A portable, handheld electrospinning apparatus is designed and constructed using off-the-shelf components and 3D-printed parts. The portable electrospinner is used to generate nanofibers with diameters ranging from 85 to 600 nm; examination of these fibers is achieved with scanning electron microscopy. This portable electrospinner has similar capabilities to standard stationary benchtop electrospinners in terms of the diversity of polymers the device is able to spin into nanofibers and their resulting size and morphology. However, it provides much more ambulatory flexibility, employs current-limiting measures that allow for safer operation and is cost effective. As a demonstration of the device’s unique application space afforded by its portability, the device is applied in direct-to-skin electrospinning to improve the aesthetics of simulated hair loss in a mouse model by electrospinning dyed polyacrylonitrile nanofibers that mimic hair. The superficial nanofiber treatment for thinning hair is able to achieve an improvement in appearance similar to that of a commercially available powder product but outperforms the powder in the nanofiber’s superior adherence to the affected area. The portable electrospinning apparatus overcomes many limitations of immobile benchtop electrospinners and holds promise for applications in consumer end-use scenarios such as the treatment of alopecia via cosmetic hair thickening. Electrospinning is a cost-effective technique for synthesizing polymeric fibers with diameters ranging from a few nanometers to tens of microns. Nanofibers electrospun with conventional benchtop electrospinners have found application in textiles ,2, energThe popularity of electrospinning is largely due to the simplicity of the equipment. Conventional electrospinners primarily consist of three components : a high An application of nanofibers electrospun by a portable electrospinning apparatus yet to be explored is the cosmetic treatment of alopecia by depositing dyed nanofibers onto the skin to simulate native human hair. Hair thinning, or male pattern baldness, affects half of all men over the age of 50 ,23 and fTwo HV DC-DC converters, one supplying a positive voltage  with respect to ground and another supplying a negative voltage , amplified a low voltage DC signal between 1 and 10 V to output an HV DC signal between 1 and 10 kV. Low voltage DC signals were generated using an ATmega2560 MCU  in series with a non-inverting operational amplifier circuit with a gain of 2 V/V  and an optocoupler . A custom-made syringe pump was used to push a polymer solution loaded in a syringe outfitted with a stainless-steel needle tip toward the HV source. The syringe pump was comprised of a 5 V DC power supply, 1 A stepper motor , the Allegro A4988 motor driver  and structural components fabricated with a 3D printer  using 1.75 mm PLA filament. The enclosure for the handheld electrospinning apparatus was fabricated as well using the 3D printer and PLA filament.A user-interface was constructed using PCB technology, a 24 × 2-character liquid crystal display  and a rotary encoder . The device was either powered by 110 V AC mains electricity via a dedicated 24 V, 2.5A AC/DC adapter  or by four 9 V batteries . Power from either the 24 V adapter or the batteries was used to drive two voltage regulators: a 12 V regulator  and a 5 V regulator . In line with the power sources, and prior to the PCB, a 2-mA fuse was used for overcurrent protection .nM 30 kDa) dissolved in an acetone/deionized water mixture (4:1 v/v) at a concentration of 4% (w/w), PVA (average wM 93 kDa) dissolved in THF at 6% (w/w), PAN  dissolved in DMF at 10% (w/w), PEO (average vM 900 kDa) dissolved in deionized water at 6% (w/w), PVDF  dissolved in a mixture of DMF and acetone (1:1 v/v) at a concentration of 15% (w/w) and PVP (average wM 1.3 MDa) dissolved in ethanol at 5% (w/w).All chemicals were purchased from Sigma-Aldrich  unless otherwise noted. Polymer solutions for electrospinning were prepared by mixing the following polymers with their respective solvents at room temperature under constant stirring for 1 h prior to electrospinning: CA (average nM 80 kDa) was dissolved in TFE over a 24 h period under constant stirring at room temperature at a concentration of 15% (w/w); PS (average wM 350 kDa) was dissolved in THF over a 12 h period under constant stirring at room temperature at a concentration of 12% (w/w).Two other polymer solutions were prepared as follows: PCL  outfitted with a 20-gauge, stainless-steel needle with blunt tip . A stainless-steel alligator clip  was used to connect the needle tip to the HV DC supply. The flow rate of the stepper motor powered syringe pump was set to 1 mL hrNanofibers and nanobeads were collected onto a grounded aluminum foil substrate placed 15 cm away from the needle tip. The connection between the collector and the ground plane of the electrospinning apparatus was maintained by an alligator clip and a cable soldered to a ground connection on the main PCB. For the experiment where nanofibers were electrospun onto skin, no ground cable was necessary to achieve nanofiber deposition directly onto the point of interest.Nanofibers collected on aluminum foil were mounted onto aluminum pin stubs  and sputter-coated with Au/Pd for 50 s at 18 mA. SEM was performed using a FEI Sirion XL830  microscope at 5 kV accelerating voltage, a spot size of 3, and a 5 mm working distance. Nanofiber diameters were measured using ImageJ  by analy−1 with a zero gap of 1 mm at 25 °C. Viscosity values are reported as the average and standard deviation of 10 measurements.The rheological properties of the polymer solutions were measured with a stress-controlled rheometer . A parallel-plate geometry was used with a plate diameter of 25 mm. Viscosity was measured at a constant shear rate of 10 sThe solution for the electrospinning of dyed PAN nanofibers was prepared by dissolving 0.2 g of PAN in 1.8 g of DMF under gentle stirring for 5 min; to this solution, 0.1 g of Procion MX Dye 150 Jet Black  was added under gentle stirring for 1 h. Hair thickening treatments were performed on deceased mice that were euthanized after their utilization in experiments approved by and in compliance with the University of Washington’s Institutional Animal Care and Use Committee’s guidelines. Square patches of fur, approximately 1 cm × 1 cm, were clipped from two mice. The commercial cosmetic powder Toppik™ was applied to the shaved area of one mouse. Dyed PAN nanofibers were directly electrospun onto the shaved area of the second mouse.A typical benchtop electrospinning system comprises three key components: an HV electric potential source, a fluid feed system, and a fiber collector. In order to fabricate a miniaturized and portable version of the benchtop electrospinner, while keeping cost low and functionality high, easy to obtain commercially available electronic components were sought in our design and 3D printed parts were used to create structural elements. The main printed circuit board (PCB) is shown in −1 via the front panel. Both the HV sources and syringe pump are powered by low-voltage batteries. Syringes filled with polymer solution are easily loaded into the electrospinner by an access hatch at the top of the device. A ground connection port is provided on the front side of the device so a collector substrate may be held at 0 V potential relative to the HV source.The apparatus can be operated at a voltage magnitude between 1 and 10 kV of either positive or negative polarity. A dual polarity voltage design was preferable due to evidence in the literature suggesting differences in the ease of synthesis , resulti−1. Thus, while the portable electrospinner cannot guard against all possibilities of receiving a shock, the current limiting measures taken ensure that the delivered current will be of such a small amount that no serious injury is likely.Due to the high voltages required for electrospinning, there are inherent safety concerns for users of any electrospinning apparatus. The matter of safety becomes especially important when designing electrospinners for use by untrained consumers. While the current rating of an electrical circuit is arguably the most salient metric of danger or safety, high-voltage circuits pose the risk of causing electric shocks regardless of the circuit’s current capacity. With these two aspects in mind—current and voltage—extra precautions were taken to limit the current drawn by the portable electrospinner. The portable electrospinner described draws a maximum 2 mA of current, and overcurrent protection is provided by a 2-mA fuse. When DC voltages in excess of 0.5 kV are involved, currents of 40 mA or less pose no serious hazard; conversely, currents of greater than 40 mA can cause involuntary muscle contractions, and currents of greater than 200 mA can induce fatal ventricular fibrillation . With a −1. The magnitude of the HV signal was adjusted via the rotary encoder on the electrospinner’s front panel until a uniform and continuous flow of nanofibers were observed to emit from the needle tip; nanofibers were collected onto a grounded sheet of aluminum foil placed perpendicular to and 15 cm away from the needle tip. To demonstrate the efficacy of the portable electrospinning apparatus, six polymer solutions were spun into nanofibers using the device. Polymers were dissolved in suitably volatile solvents and loaded into 3 mL syringes. The polymers selected include: cellulose acetate (CA) dissolved in an acetone/water mixture, poly (PVA) dissolved in tetrahydrofuran (THF), polyacrylonitrile (PAN) dissolved in dimethylformamide (DMF), poly(ethylene oxide) (PEO) dissolved in water, polycaprolactone (PCL) dissolved in 2,2,2-trifluoroethanol (TFE) and poly(vinylidene fluoride) (PVDF) dissolved in a mixture of DMF and acetone. After a brief 1 s priming period—which allowed a small bead of polymer solution to form at the needle tip of the polymer-loaded syringe—an HV DC signal was applied at the needle tip to induce electrospinning and the syringe pump was set to a constant flow rate of 1 mL hr−1 (Since the viscosity of the polymer solution significantly impacts how well the solution may electrospin into nanofibers, rheological measurements of all the polymer solutions used in this study were measured at a shear rate of 10 s−1 . These d−2 ± 1.4 × 10−3 Pa⋅s and 3.2 × 10−2 ± 1.0 × 10−3 Pa⋅s, respectively.Not only can the device be used for synthesizing nanofibers, but it may produce nanobeads through electrospraying as well. Point-of-need electrospinning designates a situation where pre-fabricated nanofiber mats would prove insufficient or cumbersome because of the large size or irregular geometry of the area to be covered or due to the need to handle the nanofibers prior to application at the desired site. Conversely, a portable electrospinning apparatus that could be used to directly deposit nanofibers onto a large, arbitrarily shaped area overcomes such limitations.One example of point-of-need electrospinning is for the cosmetic treatment of alopecia. Nanofibers may prove to be an ideal cosmetic treatment for hair loss because the morphology of nanofibers may be tuned to be similar to that of human hair. Furthermore, nanofibers of an appropriate polymer may be dyed to match a wide variety of hair shades ,32,33. AIt should be noted that the results of this experiment are intended to serve as a proof-of-concept that nanofibers may be used to simulate human hair in the cosmetic application of nanofibers to hide an area of thinning hair. Before extending such a concept to use on humans, more study is required with respect to the biocompatibility and biodegradability of the nanofiber material employed. It is possible that an improperly engineered nanofiber material—when in contact with human skin—could produce an adverse reaction with the delicate biological environment presented by humans. We have constructed and demonstrated the use of a custom-designed portable electrospinning apparatus for direct-to-skin nanofiber generation for cosmetic hair-thickening treatments. The benefits of this electrospinning device over conventional equipment include: low cost, portable and handheld design, battery powered, composed of off-the-shelf components and dual polarity HV sources. A wide variety of polymeric nanofibers can be synthesized with the handheld electrospinner. The mobility of this handheld device was showcased in a point-of-need electrospinning scenario to generate dyed nanofibers for application in a cosmetic remedy for alopecia. The deposited nanofibers concealed an area of thinning hair and delivered aesthetically similar benefits as compared with a commercially available treatment option—Toppik™—while improving upon the adherence of the applied material to the site of interest. A portable electrospinning apparatus may prove to be highly appealing in direct-to-skin nanofiber deposition for both healthcare and consumer end-user scenarios.


During polymerization, the fluorinated alcohol catalyst forms multiple dynamic hydrogen bonds with the initiator, monomer and propagating polymer chain. These cooperative hydrogen bonding interactions activate the NCA monomers and simultaneously protect the overactive initiator/propagating polymer chain-ends, which offers the whole polymerization with high activity and selectivity. Mechanistic studies indicate a monocomponent-multifunctional catalytic mode of fluorinated alcohol. This finding provides a metal free and fast approach to access well-defined polypeptides.Organocatalysis is an important branch of catalysis for various organic transformations and materials preparation. Polymerizations promoted by organic catalysts can produce polymeric materials without any metallic residues, providing charming materials for high-value and sensitive domains such as biomedical applications, microelectronic devices and food packaging. Herein, we describe a fluorinated alcohol based catalytic system for polypeptide synthesis via catalytic ring-opening polymerization (ROP) of  N-carboxyanhydride, fulfilling cocatalyst and metal free conditions with high rate and selectivity.Polymerizations promoted by organic catalysts can produce polymeric materials without any metallic residues contamination. Here the authors show a fluorinated alcohol based catalytic system for polypeptide synthesis from α-amino acid  Ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCAs) initiated by different amines and amines derivatives is considered to be the most common method for polypeptide synthesis. However, in the ROP of NCAs, the normal amine mechanism (NAM) and activated monomer mechanism (AMM), often compete with each other, which complicates the overall polymerization process, making it challenging to produce well-defined (co)polypeptides  or by exploring new reaction conditions for the traditional primary amine-initiated polymerizations . As for the ROP of NCA promoted by metal complexes, the metal residues in resultant polypeptides can cause toxicity concerns. An extra purification of the as-synthesized polymer such as dialysis is usually required. In regards to the NCA polymerizations initiated by organic initiators, low polymerization activities are usually observed, even with low monomer/initiator ratios11, showing limitations in the synthesis of polypeptides with high molecular weights and/or for ROP of NCAs with low stabilities. Therefore, developing a fast and metal-free process for the synthesis of polypeptides is very necessary and promising.Synthetic polypeptides, as mimics of natural analogues, is a unique family of bio-inspired biomaterials with broad biomedical applications including controlled drug release, gene delivery, tissue engineering, and regenerative medicine21. In an analogous manner to natural systems, organocatalysis utilizing hydrogen bonding to accelerate and control chemical reactions is of particular interest in organic chemistry and has become an active and vibrant research area in the past two decades25. Numerous classes of compounds such as binols28, silane diols30, squaramides32, (thio)ureas34, guanidine35, and amidine functionalities36 have been explored for various organic transformations. Furthermore, as an elegant alternative to organometallic and enzymatic catalysis, hydrogen-bonding organocatalysis has been a promising pathway for innovative polymer synthesis, especially for the preparation of metal-free polymers required for advanced packaging, medical and microelectronic applications42.Hydrogen-bonding is widely effective in biological systems, playing a crucial role in sustaining the elegant architecture/functionality of proteins and nucleic acids44. For example, in biochemistry trifluoroethyl alcohol (TFE) and hexafluoroisopropyl alcohol (HFIP) are used to modify the conformation of proteins47. In organic chemistry, fluorinated alcohols are widely accepted as exceptional promotion media to perform organic reactions50, including oxidation reactions with H2O2 or sodium hypochlorite53, aza-Michael reaction54, protection and deprotection of amine groups56, cyclopropanation of alkenes57, and oxirane ring-opening59. In polymer chemistry, fluorinated alcohols are effective solvents to control the stereospecificity of atom transfer radical polymerization (ATRP)63 and conventional radical polymerization65. So far, fluorinated alcohols, as small molecule metal free catalysts, rather than reaction mediums for polymer synthesis, are seldom explored. The only attempt refers to a fluorinated alchohol/(−)-sparteine bicomponent catalytic system developed by Hedrick et al. for ROP of cyclic esters, in which a fluorinated alcohol is used to activate the monomer and an organic base cocatalyst (−)-sparteine to activate the initiating/propagating alcohol. Without (−)-sparteine, the polymerization cannot happen , resulting in well-defined polypeptides. Different from the bicomponent-bifunctional catalytic process of fluorinated alchohol/(−)-sparteine system for ROP of cyclic esters66, the 1,3-Bis-HFAB catalyst doesn’t require a Lewis base cocatalyst, and it is defined as a monocomponent-multifunctional catalytic process for ROP of NCA benzene , a small fluorinated alcohol, without the assistance of a Lewis base cocatalyst, can catalyze a fast and selective ROP of NCA Fig. . During N,N-dimethylethanolamine (DMEA), as the initiator. The catalyst 1,3-Bis-HFAB and the initiator DMEA were dissolved in dichloromethane, followed by introduction of a dichloromethane solution of Glu-NCA monomer at room temperature. Under these conditions, the polymerization reaction proceeded not only at a high rate but also high selectivity, with negligible side reactions, such as chain-transfer and chain-termination. As shown in Table w/Mn = 1.07, Mn,SEC = 4.5 × 104; Table ]  of polypeptides increased linearly as a function of [Glu-NCA]/[DMEA] ratio and the molecular weight distributions remained narrow (Đ = 1.05–1.07), indicative of the living nature of the polymerization 2NCH2CH2O-[COCH(CH2CH2COOCH2C6H5)NH]n-COCF3}H+ (n = 7–23) was established, which is in good agreement with an oligomeric polypeptide having −NH2 and DMEA end-groups . These results suggest that with DMEA as an initiator, the NCA monomers can undergo rapid and selective polymerization, giving polypeptides with well-defined molecular weight and low Đ.The catalytic performance of 1,3-Bis-HFAB for ROP of Glu-NCA was evaluated by using a small aminoalcohol molecule, ion Fig. . The endlue Fig. . The chelue Fig. . Based ow/Mn = 1.05, Mn,SEC = 35.87 × 104; Table Besides possessing a high selectivity for propagating chains, the ROP of Glu-NCA promoted by 1,3-Bis-HFAB exhibits high activities. When the [Glu-NCA]/[DMEA] ratio was changed from 120 to 960 by varying the initiator concentration [DMEA] (Table 0 = 0.19 M and [DMEA]0 = 4.0 × 10−4 M), the conversion of Glu-NCA with time was monitored by in situ FT-IR at various catalyst concentrations  until full conversion was reached. Sampling was from 2800 cm−1 to 650 cm−1 at 8 wavenumber resolution and the automatic sampling interval was 10 s, which guaranteed the real-time data acquisition of polymerization  to α  configuration during growth68. The rate constant for the second propagation step is at least several times larger than that for the first. Thus, the whole polymerization rate is largely dependent on the second stage propagation. As shown in Fig. kapp = 1.50 × 10−3 s−1, 1.20 × 10−3 s−1, 9.98 × 10−4 s−1, 9.29 × 10−4 s−1, and 6.29 × 10−4 s−1) were obtained from the semilogarithmic plots Ln([M]0/[M]t) = kapp t. The plot of Lnkapp versus Ln0 is linear, and the gradient of the best-fit line is −0.3785. Therefore, the kinetic order in catalyst is equal to −0.40 . The minus order in catalyst indicates that high loading amount of catalyst in the system can cause a decrease of the polymerization rate.We further studied the reaction kinetics by using in situ FT-IR technique Fig. . At fixeion Fig. . The pol0 = 0.19 M and 0 = 1.58 × 10−3M), the conversion of Glu-NCA with time was monitored at various initiator concentrations . Again, the consumption of the monomer follows the first-order kinetics. Hence the kinetic order in initiator is 1.45 . As a result, the polymerization rate law is Rp = −d[M]/dt = kp−0.40[DMEA]1.45[NCA].The dependence of polymerization rate on initiator concentration was also determined Fig. . At fixe1H NMR comparative study of 1,3-Bis-HFAB, Glu-NCA and 1,3-Bis-HFAB/Glu-NCA mixture revealed that 1,3-Bis-HFAB didn’t induce the ROP of NCA and acted as a hydrogen-bonding donor to Glu-NCA. When 1,3-Bis-HFAB was mixed with equivalent Glu-NCA monomer in anhydrous DCM-d2 and monitored at room temperature for 24 h, no evidence of polymer was observed in the 1H NMR spectrum. Due to the hydrogen-bonding interaction between Glu-NCA and 1,3-Bis-HFAB, the -OH of 1,3-Bis-HFAB shifted downfield from 3.66 to 3.92 ppm in the 1H NMR spectrum. Only one hydroxyl proton signal was observed, indicative of a fast equilibrium on the NMR time scale  in the 19F NMR spectrum  spectroscopy Fig. . The 1H 69. However, the end-group analysis of polypeptide by 1H NMR and MALDI-TOF in the present study shows that the tertiary amine of DMEA is totally silent during polymerization  also shifted downfield slightly  and a very broad molecular weight distribution (Đ = 1.63) 2NCH2CH2NH2) in the absence of 1,3-bis-HFAB . DMEDA has both tertiary amine and primary amine in its molecular structure and can trigger the NCA polymerization via both mechanisms. The SEC characterization demonstrated that the resultant polypeptide possessed a bimodal molecular weight distribution (Đ = 1.38) and the experimental molecular weight  is more than two times higher than the theoretical molecular weight  .Since the polymer chain-end will be primary amine group after initiating by the hydroxyl group of DMEA, we further investigated the interaction of 1,3-Bis-HFAB with polymer chain-end by using 2-amino-2-phenylacetate (MAP), as a model molecule. The model molecule has a structure similar to the polymer chain end. The 4. Therefore, the hydrogen bonding formed between 1,3-Bis-HFAB and chain-end primary amine can provide a dynamical protection of the overactive chain-end amine and suppress the occurrence of side reactions in system. A control experiment was carried out by using a primary amine, n-hexylamine (HA) as initiator for the ROP of Glu-NCA, in the absence of 1,3-Bis-HFAB . The results showed that the polymerization was very slow, forming polypeptide with a broad molecular weight distribution . A further kinetics study via in situ FT-IR method showed that the polymerization process did not have a constant reaction rate  than the theoretical molecular weight . Also, the monomer could not be completely converted to polypeptides even with a very long reaction time (1 day).According to previous reports in the literature, the chain-end primary amine is prone to side reactions during polymerization, which make it challenging to synthesize polymers with complex architectures like block copolymers/DMEA complex  showed that the charge of the oxygen atom of DMEA increased from −0.497 to −0.229 after forming hydrogen-bonding with 1,3-Bis-HFAB, rendering the hydroxyl group of DMEA in a dormant state without nucleophilicity (Supplementary fig. 3)2 increased from −0.126 to 0.318 and lost nucleophilicity. The charge of nitrogen atom of –NH2 increased from −0.867 to −0.713, indicative of a decreased nucleophilicity of –NH2 -fitting charges are derived from the DFT calculation. The lowest-energy structure of 1,3-Bis-HFAB/Glu-NCA complex  demonstrated that the –OH of 1,3-Bis-HFAB formed hydrogen-bonding with the C(5) = O of NCA monomer. The charges assigned to the two hydrogen atoms of –OH groups in 1,3-Bis-HFAB decreased from 0.393/0.390 to 0.380/0.238 and the charge of the oxygen atom of NCA carbonyl group decreased from −0.350 to −0.389 , catalyst  and propagating polymer chain exist. We postulate that the catalysis proceeds by simultaneous activation of the carbonyl of a NCA monomer and dynamic protection of the polymer chain-end amine group via hydrogen bonding to 1,3-Bis-HFAB. Nucleophilic ring-opening of the NCA leads to propagation, whereby the ring-opened NCA forms the propagating amine for the subsequent addition of monomer Fig. . During 1H NMR, 19F NMR, and EDS. We purposely prepared a high molecular weight polypeptide sample from polymerization with high loading of catalyst . When the polymerization is complete, and the resultant polypeptide was precipitated using methanol, a further two-step washing process of the precipitated polypeptide with methanol can totally remove the catalyst from the final polymer. The 1H NMR, 19F NMR, and EDS spectra show no 1,3-Bis-HFAB catalyst residue in the final polypeptide . Afterwards, the cytotoxicity was evaluated by adding CCK-8 solution to each well of the plate. The cell viabilities with the extracts of polypeptide exceeded 95%  expanding NCA monomers, (2) functionalizing polypeptides and polypeptide hybrid copolymers by designing new initiators.1H, 19F, and 13C NMR spectra were recorded on a Bruker AV500 spectrometer . Chemical shifts are given in ppm and were referenced to the resonance for residual solvent (δ7.26 for CDCl3 and 5.32 for CD2Cl2). Anhydrous CD2Cl2 (99.9 atom % D) was used for the NMR study of the hydrogen-bonding interactions between catalyst, monomer and propagating chain. A mixed deuterated solvent (CDCl3/CF3COOD = 2) was used for the 1H NMR characterization of polypeptide oligomer. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra were collected on a Bruker UltraFLEX MALDI-TOF mass spectrometer in the reflector mode. 2,5-Dihydroxybenzoic acid (DHB) was used as matrices, and dichloromethane(85%)/trifluoroacetic acid (15%) was used as solvent. No cationic agent was added. Energy dispersive X-ray spectroscopy (EDS) analysis was carried out with a JSM-7500F scanning electron microscope . The polymer film sample for EDS analysis was prepared by using solution spin coating method and was metalized with a sputtered gold layer before testing. The in situ FT-IR monitoring of NCA polymerization was carried out by using TENSOR II with MCT Detector from BRUKER. An attenuated total reflectance (ATR) diamond probe was connected to the reaction flask via AgX Fiber . Sampling is from 2800 cm−1 to 650 cm−1 at 8 wavenumber resolution and the automatic sampling interval is 10 s. The real-time concentration of NCA was quantified by measuring the intensity of NCA’s anhydride peak at 1792 cm−1. Polymer characterizations were carried out by using Agilent 1260 infinity SEC instrument. The fractionation module was connected with three different detectors (from Wyatt Technology), Optilab T-rEX RI detector, ViscoStar-II viscometer and DAWN HELEOS-II multiangle laser-light scattering (MALLS) detector at a laser wavelength of 690 nm. Three 7.8 × 300 mm columns  and one guard column were used for polymer fractionation. The columns were calibrated with a series of polystyrene standards . HPLC-grade DMF (containing 0.1 M LiBr) was used as the mobile phase at a flow rate of 1.0 mL/min. The whole SEC system was equilibrated in mobile phase at 60 °C. Polymer solutions with concentrations between 5.0 and 10.0 mg mL−1 were injected with an injection volume of 200 μL. ASTRA software from Wyatt Technology was used for the data collection and analysis. The absolute molecular weights of resultant polypeptides were calculated with dn/dc values (dn/dc(PBLG) = 0.104 mL g−1, dn/dc(PZLL) = 0.123 mL g−1, dn/dc(PABLG) = 0.126 mL g−1, dn/dc(PBLG-co-PZLL) = 0.114 mL g−1, dn/dc(PBLG-co-PABLG) = 0.115 mL g−1. PBLG, PZLL, and PABLG represent polypeptides obtained from ROP of Glu-NCA, Lys-NCA and Alkynyl-NCA, respectively.) The Density Functional Theory (DFT) calculation was performed at the GGA/PBE/DNP level of theory. All of the constructed structures are fully optimized and electrostatic potential (ESP)-fitting charges are derived from the DFT calculation.−1 to 650 cm−1 at 8 wavenumber resolution and the automatic sampling interval is 10 s. When full conversion was achieved from the in situ FT-IR spectra, 0.2 mL of the reaction mixture was taken out from the reaction system and diluted to 10 mg/mL by using HPLC-grade DMF (containing 0.1 M LiBr). The solution was then used for SEC characterization to determine the molecular weight and molecular weight distribution of the obtained polypeptides. The remaining reaction mixture was poured into methanol, sonicated, and centrifuged to remove the solvent. The white precipitate was collected and further washed twice with methanol and then dried overnight under vacuum to give the final polymer. Polypeptide with variable molecular weights was prepared by tuning the relative ratios of monomer to initiator [NCA]/[DMEA]. Homopolymerization of Lys-NCA or Alkynyl-NCA was operated by following above operations at 25 °C with [NCA]0 = 0.19 M. As for random copolymerizations of Glu-NCA with Lys-NCA or Alkynyl-NCA, the overall monomer concentration [Glu-NCA + Lys-NCA]0 or [Glu-NCA + Alkynyl-NCA]0 was fixed at 0.19 M. As for the sequential polymerization of Glu-NCA with Lys-NCA or Alkynyl-NCA, the polymerization of Glu-NCA was carried out firstly. When a full conversion of the Glu-NCA was achieved, the Lys-NCA or Alkynyl-NCA monomer was quickly added into reaction system. The whole polymerization process was monitored by in situ FT-IR in order to determine the adding time of the second kind of monomer.A typical procedure for the ring-opening polymerization of NCA was performed at 25 °C in a Braun Labmaster glovebox. Before polymerization, an attenuated total reflectance (ATR) diamond probe was connected to the reaction flask via a AgX Fiber . A solution of the initiator (DMEA) and catalyst  in 4 mL of reaction solvent (DCM) was firstly added into the flask. After stirring at 25 °C for 1 min, the monomer solution of 0.4 g Glu-NCA monomer in 4 mL DCM was then quickly added into the flask to start the polymerization and the auto sampling system was switched on immediately. Sampling is from 2800 cm2 in dulbecco’s modified eagle medium . The extract of the polypeptide sample was prepared by following international standard ISO 10993-12:2012. The cellular toxicity of the polypeptide samples was evaluated as follows. HepG2 cells were firstly seeded in 96-well plates with a density of 5 × 103 cells per well and incubated overnight. Subsequently, the cells were incubated for 48 h with various concentrations of the extract of polypeptides. Afterwards, the cytotoxicity was evaluated by adding Cell Counting Kit-8 (CCK-8) solution  to each well of the plate. After incubation for 1 h, the absorbance was investigated at 450 nm using a Multiskan MK3 Microplate Reader. For fluorescence microscopy, a live-dead cell staining kit from RayBiotech, Inc. was used. The live-dead cell staining kit provides the ready-to-use reagents for convenient discrimination between live and dead cells. The kit utilizes Live-DyeTM, a cell-permeable green fluorescent dye (Ex/Em = 488/518 nm), to stain live cells. Dead cells can be easily stained by propidium iodide (PI), a cell non-permeable red fluorescent dye (Ex/Em = 488/615). Stained live and dead cells can be visualized by fluorescence microscopy using a band-pass filter.The human liver cancer cell line HepG2 was purchased from ATCC . Before experiments, the cell was kept at 37 °C in a humidified atmosphere of 5% COSupplementary InformationPeer Review FileSource Data


The effects of miR-216a overexpression and inhibition on ovarian cell proliferation, colony formation, and cisplatin resistance were investigated by MTT assay and soft agar colony formation assay. Bioinformatics analyses using TargetScan and rVista, qPCR, and luciferase assay were also used to explore and verify downstream effectors and regulators of miR-216a. Proliferation, colony formation, and cisplatin resistance of ovarian cancer cells are promoted by miR-216a overexpression but inhibited by miR-216a inhibition. PTEN is a direct target of miR-216a and PTEN expression antagonizes the tumor-promoting function of miR-216a. STAT3 is a regulator of miR-216a, and PTEN is also regulated by STAT3. miR-216a up-regulation is associated with cisplatin resistance in ovarian cancer and this effect is mediated by PTEN. STAT3 is a regulator of miR-216a. Strategies that inhibit miR-216a is a potential strategy for overcoming the cisplatin resistance in ovarian cancer.Cisplatin is the first-line treatment for ovarian cancer. However, the clinical outcome of cisplatin treatment in ovarian cancer is hindered by cancer resistance. Here we aim to explore the role and mechanism of  Ovarian cancer is the fifth most common cancer amongst women in the United States. According to the estimation by American Cancer Society, approximately 22240 women will be newly diagnosed of ovarian cancer and approximately 14070 women will die from ovarian cancer in 2018. Despite progresses made in ovarian cancer therapy, the survival of patients with advanced ovarian cancer still remains dismal, due to the lack of effective treatment strategies to impede ovarian cancer metastasis and recurrence. Cisplatin, the platinum-based treatment, has been used as the first-line chemotherapy drug for ovarian cancer since more than 30 years ago [miR-214, miR-130a-3p, and miR-25-3p have been identified as biomarkers of cancer cisplatin resistance [Recent studies have implicated a plethora of factors involved in the ovarian cancer cisplatin resistance ,4, inclusistance . These fmiR-214 is responsible for the enhanced cell survival and cisplatin resistance of ovarian cancer by targetting PTEN [miR-216a is amongst the most investigated miRNAs in cancer [miR-216a is also an important mediator of PTEN [In ovarian cancer, the essential role of a number of miRNAs have been reported ,11. Alteing PTEN . miR-216n cancer . It has  of PTEN .miR-216a expression and malignant ovarian cancer and explore the mechanism of miR-216a in regulating cisplatin resistance in ovarian cancer. The effects of miR-216a up-regulation or inhibition on the phenotypical changes in two commonly used ovarian cancer cell lines, SKOV3 and OVCA433, were investigated. We are particularly interested in the downstream effector and upstream regulator of miR-216a. The results reported in the present study could provide clear evidence on the functional role of miR-216a and potentially provide an opportunity for the diagnosis, treatment of cisplatin-resistant ovarian cancers.Herein, we strive to clarify the correlation between 2. The cisplatin resistant SKOV3 CR cells were acquired by maintaining SKOV3 cells in the presence of cisplatin over a 10-month period. The cisplatin resistance sustained when SKOV3 CR cells were grown in the absence of cisplatin for 30 passages.SKOV3 and OVCA433 were acquired from American Type Culture Collection . Cells were cultured in RPMI-1640 medium supplemented with 10% FBS, in an incubator maintained at 37°C and 5% COMiR-216a mimics and miR-216a inhibitors (miR-216a siRNA), were transfected into ovarian cancer cells to induce miR-216a up-regulation and down-regulation, respectively. The PTEN plasmid was cloned into pcDNA3.1 plasmid and transfected into the cells using Lipofectamine 2000  according to manufacturer’s protocols.The miRNAs were purchased from GenePharma . MTT assay was used to assess proliferation of cells. In brief, 2000 cells were seeded into 96-well plates at 24 h after treatment, the proliferation was monitored for 5 days. On each day, MTT reagent was added to a well and incubated for 2 h. Then, medium of the well was removed and 100 µl of DMSO was added and the absorbance was measured at 562 nm. Soft agar colony formation assay was performed according to a previously published protocol [C−ΔΔT method.RNA was extracted from cells using the miRNeasy Mini Kit  according to manufacturer’s recommendations. After purification, RNA was transcribed into cDNA using the High-Capacity cDNA Kit . Real-time PCR was then performed using the SYBR Green Master Mix . The primers used for qPCR include: PTEN sense 5′-TTGGCGGTGTCATAATGTCT-3′, antisense 5′-GCAGAAAGACTTGAAGGCGTA-3′; STAT3 sense 5′-TAGCAGGATGGCCCAATGGAATCA-3′, antisense 5′-AGCTGTCACTGTAGAGCTGATGGA-3′; GAPDH sense 5′-GAGTCAACGGATTTGGTCGT-3′, antisense 5′-TTGATTTTGGAGGGATCTCG-3′. The expression of GAPDH was used as a control. Quantitation of mRNA levels was performed using the 2Western blot analysis was used to analyze PTEN expression. Proteins in SKOV3 cells were first extracted after cells’ lysis. Protein lysates of 20 µg were then used for SDS/PAGE, followed by transferring on to PVDF membranes. Rabbit anti-PTEN primary antibody was purchased from Abcam. The HRP–conjugated anti-rabbit secondary antibody was purchased from Abcam. PVDF membranes were blocked with 1% BSA for 1 h at room temperature. The anti-PTEN primary antibody diluted at 1:1000 in PBS-T  was then added to the membrane and incubated at RT for 1 h with gentle shaking. After extensive washing with PBS-T, HRP–conjugated anti-rabbit antibody (1:20000 dilution) was added to the membrane and incubated at RT for another hour.TargetScan and rVista 2.0 were used for bioinformatics analysis. The prediction of effective miRNA target sites in mammalian mRNAs was performed using TargetScan using a previously published protocol . The evot test was used to compare differences between two groups, and two-way ANOVA was used for comparison amongst three groups or between two groups with two factors. P-values of less than 0.05 were considered statistically significant.SPSS was used for statistical analysis. Data were represented as mean ± S.D. Student’s miR-216a in ovarian cancer, miR-216a mimics or inhibitors were transfected into the SKOV3 and OVCA433 ovarian cells, followed by monitoring proliferation by MTT assay. As shown in miR-216a mimics promoted the proliferation of SKOV3 . As shown in miR-216a was markedly up-regulated in SKOV3 CR. Further, SKOV3 cells were treated with cisplatin (4 μg/ml) for varied duration (0–72 h), and consequently, an increasing miR-216a levels were seen with treatment duration . TargetScan study further identified the binding sites of miR-216a on PTEN  (P<0.001). These data implied that PTEN down-regulation is correlated with cisplatin-resistance. As another verification, we overexpressed PTEN in SKOV3 cells and observed an increased cell survival with increasing dose of cisplatin  D, and PTP<0.001) E. At dayisplatin F. Furthes effect G. Taken miR-216a in ovarian cancer. Using rVista 2.0 for binding region prediction, we uncovered that miR-216a promoter contains a binding region for STAT3   E. These miR-216a in cisplatin resistance of ovarian cancer. Thus far, miR-216a has been reported as both a tumor inducer and a tumor suppressor [miR-216a in ovarian cancer still remains elusive. Additionally, the link between miR-216a and cancer resistance has rarely been established. Previously, Xia et al. [miR-216a/217 induces epithelial-to-mesenchymal transition (EMT) through targetting PTEN, thereby promoting drug resistance and recurrence of liver cancer. The up-regulation of miR-216a, which can be detected by measuring miR-216a levels in plasma, has also demonstrated potential as a diagnostic marker in pancreatic cancer [miR-216a increases cisplatin resistance in ovarian cancer. What has not been demonstrated here is whether miR-216a possesses diagnostic value in ovarian cancer. In this context, the measurement of plasma miR-216a levels can also be adopted to study the correlation of miR-216a up-regulation and clinical outcomes of ovarian cancer patients. No effective screening method is available to detect early-stage ovarian cancer with high sensitivity and specificity. The close alliance between miR-216a up-regulation and cisplatin may allow to identify the patients who do not respond to cisplatin therapy, consequently facilitating timely and efficient treatment planning for this highly lethal disease.Here we demonstrate the important role of ppressor ,23 and tc cancer . HowevermiR-216a to inhibit miR-216a expression, which is a clinically applicable approach. Fueled by recent advances in moving siRNA therapeutics from bench to clinics [miR-216a expression. It should be noted that cisplatin resistance may also be ascribed to other mechanisms, such as reduced accumulation of the drug [Our study provides a new avenue for overcoming cisplatin resistance to improve the clinical outcomes of patient with ovarian cancers at advanced stages. Currently, the overall 5-year survival rates of patients diagnosed at stages III and IV of this disease are 32 and 18%, respectively ,25–27. T clinics , possiblthe drug , increasthe drug  and gluothe drug , as wellmiR-216a. We also analyzed a panel of putative cancer-related genes, including CDK8, MITF, SMAD7, KLF12, AG02, CDK14, and BCL-9 [miR-216a overexpression. The loss of PTEN has been implicated as an important mediator of EMT [miR-216a led to reduced apoptosis by cisplatin [We identified PTEN as the downstream effector of nd BCL-9 , but onlr of EMT , which eisplatin . This obisplatin .miR-216a in ovarian cancer regulation, we also demonstrated that STAT3, a putative oncogene in ovarian cancer [miR-216a. Previous studies have suggested the interaction between miR-216a and JAK2/STAT pathway [miR-216a. STAT3 induces cellular transformation and promotes tumorigenesis by regulating a wide array of oncogenes, including c-Myc, cyclinD1 etc. [miR-216a by binding to the promoter region of miR-216a. These data are in accordance with previous evidences that STAT3 regulates the expression of a number of miRNAs [miR-216a, PTEN was suppressed by STAT3 overexpression. In-line with this, the interaction of PTEN and STAT3 has been demonstrated previously and their levels of expression were shown to be negatively correlated [miR-216a with both of them, miR-216a is an important molecule in cancer and further investigations are warranted to further demonstrated the clinical utility of miR-216a in ovarian cancer.To elucidate the underlying mechanism of n cancer , is a re pathway ,18. HowenD1 etc. . In the f miRNAs . By regurrelated ,46. GivemiR-216a is an inducer of cisplatin resistance in ovarian cancer. Inhibition of miR-216a attenuated the cisplatin resistance. Using bioinformatics approach and further validation with qPCR and Western blot, we show that PTEN is an effector and STAT3 is a regulator of miR-216a. By elucidating the role and mechanism of miR-216a in ovarian cancer, our study provides an opportunity to overcome cisplatin resistance in ovarian cancer.Here we show that


Mycobacterium tuberculosis (Mtb) infection, 2–4 mm spherical structures called granulomas develop in the lungs of the infected hosts. These are the hallmark of tuberculosis (TB) infection in humans and non-human primates. A cascade of immunological events occurs in the first 3 months of granuloma formation that likely shapes the outcome of the infection. Understanding the main mechanisms driving granuloma development and function is key to generating treatments and vaccines. In vitro, in vivo, and in silico studies have been performed in the past decades to address the complexity of granuloma dynamics. This study builds on our previous 2D spatio-temporal hybrid computational model of granuloma formation in TB (GranSim) and presents for the first time a more realistic 3D implementation. We use uncertainty and sensitivity analysis techniques to calibrate the new 3D resolution to non-human primate (NHP) experimental data on bacterial levels per granuloma during the first 100 days post infection. Due to the large computational cost associated with running a 3D agent-based model, our major goal is to assess to what extent 2D and 3D simulations differ in predictions for TB granulomas and what can be learned in the context of 3D that is missed in 2D. Our findings suggest that in terms of major mechanisms driving bacterial burden, 2D and 3D models return very similar results. For example, Mtb growth rates and molecular regulation mechanisms are very important both in 2D and 3D, as are cellular movement and modulation of cell recruitment. The main difference we found was that the 3D model is less affected by crowding when cellular recruitment and movement of cells are increased. Overall, we conclude that the use of a 2D resolution in GranSim is warranted when large scale pilot runs are to be performed and if the goal is to determine major mechanisms driving infection outcome . To comprehensively compare the roles of model dimensionality, further tests and experimental data will be needed to expand our conclusions to molecular scale dynamics and multi-scale resolutions.Within the first 2–3 months of a  Adding further complication is that etch human suffers the development of multiple granulomas, and each granuloma has been shown to have a unique trajectory [Tuberculosis (TB) is an ancient disease that has re-emerged as the number one cause of death due to infection in the world . Despiteajectory , As granThere are several animal models that are used to study TB . Mice weGranSim, which is an ABM that was developed in two-dimensions (2D) and is a hybrid formulation of an agent-based model and differential equations that tracks spatio-temporal granuloma development in real time . Many otime see .With both 2D granuloma experimental data as well as with simulations, the question remains as to how well they capture the 3D granuloma structures. While we are not poised to address the wet lab aspect of this question in this work, we can explore the relationship between our 2D simulations and a new 3D version of the model developed herein to predict how well, and under what circumstances, the 2D representation does a good job of predicting 3D behavior. Datasets derived from NHPs and humans on a whole-granuloma scale provide data from 3D in vivo experiments that we can use to calibrate the 3D model and validate our predictions.We then are able to perform sensitivity and uncertainty analyses to predict what factors drive simulated granuloma dynamics in 3D and how they are similar to or different from those that drive dynamics in 2D. As questions pertaining to the importance and role of dimensionality are relevant to many other applications of 2D modelling found in the literature, for instance, that of tumors, this work can also prove helpful to a larger community.The questions regarding ABM modelling of lymph nodes (LNs) that we posed in our previous work  guide thIn our previous 2D study of LNs , we defiThe measures examined above describe cells locating each other in space (either 2D or 3D), and thus it is not surprising that the sensitivity analysis identified different parameters between 2D and 3D that influenced model outcomes when those factors related to movement and dynamics of cell-cell interactions. As shown by the sensitivity analysis results in our LN studies, the parameters that most strongly affect our 3D model outputs ,18 are dTo our knowledge, since our LN study above there has been only one other study that compares results between a 2D and 3D model. In , GadhamsThe purpose of the work herein is to assess whether dynamics and mechanisms driving outcomes matter in 2D versus 3D for modelling TB granulomas and what can be learned in the context of 3D modelling that is missed in 2D. Additionally, using sensitivity analysis comparisons, we aim to determine which factors hold as significant across 2D and 3D, and which do not. This can assist in the choice between using 2D or 3D models in simulations going forward, as 3D models are much more expensive to run in terms of both simulation time and real time for research goals. Thus, having a way to parse which to use under what conditions will be extremely useful.2.GranSim, our ABM of single granuloma formation during Mtb infection. The next sections briefly describe (i) granuloma immunobiology, (ii) 3D-ABM implementation of GranSim, (iii) uncertainty and sensitivity analysis and (iv) model calibration steps.We integrated experimental data and computational model simulation to build and calibrate a 3D version of 2.1.GranSim. Then we show how the biology is transformed into computational and mathematical constructs that recapitulate those features and allow us to postulate on the mechanisms driving granuloma formation and development.The immunobiology of Mtb infection has been described in many previous studies, both from our group and from others ,20,21. H2.1.1.Upon inhalation of Mtb, the host mounts an immune response which results in the localization of immune cells, namely lymphocytes, phagocytes (primarily macrophages) and polymorphonuclear cells (neutrophils and eosinophils), and fibroblasts in the lungs . Each ceGranSim represents a platform for studying the dynamics of individual granulomas in silico. The model can be used to generate insights into the emergence of such complex and consistent structures and how they function [Multiple granulomas can form within a single lung upon infection. However, it is noteworthy that granulomas evolve largely independently from one another, and function .2.1.2.GranSim is a hybrid ABM that describes the formation and function of a granuloma during Mtb infection in the lung. GranSim captures the cellular behavior of macrophages , T lymphocytes , CD8+, and regulatory), and bacteria . It also incorporates molecule dynamics, principally degradation and diffusion of the relevant cytokines Tumor Necrosis Factor (TNF), Interferon gamma (IFN-γ) and interleukin 10 (IL-10) and the chemokines ; these are modelled using partial differential equations. Bacteria can be simulated either as agents or as continuous entities.GranSim has been developed and curated since 2004 in 2D [2. The size of each micro-compartment has been determined by matching the grid compartment to the average size of the largest immune cell, macrophages, reconciling their average speed with the iteration clock of GranSim (10 min) [Although granulomas are 3D structures in the lung, due to the high computational costs associated with simulating in 3D, 04 in 2D ,13,23–28(10 min) . Each mi(10 min) , even wh(10 min) . The dif3 (see sourceDensity and intiDensity). More details on GranSim rules and specifications can be found in This study represents the first attempt to simulate a 3D TB granuloma in the lung . The ini3 see  and resiGranSim is integrated with the 2D version in a single source code (see [Our implementation of the 3D version of ode (see ). This uIn order for our software to be cross-platform among Linux, Windows, and MacOS X, we used the Qt application framework for individual runs . The Qt 2.1.3.We used DataTank, an object-oriented programming environment providing large dataset support and visualization tools, to generate many of the figures herein  scheme. Sensitivity analysis was performed by a generalized correlation coefficient, namely the partial rank correlation coefficient (PRCC). The details on LHS and PRCC procedures are available in our review  and webs2.3.To calibrate our 3D model, we used published experimental data on bacterial loads per granuloma  in NHPs ,37. At aThe model calibration protocol used here aims to limit the stochastic variability embedded in the ABM implementation. Stochastic variability means that solving the ABM with the same parameter set  will not return the same outcome as in deterministic systems  in silico granulomas passed both steps  the equiata  per granuloma at time points: days 7, 14, 28, 50, 60, 90, and 100 post infection . growthRatelntMtb) is consistently selected at the most important mechanism driving; CFU in single granulomas in both 2D and 3D, with values ~0.9.Both the 2D and 3D PRCC results consistently predict the same most important parameters, and their magnitudes and signs are consistent across the two resolutions. It is important to note that this consistency of PRCC results is mostly true for positive PRCCs, which are also the largest ones. For example, the intracellular Mtb growth rate  or the maximum probability of recruiting either a macrophage (maxRecPro.) or a Tgamma cell (Tgam_maxRecProb): are consistently important in the 2D setting (negative correlation), suggesting that a potential crowding effect, caused by the emergence of many cells in the granuloma, might affect recruitment much more in 2D than in SD. This finding; is confirmed by this PRCC (negative correlation) for the parameter probMoveToMac , as it is consistently significant in 2D but absent in 3D. A crowding effect is likely stronger in 2D due to the lower degrees of freedom for agent movement in its Moore Neighborhood (8 vs. 26 possible directions or not moving). Enhancing recruitment as well as facilitating movement of T cells in 2D seems an effective mechanism to reduce bacterial levels. In contrast, the 3D setting does not gain any significant advantage when these two mechanisms are increased .Parameters that directly affect cell recruitment, such as the vascular sources of the lung grid A regulatory mechanism triggered by cell recruitment that appears to be consistently at play in both 2D and 3D after 4 weeks post infection is the (max) recruitment of regulatory T cells: the more Tregs are recruited, the higher the CFU per granuloma. This effect is more pronounced in 3D.3.2.2.(kApoptosis) and the TNF threshold for TNF-induced apoptosis (thresholdApoptosisTNF), as well as the scaling factor, boundTNFR1 (estIntPartitionTNF), for coarse grained internalization. The TNF degradation rate constant (kDeg) and a scaling factor that accounts for the coarse-grained TNF dynamics (estConsRateTNF) only significantly affect bacterial levels after ~50–60 days post infection. The rate of NFkB activation (kNFkB) also significantly affects bacterial numbers after 50–60 days post infection, but only in 3D. Although each of these TNF-related mechanisms show significant PRCC values, their magnitudes are not as strong as the PRCC associated with the intracellular bacteria growth rate that dominates all of the PRCC sensitivities . Taken together, these results suggest a combined effect that points to the need for a better representation of the TNF molecular scale dynamics in 3D, as we discussed in detail in several of our previous 2D studies [Several TNF-related parameters show a significant PRCC value, both in 2D and 3D, suggesting an important role associated with TNF regulation. TNF is a molecule that does 4 key things in TB immunobiology, and thus in our models: (1) TNF acts to enhance recruitment of immune cells during infections, (2) TNF activates macrophages that make chemokines, (3) TNF allows for bacterial killing by macrophages, upon activation, and (4) TNF itself can induce death (apoptosis) of infected cells also killing intracellular bacteria. Overall, we find that every mechanism/parameter that is aimed at increasing TNF levels on the grid is associated with lower bacterial levels. Some mechanisms are important early during infection (within the first 4–8 weeks), such as the rate of TNF-induced apoptosis  studies ,24,39.3.2.3.Effective visualization of a biological system, whether through experimental or computational methods, is often crucial to further our understanding. Particularly in the case of biological systems at small scales, mathematical modeling and visualization of simulated data can lend insight when intuition or experimental methods are limited. Visualizing model output may expose non- or counter-intuitive spatial configurations, temporal processes, or agent behaviors. In Figures GranSim generates high-resolution cytokine gradient data and returns the molecule concentrations in each grid compartment as cytokines diffuse over time. Through visualizing and analyzing this data, we can gain better intuition about how cytokines diffuse within the grid—for example, where  and when  over the course of the simulation the highest concentrations are present. Thus, we are better equipped to generate informed hypotheses regarding phenomena such as the relationship between cytokines and agent movement. A 3D computational model affords us the ability to visualize concentration gradients in 3D both spatially and temporally. Whereas experimental datasets are more limited, GranSim  calibrate a 3D ABM describing TB granuloma model and (ii) assess if a 2D ABM representation that uses the same parameter values will reach similar results, at least in terms of matching available experimental datasets and returning similar sensitivity analysis results.GranSim is an ABM, and thus by construction is a discrete, stochastic formulation. We are also working on automating the second step for our model calibration, namely to sift out granulomas that properly form. Dissecting spatial structures that match our prototypical granuloma architecture will likely rely on more sophisticated techniques for volume matching or image comparisons. The goal will be to design and implement metrics with high specificity and sensitivity, in order to automatically screen among thousands of granulomas evolving over time.Finally, when switching from a 2D to a 3D ABM environment, a more realistic representation of the lung vascularization  will enhance the model without significantly affecting the main outcomes. The downside could be the increased computational costs due to more agents entering the grid. The latter challenge could be overcome by introducing stricter thresholds for recruitment (without changing the recruitment probabilities). More experimental data will be needed to balance higher lung vascularization and cell densities within TB granulomas.Supplementary file 1


RL was up to −48.1 dB at 10.5 GHz with a thickness of only 2 mm. Meanwhile, the absorption bandwidth of RL values below −10 dB was 9.4 GHz  in the thickness of 1.5–3 mm. The enhancement is attributed to the modification of PEDOT and the unique structure of nanofibers. On one hand, the deposition of PEDOT nanofibers on the surface of GNs decreases the conductivity of graphene, and makes impedance match better. On the other hand, the unique structure of PEDOT nanofibers results in relatively large specific surfaces areas, providing more active sites for reflection and scattering of EM waves. Therefore, our findings demonstrate that the deposition of conducting polymers on GNs by non-covalent bond is an efficient way to fabricate strong EM wave absorbers.Graphene sheets (GNs) have high conductivity, but they exhibit weak electromagnetic (EM) wave absorption performance. Here, poly  (PEDOT) nanofibers were decorated on the surface of GNs in which the residual defects and groups act as the active sites and therefore are beneficial for the deposition of PEDOT nanofibers. The SEM images display that PEDOT nanofibers are successfully decorated on the surface of GNs through in situ polymerization. The diameter of the PEDOT nanofibers were ranged from 15 to 50 nm with hundreds of nanometers in length. The EM wave absorption properties of graphene, PEDOT, and GNs-PEDOT were also investigated. Compared to pure graphene and PEDOT, the EM wave absorption properties of GNs-PEDOT improved significantly. The maximum value of  In recent decades, electromagnetic interference (EMI) problem has emerged due to the increasing use of electronic devices and communication facilities in most areas, leading to complex electromagnetic environment. An effective way to solve the problem is developing EM wave absorbing materials, which can absorb EM wave effectively and convert EM energy into thermal energy or dissipate the EM waves by interference. Therefore, considerable efforts have been made to develop strong, lightweight, and broadband absorbing materials –3. Typic3O4-PEDOT microspheres with EDOT/Fe3O4 ratio of 20 [Graphene, a novel two-dimensional carbon material, has triggered enormous interest due to its fascinating electrical properties , 13. Graio of 20 . To the In this paper, PEDOT nanofibers were directly grown on the surface of GNs by non-covalent interactions through in situ polymerization. The maximum reflection loss of the nanocomposites reaches −48.1 dB, and the absorption bandwidth exceeding −10 dB is 3.1 GHz with a thickness of only 2 mm. Meanwhile, the absorption bandwidth corresponding to reflection loss below −10 dB is 9.4 GHz  in the range of 1.5–3 mm. Moreover, the addition amount of the nanocomposites into the paraffin matrix is only 25 wt%. Thus, GNs-PEDOT nanofibers are very promising as lightweight EM wave-absorbing materials.2SO4 were added. Then, (NH4)2S2O8, the oxidation, was added in the above solution. Thirdly, the mixture was cooled down to 0 °C and stirred for 24 h. The obtained product was washed with water until pH 7 and dried at 60 °C for 12 h.Graphene oxide (GO) was prepared by Hummers method . The nan2). The electromagnetic parameters of samples were measured in a HP8753D vector network analyzer at the frequency range of 2–18 GHz. The samples were prepared by mixing 25 wt% of the nanocomposites with a paraffin matrix uniformly. The mixture was then pressed into toroidal-shaped samples . The input power level of the incident microwave is −5.0 dBm.The molecular structures of GNs-PEDOT nanofibers were observed on Fourier transform infrared spetctroscopy . Raman spectroscopy were characterized by Renishaw in Via Raman Microscope. The chemical states were investigated by X-ray photoelectron spectroscopy . The morphology were characterized by field-emission scanning electron microscope  and transmission electron microscope 2S2O8, EDOT monomers began to polymerize; thus, PEDOT nanofibers were formed. Figure −1 are attributed to the C=C/C–C stretching vibrations of the thiophene rings. The peaks at 1196, 1141, and 1081 cm−1 are ascribed to the C–O–C stretching, and the bands at 980, 840, and 692 cm−1 are ascribed to the C–S bond in the thiophene rings, respectively. The results indicate that PEDOT is successfully grafted on the surface of GNs [−1 and a G band at 1590 cm−1. For GNs-PEDOT, apart from the D and G bands, the bands at 1434 and 1515 cm−1 are attributed to C=C stretching, whereas the bands at 575 and 989 cm−1 are assigned to oxyethylene ring deformation, and the band at 701 cm−1 is related to symmetric C–S–C deformation, suggesting the formation of PEDOT [The formation mechanism of GNs-PEDOT is schematically depicted in Fig. e of GNs . Figure of PEDOT . In addiThe morphology of the samples are shown in Fig. p XPS spectra  and 165.1 eV (S 2p1/2) with an energy splitting of 1.1 eV, suggesting the formation of doped PEDOT.XPS was used to verify the surface composition of GNs-PEDOT and the results are presented in Fig. see Fig. c show thε′) and imaginary part (ε′′) of the relative complex permittivity, as well as the real part (µ′) and imaginary part (µ′′) of the relative complex permeability of GNs-PEDOT were investigated. In Fig. ε′ values varied from 17.5 to 10.8, and the ε′′ values decrease from 7.3 to 3.6 with several fluctuations in the frequency range of 2–18 GHz. In Fig. µ′ values are in the range of 0.9–1.1 and the µ′′ values are less than 0.2 over 2–18 GHz. The dielectric loss tangent (tanδε = ε′′/ε′) and magnetic loss tangent (tanδµ = µ′′/µ′) are shown in Fig. δε values are higher than tanδµ in the range of 2–18 GHz, suggesting that GNs-PEDOT mainly depends on the dielectric loss.To understand the EM wave absorption mechanisms, the real part (RL) were calculated according to formula ,,RL) werRL is only −8.9 dB at 9.5 GHz. Similarly, pure PEDOT has weak attenuation to EM wave, and the maximum RL is only −14.5 dB at 7 GHz. After coating PEDOT nanofibers on the surface of GNs, the GNs-PEDOT nanofibers show better EM wave absorbing properties in terms of both maximum RL value and absorption bandwidth. The maximum RL is up to −48.1 dB at 10.5 GHz, and the absorption bandwidth corresponding to the RL values below −10 dB is 3.1 GHz (from 9.2 to 12.3 GHz) with a thickness of 2 mm. Furthermore, it is obvious that the maximum RL values are less than −17 dB with the thickness of 1.5–3 mm. The results also demonstrate that GNs-PEDOT nanofibers present better EM wave absorption properties than PEDOT microspheres [In Fig. ospheres , flake-lospheres  and grapospheres . The enhospheres .In summary, GNs-PEDOT nanofibers were synthesized via in situ polymerization method, and their EM wave absorption properties were investigated. TEM images indicate that PEDOT nanofibers were grafted on GNs, and the diameter ranged from 15 to 50 nm. The results state that poor EM absorption properties of GNs can be significantly improved by deposition of PEDOT nanofibers on the surface of the GNs. The maximum reflection loss of GNs-PEDOT is −48.1 dB with a thickness of only 2 mm, and the absorption bandwidth corresponding to reflection loss below −10 dB is 9.4 GHz  in the thickness of 1.5–3 mm. Thus, the deposition of dielectric nanostructures on the surface of the GNs is an efficient way to fabricate lightweight materials for strong and lightweight EM absorbers.