bolometer could detect electromagnetic radiation of nearly any wavelength and withstand high operating temperatures. However, graphene has performed poorly in conventional bolometry8—where the electrical resistance serves as the readout for absorbed power—because its electrical resistivity is relatively insensitive to temperature9. Although graphene has shown promise in hot-electron bolometry14, in which a weak electron–phonon interaction generates a thermally insulated electron gas with a low electronic heat capacity, these implementations require cryogenic temperatures and lack portability.The speed-sensitivity trade-off can be evaded by decreasing the bolometer heat capacity, as the speed is also inversely proportional to heat capacity. Being just one atom in thickness, graphene offers a tantalizing prospect for ultrasensitive and ultrafast bolometry15, absorbed power is measured by monitoring changes in a miniaturized mechanical structure, such as the deflection of a microbeam16. In our GNB  elastic modulus, f0 is the initial frequency, and ΔT is the temperature change , the ΔT is amplified by the thermal resistance (RT), as determined by Fourier’s law ΔT = PabsRT. The RT of suspended graphene is abnormally large19, but to enhance RT further, we patterned the suspended graphene20 into a trampoline geometry22 with narrow, tapered tethers . In nanomechanical bolometryane Fig.  , as shown in Fig. 24 by applying an a.c. voltage between the graphene and the backgate , Young’s modulus (Y), and initial stress (σ0). We track the frequency during light illumination with frequency modulation detection26, which uses a phase-locked loop (PLL) with the phase locked on resonance. A key advantage of using frequency modulation is that the GNB response BW is not determined by the resonance linewidth, as it is with amplitude modulation detection. The PLL BW allows tracking up to ~50 kHz. For frequency-shift measurements, we maximize the signal-to-noise ratio in several ways. First, we use the scanning interferometer to obtain a 2D spatial map of the vibrational amplitude of the resonator. A map for a trampoline  of the GNB is calculated with the expression f is the frequency noise, t is the measurement time, and Rf is the frequency-shift responsivity , defined as Rf, we illuminate the GNB membrane with an amplitude-modulated heating laser (532 nm) and measure f0 with a PLL. A time recording of f0 when the GNB is exposed to sinusoidally modulated light is shown in Fig. Pabs = 4.4 nW. Here we assume the absorption is 2.3% of the incident power12. The shift Δf0 is inferred from a sine fit  and therefore f0 was observed in all GNB devices. In Fig. w) for nine different trampolines and three different drumheads; for drumheads, w is given by one-fourth the circumference. The trampoline width (w) is indicated in Fig. w ranging from 200 nm to 1.4 µm and GNBs with a d of 6 and 8 μm. In general, the drumheads had Rf values about 1% that of trampolines. Our most sensitive device, a 6 µm diameter trampoline with 200 nm wide tethers, had Rf ~ 300,000 W−1, a factor 100 greater than state-of-the-art nanomechanical bolometers15. As seen from Fig. Rf increases with smaller w for trampolines.The noise-equivalent power  and not by thermomechanical noise28. In this case, the frequency noise is not generally reduced with a larger quality factor30.As a measure of the fractional noise, σlate see  from temlate see . RepreseRf and the Allan deviation (measured at 100 Hz), we calculate the noise-equivalent power η for each device and plot η vs. w, shown in Fig. η decreases with narrower tether width. A trampoline with a tether width of 200 nm exhibited the best power sensitivity, η = 2 pW Hz−1/2 (at 1 kHz BW), which is also the lowest reported value of noise-equivalent power for a room-temperature graphene bolometer to date12. The η is much larger for drumheads; the largest value (η ~ 1 nW Hz−1/2) is over 200 times greater than the most sensitive trampoline. From these trends, it is clear that reducing the tether width provides a straightforward means to lower, and thus improve, the GNB’s η.Combining η assumes 2.3% absorption. However, cavity effects and surface contaminants could lead to large deviations from 2.3%. Our cavity modeling . The absorbed power, I = Pabs, obeys Fourier’s heat law, ΔT = PabsRT, where ΔT is the temperature difference between the graphene and the surrounding substrate  By using first-order thermal expansion, we relate ΔT to the mechanical strain in the GNB membrane to calculate Δf0. For an absorbed power modulated at angular frequency ω, the model provides an expression for the frequency-shift responsivityα is the thermal expansion coefficient, v is the Poisson ratio, σ0 is the initial in-plane stress, and Y is the 2D elastic modulus. The full details of the thermomechanical model are provided in Supplementary Note Rf is independent of incident power, in accord with the measurements given in Fig. ρT is the 2D thermal resistivity of graphene, and l and w are the tether length and width, respectively, Eq. Rf vs. w for trampolines given in Fig. R-value of 0.74. Moreover, the model predicts η ∝ w, which is also in agreement with our measurements . In both cases, the agreement is good, despite some variations in σ0 and l.The observations of Rf(ω), which we get by sweeping the modulation frequency, ω, of the heating laser at fixed power and measuring Δf0 with the PLL and a second lock-in (see Supplementary Note Rf(ω) with Eq. Rf spectrum for a trampoline GNB is illustrated in Fig. Rf(ω) obeys the circuit model very well.Another important metric in a bolometer is the response BW, which determines its ability to detect transient signals and fast variations of the radiation intensity. We characterize the BW in two ways. First, we infer the BW from the 3 dB roll-off of entclass1pt{minimaRf, but are limited by the measurement BW of the PLL. To overcome these speed limitations, our second approach infers the BW from the off-resonant thermomechanical out-of-plane displacement of the graphene membrane, which occurs when thermal stress tightens and locally flattens the membrane31. In the limit of small displacement and first-order thermal expansion, the mechanical displacement amplitude will be proportional to the change in temperature, ω will obey our thermal circuit model and will have the same frequency dependence as Rf(ω), as given in Eq. A(ω) (see Supplementary Note A(ω) to our model, we extract the thermal response time A(ω) along with the model fit (black traces) for a trampoline device with Rf(ω) and A(ω), finding excellent agreement , showing an excellent agreement with the prediction BW ∝ η . In this data, all parameters are constant except for σ0 and A, but the larger A devices tend to have lower stress σ0 so the effects cancel to preserve the linearity. Accordingly, for a given sensitivity, a smaller device area A will boost the speed. Supplementary Note The BW and the noise-equivalent power are expected to be directly proportional, regardless of the device geometry. Specifically, our model predictsmentclass2pt{minimmentclass2pt{minim12. Assessing the speed and sensitivity together, our lowest η = 2 pW Hz−1/2 compares favorably to the state-of-the-art in room-temperature bolometry, currently set by vanadium oxide and nickel-resistive microbolometers36 ~1–10 pW Hz−1/2, while often outperforming the BW of these systems by several orders of magnitude. Moreover, the drumhead GNBs, although not as sensitive, are 103–105 times faster than modern vanadium oxide bolometers. Using a standard figure-of-merit (FOM)35—which assesses the combination of speed and sensitivity normalized by the device geometry and absorption , our GNB has already matched these record-low FOM values.The response BW and noise-equivalent power demonstrated by our GNB rivals modern high-performance bolometers. Comparing to the sensitivity of previous graphene-based hot-electron bolometers, the GNB is nearly 1000 times more sensitive at room temperature7 and graphene nanomechanical resonators have been shown to operate up to 1200 K37. During our experiments, we tested this thermal stability by irradiating the GNB with a laser power of ~400 μW, a level which we estimate would have increased the GNB temperature as T = 920 K or T ~ 1213 K, and yet the device remained fully operational and undamaged. Although our experiments used a hybrid electronic/optical scheme for actuation and readout, the GNB can be used in an all-optical platform20, eliminating the need for on-chip electronics that could degrade at high temperature. Thus, in contrast with most photodetection technologies, our GNB platform is suitable for relevant high-temperature applications, including safety and security applications such as firefighting and industrial process monitoring, and in scientific experiments that take place at high temperature, such as close-proximity solar imaging.The GNB may enable photodetection applications at very high temperatures because of graphene’s extreme thermal stability. Graphene can sustain temperatures up to at least 2600 Kη) of our GNB through practical modifications to material properties and device geometry. Our modeling shows that η ∝ σ, where σ is stress, so using lower stress graphene38 would improve η. Increasing the optical absorption directly improves η to incident power. The GNB absorption could be increased to near unity by placing the bolometer in an optimized optical cavity15, at the expense of reduced spectral BW, or by depositing an absorptive material on the GNB, which would also reduce the spectral BW as well as the speed. The simplest way to improve η is to reduce the tether width, which can be narrowed down to ~10 nm using FIB (ref. 20), or to use FIB to create lattice defects in the tethers39, thereby increasing RT. Taken together, these changes could bring the noise-equivalent power down to the regime of femtowatt sensitivity with 100 Hz response BW.Although we have used the GNB to achieve record bolometric sensitivity at room temperature, it is possible to further improve the noise-equivalent power  and the steady-state frequency shift (from the added mass). As a consequence of independently measuring the mass and energy of a particle, the GNB would provide a means to measure the momentum of atoms and elementary particles.Sensing is an important application of nanoelectromechanical systems (NEMS). In general, the lower the mass of the NEMS device, the more sensitive it will be. By employing low-dimensional materials  to operate in the limit of ultralow mass, NEMS sensors have achieved record sensitivity to mass, electrical charge, and force−1/2 and a BW up to 1 MHz, thus demonstrating a previously unattainable sensitivity in room-temperature, graphene-based bolometry and greatly outperforming the speed of state-of-the-art room-temperature bolometers. By using graphene, we have demonstrated bolometry in the ultimate lower limit of lattice heat capacity and, consequently, have circumvented the speed-sensitivity trade-off that plagues bolometry. Our GNB fills a vital need in portable medical and thermal imaging, THz spectroscopy, and astronomy for fast, sensitive, and spectrally broadband bolometers and bolometer arrays that operate at and far above room temperature. Furthermore, as the GNB detects power via a nanomechanical mechanism, our work opens the possibility of multi-mode NEMS sensing, which may provide a useful tool in material science, nanoscience, and particle physics to simultaneously measure the energy of a particle along with its electrical charge and mass.In conclusion, we have measured visible light using a graphene nanomechanical resonator by tracking the frequency shifts of the resonator that are induced by light absorption. Using our GNB, we achieve a sensitivity of 2 pW Hz++ wafers at 1100 C. Next, we patterned 6–8 µm diameter holes with AZ1512 photoresist and a direct write laser photolithography system. We etched 600 nm deep into the oxide with a dry inductively coupled plasma etch using a plasma of CHF3 and argon. By leaving some of oxide intact, any collapsed graphene could not cause a short between the suspended graphene and the Si++. We then patterned metal electrodes using another AZ1512 direct write photolithography step. Next, we evaporated 5/50 nm Ti/Pt using electron beam evaporation followed acetone liftoff with sonication.We fabricated suspended graphene mechanical resonators using standard semiconductor processing techniques. We began by growing 1 µm of wet thermal oxide on Si2. The PDMS was then peeled away and the PMMA was removed in flowing Ar and H2 at 350 °C for 3 h. Graphene was scratched off the perimeter of the substrate to prevent shorting to the Si++ gate.A semi-dry polymer-supported transfer technique was used to place a large sheet of commercial monolayer graphene on Cu foil (Graphenea) over the exposed holes and metal contacts. First, a ~3 μm-thick layer of PMMA A11 was spun onto the Graphene/Cu. The graphene on the backside of the foil was removed with oxygen plasma. Then, a 1 mm-thick piece of polydimethylsiloxane (PDMS) with a ~1 cm diameter hole punched through the middle of it was placed on top of the Graphene/Cu stack. A thin plastic backing was left on the PDMS to increase the rigidity of the film. The Cu foil was etched on a solution of ammonium persulphate (40 mg/ml). The relatively rigid PDMS/PMMA/Gr stack was picked up with tweezers and placed in three sequential water baths before being removed and dried in air. Concurrently, the target substrate with holes was prepared by cleaning it in oxygen plasma before placing it on a hot plate at 155 °C. The now dry PDMS/PMMA/Graphene stack was placed on top of the hot substrate with the through hole covering the entirety of the chip. The substrate was left for ~16 h to improve adhesion between the graphene and the SiO+ source. The ion beam current and voltage were 1.1 pA and 30 kV, respectively. To fabricate a trampoline, four circle outlines were cut into a graphene drumhead using a single beam pass and a dwell time of 1 ms, which was enough to etch a line completely through the suspended graphene sheet. The high tension in the graphene sheet causes the graphene inside the circular cut to pull away from the trampoline resonator and collapse into the cavity. The FIB fabrication technique has a yield of near 100%, with device failures typically due to holes or other defects present in the graphene prior to milling. Although the FIB milling likely induces additional disorder in the graphene sheet, it still maintains its excellent electrical, mechanical, and thermal properties.We shaped the graphene into trampolines with a focused ion beam. FIB shaping was performed in an FEI Helios 600i SEM-FIB with a GaP < 10−5 Torr. Measurement laser powers were kept <1 μW to minimize any heating caused by the measurement. Reflected light was measured with a silicon avalanche photodiode and recorded with a lock-in amplifier. The lock-in amplifier was used to apply an AC voltage to actuate motion in the suspended graphene resonators. A built-in PLL tracked changes to resonant frequencies due to radiation-induced heating or inherent frequency fluctuations. Heating radiation was applied with a 532 nm diode laser modulated with an acousto-optic modulator. The Mechanical motion was measured with optical interferometry with a 633 nm HeNe laser. All measurements were performed at room temperature under a vacuum of Supplementary InformationPeer Review FileSource Data


The effect of soybean soluble polysaccharide (SSPS) on the formation of glucono-δ-lactone (GDL)-induced soybean protein isolate (SPI) gel was investigated. Electrophoretic analysis showed the SSPS did not change the electrophoretic behavior of SPI during the formation of SPI gel. However, infrared analysis indicated the β-sheet content increased, and the contents of random coil and α-helix decreased in both cooked SPI and SPI gel. The SSPS and SPI might conjugate via the Maillard reaction according to the results of grafting degree, color change, and infrared analyses. The main interactions during the formation of SPI gel changed from non-covalent to electrostatic interaction after adding SSPS. Sulfhydryl group content also increased in both cooked SPI and SPI gel. The water-holding capacity and gel strength of SPI gel decreased as the SSPS concentration increased. Larger aggregate holes were observed in the microstructure of SPI gel at higher SSPS concentration. Thus, SSPS could covalently conjugate with SPI and influence the formation of hydrogen bonds, disulfide bonds, and electrostatic interaction among SPI molecules to eventually form a loose gel network. Soy protein isolate (SPI), widely known for its diverse functionalities and health benefits, is commonly utilized in the food industry. The gelation is one of the most important functional properties of soybean protein in food . GeneralSoybean soluble polysaccharide (SSPS), a type of acidic polysaccharide that can be extracted from soy residue (okara), is composed of a main backbone involving long rhamnogalacturonan, branched by β-1,4-galactan, α-1,3 or 1,5-arabinan chains, and short homogalacturonan  6]. Whe. Whe6]. 4-induced soybean protein gels. Previous studies have reported the effect of sugars on the rheological and textural properties of SPI gels. Few studies have been devoted to the formation of acid-induced SSPS-SPI tofu-type gels. The purpose of this study is to provide insights into the effect of SSPS on the formation of GDL-induced SPI gels at a molecular level by correlating with the molecular interactions and the textural and microstructural properties.Recently, the influence of polysaccharide on the properties of soy protein isolate (SPI) gels have attracted extensive attention. Gu et al.  comparatSPI was purchased from Shansong Biological Products Co., Ltd., Shandong, China. SSPS was provided by BoMei Biotechnology Co., Ltd. . GDL was obtained from Xingzhou Medicine Food Co., Ltd. . O-phthaldialdehyde (OPA), glycine, sodium dodecyl sulfate (SDS), Coomassie Blue G-250, and 8-anilino-1-naphthalene sulfonic acid (ANS) were purchased from Aladdin Co., Ltd. . All chemical reagents used in this study were of reagent grade.w/v) and different SSPS concentrations  were selected to prepare tofu-type gels. Briefly, mixed powders and deionized water were further mixed using a magnetic blender  for 2 min. The suspension was heated at 75 °C for 10 min and was then heated at 90 °C for 10 min. The hot slurry was placed in an ice-water bath until the temperature decreased to 20 °C. GDL  was dissolved in deionized water (10 mL) and then added as a coagulant into the cooked SPI suspension and stirred for 2 min using a magnetic stirrer [Solutions with 10% SPI concentration . Aliquots (10 μL) of the prepared samples were added into the gels. The electrophoresis was run at a constant voltage of 120 V. After separation, the gels were stained by using Coomassie Blue G-250 The gel image was scanned using an image scanner  and analyzed using Quantity One software .Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) was performed according to the Laemmli method ,13 invol2EDTA, 0.09 mol/L glycine, and 0.086 mol/L Tris, pH 8.0) and stirred at ambient temperature for 2 h. SPI solutions were centrifuged at 12,000× g at 4 °C for 30 min. The supernatant was used for the measurement of the content of sulfhydryl groups. Three milliliters of supernatant was mixed with 0.03 mL Ellman’s reagent solution (4 mg DTNB/mL buffer) and was kept at ambient temperature for 15 min. The absorbance was then measured at 412 nm. The buffer was used instead of sample solutions as a reagent blank. A protein blank was used in which 0.03 mL buffer was replaced by Ellman’s reagent solution.Ellman’s reagent (DTNB) was used to determine the content of free sulfhydryl groups of lyophilized samples of cooked SPI suspension and SPI gels, according to a previous method . Sample w/v). A 200 μL quantity of sample solution was mixed with 4 mL OPA reagent and was then was maintained at 35 °C for 2 min in a water bath. Absorbance was measured at 340 nm with a Thermo Scientific Microplate Reader . SPI without adding SSPS was used as a control. DG was calculated according to the following equation:A0 is the absorbance of a sample without SSPS, and A1 is the absorbance of the sample with SSPS addition. All samples were measured in triplicate.The DGs of SPI gels were determined indirectly by the OPA method . Samples−1 with an FTIR spectrophotometer . Each sample was scanned 32 times with a resolution of 4 cm−1.Based on a previously reported method , 2 mg ofSurface hydrophobicity of samples was determined according to a previous method , using 12EDTA, pH 8.0); and S3, S2 containing 0.5% SDS and 6 mol/L urea. The resultant solutions were incubated at 25 °C for 24 h in a shaking water bath and then were centrifuged at 12,000× g and 25 °C for 30 min. The protein concentration of the supernatant was estimated by the Lowry method using a bull serum albumin standard. Absorbance at 650 nm was measured using a Varioskan Flash . All determinations were conducted in duplicate. Protein solubility (%) was expressed as 100 times the protein content of the supernatant divided by the total protein content.The investigation of noncovalent and covalent interactions involved in the cooking of SPI suspension and the formation of SPI gels was performed by dissolving the samples in different solvents . SolventWt is the total weight of native SPI gels and Wr is the weight of SPI gels that were centrifuged.Samples with different treatments were subjected to WHC measurement according to a previous method  with sliThe morphology of SPI gels was observed with a scanning electronic microscope (SEM) . Before observation, samples were dried to a critical point by a method of Ramlan et al. . SamplesThe gel strength of SPI gels was measured using GMIA (2013) Standard . SPI gelL*), redness/greenness (a*), and yellowness/blueness (b*) color scale.The color of SPI gels was determined using a colorimeter . An inbuilt standard white plate was used for the instrument calibration. The measurements were recorded as lightness . Values given in the tables and figures are the means of triplicates, and error bars indicate the standard deviation. Statistical significance of differences among means was analyzed by analysis of variance test and Duncan’s multiple range tests by SSPS Statistics, version 17.0 .The protein composition of cooked SPI suspension and SPI gels at various SSPS concentrations was analyzed by SDS–PAGE. As shown in −1 was used to determine the protein secondary structure [−1 was caused by the stretching vibration of O–H and C–C bonds of sugar, indicating that SPI underwent glycosylation modification [−1, the O–H stretching vibration of cooked SPI suspension and SPI gels caused the emerging of absorption peaks [To analyze the effect of SSPS on the secondary structure of SPI, FTIR spectra were processed by splitting and fitting using PeakFit 4.12 . As previously reported, the shape of the amide I band located at 1600–1700 cmtructure ,23. As sfication . Within on peaks . The O–HAs exhibited in Above all, SSPS did not change the primary structure of SPI but affected the profiles of the secondary structure. The addition of SSPS resulted in increasing the content of the β-sheet and reducing the contents of α-helix and the random coil of cooked SPI suspension and SPI gels, which might have finally resulted in a loose gel network.Solubility of protein is the specific behavior of its hydration, which can be improved by increasing the exposed surface charge of polar groups in the depolymerization/subunit extension of protein molecules or molecular polymers . It was According to In addition, protein solubility of SPI gels in S2 was higher than that of SPI gels in S1, suggesting that the electrostatic interaction was significant for the SPI gel network in all samples. The solubility of the protein in S2 buffer increased obviously after adding SSPS, which meant that electrostatic interaction was the important factor for the formation of the SPI gel network. After the addition of SSPS, S2–S1 was more than 50%, and S3–S2 decreased significantly, indicating that electrostatic interaction was an important factor in the formation of the SPI gel network. In addition, S3–S2 of the SPI gels decreased after the addition of SSPS, reflecting the decrease of the non-covalent interaction in SSPS–SPI gels. This might be due to their different microstructure .Changes in the secondary and tertiary structures of protein affect its spatial conformation and surface charge density. The level of surface hydrophobicity indicates the unfolding degree of protein, which leads to the exposure of non-polar amino acids within the molecule . During The surface hydrophobicity of the cooked SPI and SPI gels had a similar change trend , indicatThe sulfhydryl group is derived from disulfide bonds, which plays an important role in stabilizing the tertiary structure of proteins. Therefore, sulfhydryl group analysis is an indispensable method to explore the structural and functional changes of protein after denaturation. The sulfhydryl group contents of cooked SPI suspension and SPI gels at different SSPS concentrations are shown in The determination of free amino groups is used to estimate the progress of the Maillard reaction ,35. The Color is not only an important characteristic of gel quality but also an index to estimate the degree of the Maillard reaction . It is wWHC is an important characteristic of food products, especially for gels . WHC refThe gel strength is one of the most important characteristics of gels. As exhibited in As shown in The possible mechanism for the effect of SSPS on the GDL-induced SPI gelation is shown in These results pointed out that the addition of SSPS had a profound influence on the gelling properties of SPI gels by decreasing their WHC and gel strength and changing their microstructure. The changes in functional features mentioned above could be due to the changes in structural and functional properties induced by SSPS. In summary, SSPS increased the β-sheet content and decreased the contents of random coil and α-helix in both cooked SPI and SPI gel, and increased hydrophobic interactions of SPI gels. SSPS might conjugate with SPI via the Maillard action. Furthermore, the addition of SSPS reduced the content of the sulfhydryl group of cooked SPI suspension and changed the main intermolecular forces of the gel network from non-covalent bonds to electrostatic interactions. There were fewer gel aggregates formed and a structure of looser and increased porosity in SPI gels. However, the effect of glycosylation products between SSPS and proteins on the formation of SPI gels needs further study.


A large body of research has revealed the challenges that disproportionately affect women as they climb the academic ladder. One area that has received relatively little attention is women’s experiences at academic conferences, which are often integral to academics’ professional development. As conferences are attended by professional colleagues and influential players in specific fields, the professional consequences of any gender bias in criticism are likely to be amplified at such venues. Here, we explore the degree to which the likelihood of audience members asking a question and offering criticism is associated with the gender of a presenter. Audience questions were tabulated during the authors’ visits to the three American Anthropological Association Annual Meetings. The results suggested that men were indeed marginally more likely to ask a question, both when considering all types of questions and when considering only critical questions. However, there was no evidence that they differentially targeted women for these questions. Future research might explore what motivates assertive and critical speech in men and women and how their experiences in receiving it might differ, particularly in academic settings in which critical speech might be considered more acceptable. Women continue to be underrepresented in numerous academic fields, and importantly, remain underrepresented in higher academic ranks –5. A larOne area where women likely face hurdles is at academic conferences . ConfereAs one of the few venues in which academics have the opportunity for face-to-face conversation, women might experience another form of gender bias which they are free from in academic writing—bias that relates to gender differences in patterns of speech –12. One Gender and SpeechThe study of gendered patterns of speech has a long and complex history. Early attempts were founded upon little more than anecdote and speculation, much of which was rooted in sexist stereotypes of the time e.g., –17). The. The17])and is associated with gender, a spurious gender effect might be reported unless social status is appropriately accounted for [Much of the variance in the literature was due two major challenges that muddled research design—those relating to function and to context . Regardinted for . There anted for , 23. ThiAnother challenge of the tabulation approach was simply the results: meta-analyses run over large numbers of empirical studies ultimately revealed mostly negligible effects , 23, 24.A phenomenon that is widely discussed in the media today, and which is rooted squarely in this dominance viewpoint, is that of “mansplaining” . This isThe AAA has 9,218 members, and while demographic data is not collected in the application process, a recent survey of members suggests that women constitute a majority of the membership at 61% (the remaining include 36% men and 2% not selecting a binary option) . This fiWhile women in anthropology have greatly increased their representation through time, they still appear to disproportionately face challenges in their professional development. Female anthropology professors reported less satisfaction with university support, mentor support, and departmental collegiality than their male colleagues. They also report greater challenges due to gendered expectations of labor distribution, both with more administrative duties in the office and family responsibilities in the home .Similarly, women and men likely experience academic conferences differently. For professional anthropologists, the AAA Annual Meeting is the largest annual conference with attendance that tends to range in the upper thousands . ResearcThe most common format for sessions at the AAA Annual Meeting is for each speaker to present during a 15 minute time slot and for all questions to be held until all speakers have presented, at which time the questions are asked publically. Such a context offers a unique opportunity for exploring speech content, as it is a natural setting in which speech is presented in discrete segments and directed to specific receivers. Furthermore, any bias is likely to be amplified in effect, as the critical/explanatory speech is witnessed by many individuals and by those who often carry disproportionate influence in particular fields. However, like in much of the literature, these types of interactions are complicated by relational and motivational ambiguities. Although speaking on a topic establishes one as a de facto authority, audiences are also often comprised of like-interested specialists, and the true status relationship might be lost on observers. Similarly, an academic conference is one of the few venues in which critical speech might actually be sought or encouraged as researchers hone methodological approaches and negotiate inferences, making the motivation for critical speech ambiguous.Regardless of these complications, a number of hypotheses (H) and associated predictions (P) can be derived:(H1) If men speak more in mixed groups than women , then(P1) men should be more likely than women to ask questions in general in mixed audiences.(H2) If men are more likely to assume authority on a given topic and speak accordingly in interactions with women , we can (P2a) men should be more likely to offer any question/comment to a female presenter than to a male presenter, and(P2b) men should be more likely to offer any question/comment to a female presenter than a female audience member would.Using a more strict definition of assuming authority, we can also predict that(P3a) men’s questions/comments to female presenters will be more likely to be critical than those to male presenters, and(P3b) men’s questions/comments to female presenters will be more likely to be critical than those from female audience members.These effects combined should result in(P4a) men being more likely to offer a critical question/comment to female presenters than to male presenters, and(P4b) men being more likely to offer these questions/comments than female audience members.The reciprocal predictions of H2  will be explored to assess whether any gender bias flows in the opposite direction as well.Data was collected during the American Anthropological Association Annual Meetings (AAAs) in 2014 , 2015 , and 2016 . Convenience samples of sessions were attended. Sessions with relatively even numbers of male and female presenters and with discussions scheduled as the final block were sought, although other factors, such as proximity and even coder interest influenced selection. At the AAA conference, each session is typically sponsored by one of 40 different AAA sections, which are organized around specific topics . The sessions that were sampled include 20 different section sponsors, with the most sampled section including six sessions. While the sample is clearly not a probability sample, we were not attempting to characterize the nature of the conference as a whole, but instead examine within-session treatment effects. Thus we do not believe this hampered the study’s inferential value.If questions were solicited after a specific presentation, only data from that presentation was included. In most sessions, however, questions were reserved until the final discussion block. No details of participants other than gender were recorded, and speech was only categorized and not recorded. Human subjects review and approval, including a waiver of consent, was granted by the Texas A&M University Institutional Review Board under protocol IRB2015-0662. Permission was also granted by AAA Annual Meeting coordinators.Coders positioned themselves toward the back of the room so as to be able to view and hear as best as possible, but still remain inconspicuous. Prior to the question round, the number of men and women in the audience was tabulated. Although gender was not difficult to ascertain in the vast majority of cases, if a dichotomous gender category was not being performed or immediately evident, individuals were assigned an “other / unknown” category. For each round, the number of hands raised by men and women was recorded, as well as the gender of the person selected, the speaker targeted for their comment, and the nature of the question. Occasionally questions were directed to the group of presenters, instead of one individual, which was also recorded. Individuals were monitored to determine if the same audience member asked more than one question in order to record the data accordingly.Questions were categorized into any combination of seven categories . This process was repeated until the question/answer portion ended. For this study, a question/comment was deemed to be “critical” if it included a Criticism , Alternative Interpretation , or Reference . In contrast, questions were deemed “non-critical” if they were only categorized as one or more of the following: Request to Expound , Request to Clarify , Compliment , and Other (any major comments that do not fit under these categories).All data coders  trained using online recordings of academic presentations which included question rounds. Seven presentations with 26 questions were coded by all data coders. This resulted in an average pairwise agreement of 82.1% and a Fleiss’ kappa of 0.764 for Criticism, 92.3% and 0.639 for Alternative Interpretation, and 87.2% and 0.624 for Reference (the three categories of importance for this paper). These represent acceptable levels of agreement in all reported benchmark configurations, but still warrant some caution in interpretation –35.The data were organized in four ways in order to test the four types of comparisons. To test for differences in general frequencies of questions (P1), the data were arranged such that each individual represented a single row. To test for differences in how audience members responded to male versus female presenters (P2a and P4a), the data were arranged so that each row represented an audience member “opportunity.” For example, if an audience member had the opportunity to ask a question to a male presenter, that would constitute a row, and if that audience member also had the opportunity to ask a question to a female presenter (in a mixed-gender discussion panel), then that would constitute a second row for that audience member. Audience members thus had either one or two rows, each with an outcome for whether or not the audience member asked any question (P2a) or a critical question (P4a). The data were analyzed using a mixed logistic regression model using PROC GLIMMIX in SAS 9.2 with Audience ID as a nested random effect within the random effect for Session. Controls for the size of the audience, whether or not the question round was during a panel-wide discussion, and the number of panelists of the gender of analysis, were initially included and then eliminated if not significant.For tests of whether questions were more likely to be critical based on the gender of the asker and the speaker (P3a and P3b), the data were arranged such that each row represented a single question, and the outcome was a dichotomous variable signifying whether the question was categorized as critical. For P3, only questions to single individuals or single-gender groups were included, as those directed to mixed-gender groups could not be coded as being directed to either a male or female. For P4, however, a question asked to a mixed-gender group of presenters would be included in both the database limited to questions asked to women and the database limited to questions asked to men. The data were analyzed using a mixed logistic regression model with no controls and with Audience ID and Speaker ID as crossed random effects. However, the inclusion of Audience ID resulted in convergence problems, suggesting that it captured very little variance, so it was omitted.Finally, for tests exploring whether the gender of the audience member was associated with the likelihood of asking any question (P2b) or a critical question (P4b), the data were arranged such that each row represented a single individual. The outcome variable represented whether or not they asked any type of question for P2b or a critical question for P6b. The data were analyzed using a mixed logistic regression model with Session as a random effect. A number of controls were initially included and then eliminated sequentially if not significant. These included the size of the audience, whether or not the question round was during a discussion, and the number of panelists in the session who were of the gender of analysis .A total of 45 sessions were attended in which the audience was invited to ask questions. In 36 of these, the questions were solicited during a panel-wide discussion, and for 9, they were solicited after a single presentation (data from subsequent presentations in these sessions was not used). This led to a dataset that includes 195 speakers  and 848 audience members  from whom 175 questions were asked . Only five individuals raised their hands and were not called on (three men and two women).2 = 2.785, p = 0.095). In order to account for the fact that certain sections were oversampled, we also calculated a weighted proportion of the study sample by calculating proportion female within each section and then weighting them by the total number of members within that section. This did not substantially affect the estimates or the analyses , which suggests that the convenience sample of sessions is fairly representative of the gender distribution of the organization.Including all 45 sessions observed for the current study, the overall audience  and speaker composition  did not significantly differ from the membership survey results (both comparisons approached significance at p<0.1). Similarly, the gender composition of speakers in our sample trended male, but not significantly so, compared to a random sample of all speakers at the three conferences  for simplicity. While the variance of the random effects ranged from 0.025 to 2.097, we feel using standard logistic models is appropriate, as excluding the random effects had no impact on the direction or significance status of any predictors (the one significant main effect mentioned above does change to one-tailed significant)  had no impact on the likelihood of female audience members asking a question. However, male audience members were significantly more likely to ask questions in rooms with ewer men . IncludiIn this study, we found that men were marginally more likely to ask questions/make comments in a conference setting; this was true when considering all questions or just critical questions. This is in line with results from similar studies exploring gender differences in speech patterns . HoweverDue to the structure of AAA sessions, the analytical approach taken in this paper was more complex than originally intended. In AAA sessions, presentations last 15 minutes and all questions are typically held until the final block of the session. Thus, individuals would be simultaneously presented the opportunity to ask numerous male and female presenters a question. The authors attempted to model the likelihood of an audience member asking a question for each possible audience member/speaker dyad, with audience member and speaker as crossed random effects. This created a very large dataset and most of the resulting analyses resulted in convergence problems. This unnecessary complexity could be avoided in future research by focusing on conferences in which it is standard to solicit questions after each presentation.It is important to note that many “critical” comments may actually be offered out of a spirit of collaboration and helpfulness. Indeed, one can argue that one of the primary purposes of presenting at an academic conference is to seek constructive, critical commentary. Having participated in numerous conference proceedings, however, the authors can attest that while criticisms often result in positive changes to one’s work, they also tend to evoke a negative emotional response in the moment. Despite possible positive intentions and impacts on research, it is likely that higher rates of critical comments, or a more negative reaction to them, would have a negative impact on one’s experience at conferences and erode one’s self-confidence, potentially leading to an imposter syndrome.Similarly, it is impossible to ascertain perceived status differences, which likely would impact how one experiences the criticism (and represents a defining element of the mansplaining phenomenon). However, it would be surprising if presenters on a particular topic held less expertise than the average question asker.Finally, it is possible that women and men experience criticism differently. If the perceived threat of criticism is in any way calibrated to one’s own propensity to offer it, then women might experience more of a negative response upon being criticized. Evidence suggests that women perceive related behavior, such as interrupting  and angeWhile this study suffers from some of the elements that have been critiqued in earlier tabulation studies, its focus on speech content, and particularly on critical content, fills an important void in the literature. We provide further evidence that men exhibit a tendency towards assertive and critical speech compared to women. However, results do not suggest a bias in men’s targets of such speech at a major academic conference, failing to support the widely-discussed phenomenon of mansplaining in this context. It is important to note that this study cannot be generalized to speech in general. As previous research has shown, environmental context and relational characteristics are often essential to understanding the relationship between gender and speech. In this study, we focused on an academic conference because of the importance of such venues in professional development, the augmented impact any biases would result in at such venues, and the discrete nature of speech offered during question rounds. Our understanding of this phenomenon would obviously be enhanced by expanding the range of contexts in the future. Furthermore, future studies could improve upon the current research design by interviewing recipients of critical speech to determine the recipients’ perceptions of the exchange as well as any perception of status difference.Ultimately, we believe that in the exploration of gender discrimination and biases, quantitative studies are an important complement to more experientially-focused research. Such studies can further elucidate the frequency and impacts of discriminatory patterns, highlighting areas that might be in greater need for advocacy; they can assess the effectiveness of such advocacy and chronical societal changes in positive or negative directions. For such complex and socially important topics, the holistic approach that anthropology is founded upon is essential.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


It is shown that the commonly assumed bilinear softening law of the cohesive interface could lead up to 55% error in the predicted separation of the interface.The mechanical behavior of graphene/polymer interfaces in the graphene-reinforced epoxy nanocomposite is one of the factors that dictates the deformation and damage response of the nanocomposites. In this study, hybrid molecular dynamic (MD) and finite element (FE) simulations of a graphene/polymer nanocomposite are developed to characterize the elastic-damage behavior of graphene/polymer interfaces under a tensile separation condition. The MD results show that the graphene/epoxy interface behaves in the form of elastic-softening exponential regressive law. The FE results verify the adequacy of the cohesive zone model in accurate prediction of the interface damage behavior. The graphene/epoxy cohesive interface is characterized by normal stiffness, tensile strength, and fracture energy of 5 × 10 The ever-increasing demands for the usage of graphene-reinforced polymer composites in advanced instruments such as gas sensors, electrodes for batteries, solar cells, etc., require in-depth mechanical characterization of these nanomaterials ,2,3. GraWhile numerous investigations have been performed on the mechanical characterization of the graphene, polymers, and graphene-polymer nanocomposites ,20,21,22The cohesive zone model (CZM) is often used to simulate the elastic-damage behavior of the interface in composites at different length scales ,32,33,34The objective of this study is to establish an accurate methodology for predicting the properties and mechanical response of the graphene/polymer interface in graphene-reinforced epoxy nanocomposites employing both the MD and FE simulation procedures. In the modeling process, the formation of chemical bonding at the graphene/epoxy interface during the curing of the nanocomposite is simulated using the MD approach. An in-house code is developed to simulate the polymer cross-linking process during the curing of the nanocomposite. Consequently, the characteristic of the graphene/epoxy cohesive interface under the tensile separation loading is established. The resulting properties of this nanoscale cohesive interface are then prescribed for the cohesive elements of the FE simulation approach. This enables the accurate FE prediction of the properties and the mechanics of deformation of the graphene-reinforced epoxy nanocomposites in the multiscale simulation.The elastic and damage behaviors of the graphene/polymer interface in the nanocomposite were determined through the MD simulation of the nanocomposite. For this purpose, a representative volume element (RVE) of the graphene-polymer nanocomposite with the specifications provided in 3 in order to restrict the formation of unreal configuration and huge forces which occur due to the ring spearing and atoms overlapping [The initial molecular model of the polymer was created in PACKMOL software. The model was then submitted to the large scale atomic/molecular massively parallel simulator (LAMMPS) software to model the curing process of the polymer ,42. The rlapping ,44. The rlapping ,45,46. FIt was assumed that the primary and secondary amines have the same reactivity with the reaction cutoff distance of 5 Ǻ.The system was checked for the bond formation at every 50 ps after each equilibration.The constants in the equation of the newly-formed bonds were decreased in the first equilibration level and progressively elevated to the real values .The outputs were examined to update the topology parameters according to the new bonds.An annealing process was implemented to release the residual stresses. In this process, the temperature was increased to 450 K and then cooled to 300 K gradually over 2 × 106 time-steps.Steps 2 through 5 were iterated to obtain about 80% cross-linking.Finally, the last NPT equilibration in an interval time of 3 ns was applied to the RVE.In the modelling of the nanocomposites RVE, the graphene sheet was inserted along the x-y plane such that its center-of-mass is placed on the center of the simulation box, while the polymer molecules were positioned around the graphene sheet. Subsequently, the curing process was applied through the static cross-linking procedure ,47,48. TThe MD simulation procedure of the curing process is verified by comparing the predicted elastic properties of the epoxy polymer with those of measured values. In this process, an RVE of the epoxy polymer is created and cured to establish the intrinsic properties. The cured epoxy model is then loaded in tension to establish the force-displacement response in the elastic range, thus the elastic properties of the polymer could be determined. The details of the modelling steps are discussed elsewhere . The cloThe MD simulation of the graphene-polymer nanocomposite under tensile loading was performed using LAMMPS software  with a pl angles .2 carbon atoms of graphene and all of the polymer atoms in the condensed graphene-polymer nanocomposites [ii) is described using the L-J potential as:i and j were calculated based on the sixth-power mixing rule, as:The polymer matrix and the Van der Waals interactions between the graphene and polymer atoms were simulated based on the ab initio polymer consistent force field (PCFF) ,53,54. Tmposites ,54. In ting rule . Therefo−7 ns−1, the other faces were maintained under one atmospheric pressure [The RVE was modelled as a long structure of embedded graphene in the epoxy matrix, with a specified periodic boundary condition , as showpressure ,57.pqτ, were calculated using Virial expression as [m and ν are the mass and velocity of atom i respectively, i and j indices. The vector pqτ represents the stress components xxτ, yyτ, zzτ, xyτ, xzτ, and yzτ.Following each loading step, the stress components, ssion as ,59:(4)τpThe MD model of the graphene-polymer nanocomposite, as shown in The mechanical behavior of the interface between two continuum bodies can be simulated using the CZM with a bilinear traction-separation law for single-  and mixed-mode loading conditions ,35. Undeiδ are the traction and separation at an interface point, and indices 3, 1, and 2 refer to mode I  load, mode II, and mode III (shear) loadings, respectively. The parameter ik represents the cohesive stiffness of the interface in the respective load direction. In this study, the pure tensile loading mode was considered, thus the traction-displacement relation could be simplified to The initial linear elastic behavior of the interface is described by:d varies from “zero” for the pristine interface to “one” indicating the onset of interface of damage. In the pure tensile mode, Equation (6) was simplified to iG, in each loading mode could be calculated by the following equation:α is a non-dimensional interface parameter that determined by the rate of damage evolution. This parameter was calculated from the exponential decay curve of the interface softening behavior through the MD simulation of the graphene-polymer interface.A quadratic stress-based criterion was used to indicate the damage initiation in the mixed mode condition as:te, GIC, . This isfined as :(8)dpi=1The geometrical model of the RVE consisting of a graphene sheet bonded by two blocks of epoxy polymer is shown in The hyper-elastic behavior of the epoxy and the properties of the graphene used in the FE simulation were reported elsewhere . The proThe computational results of the graphene-polymer nanocomposite model, performed in MD and FE simulation environments, are presented and discussed in the following subsections. The mechanics of deformation of the graphene/polymer interface under the tensile loading is described. The validation aspect of the FE simulation approach in capturing the physics of the interface failure process is deliberated.12 ng/nm3, as illustrated by the dashed line and without considering the density readings below 0.65 × 1012 ng/nm3. It is noted that a 0.3% variation of the average density of the epoxy is calculated over the sampling distance of 7 nm, particularly contributed by the higher density gradient next to the graphene/polymer interface. The polymer density diminishes to zero across a small gap to the location of the graphene. This density gradient indicates the non-uniform distribution of the polymer atoms in the nearby graphene/polymer interface.The density profile of the epoxy polymer across the cured polymer/graphene/polymer phases of the RVE model is shown in 2 . A zero-density gap with a thickness of 0.40 nm was determined at the central location of the nanocomposite RVE. The graphene thickness was assumed to be similar to the distance between the graphene sheets in the graphite structure (0.34 nm) and located in the middle of the nanocomposite RVE. Thus, the graphene/epoxy interface thickness on each side of the graphene is determined as having the thickness of 0.03 nm. The epoxy matrix on each side of the graphene was considered as 3.3 nm-thick with the cross-sectional area of 4.52 × 4.54 nm2 see  for the −10 aPa is achieved. The stress decreases exponentially with the continuous displacement. A similar cohesive stress-displacement response with a nonlinear spring model was predicted for the interface of a pristine graphene/one-layer polymer using the MD approach [The nanomechanical behavior of the graphene/epoxy interface, predicted using the MD simulation, is shown by the stress-displacement curve in approach . At the ce curve b. Beyondα = 7.74.Analogously, while the initial deformation of the graphene/epoxy interface is elastic, beyond the displacement corresponding to the peak stress the decreasing stress-displacement curve also exhibits an exponential decay form, and constitutes of both recoverable and irrecoverable components a. Only wGraphene/epoxy interface properties: The elastic properties of the interface were also determined from the MD-calculated stress-displacement curve of k3, of the interface. The area bounded by the curve represents the critical strain energy release rate, ICG, for the tensile loading mode, which could be calculated by Equation (7). These properties along with the parameters of the exponential softening law are used to define the graphene/epoxy cohesive interface behavior for the FE simulation. The property values are listed in The FE-calculated stress-displacement response of the graphene/epoxy cohesive interface using the properties listed in In the absence of the MD-predicted exponential regressive behavior, a bilinear softening law is often employed as an estimate of the cohesive interface behavior. The response of the FE model with a bilinear softening law specified for the cohesive interface was examined. The CZM properties of tensile strength, penalty stiffness, and the critical Mode I strain energy release rate is maintained in the simulation to acknowledge the same physical interface. The resulting stress-displacement curves are compared in δo. The calculated evolution of the DDE follows the prescribed form of the regressive softening process, as illustrated in the figure. The initial rate of the energy dissipation is faster for the cohesive interface with exponential regressive than that of the linear softening law, as reflected by the initial slope of the curve. The evolving DDE saturates to a constant level when reaching the separation of the graphene/epoxy cohesive interface.The energy released by the graphene/epoxy interface during the interface failure process under the applied tensile loading is termed the damage dissipation energy (DDE). The evolution of the total DDE, for all the cohesive interface elements of the model with the two different softening laws of the cohesive interface, is compared in α = 7.74. The assumed bilinear softening law of the cohesive interface, commonly employed in the microscale FE simulation of the nanocomposite behavior, could lead to erroneous results when compared to the MD-calculated exponential regression law. This was demonstrated by the different traction-displacement curves and the evolution of DDE to tensile separation of the graphene/epoxy interface of the nanocomposite RVE. A 55% difference in the predicted displacement at the separation of the interface was calculated when the bilinear softening law was assumed, compared to that using the exponential regressive damage law of the cohesive interface.The deformation and failure process of the graphene-polymer nanocomposites could be quantified through the FE simulation with the cohesive interface elements. The missing intrinsic properties and behavior of the cohesive interface could be determined from the MD simulation of the curing and subsequent loading of the nanocomposite RVE. This paper has demonstrated the procedures in establishing the intrinsic regressive softening law for the cohesive interface of the graphene/epoxy nanocomposites. Results show that the graphene/epoxy interface has a thickness of 0.03 nm, based on the density variation of the polymer across the graphene/epoxy interphase. Upon tensile loading of the RVE, an exponential-decay form of the cohesive interatomic forces was established. Analogously, the cohesive interface was described by the exponential regression law with the exponent,


Consolidation treatment with an anti-PD-L1 antibody, durvalumab, following concurrent chemo-radiotherapy (cCRT) has become a new standard of care for locally advanced non-small cell lung cancer (NSCLC). The rationale of PD-L1 blockade after cCRT is based on preclinical evidence suggesting that chemotherapy and radiotherapy up-regulate tumoural PD-L1 expression, which has not been shown in clinical studies.To examine alteration in tumoural PD-L1 expression  and density of stromal CD8-positive tumour-infiltrating lymphocytes (CD8 + TILs) after cCRT, paired NSCLC samples obtained before and after cCRT were reviewed in comparison with those obtained before and after drug therapy.P < 0.001), but not after drug therapy. There was no significant correlation between baseline TPS and post-cCRT TPS. CD8 + TIL density was significantly increased after cCRT , and higher post-cCRT CD8 + TIL density was associated with a higher pathologic response and with a favourable survival (P = 0.019).PD-L1 expression was significantly up-regulated after cCRT (median TPS, 1.0 at baseline versus 48.0 after cCRT; Tumoural PD-L1 expression was up-regulated after cCRT, which provides pathologic rationale for PD-L1 blockade following cCRT to improve prognosis. Stromal CD8 + TIL density was also increased after cCRT, and higher post-cCRT CD8 + TIL density was a favourable prognostic indicator. However, the prognosis achieved with cCRT remains un-satisfactory with the 5-year overall survival (OS) rate of 15%.2 Recently, a randomised controlled trial demonstrated that the consolidation treatment with durvalumab, an antibody against programmed death ligand-1 (PD-L1), provided a significant survival benefit among patients who did not have disease progression after cCRT.4 Based on the results, durvalumab treatment following cCRT has become a new standard of care for locally advanced and unresectable NSCLC patients with good performance status (PS).5Non-small cell lung cancer (NSCLC) accounts for ~85% of lung cancer, which is the leading cause of cancer deaths worldwide.6 and radiotherapy8 up-regulate PD-L1 expression on tumour cells. Up-regulation of tumoural PD-L1 leads to acquired resistance to radiotherapy, which may be overcome by PD-L1 blockade.8 However, there has been reported no clinical evidence showing up-regulation of tumoural PD-L1 expression after cCRT. Only one clinical study to examine alteration in PD-L1 expression by using paired NSCLC samples obtained before and after cCRT showed a significant decrease in PD-L1 expression after cCRT,9 which is contrary to preclinical results.8 In the present study, to assess the impact of cCRT on tumour immune microenvironment in NSCLC, we examined alteration in tumoural PD-L1 expression and in density of stromal CD8-positive tumour-infiltrating lymphocytes (CD8 + TILs) in paired histologic samples that had been obtained before and after cCRT in comparison with that in paired specimens obtained before and after drug therapy.The rationale of addition of PD-L1 blockade following cCRT is based on preclinical evidence suggesting that chemotherapyNSCLC patients who received pre-operative treatment followed by surgery from January 2008 through December 2017 at our institute were retrospectively reviewed. Patients were eligible when paired tumour specimens obtained before and after preoperative treatment were available for pathological evaluation and for immunohistochemistry (IHC) to examine tumoural PD-L1 expression and stromal CD8 + TILs. No patients received radiotherapy alone. Seven patients who achieved pathologic complete response with preoperative treatment were excluded, because tumoural PD-L1 expression could not be evaluable. A total of 23 patients who underwent cCRT were included and were reviewed in comparison with 18 patients who underwent drug therapy alone as preoperative treatment  by the Japan Lung Cancer Society’ as follows: therapeutic effect 0, no therapeutic effect; 1, residual viable cancer cells detected in ‘≥1/3’ of resected tumour; 2, residual viable cancer cells detected in ‘<1/3’ of resected tumour; 3, no residual viable cancer cells.11Clinical stage (c-stage) was determined according to the current tumour, node, metastases (TNM) classification.14 Serial 4-µm sections were cut from each formalin-fixed and paraffin-embedded tumour specimen. For evaluation of PD-L1 expression, sections were heated in 1 mmol/L ethylenediaminetetraacetic acid (pH 8.0) at 98°C for 15 min for antigen retrieval, and were incubated in 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidase. After blocking with Protein Block Serum-Free  for 30 min, sections were incubated with a rabbit anti-PD-L1 monoclonal antibody  diluted at 1:200 for 1 h at room temperature. Sections were then washed and incubated with SignalStain Boost IHC Detection Reagent HRP Rabbit  for 30 min. Thereafter, they were visualised with DAB + Liquid (Agilent Technologies) and counterstained with haematoxylin. Paraffin-embedded cell pellets  served as controls for PD-L1 immuno-staining. For evaluation of CD8-positive lymphocytes, slide was stained with an anti-CD8 antibody  using the DAKO Autostainer Link 48 (Agilent Technologies) according to the manufacturer’s protocol.PD-L1 expression on tumour cells and density of stromal CD8 + TILs were evaluated with IHC.14 When a discrepancy was found between the two investigators, the slide was reviewed via their simultaneous examination using a double-headed microscope to achieve a consensus.Each slide was independently evaluated by two of the investigators (K.Y. and F.T.) without the knowledge of any clinical data. The percentage of tumour cells with membrane staining for PD-L1 was recorded and represented as tumour proportion score (TPS). A minimum of 100 tumour cells were evaluated to calculate TPS. The percentage of CD8-positive lymphocytes among total nucleated cells in the stromal compartments were defined as the CD8 + TIL density.Proportions of categorical data were compared by the chi-square test or the Fisher’s exact test, as appropriate. Continuous data were compared with a non-parametric test (Wilcoxon signed rank test for paired data or Mann–Whitney U-test for un-paired data). Spearman’s rank correlation coefficients (two-sided) were used to evaluate correlations between two parameters. Receiver operating characteristic (ROC) curve analyses were performed to determine the optimal cut-off value of TPS for PD-L1 expression and CD8 + TIL density.A telephone inquiry was made if the patient did not come to our clinic for a routine follow-up. The Kaplan–Meier method was used to estimate probability of recurrence-free survival (RFS) and OS, and survival differences were analysed by the log-rank test. The hazard ratio (HR) and 95% confidence interval (CI) were calculated for each variable.P-values < 0.05. All statistical analyses were performed with the SPSS version 21 software .Differences were considered to be statistically significant for Among 23 patients who received cCRT, the majority of patients had c-stage III disease. All patients received platinum-based chemotherapy in combination with concurrent radiotherapy with the median dose of 60 Gy followed by complete resection. Seventeen (73.9%) of 23 patients achieved therapeutic effect 2 with cCRT. In the drug-therapy group, only five (27.8%) of 18 patients achieved therapeutic effect 2 with pre-operative treatment (Table P < 0.001) but was not after drug therapy , whereas baseline TPS was similar . There was no significant correlation between baseline TPS of and post-treatment TPS in the cCRT group or in the drug-therapy group  showed an increase in TPS, and the other two patients showed a decrease. After drug therapy, 10 patients showed an increase in TPS, whereas the other eight patients showed a decrease. TPS was significantly increased after cCRT . PD-L1 expression was significantly increased after cCRT regardless of pathologic response .In the cCRT group, PD-L1 expression was significantly increased after treatment not only in patients with positive PD-L1 expression at the baseline  but also in patients with negative PD-L1 expression at the baseline   and was also increased after drug therapy  , whereas baseline CD8 + TIL density was similar in both groups . There was no significant correlation between baseline CD8 + TIL density and post-treatment CD8 + TIL density in the cCRT group or in the drug-therapy group  showed an increase in CD8 + TIL density, and only one patient showed a decrease. After drug therapy, 16 patients showed an increase in CD8 + TIL density, and only two patients showed a decrease. CD8 + TIL density was significantly increased after cCRT , whereas no significant difference in baseline CD8 + TIL density between two groups was observed (P = 0.309).In the cCRT group, CD8 + TIL density was significantly increased after treatment regardless of pathologic response .Prognostic implication of tumoural PD-L1 expression status and stromal CD8 + TIL density was assessed in 23 patients who underwent cCRT followed by surgery. ROC curve analyses revealed that only the post-cCRT CD8 + TIL density provided a significant prognostic value to predict death or tumour recurrence with the optimal cut-off value of 40  showed significantly favourable RFS and OS than those with lower post-cCRT CD8 + TIL density . CD8 is predominantly expressed on cytotoxic T cells (CTLs) that play central roles in cell-mediated immune attack, whereas it can be expressed on natural killer and dendritic cells.is Figs. –5. In this Figs. , or provis Figs. –5. Accor24 It has been generally considered that the main mechanism of killing tumour cells by ionising radiation is the direct DNA damage. However, several preclinical studies have revealed that radiation therapy may induce anti-tumour immune response through promoting release of neo-antigen from tumour cells.27 For example, Takeshima and co-workers showed that tumour-inhibitory effect of radiation was almost completely abolished in tumour-bearing mice when CD8-positive T cells were depleted.23 These preclinical results suggest that the immune attack by CD8 + TILs is an important mechanism of anti-tumour effect of radiation therapy. Therefore, when irradiated, tumour cells may evade from immune attack by CD8 + TILs through several mechanisms, which leads to resistance to radiotherapy. The PD-1/PD-L1 axis is the most important mechanism responsible for immune evasion of tumour cells.28 PD-L1 expressed on tumour cells binds to PD-L1 on CTLs, which results in down-regulation of immune activity of CTLs. PD-L1 on tumour cells may be up-regulated in response to immune attack induced by radiotherapy, which has been demonstrated in preclinical models.8 The present study is the first clinical study showing up-regulation of PD-L1 after cCRT .4 In the present study, the majority of patients showed an increase in TPS after cCRT , which is reasonably explained by experimental results showing that CTLs play a crucial role in anti-tumour effects of radiation.12 However, results in the present study shall be validated in a prospective multi-institutional study with a validated IHC assay. Third, there are vast heterogeneity in patient characteristics such as radiation dose and chemotherapy regimen employed in cCRT. The conflicting results between the present study showing increased PD-L1 expression after cCRT and in the previous study showing decreased PD-L1 expression9 may be caused by differences in several patient characteristics. Tumoural PD-L1 status is employed in routine clinical practice as a predictive marker in blockade of PD-1/PD-L1 axis for advanced NSCLC.31 However, the prognostic value of PD-L1 status remains controversial, and some studies indicated a poor prognosis in patients with strong PD-L1 expression but others indicated a favourable prognosis.33 In the present study, pre-treatment or post-treatment PD-L1 status provided no significant prognostic value in patients who underwent cCRT followed by surgery, whereas high TPS seemed to be associated with a favourable OS . A prospective multi-institutional study revealed that the E1L3N assay showed similar results to trial-validated PD-L1 IHC assays (28–8 and 22C3 assays). OS Fig. . The proIn conclusion, PD-L1 expression on tumour cells and stromal CD8 + TIL density were significantly increased after cCRT. There was no significant correlation between pre-treatment and post-treatment tumoural PD-L1 or stromal CD8 + TIL density. The higher CD8 + TIL density after cCRT was associated with a higher pathologic response and with a favourable survival.Supplementary Infirmation


Bacillus cells faced with unfavorable environmental conditions undergo an asymmetric division process ultimately leading to the formation of the bacterial spore. In some instances the spore serves as an infectious agent; such is the case with the spore of Bacillus anthracis and the disease anthrax. Spores are resistant to a variety of environment conditions including traditional decontamination techniques due to the formation of specialized cellular structures. One such structure, the spore cortex, is a thick layer of modified peptidoglycan that contributes to spore dormancy through maintenance of the dehydrated state of the spore core. During spore germination, degradation of the cortex is required to facilitate complete hydration of the core and a return to vegetative growth. Degradation of the cortex is accomplished through the action of germination-specific lytic enzymes. One of these enzymes, SleB, has been previously shown to require the presence of the YpeB protein for its stable incorporation and subsequent function in spores of B. anthracis. The focus of the present study is to identify protein interactions of YpeB through in vivo chemical cross-linking and two-hybrid analysis.Conserved residues within YpeB PepSY domains were altered to facilitate implementation of a site-specific chemical cross-linker, 4-Azidophenacyl bromide. Analyses of crosslinked-spore extracts suggests that YpeB exists as a dimer or larger multimer within the spore, potentially mediated through interactions of the C-terminal domains. Spores expressing stable truncated forms of YpeB were crosslinked and corresponding truncated dimers were detected. Further characterization of individual YpeB domains using bacterial two-hybrid analysis indicated a possible role for both N-and C-terminal domains in YpeB oligomerization.The YpeB protein likely exists as dimer or higher-order multimer in the dormant spore. Both the N- and C-terminal YpeB domains contribute to multimerization. SleB likely also exists as an oligomer, and SleB and YpeB may be found together within a protein complex. Disassembly of this complex during spore germination likely allows SleB to become active in spore cortex degradation. Further study of this protein complex may contribute to the development of methods to inhibit or stimulate germination, allowing more effective spore decontamination.The online version of this article (10.1186/s12866-019-1544-1) contains supplementary material, which is available to authorized users. Bacillus anthracis has the potential to cause widespread illness and severe disease through multiple routes of infection. As with many other disease-causing endospore-producing bacterial species, the bacterial endospore serves as the infectious agent of the disease anthrax [2+-dipicolinic acid (DPA). [B. anthracis poses, especially as a bioterrorism agent. anthrax . This is anthrax –3. The g anthrax . Dormancd (DPA). . These f2+-DPA are released from the spore core and partial rehydration of the core begins [B. anthracis within the host is required for production of the anthrax toxins and ultimately progression of the disease [When the dormant spore senses an environment with favorable nutrient availability, such as within a host, it will rapidly germinate, returning to a vegetative growth state. Germination is initiated following sensing of germinants by receptors at the inner spore membrane, after which large stores of Ca disease .B. anthracis encodes four of these enzymes, but the majority of the cortex degradation has been demonstrated to be completed through the action of partially redundant enzymes SleB and CwlJ1 [2+-DPA [The cortex is degraded by germination specific lytic enzymes (GSLEs). nd CwlJ1 , 7. Thesnd CwlJ1 –13, whicnd CwlJ1 , 15. Cwl [2+-DPA , 17.Bacillus species, both SleB and YpeB are expressed from a conserved operon and this is also true of several Clostridium strains [B. subtilis and B. anthracis [G and are translocated across the inner spore membrane via N-terminal signal sequences [In  strains . Previou strains , 19. Furnthracis , 20–22. equences . The sigequences –25. Giveequences . It has equences , while aBacillus spore.The goal of the current study was to further characterize the relationship between YpeB and SleB within the dormant spore. In vivo peptide cross-linking was used to study potential interactions of YpeB, identifying interactions that may form only within the unique environment of the dormant spore. Bacterial two-hybrid analysis was used to detect domain-specific interactions. Both methods indicate YpeB oligomer formation, which may be required for stable incorporation of SleB and subsequent germination of the ypeB orthologs. Each of the selected residues was then mutagenized, substituting the wildtype codon with that for cysteine, and YpeB-cysteine alleles were recombined into the chromosome of a ∆ypeB strain. These alleles also carried a C-terminal hexa-histidine tag for protein purification purposes. This tag has previously been shown to not interfere with YpeB function [ypeBS358C-6His and ypeBK437C-6His were nearly identical to the wild type in these regards , specifically residues beyond the first PepSY domain, for stabilization of SleB in the developing spore 3–446, sp, severalSulfhydryl specific cross-linking was conducted using the cross-linker p-azidophencyl bromide (APB), which is a heterobifunctional cross-linker with a sulfhydryl specific α-bromo-ketone motif in addition to a non-specific photoactivatable azide, separated by a spacer arm of 9 Å. In total, 12 YpeB-Cys allele-carrying strains, corresponding to 12 different amino acid substitutions across the C-terminal PepSY domains  and what appears to be a truncated YpeB dimer (~ 60 kDa). Extracts of YpeB∆25–203 K437C in WT background suggest the possibility of a YpeB∆25–203-WT YpeB heterodimer (~ 80 kDa) in addition to complexes previously visualized. We next sought to confirm that this newly visualized band contained the 6x-His-tagged YpeB. Spores encoding YpeB∆25–203 K437C were cross-linked, and 6x-His-tagged proteins in extracts were concentrated using a Ni2+ NTA affinity column. Column elutions were visualized with western blotting  and C-terminal domains (YpeBC 203–446), full-length YpeB  (YpeBFull 21–446), and the SleB C-terminal catalytic domain (SleBCat 125–253) [E. coli, which was plated on MacConkey agar supplemented with maltose, where positive domain interactions were visualized by red colony coloration. In agreement with cross-linking results, bacterial two-hybrid assays indicated that YpeBFull self-associated.   were cloN domain alone exhibits strong self-association, while the YpeBC domain alone does not. However, YpeB-Cys substitutions at some positions in the C-terminal domain can be crosslinked to other YpeB molecules, suggesting close approach of C-terminal domains within the dormant spore, though without a sufficiently strong role in dimerization to be detected using the two-hybrid system. The C-terminal domain alone can interact with full-length YpeB, suggesting that either both domains are required for stable interaction with the isolated C-terminal domain, or that multimerization of the full-length protein allows further interaction with the isolated C-terminal domain.This study further characterized interactions of YpeB within the dormant spore through in vivo chemical cross-linking and the roles of individual domains of YpeB in potential multimer formation using bacterial adenylate cyclase two-hybrid assays. Both in vivo crosslinking and two-hybrid analyses indicate that YpeB forms a dimer or higher-order multimer. The YpeB. Both N- and C-terminal YpeB domains have been demonstrated to be required for SleB stabilization [YpeB was not demonstrated to form cross-links to SleB from selected residues within the YpeB C-terminal PepSY domains. This may indicate that the YpeB C-terminal domain, although required for stabilization of SleB , is not lization , 21, howlization . Cross-lB. megaterium has been solved [S358C, one of the residues demonstrated to be reactive in our cross-linking experiments lies directly within this potential binding-pocket. YpeBK437C, the other reactive residue, lies just beneath the channel but is still surface exposed. It is not immediately obvious how multiple C-terminal domains of YpeB may interact relative to this pocket.The structure of the YpeB C-terminal PepSY domains of n solved . Authorsn solved . YpeBS35Previous work identified conserved amino acid residues in YpeB PepSY domains required for YpeB, and subsequent SleB, stabilization in dormant spores . In contAlthough YpeB cross-linked to SleB was not detected, the list of residues tested was by no means exhaustive and it is entirely possible that the two proteins do interact, especially via the YpeB N-terminal domain . YpeB maThe YpeB protein likely exists as a dimer or higher-order multimer in the dormant spore. Both the N- and C-terminal YpeB domains contribute to multimerization. SleB likely also exists as an oligomer, and SleB and YpeB may be found together within a protein complex. Disassembly of this complex during spore germination likely allows SleB to become active in spore cortex degradation. Further study of this protein complex may contribute to the development of methods to inhibit or stimulate germination, allowing more effective spore decontamination or more effective use of spores as applied microbial agents.ypeB. PCR products were then cloned into the ypeB complementation plasmid (pDPV424 [B. anthracis through conjugation as described previously [Site-directed mutagenesis by overlap extension PCR  was perf(pDPV424 ) via res. Ligations were carried out to insert ypeB or sleB domain sequences in frame with N-terminally fused p18 or p25 domains of adenylate cyclase. Plasmids were then co-transformed into BTH101 (Euromedex) to test potential interactions.Construction of strains for two hybrid assays was performed as follows. Desired B. anthracis spores were prepared in liquid Modified G medium [G medium  with antG medium . BrieflyTen optical density units of decoated spores were suspended in PBS pH 7.5 and APB crosslinker (Sigma) was added to a final concentration of 5 mM. Decoated spores were incubated with APB at 37 °C for 30 min in the dark. Samples were then exposed to UV light for an additional 15 min at room temperature. Following UV exposure, cross-linked spores were centrifuged at 10,000 x g for 1 min and the supernatant was removed. Cross-linked spore pellets were stored at − 80 °C until later use.Spores were lyophilized and then were broken mechanically with 100 mg 0.1 mm glass beads using Wig-L-Bug bead beaters for 20 pulses of 30 s each at 4,200 rpm. Samples were stored on ice between cycles. Proteins were extracted with 0.125 M Tris-HCl pH 6.8, 4% SDS, 10% β-mercaptoethanol, 10% glycerol, 0.004% bromophenol blue and run on SDS-polyacrylamide gel electrophoresis. YpeB and SleB were detected via western blot as described previously [Following cross-linking of 200 OD units of decoated spores, frozen pellets were lyophilized and broken as described above. Broken spores were suspended in Urea Binding Buffer  and incubated at 4 °C for 2 h. The samples were centrifuged at 6,800 x g for 10 min, and the soluble fraction was collected, filtered, and loaded onto a 1 mL Ni Sepharose HisTrap HP  column equilibrated in Urea Binding Buffer. Bound YpeB-His6 was eluted with Urea Elution Buffer . Fractions were stored at − 80 °C for western blot analysis.Protein interactions were screened via spotting 2 μl of co-transformed overnight culture on MacConkey agar . Spotted plates were incubated for 48 h at 30 °C. Positive interactions were visualized by acidification of the media resulting in production of red coloration.Additional file 1YpeB-cysteine mutant strain functional screens. Examination of Cys-substituted YpeB protein functionality in germination rate, proteolysis during germination, assembly into the spore, and stabilization of SleB in the spore. (PDF 358 kb)