Although the effect of humidity on the tribological performance of many materials and lubricants is evident, many studies are conducted without sufficient systems to accurately monitor and control the humidity level throughout testing. In this paper, a humidity controlling system was developed to enable continuous monitoring and precise control of the humidity at trace moisture levels. The climate controller was validated in a tri-pin-on-disc tribometer with excellent performance and can be fitted to most climate chambers. To further improve the control of operating conditions during tribotesting, a thermodynamic simulation of the contact temperature was developed.  Specifications tableMany papers have proven the remarkable effect of sliding environment on friction and wear of various materials and lubricants, where the type of gas and humidity can greatly change the behaviour of friction and wear Tribometers are usually designed to suit a wide variety of configurations and users. Therefore, specific needs such as accurate humidity control in trace moisture, i.e. extremely dry, environments can be hard to find in a commercial tribometer. From literature, several different methods have been described in various levels of detail. Some of the described climate control systems are quite complex and likely very expensive Depending on the test setup, continuous monitoring and controlling of the humidity can be vital to guarantee correct humidity levels throughout testing. Absorbed or incorporated moisture in the sliding material can be released to the sliding environment due to frictional heat and wear and water vapor can migrate into the test chamber over time, increasing the humidity in the climate chamber. This slight rate of humidity increase can be devastating for precise humidity tests.A climate controller was developed to monitor and control humidity at trace moisture levels with high accuracy, low gas consumption and inexpensive components. Key advantages of the controller are that it is closed-loop controlled, meaning no manual adjustments, and that it is built as a portable unit so that it can be used on most enclosed tribometers and climate chambers. The humidity controller was validated using an enclosed tri-pin-on-disc tribometer with high repeatability.Contact temperatures in tribometers are by its nature extremely difficult to measure. Usually, temperatures measured close to the contact are used as an estimation of the interface temperature and to control the contact temperatures for elevated temperature conditions. However, the difference between measured near-contact temperature and actual contact temperature may vary depending on operating conditions and resulting coefficient of frictions. This could lead to misleading comparisons of tests due to variations in contact temperature. For better control of the contact temperature in the sliding interface a thermodynamic simulation model was developed.The tri-pin-on-disc was based on a Phoenix TE 92 rotary tribometer The developed tri-pin-on-disc configuration can be seen in The test pin holder consists of two parts assembled with a centered steel ball between them, which allows the bottom part holding the three test pins to align against the counterface. The upper part has a machine taper to secure it to the rotating shaft. The torque is transmitted from the upper part of the test pin holder to the bottom by three symmetrically placed screws. Some amount of clearance between the upper part and screws exists to enable self-alignment. The test pins are 8 mm in diameter and have an initial length of 10 mm. The pins are put in slitted steel sleeves prior to mounting and then screwed tight to the pin holder by set screws. The test pin holder is designed to protect the displacement sensor from wear debris and also prevent the holder from damaging the sensor.The tribometer test cell is enclosed by an o-ring sealed lid and a flexible sealing, consisting of a strip of plastic from a plastic bag and packing tape, between the lid and bearing housing to allow some vibrations and radial movement. Other types of sealing, such as a rotary shaft seal would cause interference with the friction measurements and limit the natural response of the sliding system. The rotating shaft is hollow on the TE 92 to enable the removal of the upper specimen. During tests, this hole is sealed on top using a threaded screw with a washer and o-ring. Three threaded holes were added to the reservoir in which tube connections were fitted. These were used for gas supply and gas circulation, which is explained in detail in the humidity controller section.The displacement sensor is a high precision capacitance sensor, see specifications in The thermocouple is inserted in a 1.5 mm in diameter hole in the side of the counterface disc. The hole is located 1.5 mm below the countersurface and reaches into the center of the sliding contact, providing close temperature measurements. The TE 92/6 reservoir is equipped with two heaters, illustrated in A thermodynamic simulation with the setup depicted in The simulation was simplified as an axis symmetrical system, The input parameters in The two heat cartridges depicted in The mass flow in and out of the small space formed between the inside of the counterface disc and the test pin holder during operation is considered small and hence the convective cooling of the inner surfaces is neglected and modeled as thermal insulators in the simulation model.In accordance with the temperature control in the test rig, the main principle of the simulation model is to keep the temperature at the near-contact thermocouple As proven, the simulation model accurately estimates the contact temperature for tested operating conditions and coefficient of friction ranging between 0.05 and 0.15. Good estimations of the contact temperature are also expected for higher coefficients of friction, although the error will slightly increase with the coefficient of friction. This is mainly due to a simplification in the model that the gas temperature in the climate chamber is constant and independent of the coefficient of friction. However, in reality the gas temperature decreases with increased friction, which causes the error of Temperature conditions and average friction torque from tests against the 34CrNiMo6 counterface disc in the research paper Since the heat distribution is parabolic and the distance between the thermocouple and the sliding contact is much smaller than the radius of the test pins, it is reasonable that the temperature difference is small for the given inputs. Moreover, the increasing difference between the measured and simulated temperature with higher friction is also logical. Higher friction torque results in higher heat flux in the contact zone and less applied heat, if any, from the heaters, resulting in a steeper temperature gradient as can be seen by comparing It can be concluded that the difference between the maximum contact temperature and measured near-contact temperature under elevated temperature conditions can be neglected for mild sliding conditions. The temperature difference increase with the coefficient of friction but is only significant for very severe sliding conditions.To minimize the post-processing time of the data from the displacement sensor and dew point transmitter, the existing data acquisition system (SLIM) of the TE92 tribometer was used. Two analog inputs with 12 bits resolution were available and could by small means be adjusted for the 0–10 V signals. The dew point meter was calibrated to the SLIM simply using a laboratory power supply and the displacement sensor was calibrated using a stand and precision gauge blocks with different heights.The components used for the humidity controller can be seen in This system both monitor and actively control the humidity in the tribotest climate chamber. The humidity in the chamber is measured by circulating the environmental gas between the test chamber and a dew point meter by an eccentric diaphragm pump. The gas is pumped through a set of filters (5 to 0.01 µm) to protect the dew point meter from wear particles before entering the sampling block of the dew point meter. The dew point transmitter feeds a signal to both the data acquisition system of the test rig for continuous data logging and a Siemens LOGO8! PLC for feedback control of the humidity. Gas is supplied to the system through a restrictor valve to achieve sufficiently low gas flow. The pressure of the gas supplied to the restrictor valve is controlled by an electro-pneumatic (E/P) pressure regulator connected to a gas cylinder. A ball valve bypasses the restrictor valve to enable flushing of the test chamber to quicker reach target humidity after being exposed to the ambient environment.For the specific tri-pin-on-disc test setup one restrictor valve with the 0.1 mm orifice gave slightly too high gas flow. This means that the pressure regulator only used a small portion of the total range during operation, leading to a somewhat rough controller. A lower flow allows the pressure regulator to operate in a wider pressure span which means that each discrete change in the controller output results in a smaller change in the flow and a smoother controlling system can be achieved. A restrictor valve with a smaller orifice would have been ideal, but in this case, a second restrictor valve of the same size connected in series was sufficient to reduce the flow due to the added pressure loss.As illustrated in The setting of the controller parameters depends largely on the characteristics of the system, i.e., the nature of humidity, the amount of leakage of the test chamber and the maximum flow of supplied gas. The gas flow is determined from the pressure range of the pressure regulator and the size of the orifice in the restrictor valve. To be able to control the humidity by the change of supplied gas flow, some amount of leakage or a designated outlet port needs to exist in order to evacuate excessive moisture or draw in external moisture based on needs. This humidity controller was meant to be used continuously for long periods. Therefore, the gas consumption needed to be minimized, whereof the test setup was designed to allow a very limited leakage.A controller system that could both handle the initial big difference between the process value (PV) and setpoint (SP), namely the controller error, during start-up and also smoothly tackle disturbances in the system proved to be hard to find due to the characteristics of the system. Reducing humidity takes time, reaching a moisture content of as low as 10 ppm takes several hours to reach from a humid state if a vacuum chamber and vacuum pump are not utilized. Water gets stuck in the filters and possibly in nooks in the test setup, making it a time-consuming process to reach trace moisture levels. To achieve a smooth and reliable controller at trace moisture levels the system needed to be flushed with nitrogen until the moisture content was reduced to about 70–90% of the target value. The initial flushing was executed in two steps; first flushing at full flow for some tens of seconds with the regulator set to about 0.05 to 1 bar just prior to fully sealing the climate chamber with flexible plastic and tape, and lastly by reducing the flow and keeping a slight overpressure after the chamber is fully sealed. The duration of the second step depended on the initial conditions and the setpoint and could vary between some tens of seconds to several minutes. The gas flow in the second step was adjusted with the ball valve by slowly opening it until the flexible plastic seal expanded slightly due to the overpressure in the climate chamber.The Ziegler-Nichols method V) using the saturation vapor pressure formula given by Huang Due to the characteristics of the system, a short integral time V of target humidity, but for this setting, signs of humidity influencing phenomena became evident. In A lower proportional gain of The fast controller settings resulting in the straight humidity curves in An important modification to the PI regulator was to add a gain, B002 in v with excellent results. Humidity levels down to 5 ppmv are most likely achievable but for levels above 100 ppmv a humidifier is needed to get stable humidity. A simple and inexpensive solution can be used to humidify the climate chamber and still enable control by the flow of supplied gas. By putting a saturated salt solution in a wide-bottomed glass container with a gas-tight lid with two tube connectors, the gas can be humidified on its way back from the dew point meter by connecting the salt solution container between the circulation pump and climate chamber. However, this method would need some trial and error to choose an appropriate salt and sufficiently large surface area between the saturated salt solution and gas. One approach would be to choose a saturated salt solution The humidity controller has been tested in a range of 10 to 100 ppmAs seen in The humidity controller could easily be upgraded to also measure the amount of oxygen in the test chamber by installing a trace oxygen sensor in series with the dew point meter. E.g., the thermocouple fitted between the filter rack and dew point meter, The developed method for controlling humidity at trace moisture levels has proven to be accurate, stable and well-functioning. Due to the simplicity and portability, the humidity controller can be moved and optimized to measure and control the humidity in most enclosed tribometers and climate chambers. Components can also be added or replaced to fulfill special requirements such as measuring the amount of oxygen in the gas, supply a mixture of two gases or control higher humidity levels than described in this paper.For tribotesting, it is important to conduct tests under operating conditions similar to the actual application. Contact temperatures in sliding systems are often difficult to measure, wherefore thermodynamic simulations can be used to approximate the contact temperature from known parameters. The thermodynamic simulation method described in this paper has provided valuable information of the contact temperature and thermal transfer in and out of the test setup, e.g., the insignificant temperature difference between the near-contact thermocouple and the contact. The thermodynamic simulation model was validated with different inputs and temperature measurements from two separate locations to certify that the model is compliant with the real test setup. The results showed that the difference between measured near-contact temperature and contact temperature follows a linear relation with the friction torque, where Similar methodology can be used to develop thermodynamic simulation models for contact temperatures in other types of tribometers, given that near-contact temperatures are possible to measure and additional measurement points that can be used to optimize and validate the model.Co-submissions are papers that have been submitted alongside an original research paper accepted for publication by another Elsevier journalThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Natural fiber reinforced composites (FRC) are of great interests, because of their biodegradability, recyclability, and environmental benefits over synthetic FRC. Natural jute FRC could provide an environmentally sustainable, light weight, and cost-effective alternative to synthetic FRC. However, the application of natural jute FRC is limited because of their poor mechanical and interfacial properties. Graphene and its derivatives could potentially be applied to modify jute fiber surface for manufacturing natural FRC with excellent mechanical properties, and lower environmental impacts. Here, we review the physical and chemical treatments, and graphene-based modifications of jute fibers, and their effect on mechanical properties of jute FRC. We introduce jute fiber structure, chemical compositions, and their potential applications first. We then provide an overview of various surface treatments used to improve mechanical properties of jute FRC. We discuss and compare various graphene derivative-based surface modifications of jute fibers, and their impact on the performance of FRC. Finally, we provide our future perspective on graphene-based jute fibers research to enable next generation strong and sustainable FRC for high performance engineering applications without conferring environmental problems.  Materials science; Mechanical processing; Nanotechnology fabrication Sustainable and biodegradable natural fiber reinforced composites (FRC) have received significant interests in recent years, because of their lower environmental impacts , includiThere are various physical and chemical treatments that have been carried out to remove the noncellulosic materials and improve the mechanical properties of the jute fiber and their composites. Among them, the alkali treatment is the most popular surface treatment which removes noncellulosic materials and impurities from the interfibrillar region of jute fiber. Thus such treatment makes the fibrils more capable of rearranging themselves along the direction of tensile deformation and provides a better load sharing capability between themselves to contribute to higher stress development during the tensile test . The survia microfluidization technique, and used for smart composites applications (via forming either bonding (GO) or mechanical interlocking (G) between fibers and graphene-based flakes. In addition, graphene-based jute FRC have been developed for high-performance composites multifunctional smart composites applications, as demonstrated by effective electro-magnetic interference shielding , graphene oxide (GO), and reduced graphene oxide (rGO) have attracted tremendous attention for high-performance composite applications because of their incredible mechanical properties. Graphene derivatives (GO and rGO) could be produced in a huge quantity in their stable dispersions. In addition, such materials provide good chemical reactivity and handling characteristics because of their intrinsic functional groups . Furtherications . Previouications  demonstrhielding  and de-ihielding . Such deAlthough there have been many previous reviews focusing on jute fibers and their composites , there rCorchorus capsularis (white jute) and Corchorus olitorius (Tossa jute)  . For sucsa jute) . The jutsa jute) . Jute isvia a biological method or a chemical method with the help of chemicals including ammonium oxalate, sodium sulphite, etc. However, the chemical retting process is expensive  is extensively used for the alkali treatment of jute fiber. The alkali treatment of jute fiber removes noncellulosic materials such as hemicellulose, lignin, wax, and oils that surround the external surface of the jute fiber. Alkali treatment of the jute fibers eliminates the moisture-related hydroxyl groups which decrease the hydrophilic nature of the fiber. When NaOH reacts with jute fiber, it produces water molecules, and Na-O- combines with the cell wall of the fiber to produce fiber-cell-O-Na groups referring to The alkali treatment directly affects the jute fiber properties which removes noncellulosic materials and makes the fiber more capable of rearranging themselves along the fiber direction and as well as improving the fiber matrix adhesion. This permits a better load shearing capability, which results in higher tensile strength in the composites .The NaOH treatment of the jute fiber is performed by dipping the fiber in a NaOH solution for a certain time and temperature. The efficiency of NaOH treatment of jute fiber depends on the NaOH concentration, treatment time, temperature, and material liquor ratio. A previous study  investigMechanical properties of the alkali-treated jute fiber reinforced thermoplastic and thermoset composites have been studied extensively, In other work, flexural properties of the 5% alkali-treated jute fiber/unsaturated-polyester resin composites for different treatment times  were investigated . FlexuraThe silane molecules have different functional groups at both ends. One such functional group reacts with hydrophilic groups of the jute fiber and the other with hydrophobic groups in the polymer matrix to form a bridge between them . For thiIn one study , the effMany researchers investigated the plasma treatment of jute fibers to manufacture the jute FRC with improved mechanical properties . Plasma In another study , the eff2O2) bleaching was carried out to modify short jute fibers, and the effect of such treatment on composites’ tensile properties was investigated (2O2 (10 mL/L) treatment. The composites were manufactured with different weight%  of treated and untreated jute fiber content with PLA matrix via an injection molding technique. Jute/PLA composites with 10% NaOH treated fibers followed by H2O2 bleaching at 20% fiber loading provided ∼7.5% and ∼40% higher tensile strength and modulus, respectively, than those of untreated jute fiber/PLA composites.Alkali treatment followed by hydrogen peroxide  with graphene and its derivatives  and a higher concentration compared to other LPE processes as the force acts over the whole volume of the liquid, whereas , bath sonication, tip sonication, and shear exfoliation generally produce graphene of lower concentration as well as lower yield (up to 2%) by weight (Yw). Electrochemical expansion process offers much better yield by weight (YW >70%). To enjoy the distinctive properties of graphene it is important to produce and maintain graphene as individual sheets. However, graphene sheets tend to agglomerate and even restack to form graphite through van der Waals interactions , thermal reduction and electrochemical reduction can be used to achieve different properties in rGO depending on various final applications  is a versatile, scalable, sustainable, and cost-effective method to produce single-layer graphene, and therefore used widely . Basical2 . Among oractions . Thereforactions . Variousications , 2021b. ications . The proications  for obtaications .Lower crystallinity, hydrophilicity, and inherent electrical insulation properties of jute fiber results in lower mechanical, poor interfacial, and electrical properties of jute fiber reinforced composites that limits their application as multifunctional composites . Few carvia simple dip-coating method, The surface modification of jute fibers with graphene oxide (GO) , reducedThe wide-scan X-ray photoelectron spectroscopy (XPS) spectra in Scanning electron microscope (SEM) images show smooth untreated jute fiber surface G, becausvia suitable bonding. Similarly, there was a large increment in IFSS of G-flakes coated jute fibers  of the untreated, alkali-treated, GO, rGO, and G-coated jute fibers were also investigated . A single fibers , which me fibers .Figure 5via mounting the fiber on the paper frame. The single fiber tensile test shows the increase of tensile strength from 295 MPa to 575, 814, and 474 MPa, and Young's modulus from 30 GPa to 48, 78, and 52 GPa for 1% GO, 0.5% rGO, and 10% G flakes coated jute fibers  jute/epoxy composites were prepared with alkali-treated and graphene materials-coated jute fibers via vacuum-assisted resin infusion (VARI) method. The longitudinal tensile strength, Young’s modulus, and strain% of jute/epoxy composite were increased with the increase of concentration of GO up to 0.75mg/mL because of the nano-engineering effect of graphene materials on jute fibre but after this concentration of GO deteriorate the tensile properties of composites because of the agglomeration of GO flakes, Although mechanical properties of individual fibers have been increased significantly us study , we addrIn another study, mechanical and thermal properties of the graphene oxide nanoplatelets (GONPs) modified jute/polypropylene composites were investigated and compared with untreated jute/PP composites . FirstlyThe mechanical behavior of rGO filled jute/epoxy composites was investigated at different temperatures to quantify the effect of temperature and rGO filler on the mechanical properties of the composites . The ten2@GONPs) at different loadings  on the flexural and high-velocity impact properties of jute fiber/epoxy composites was also investigated (2@GONPs/jute/epoxy) were prepared using the static pressing assisted hand layup method. The most promising results were obtained with 0.3 wt.-% of SiO2@GONPs modified jute/epoxy composites. The study found that the flexural strength, energy absorption capability, and impact limit velocity of the 0.3 wt.-% SiO2@GONPs modified jute/epoxy composite improved by ∼40%, ∼61%, and ∼28%, respectively, from those of the neat jute/epoxy composites. In other study, the effect of two different nanofillers such as graphene oxide (GO) and functionalized graphene (FG) on the mechanical properties of the Jute/epoxy composites have been investigated at different nanofiller contents  (The influence of GONPs and silica-decorated graphene oxide (SiOstigated . The mul1 wt.-%) . It was Drilling and milling processes are widely used during the assembly of composite parts with other components . The survia physical and chemical treatments of jute fibers, and also incorporating graphene-based 2D materials. A wide range of studies has been carried out to improve the interfacial adhesion between jute fibers and polymer matrix such as physical (plasma), chemical , or combined physical and chemical  or different chemical  treatments. Such treatments have improved the mechanical and interfacial properties of the composites. However, this improvement is not sufficient to use developed composites as an alternative to synthetic FRC. A combined physical and chemical treatment using graphene-based materials modified jute fiber/epoxy composites showed a significant increase in the tensile and interfacial properties and comparable specific properties to those of glass fibers with added multifunctionalities. There has been very limited research that used graphene-based materials to modify jute fiber and manufacture FRC. Therefore, more research is needed for better understanding of the interactions among graphene-based materials, reinforcing fibers and matrix materials to promote this novel class of composites materials for industrial applications.With growing environmental concerns with petrochemical-based synthetic fibers , researcThere is a growing interest in sustainable, biodegradable, and lightweight smart materials for structural composites applications, that would offer unprecedented combinations of stiffness, strength, toughness and multifunctionality. Most of these researches have used petrochemical-based or nonbiodegradable polymer matrices to manufacture jute FRC that are not fully green (100% bio-based) composites. The application of these bio-based nonbiodegradable composites has been increasing in the automotive and other manufacturing industries. Some green composites are manufactured using bio-based and biodegradable resin but the application of such materials is limited in the automotive and other structural applications, because of their poor mechanical properties. Efforts should be put into developing suitable bio-based and biodegradable resin for manufacturing green and environmentally sustainable high-performance natural FRCs. Such composites could potentially help reducing non-degradable plastic waste and improve the overall carbon footprints associated with composites industries. Based on the articles discussed in this review, the researchers should pay attention toward the development of the next generation of high-performance green composites by incorporating the graphene and other 2D materials in the jute fiber reinforced composite.


TDP-43 is a nuclear RNA-binding protein that forms neuronal cytoplasmic inclusions in two major neurodegenerative diseases, ALS and FTLD. While the self-assembly of TDP-43 by its structured N-terminal and intrinsically disordered C-terminal domains has been widely studied, the mechanism by which mRNA preserves TDP-43 solubility in the nucleus has not been addressed. Here, we demonstrate that tandem RNA recognition motifs of TDP-43 bind to long GU-repeats in a cooperative manner through intermolecular interactions. Moreover, using mutants whose cooperativity is impaired, we found that the cooperative binding of TDP-43 to mRNA may be critical to maintain the solubility of TDP-43 in the nucleus and the miscibility of TDP-43 in cytoplasmic stress granules. We anticipate that the knowledge of a higher order assembly of TDP-43 on mRNA may clarify its role in intron processing and provide a means of interfering with the cytoplasmic aggregation of TDP-43. In comparison to other proteins, many RNA-binding proteins (RBP) harbor low-complexity domains (LCD) that initiate weak multivalent interactions leading to the assembly of liquid-like membraneless organelles, notably in the mammalian cell nucleus . MembranHowever, despite the link between the TDP-43 LCD and several neurodegenerative diseases, the precise mechanism leading to the formation of cytoplasmic aggregates of TDP-43 remains obscure. The known mutations located in the TDP-43 LCD, associated with neurodegenerative diseases, still allow normal cognitive functions in adults before aging . TherefoIn other models, the recruitment of TDP-43 in stress granules rather plays a negative role in neurodegenerative diseases by acting as crucibles in which TDP-43 is concentrated . Howevermsl2 mRNA , to long GU repeats. A cooperative binding to RNA is not unique to TDP-43 but is also found in many other RBPs. Indeed, most RBPs have a low specificity for short sequences of few nucleotides . A coopesl2 mRNA . In addisl2 mRNA . Our intTo understand through which molecular mechanisms TDP-43 targets pyrimidine-rich introns in cells as revealed by CLIP experiments, we considered whether a cooperative association of TDP-43 may take place in long GU-rich repeats to secure the binding of several TDP-43 proteins. The structure of TDP-43 is generally represented with three distinct functional domains: a structured N-terminal domain (NTD), two central RRMs, and a long unstructured C-terminal (LCD) . Since tThe presence of higher order assemblies of TDP-43 was first evidenced by cross-linking assays in the presence of a loop containing 24 GT repeats that mimics long GU-rich mRNA sequences functionally targeted by TDP-43  and can To further characterize the higher order assembly of TDP-43 bound to 24 GT repeats, gel mobility shift assays were performed with RRM1–2 in the presence of 24 GT repeats . In addi1/Kd2. Kd1 and Kd2 represent the dissociation constants of the first and second proteins interacting respectively with GT repeats, as measured by ITC  than for RRM1–2 (Kd1/Kd2 = 128).Whereas electrophoretic mobility assays and cross-linking analyses reveal the formation of TDP-43 multimers on 24 GT repeats, an assessment of its cooperative binding is missing. Therefore, we applied isothermal titration calorimetry (ITC) . The ITCd by ITC . Since KTogether, biochemical and ITC data provide compelling evidence of a cooperative binding of TDP-43 RRM1–2 to GT repeats.To provide insights into the structural mechanism leading to the cooperative association of RRM1–2 to GU or GT repeats, we performed a comparative analysis by NMR spectroscopy of RRM1, RRM2, and RRM1–2 residues in interaction with GU sequences of different length . In agreement with the results from ITC experiments, a single RRM1–2 binds to 6 GU repeats mostly through conserved RNA-binding residues . Twelve To identify TDP-43 RRM1–2 residues involved in the cooperative binding of TDP-43, we selected residues displaying significant CSPs in the presence of 12 GU repeats compared to 6 GU repeats which can accept only one RRM-1–2 . AnotherAccording to the above-mentioned selection rules, 28 residues likely critical for the homotypic interactions leading to the formation of RRM1–2 multimers in the presence of GU repeats were selected and represented in the RRM1–2 primary structure .Even if CSPs may be useful to identify critical residues and delineate an interface between two RRM1–2 monomers in the presence of GU repeats, the active contribution of the selected residues to TDP-43 cooperativity remains to be validated. Indeed, unstructured residues may be particularly sensitive to environmental changes following RRM1–2 multimerization without being directly involved in any interaction. Other methods have therefore to be used to decipher which of the identified residues actively participate in the cooperative binding phenomenon of RRM1–2 to RNA, notably in a cellular context.To probe the putative functional role of the selected residues, after our NMR analysis , in the To analyze the role of the 28 TDP-43 residues selected from our structural analysis, the mixing of 34 TDP-43 mutants with wild-type TDP-43 was probed with the MT bench assay . As a poIn summary, the MT bench assay identified nine residues out of the 28 residues selected by NMR which impact significantly the mixing of TDP-43 with itself in cells . HoweverWith the aim to delineate an intermolecular interface, we first decided to use TDP-43 mutants that would be defective in their cooperative association to mRNA in gels shift and ITC assays. A comparison between wild-type and mutant RRM1–2 could then help to identify an interface by using a comparative NMR analysis. We focused our attention on the RRM1 loop 3, as many residues located in this region are critical for the mixing of TDP-43 mutants with wild-type form in cells. The two mutants, T141A/G142A and G146, were selected based on the strongest effect on their demixing with wild-type TDP-43 in cells and the Q213A mutant was added as a negative control. After their production and purification, all the three mutant proteins were soluble . The dou1/Kd2 value compared to wild-type RRM1–2  (data not shown). Noteworthy, by applying NMR and SAXS approaches together with MD calculations, the identified intermolecular interface is also observed in RNA/RRM1–2 complexes when using a different RNA oligonucleotide, 5′-GUGUGAAUGAAUGUGUGUGUGUGU-3’  . Size-exleotides , here GUleotides . The cal of 1.28 . This mo of 1.28 . The RRM of 1.28 . Among t of 1.28 . In the  of 1.28 , which iUGUGU-3’ .The striking reappearance of V220 in the NMR spectrum of T141A/G142A mutant interacting with 12 GU repeats also points toward an interaction between the RRM2 pocket and RRM1 loop 3 . In turnThe results of SAXS, NMR, and MD experiments thus point toward an interface between the RRM2 pocket around V220 and the RRM1 loop 3, when two RRM1–2 monomers interact with 12 GU repeats.Given the lines of evidence pointing toward an interaction between the pocket around V220 in RRM2 of the first monomer and the RRM1 loop 3 of the second monomer, we devised a direct strategy to probe the relevance of this interaction in a cellular context. We noticed the close proximity of residues K137 and R208 in the dimer from the MD structure . HoweverTherefore, these results support the spatial proximity between K137 and R208 in TDP-43 multimer, which is consistent with an intermolecular interaction between the RRM1 loop three and the V220 pocket in the RRM1–2 dimer.After the identification of a cooperative binding of TDP-43 to GU and GT-repeats, we asked whether the TDP-43 multimerization on GU- or GT-repeats may antagonize the formation of full-length TDP-43 aggregates in vitro. An intermolecular interaction of the RRM2 pocket around V220 with RRM1 loop 3 of the second monomer could notably limit a head-to-tail assembly  that is In contrast with wild-type TDP-43, the solubility of G146A TDP-43 mutant thus cannot be increased significantly in the presence of mRNA in vitro, most probably because of an impaired cooperative binding to mRNA.As TDP-43 cooperativity antagonizes the formation of TDP-43 aggregates in vitro, we explored whether the cooperative binding of TDP-43 to mRNA in cells plays a central role in the formation of reversible compartments. HA-tagged wild-type and mutant TDP-43 were therefore expressed in HeLa cells to analyze by optical microscopy observations  their spTogether, these results support a preserved solubility of wild-type TDP-43 and Q213A mutant and the formation of cytoplasmic inclusions for cooperativity-defective mutants, G146A and K140A/T141A.2O2, an oxidative stress agent, which, on its own, poorly induces stress granule assembly at the concentration used in this study .The recombinant His6-tagged RRM1, RRM2, and RRM1–2 fragments from human TDP-43, respectively) were grown at 37°C in 2YT-ampicillin medium (1 L culture) (non-labeled proteins) or in minimal medium M9 supplemented with 15NH4Cl (labeled proteins). When the optical density of the culture reached 0.7, IPTG was added at a final concentration of 1 mM, and growth was continued for 3 hr. Cells were harvested and washed with 20 mL of cold 25 mM Tris–HCl buffer, pH 7.4, containing 1 mM TCEP, 1 mM PMSF, and EDTA-free protease inhibitor Cocktail (Roche) and 1.5 M KCl (buffer A). The cell pellet (4.5 g wet wt) was suspended in 10 mL of the same buffer, and cells were disrupted by sonication on ice . The resulting suspension was centrifuged at 4°C for 30 min at 150,000× g in a TL100 Beckman centrifuge. The supernatant was used for purification experiments.Cells carrying plasmids, pTDP-RRM1_101–192, pTDP-RRM2_176–277, and pTDP-RRM1-2_101–277 (encoding for the recombinant His2+-NTA-agarose (Qiagen) (20 mg of proteins/mL of resin) pre-equilibrated in buffer A. After incubation, the resin was transferred to an Econo-Pac chromatography column (Bio-Rad). The polymer was then washed extensively with buffer A containing 20 mM imidazole. The elution of the protein was obtained by increasing step by step the concentration of imidazole, from 40 to 250 mM, in buffer A. Pure protein-containing fractions (100–250 mM imidazole) were pooled and incubated with a His6-tagged TEV protease to cleave off the His6-tag peptide from the target protein. The protease (15 µg) was mixed with 1 mg of target protein (0.5 μM TEV to ∼30 μM protein) in buffer A containing of 1 mM DTT and 1 mM EDTA. All digestions were conducted for 16 hr at room temperature. A PD-10 column  was used to remove imidazole and to exchange buffer. Then, TEV protease and His6-tag peptide from target protein were trapped on Ni-NTA agarose column and target protein was recovered in pass-through (nonbinding) fraction. The protein was concentrated to 2 mL and conserved in 20 mM Tris–HCl buffer, pH 7.4, containing 25 mM KCl and 1 mM TCEP by using a PD-10 column. For NMR experiments, the 15N-labeled proteins were stored in phosphate buffer 15 mM pH 6.8 containing 25 mM KCl and 1 mM TCEP by using a PD-10 column . The final preparations were stored at –80°C.TDP-43 fragments were purified basically following the manufacturer’s recommendations (Qiagen). Imidazol (10 mM) was added to soluble fractions described above and incubated for 2 hr at 4°C with NiE. coli cells carrying plasmid pET-WT_TDP-43 and pET-G146A_TDP-43, were grown at 37°C in 2xYT-ampicillin medium (1 L culture). When the optical density of the culture reached 0.7 OD at 600 nm, IPTG was added to a final concentration of 1 mM, and growth was continued for 3 hr. Cells were harvested and washed with 20 mL of cold buffer A. The cell pellet was suspended in 10 mL of the same buffer, and cells were disrupted by sonication on ice . The resulting suspension was centrifuged at 4°C for 30 min at 150,000× g in a TL100 Beckman centrifuge. Then, the pellet was resuspended in 10 mL of the buffer A containing 6 M Urea, and 10 mM Imidazole and incubated for 120 min at 4°C. The resulting suspension was centrifuged at 4°C for 30 min at 200,000× g in a TL100 Beckman centrifuge. The supernatant was stored at −20°C and used for purification experiments. The His6-tagged proteins were purified as follows: soluble fractions described above were incubated for 2 hr at 4°C with Ni2+-NTA-agarose (15 mg of protein/mL of resin) pre-equilibrated in buffer A containing 6 M Urea, and 10 mM Imidazole. After incubation, the resin was transferred to an Econo-Pac chromatography column (Bio-Rad). The resin was then washed extensively with buffer A containing 6 M Urea, and 20 mM imidazole and elution of the protein was obtained by increasing step by step the concentration of imidazole, from 40 to 250 mM, in buffer A. The purity of the resulting protein preparations was monitored at all stages by SDS–PAGE. The pure protein-containing fractions (100–250 mM imidazole) were concentrated to 2 mL and then dialyzed overnight against 100 volumes of buffer containing 20 mM Tris–HCl, pH 7.4, 0.5 mM DTT, 6 M urea, and 200 mM NaCl. The final preparations were stored at −20°C.Full-length forms of TDP-43, wild-type, and G146A mutant were purified as previously described for full-length wild-type form . BL21(DERRM1–2 coding gene from human TDP-43 was carried out directly on the pTDP-RRM1-2_101–277 expression plasmid by using the ‘Quikchange II XL site-directed mutagenesis kit’ from Stratagene and appropriate oligonucleotides (Eurofins Genomics). The introduced mutations were checked by DNA sequencing (Eurofins Genomics). Overexpression and purification of mutated forms were performed following the method described above.Site-directed mutagenesis of the The protein purity was monitored at all stages of the purification by SDS–PAGE .GATATAGAGGTAAGATAG-(GT)24-CTATCTTACCTCTATATC] expected to form a stem-loop structure (named (GT)24-loop) with 24 GT dinucleotide repeats (GT)24 being the loop. The stem structure facilitates the ethidium bromide visualization on a poly-acrylamide gel. Briefly, the mixtures were incubated in 10 μL of binding buffer  at room temperature for 20 min. Free and bound-to-protein oligonucleotides were separated in a 10% poly-acrylamide gel in 0.5× TAE buffer at 80 V for 75 min on ice. Finally, gels were stained with 0.5 μg/mL of ethidium bromide.Increasing amounts of previously purified RRM1, RRM2, or RRM1–2 protein fragments were incubated in the presence of 10 pmol of the oligonucleotide  The authors successfully demonstrate that TDP43 cooperatively binds GU-rich sequences and identify the residues responsible for that interaction, which addresses an outstanding question in the TDP43 field and is likely of interest to potential readers. However, the work could be improved by additional evidence that impaired cooperative TDP43 binding is relevant to the formation of TDP43 aggregates. No distinction is made between cooperative binding to RNA and TDP43 RNA binding in general, and TDP43 overexpression in HeLa cells under two forms of stress .We provide the kdFinally, we suggest a different model system to study the impact of impaired cooperative binding on TDP43 aggregation. Stress granules are transient structures, and there is no definitive evidence that they seed TDP43 aggregates. Overexpression of TDP43 isoforms in immortalized cell lines or a disease-relevant neuronal context (e.g. primary rodent neurons) is sufficient to induce aggregate formation, and would more clearly demonstrate the impact of TDP43 cooperative binding on aggregate formation.We agree with the reviewer. As indicated in the manuscript, stress granules may have a positive or neutral impact on TDP-43 aggregation in the cytoplasm. Though this is a very interesting subject of research, the presence of stress granules in neurons of ALS patients is not yet certain. Here we used cellular stress because we obtained a robust aggregation in cells expressing TDP-43 mutants. However, as suggested by reviewer 2, we also performed an analysis of TDP-3 aggregation in the cytoplasm after the expression of TDP-43 mutants without additional stress. Though most cells display a homogenous distribution of TDP-43 in the cytoplasm and nucleus , we measured a significant aggregation of TDP-43 mutants. In contrast with wild type TDP-43, aggregates of TDP-43 mutants are distinct from stress granules and are resistant to cycloheximide pretreatment. These new data are shown in the revised manuscript .Reviewer #2:[…] There are a several areas of improvements that could be considered by the authors:a) first of all, and most importantly, it would have been very interesting to see what happens to the cooperativity in the presence of substrates other than UG-repeats. For example, the sequence known as CLIP34nt  plays an important role in TDP-43 auto regulation. Would the cooperativity of TDP-43 on this substrate differentiations noticeably from UG24-repeats?We performed new ITC experiments to analyze the cooperative binding of TDP-43 RRM1-2 to CLIP34nt (DNA), as suggested by the reviewer. In the case of CLIP34nt, we observed three distinct plateaus supporting the binding of three protein units. Due to the number of binding events (at least three) during titration, the fitting of the experimental data becomes more challenging  . Data fWe indicate in the discussion of the revised manuscript that exploring the role of nucleic acid sequence on the cooperative behavior of TDP-43 is of interest to understand the formation of TDP-43 clusters on specific mRNA sequences.b) Disease-associated mutations are few in the RRM1 and RRM2 regions but some have been described (ie. K181E). Could some of these be considered as affecting the cooperative binding process of TDP-43. This should be discussed.In our extensive screening, we did not identify any pathological mutation as critical for the cooperative association of TDP-43 to RNA. Given the strong phenotype observed in vitro cultured cells with cooperativity-defective mutants such as G146A, we may expect, in a functional point of view, that these mutations would prevent development of an adult brain in animals.Consequently, while we have no evidence that, for example, K181E would impact the cooperative binding to RNA, we cannot exclude that K181E may have a very slight impact on the cooperative binding which in turn would contribute to the onset of ALS. We need to increase the sensitivity of our detection scheme to find whether other residues may not slightly impact TDP-43 cooperativity through an allosteric mechanism for instance. A sentence was added in the discussion regarding this point.Finally, the rather less detailed part of the manuscript is represented by the nuclear and cytoplasmic condensates of the mutant TDP-43s in cells. Although quite interesting, these condensates could have been characterised better with just small additional effort. For example:c) The TDP-43 condensates observed in the nucleus under oxidative stress conditions co-localize with some specific nuclear structures such as nuclear stress bodies or other well-known nuclear bodies?.We performed an additional experiment to determine whether Sam68, an mRNA-binding protein found in nuclear stress bodies is recruited in G146A nuclear aggregates. SAM68 was not detected in G146 nuclear inclusions.d) In Figure 7b, it is not clear how the cytoplasmic TDP-43 condensates do not relate to stress granules. In the manuscript it is stated that they do not co-localize with mRNA which would suggest this to be the case. However, have the authors performed some staining with stress granule markers  to confirm that TDP-43 does not co-localize (or not) with these structures?We agree that is important to characterize these aggregates. We observed that nuclear and cytoplasmic aggregates of TDP-43 mutants are not enriched in mRNA (poly-dT probes), in FUS . In addition, we shown that wild type TDP-43 co-localized with G3BP-1 positive stress granules (mRNA-rich and sensitive to cycloheximide). In contrast, TDP-43 cytoplasmic aggregates are indeed not positive to anti-G3BP-1 and anti-FMRP antibodies, not enriched in mRNA and unsensitive to cycloheximide.e) Finally, it would have been interesting to know whether these TDP-43 condensates triggered by the key mutations  were also positive for phosphorylation at the 409/410 Ver residues.Cykowski et al., 2018, doi: 10.1186/s40478-018-0528-y). We found a significant staining of TDP-43 rich stress granules when wild type TDP-43 is expressed. In addition, G146A and T141A/G142A cytoplasmic or nuclear aggregates are also positive to this antibody.We performed these novel experiments with a commercial anti-phospho TDP-43 antibody (409/410 Ser, see However, we considered that the link between TDP-43 phosphorylation and the recruitment of TDP43 in stress granules or the formation cytoplasmic inclusions deserves a detailed study on its own. We therefore preferred to add a sentence about the relevance of studying TDP-43 phosphorylation in the discussion.


While immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treatment, only subgroups of patients show durable responses. Insight in the relation between clinical response, PD-L1 expression and intratumoral localization of PD-L1 therapeutics could improve patient stratification. Therefore, we present the modular synthesis of multimodal antibody-based imaging tools for multiscale imaging of PD-L1 to study intratumoral distribution of PD-L1 therapeutics.111In-labeled constructs in 4T1 tumor-bearing mice. Comparing our site-specific antibody-conjugates with randomly conjugated antibodies, we found that antibody clone, isotype and method of DTPA conjugation did not change tumor uptake. Furthermore, addition of sulfo-Cy5 did not affect the biodistribution. PEGylated Fab fragment displayed a significantly longer half-life compared to unPEGylated Fab and demonstrated the highest overall tumor uptake of all constructs. PD-L1 in tumors was clearly visualized by SPECT/CT, as well as whole body fluorescence imaging. Immunohistochemistry staining of tumor sections demonstrated that PD-L1 co-localized with the fluorescent and autoradiographic signal. Intratumoral localization of the imaging agent could be determined with cellular resolution using fluorescent microscopy.To introduce imaging modalities, a peptide containing a near-infrared dye (sulfo-Cy5), a chelator (DTPA), an azide, and a sortase-recognition motif was synthesized. This peptide and a non-fluorescent intermediate were used for site-specific functionalization of c-terminally sortaggable mouse IgG1 (mIgG1) and Fab anti-PD-L1. To increase the half-life of the Fab fragment, a 20 kDa PEG chain was attached via strain-promoted azide-alkyne cycloaddition (SPAAC). Biodistribution and imaging studies were performed with A set of molecularly defined multimodal antibody-based PD-L1 imaging agents were synthesized and validated for multiscale monitoring of PD-L1 expression and localization. Our modular approach for site-specific functionalization could easily be adapted to other targets.The online version contains supplementary material available at 10.1186/s12951-022-01272-5. Over the past decade, immunotherapy, in particular blockade of immune checkpoint molecules such as Programmed Death 1 (PD-1) and its ligand PD-L1 using antibodies, has revolutionized the field of cancer therapeutics due to unparalleled responses in a range of cancers . DespiteSeveral preclinical –21 and cHere, we demonstrate that combining our CRISPR/HDR hybridoma engineering technology with chemical synthesis provides a plug-and-play platform to create molecularly defined antibody-based multimodal, multiscale PD-L1 imaging agents. To this end, we functionalized engineered mouse IgG1 (mIgG1) and Fab aPD-L1 site-specifically with a peptide containing both the radionuclide chelator diethylenetriaminepentaacetic acid anhydride (DTPA) and near-infrared (NIR) dye sulfo-Cy5 , while blood concentration and kidney uptake were significantly lower . Site-specifically conjugated isotype-switched mIgG1 with either DTPA alone (mIgG1-IH20) or with the additional fluorophore (mIgG1-IH18) did not show a significant change in blood clearance or liver uptake, compared to randomly labeled WT rIgG2a antibody. Spleen uptake of mIgG1-IH18 was increased compared to mIgG1-IH20 . Duodenum uptake was slightly decreased for IH18- and IH20-labeled antibodies  compared to the MIH5 WT antibody (10.94 ± 0.52% ID/g). Taken together, isotype switching and site-specific conjugation of PD-L1 targeting antibody with IH18 and IH20 was feasible without major changes in the tumor targeting.First, using our molecular toolbox Fig. A and B wIH18, we next aimed to use our platform to produce multimodal imaging conjugates with different pharmacokinetic properties. In addition to mIgG1-IH18, we produced c-terminal sortag-histag carrying aPD-L1 Fab fragments 2+) 659.28, found 659.30. C52H84N16O20S23+ ([M + 3H]3+): 439.85, found 439.94. After purification and lyophilization, IH20 was obtained as a white powder (yield 43%).The resulting IH18 by modification of IH20 with sulfo-Cy5, IH20  was dissolved in DMF (60 mg/mL) and mixed with 1.1 eq.  sulfo-cyanine-5-maleimide  in PBS . This mixture was agitated overnight at RT. The resulting IH18 peptide was purified using RP-HPLC (5–40% ACN/H2O in 35 min), and analyzed using LC–MS. HPLC rt: 27.27. LC–MS (ESI +) m/z calcd for C90H128N20O29S42+ ([M + 2H]2+) 1041.40, found 1041.80. C90H128N20O29S43+ ([M + 3H]3+): 694.60 found: 694.84. After lyophilization, IH18 was obtained as a blue powder .To yield IH18 or IH20 for mIgG1 PD-L1 and 50 eq of IH18 or IH20 for Fab PD-L1. After termination of the reaction with EDTA , the reaction mixture was incubated with 200 µl Ni–NTA beads for 20 min at RT, in order to remove unreacted antibody and Sortase. Beads were separated from the reaction mixture using empty spin columns , and washed twice with wash buffer  and twice with PBS. Reaction mixture and wash fractions were combined and purified using size-exclusion chromatography (SEC), with 1 mM EDTA in PBS as buffer (10 mL/min). Fractions containing the product were combined and concentrated using Amicon Ultra-15 Centrifugal Filter Units, MWCO 10 kDa . Buffer exchange was performed using sterile PBS. Antibody concentration was determined on NanoDrop, using the UV–vis program and measuring at 280 nm for IH20 and at 280 and 646 nm for IH18. Protein purity was assessed under reducing conditions using SDS-PAGE gel electrophoresis (12% acrylamide), comparing starting material, product and product clicked with 5 kDa mPEG-DBCO to verify functionalization of all heavy chains. Per construct, several batches were produced and these batches were combined before performing in vitro and in vivo experiments to guarantee batch uniformity. Quantification of protein purity was achieved using densitometry. Densitometry was performed in ImageJ, and defined as (product bands − background)/(non-product bands − background) × 100%. Purity mIgG1 PD-L1-IH18 = 85%, Fab PD-L1-IH18 = 96%.After optimization, batch sortagging was carried out using 0.5–1.0 mg of antibody (fragment) and 25 eq of 20kDa = 89%.Fab PD-L1-IH18 was conjugated to 20 kDa PEG by adding 10 Eq. (94.0 nmol) of mPEG-DBCO  in PBS (3.33 mM) directly to Fab PD-L1-IH18 in PBS . This mixture was incubated for 90 min at 37 °C, buffer exchange was performed with 0.02 M acetate buffer  using Amicon Ultra-4 Centrifugal Filter Units, MWCO 10 kDa  and subsequently purified using a 1 mL HiTrap SP FF  cation exchange column on an NGC Quest Plus Bio-Rad system operating at 0.5 mL/min. After equilibrating with buffer A for 8 column volumes, a linear gradient of 0.5 M NaCl in buffer A was applied (0–0.5 M NaCl in 30 column volumes). PEGylated Fab PD-L1-IH18 were typically eluted after about 8 column volumes (0.15 M NaCl). Fractions containing the purified product were concentrated using Amicon Ultra-4 Centrifugal 10 kDa MWCO filter units  and sterile PBS as a buffer, and analyzed by 12% SDS-PAGE. The concentration was determined using Nanodrop. This method was verified as reliable by use of a BCA assay . Quantification of protein purity was achieved by performing densitometry, as described above. Purity Fab PD-L1-IH18-PEG3 in PBS, pH 5.5 were added to each antibody and incubated for 1 h at RT. Non-conjugated p-SCN-DTPA was removed from the reaction mixture by dialysis against 5 L 0.25 M NH4Ac  using Slide-a-Lyzer Cassettes (ThermoFisher Scientific). After dialysis, concentration in all samples was determined via spectrophotometer.To prevent contaminants from disturbing radiolabeling, 1 mg of mIgG1 PD-L1-srt-his and rIgG2a WT antibodies were dialyzed against 5 L sterile PBS  using Slide-a-Lyzer Cassettes (ThermoFisher Scientific). Subsequently, a 15 eq. of S-2-(4-Isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid  and 1/10 reaction volume 1 M NaHCO111In  in 0.5 M MES buffer (pH 5.4) for 20 min at room temperature under metal-free conditions as described previously [111In was complexed by adding 50 mM EDTA to a final concentration of 5 mM. Labeling efficiency was determined using thin-layer chromatography on silica gel chromatography strips (Agilent Technologies), using 0.1 M citrate buffer  as mobile phase. Samples with a labeling efficiency below 95% were purified using a PD-10 column  eluted with PBS. Radiochemical purity of 111In-labeled antibody-IH18 conjugates exceeded 95% in all experiments.Antibody-IH18 conjugates were incubated with eviously . Non-che20kDa or a mIgG1 isotype control . Concentrations of these constructs ranged from 0.1 pM to 1000 nM for antibodies and 0.4 pM to 6000 nM for Fabs. After 20 min of incubation at 4 °C, 30 μL of commercially available MIH5 PD-L1-PE  was added . After another 30 min of incubation at 4 °C, cells were washed and resuspended in PBA and measured on FACS. The IC50 was defined as the antibody-conjugate concentration that was required to inhibit binding of the commercially available fluorescently-labeled antibody by 50%.30,000 Renca cells were seeded per well in a 96-well V-bottom plate, and stained with 30 μl of mIgG1 PD-L1-IH18, Fab PD-L1-IH18, Fab PD-L1-IH18-PEG2. Non-specific binding and uptake were determined by co-incubation with 16 nM unlabeled WT PD-L1 (rIgG2a). After incubation, cells were washed and incubated with acid wash buffer  for 10 min at 37 °C to remove the membrane-bound fraction of the antibody conjugates. Cells were washed with PBS, and the acid wash and PBS were combined into one fraction containing the membrane-bound activity. Finally, cells containing the internalized activity were lysed with 0.1 M NaOH and harvested. Samples were measured in a gamma counter  to determine the internalized and membrane-bound activity.Renca cells were cultured to confluency in 6 wells plates and incubated for 1, 3 or 24 h with 105 pM radiolabeled mIgG1, 323 pM Fab or 86 pM PEGylated Fab in RPMI-1640 containing 1% BSA at 37 °C in a humidified atmosphere with 5% CO6 4T1 cells (ATCC) by injection into the mammary fat pad. Mice were block-randomized across the experimental groups based on tumor size. Experiments started when average tumor size reached 0.3 cm3.All experiments were performed in accordance with the revised Dutch Act on Animal Experimentation (2014) approved by the central authority for scientific procedures on animals and animal welfare body of the Radboud University, Nijmegen. Mice were housed in individually ventilated cages with a filter top  under pathogen-free conditions with cage enrichment present , and were fed and watered ad libitum. Biotechnicians were blinded to all groups and compounds administered. Studies were performed with 6–8 weeks old BALB/c mice . In all experiments mice received 1 × 10111In-labelled tracer . At 1, 2 and 24 h after injection with Fab PD-L1-IH18 or Fab PD-L1-IH18-PEG20kDa, or 4, 24 and 72 h for mIgG1, mice were sacrificed using CO2/O2-asphyxiation. To determine the biodistribution of each construct, tumor and normal tissues  were harvested, weighed and measured in a gamma counter, as well as 1% standards of the injected dose. Values are reported as percentage injected dose per gram (%ID/g).For each construct, 3 groups of 5 mice received an intravenous injection of 0.4 pmol 111In-labelled tracer . For Fab, scans were acquired at 4 h and 24 h post-injection (p.i.), for Fab-PEG at 4 h, 24 h and 48 h, and the full length antibodies were imaged at 24 and 72 h p.i.. Images were acquired for 45 min under general anesthesia  with the U-SPECT-II/CT  using a 1.0 mm diameter pinhole mouse high sensitivity collimator, followed by CT scan  for anatomical reference. Scans were reconstructed with MILabs reconstruction software using a 16-subset expectation maximization algorithm, with a isotropic voxel size of 0.2 mm and 1 iteration. SPECT/CT scans were analyzed and maximum intensity projections (MIP) were created using Inveon Research Workplace software (Siemens).For each construct, mice (n = 5/group) received 0.4 pmol Snap frozen, unstained tumor section (10 µm) were scanned on a fluorescence flat-bed scanner at pixel resolution of 4 µm and with an excitation/filter setting of 690/700 nm, quality setting ‘high’  and subsequent sections were exposed to a Fujifilm BAS cassette 2025 (Fuji Photo Film) for 3 days. Phosphor luminescent plates were scanned using a phosphor imager  at a pixel size of 25 × 25 µm. Images were acquired with Aida Image software.PD-L1 expression in tumors was determined by chromogenic detection using fresh frozen tissue sections fixed in PBS 45% acetone 10% formalin. In short, sections were pre-incubated with 10% normal rabbit serum, endogenous biotin and avidin were blocked, and endogenous peroxidase activity was blocked with 3% H2O2. Subsequently, anti–mPD-L1  was applied and tissues were incubated overnight at 4 °C. Next, sections were incubated with biotinylated rabbit anti-goat IgG  followed by incubation with avidin–biotin-enzyme complexes . Finally, 3’,3’-diaminobenzidine (DAB) was used to develop the tumor sections prior to mounting slides.Furthermore, fluorescence detection was performed to co-localize mIgG1 PD-L1-IH18 with PD-L1 expression. Fresh frozen tissue sections were fixed using buffered formalin-acetone fixative and pre-incubated with 10% normal donkey serum or 10% normal goat serum. Subsequently, slides were incubated overnight at 4 °C with anti-mPD-L1  or isotype control goat IgG . Next, donkey-anti-goat-Alexa488  was applied for 30 min and DAPI  for 2 min at RT. Sections were mounted with fluoromount  and fluorescence imaging was performed with a Leica DMI6000 epi-fluorescence microscope fitted with a 63 × 1.4 NA oil immersion objective.Statistical analyses were performed using GraphPad Prism version 5.03  for Windows. Data are presented as mean ± standard deviation. Differences in uptake of the radiolabeled tracers were tested for significance using a one-way ANOVA with a Bonferroni test. A p-value below 0.05 was considered significant.Additional file 1. Supplementary figures and tables.