The enabling of in-space cryogenic engines and cryogenic fuel depots for future manned and robotic space exploration missions begins with technology development of advanced cryogenic fluid management systems upstream in the propellant feed system. Before single-phase liquid can flow to the engine or customer spacecraft receiver tank, the connecting transfer line must first be chilled down to cryogenic temperatures. The most direct and simplest method to quench the line is to use the cold propellant itself. When a cryogenic fluid is introduced into a warm transfer system, two-phase flow quenching ensues. While boiling is well known to be a highly efficient mode of heat transfer, previous work has shown this efficiency is lowered in reduced gravity. Due to the projected cost of launching and storing cryogens in space, it is desired to perform this chilldown process using the least amount of propellant possible, especially given the desire for reusable systems and thus multiple transfers. This paper presents an assessment of two revolutionary new performance enhancements that reduce the amount of propellant consumed during chilldown while in a microgravity environment. Twenty-eight cryogenic transfer line chilldown experiments were performed onboard four parabolic flights to examine the independent as well as combined effect of using low thermally conductive coatings and pulse flow on the chilldown process. Across a range of Reynolds numbers, results show the combination significantly enhances performance in microgravity, with a reduction in consumed mass up to 75% relative to continuous flow for a bare transfer line. However, there are challenging aspects when working with cryogens due to inherent thermo-physical properties. Particularly for the current work, the low normal boiling point (NBP), low surface tension, and high susceptibility to parasitic heat leak leads to unwanted boiling and two-phase flow during propellant transfer.The enabling of in-space cryogenic engines and cryogenic fuel depots for future manned and robotic space exploration missions begins with technology development of advanced cryogenic fluid management (CFM) systems upstream in the propellant feed system. Cryogenic propellants offer significantly higher performance relative to storable counterparts, such as hydrazine, owing to a higher specific impulse and higher energy density. Further, safety and environmental concerns over the use of toxic storable propellants have led to the ongoing examination of more “green” propellants such as liquid methane as alternate fuel sources. Aside from nuclear thermal propulsion systems3, defined as an Earth-orbiting propellant storage vessel that would house cryogenic propellant to allow spacecraft to refuel, have four stages: (1) acquisition of the storage tank liquid, (2) chilldown of the connecting transfer line hardware, (3) chilldown of the receiver tank, and (4) fill of the receiver tank, all in the microgravity of space; this paper focuses on the second stage, chilldown of the transfer line. Meanwhile, cryogenic engines also require acquisition of the storage tank liquid and chilldown of the transfer line. Cryogenic fuel depots will require very high liquid volume fill fractions in the customer receiver tank, and most in-space engines require single-phase liquid up to the injectors. Therefore, without advanced CFM technologies upstream in the feed system and storage tank, vapor ingestion is inevitable, which can lead to combustion instabilities within the engine. Further exacerbating the transfer process in microgravity is the unknown location of the liquid and vapor phases in the tank as well as reduced heat transfer.Cryogenic fuel depots8. Due to the projected cost of launching and storing cryogens in space, it is desired to perform this chilldown process using the least amount of propellant as possible, especially given the drive towards reusable systems and thus multiple transfers.Before single-phase liquid can flow to the engine or customer spacecraft receiver tank, the connecting transfer line must first be chilled down to cryogenic temperatures. Chilldown, or quenching, is defined as the transient process of cooling hardware down to cryogenic temperatures so that vapor-free liquid can eventually flow between two points of interest. The most direct and simplest method to quench the line is to use the cold propellant itself. When a cryogenic fluid is initially transferred through a system, the tube walls and hardware  undergo a transient chilldown prior to reaching a steady state of operation. Chilldown thus involves unsteady two-phase heat and mass transfer and flow boiling. While boiling is well known to be a highly efficient mode of heat transfer, previous work has shown this efficiency is significantly lowered in reduced gravity, both for room temperature fluids as well as cryogens9. Key contributions in 1-g were provided by23, which investigated the effect of mass flux, inlet state, pressure, and flow direction on cryogenic tube chilldown, predominately using liquid nitrogen (LN2) and liquid hydrogen (LH2). Since 2018, five more cryogenic quenching studies on bare tubes that passed the data filtering criteria from ref. 9 by Jin et al. have been added to the cryogenic database to cover low Reynolds (Re) number LN224, liquid argon25, and liquid oxygen26 chilldown experiments while28 added high Re number chilldown tests with LH2. Hartwig et al.29 recently summarized cryogenic quenching flow boiling trends over the consolidated literature, across multiple flow regimes, mass fluxes, inlet states, and gravity levels. For all cryogens, the chilldown process is highly dominated by the film boiling regime for bare tubes30 . When a cryogen is introduced into a warm tube, especially at high mass flux and low inlet equilibrium quality, a vapor film blanket surrounds the liquid core which acts as an insulator that inhibits heat transfer between cold liquid and warm tube. At lower mass flux and saturated inlet states, dryout occurs over a longer distance along the tube as in the case of traditional fluids31. Film boiling heat transfer is a highly inefficient process relative to transition and nucleate boiling. In most instances, film boiling can persist for >85% of the total time needed to chill the tube down to the saturation temperature of the cryogen. Once the Leidenfrost point is reached, chilldown proceeds into transition boiling, nucleate boiling, and then single-phase liquid convective flow. In microgravity, this poor heat transfer is exacerbated by the lack of buoyancy force; cryogenic film boiling heat transfer was shown to be 25% lower at low to modest Re flows relative to 1-g8. At very high Re, inertial forces can overcome gravitational forces such that gravity no longer affects flow boiling32 .Numerous cryogenic flow boiling quenching experiments have previously been conducted on bare tubes, the results up to 2018 of which are summarized in ref. 34 and China35 independently confirmed that a Teflon coated tube could reduce chilldown times up to 75% over an uncoated stainless steel (SS) tube using LN2. Both researchers also investigated the effect of the Teflon coating thickness on chilldown performance, and both showed that thicker coatings led to faster chilldown times. However, as the coating thickness increased further there was an apparent point of diminishing returns because the chilldown curves  converged at the highest tested thicknesses. Coated tubes offer hope to combat the intrinsically poor film boiling heat transfer in microgravity36.To overcome this hurdle in poor performance, researchers have recently investigated low thermally conductive materials applied to the inner tube walls and the effect of such coatings on the chilldown process. The coating acts as an insulator between the cold propellant and warm wall, resulting in an inner wall surface temperature that reaches the Leidenfrost point without cooling the entire tube mass. Recent 1-g experiments conducted in the United States237 and LN238, in pulse flow, the inlet valve is cyclically opened and closed with a specified duty cycle (DC) and pulse width until the desired degree of chilldown is reached. The advantage of pulse flow is lower mass consumption over traditional continuous flow due to more efficient usage of latent and sensible energy of the fluid, with the disadvantage being potential valve fatigue and/or failure and added complexity in operation.A second way to enhance poor chilldown performance is to use pulse flow. Demonstrated using both LH2 transfer line chilldown experiments were performed onboard a parabolic flight that simulated space microgravity conditions to examine the independent as well as combined performance gains of using low thermally conductive coatings and pulse flow on the chilldown process. While previous experiments have reported the effects of pulse flow and coatings on transfer line chilldown in Earth-gravity, this is the first report of pulse flow and the combined effect of pulse flow with a coated tube in a microgravity environment.The purpose of this paper is to present an assessment of two new performance enhancements that reduce the amount of propellant consumed during chilldown while in a microgravity environment, and to investigate if the mass savings holds in microgravity. Twenty-eight LNTable 39 can be used to determine inner wall temperature for bare tubes, due to the unknown thermal contact resistances between the coated layer and tube inner wall as well as among adjacent coated layers, inner wall temperature could not be determined for coated tubes. Therefore, outer wall temperature data had to be used to determine end of chilldown. Three chilldown criteria were explored: (1) the averaged exit outer wall temperature was compared to the liquid saturation temperature , (2) the first derivative of outer wall temperature (with respect to time) reaching and remaining near 0 K/s , and (3) a peak value in the second derivative of outer wall temperature (with respect to time) which would indicate the slope change in the chilldown curve occurring at onset of nucleate boiling (ONB). The first method was found to be unreliable due to inaccurate chilldown time estimations attributed to the significant difference between inner and outer wall temperature at higher layers of coating. The third method also yielded inaccurate chilldown time estimations attributed to the absence of a true global maxima in the second derivative at higher layers of coating. Therefore, the second method using the first derivative , slightly conservative but consistent across all scenarios, was used to determine the end of chilldown in all test cases.Chilldown time was determined as follows: In practice, the most stringent chilldown criteria would be determined from a measured stream temperature downstream of the test section reading lower than the saturation temperature based on the downstream pressure; however, this measurement was not available for the current tests. Based on boiling heat transfer theory, nucleate boiling would end when the inner surface temperature drops below that of the onset-of-nucleate boiling (ONB). As a result, the wall heat flux would switch from higher boiling heat flux to much lower single-phase convective heat flux that would reflect a change on the outer wall surface temperature gradient with time. A computed inner wall temperature could also not be used to determine end of chilldown; while the inverse conduction method of Burggraf2 mass at the end of chilldown as read by the flow meter downstream of the test section:tend is the end of chilldown time and 2 mass flow rate measured by the gas flow meter. Steady state Reynolds (Re) number (defined at the end of chilldown when single phase liquid flow is established) and mass flux were evaluated using inner diameter and saturation conditions based on the measured test section pressure:Chilldown mass was the total consumed LN39 was used to determine inner wall temperature and transient radial heat conduction through the tube as follows:ρ, CP, and k are the tube density, specific heat, and thermal conductivity, respectively, ri and ro are the inner and outer radii, and To is the outer wall temperature. Heat transfer coefficient was then computed as follows:Ti is the inner wall temperature which comes from Burgraff’s method, and Tsat is the saturation temperature based on the measured pressure. The method to calculate the different heat fluxes in Eq. 22 and ref. 27.For uncoated tubes, the method of Burgraff41. The high wall surface temperature causes the liquid to completely vaporize before reaching the surface resulting in an inner liquid core and outer annular vapor core. This vapor blanket along the wall insulates the warm pipe from the cold liquid, causing the temperature of the pipe to decrease, albeit slowly. FB is the least efficient quenching mechanism. As the transfer line chills down, the system approaches the LFP, or rewet temperature, where heat flux is at a minimum (during boiling). Heat transfer here is a minimum due to the inefficiency of heat transfer between cold vapor and wall. LFP is also characterized by the onset of a rapid drop in wall temperature. As shown in Fig. 31. As the wall cools further, the tube inner surface approaches the ONB, characterized as the point at which the system evolves from nucleate two-phase cooling to single-phase liquid convection and an obvious slope change in the chilldown curve. Vapor-free liquid marks the end of the chilldown test. The single-phase cooling causes the wall temperature to drop slowly to the liquid saturation temperature and then remain steady as heat transfer reduces to near zero. In microgravity, circumferential TCs at each station have almost identical chilldown behavior at any axial distance from the inlet; stratification effects normally seen for horizontal tubes in 1-g disappear, leading to axisymmetric flow patterns through the tube, and thus uniform chilldown circumferentially.Figure Figure Trends are as follows: First, coating the inner wall of the tube drastically affects the chilldown behavior and leads to faster chilldown times. The low thermally conductive Teflon layer acts as an insulator between cold fluid and warm wall; the inner surface temperature chills down quickly without cooling the entire tube mass. The lower inner wall surface temperature earlier on means that the Leidenfrost point is reached faster such that the liquid can stay in contact with the tube for the heat transfer to be in TB and NB that reduces the poor heat transfer film boiling time; this is substantiated by the drastic slope change for 4 L and 7 L tube indicating the LFP is reached earlier on relative to the bare tube. Note that Fig. Third, however, there is an apparent point of diminishing returns; this trend of improved chilldown performance upon addition of coating is reversed when the number of coating layers is increased from 4 to 7 because the chilldown time is faster for 4 L (4.7 s) compared to 7 L (8.2 s) case. Similarly, from 4 L to 7 L, the propellant mass savings and chilldown efficiency are reduced. This crossover in performance and possible existence of an optimal coating layer is explained by counteracting heat transfer mechanisms: (1) the low thermal conductivity of the coating layer facilitates the faster temperature drop of tube inner surface by restricting heat transfer between inner surface and bulk of the metal tube and (2) the low thermal conductivity coating also creates a thermal resistance that restricts the heat conduction between bulk of the tube and cooling fluid. With these contrasting mechanisms at play, the thickness of the coating must be such that it is thick enough to quickly lower the tube inner surface temperature while being thin enough to facilitate fast wall chilldown. However, the presence of the coating accelerates chilldown as evident in any comparison between bare and coated tube at similar thermodynamic conditions.233 and LH237. Fourth, for this particular comparison, Fig. Figure Figure 8 and second-generation36 systems. As before, the system is intended for both ground and flight experiments. Figure The authors have completed four successful cryogenic line chilldown parabolic flight campaigns including the current campaign between 2015 and 2020 and are familiar with system designs, troubleshooting, issues, and failures that arise with microgravity flight testing. The fourth-generation system was modified based on flights from the first-2 was supplied to the system from an 80-liter vacuum-jacketed dewar, with a relief valve set at 861 kPa. A gaseous nitrogen (GN2) cylinder initially pressurized at 15 MPa was used to pressurize the dewar to a set value for each test, which ranged between 90 and 830 kPa absolute pressure. Dewar pressure was managed by a pressure regulator that controlled the dewar pressure to within 35 kPa of the set value during each test. Depressurization was carried out by opening the globe valve 2 (GV2) and the three-way ball valve 1 (3V1) to allow ullage gas to vent to the atmosphere.LN2 both for prechilling the plumbing upstream of the test section and for conducting the actual chilldown experiment. LN2 was delivered through valve GV3 that was connected through a 1.2 m long, 1.27 cm outer diameter (OD), 1.18 cm inner diameter (ID) 304 (stainless steel) SS braided hose to the inner tube of the precooler (or subcooler) shell-tube heat exchanger shown in Fig. 2 in the transfer line, since the saturation temperature of the shell side was always lower than the tube side, and most importantly (3) to ensure single-phase liquid at the inlet of the test section. The liquid level of the nitrogen pool was monitored by three thermocouples (TC) inside the subcooler, two at the shell-side and one at the outlet to Vap1. The temperature readings of these TCs were displayed on a laptop in real-time. The level of the LN2 pool was inferred from the TC insertion depth. A 2.5 cm ID port allowed evaporating liquid to escape the subcooler. The fluid was directed to an electrically heated “vaporizer” Vap1 which vaporized any entrained liquid and warmed the vapor to above 273 K before entering the atmosphere. Two layers of 6.35 mm thick aerogel insulation were wrapped around GV2, the hose upstream of the subcooler, the subcooler itself, 3V2, 3V3, and the 3 cm length of tube between 3V3 and the subcooler to minimize heat leak into the system upstream of the test section.The dewar was used to supply the LNDuring the prechilling process, the liquid exiting the inner tube of the subcooler was directed by two “T-type” 316SS 1.27 cm ID three-way ball valves (3V3 and 3V2) to a fill-port on top of the outer vessel of the subcooler. A 3 cm long, 1.270 cm OD, and 1.168 cm ID 304SS tube connected 3V3 to the subcooler. A pressure transducer and TC labeled “PT”, “TC” in Fig. The needle valve downstream of the test section (NV1) was used to provide fine-tuning of the mass flow rate so that tests could be run at different flow rates for the same dewar pressure setting. The flow was routed from the needle valve by a SS tube to two separate vaporizers (labeled Vap2 and Vap3) that were electrically heated to vaporize the liquid-vapor two-phase flow. To enhance the heat transfer in the vaporizer, eight 1.27 cm OD copper tubes were packed inside the vaporizer in an octaweb configuration. One electrical heating tape was wrapped around the vaporizer to heat it to 550 K before each test. A TC was placed on the outer surface of each heating tape to monitor the temperature in real time. The flow out of Vap2 and Vap3 entered two separate, identical gas flow meters (Gas Flow Meters 1 and 2) that each had a capacity of 3000 standard liters per minute. The flow was then directed to the airplane vent ports downstream the flow meters.42) test sections were individually flight tested: a bare tube with no coating, and a tube with a 4 layer and 7 layer coating. For the coated tubes, the SS tube was coated with low-thermal conductivity thin Teflon layers on the inner surface. Specifically, the coating material was made of Fluorinated Ethylene Propylene (FEP) produced by DuPont and classified by DuPont as Teflon 959G-203 that is a black color paint. The coating was applied by using a pour and drain process. After each pour and drain, the fresh film layer was cured in a furnace through a standard sintering procedure before adding another layer by the same pour and drain procedure. As a result, the final thickness of the coated layer depends on the total number of layers processed; for example, the 4 L coating went through the pour and drain process four separate times. To measure the coating layer thickness, high resolution computer tomography x-ray scans of the tube cross sections were obtained using a Phoenix v|tome|x M system in the Nano Research Facility at the University of Florida. Scanning was carried out using a 240 kV X-ray tube and a tungsten-on-beryllium target, with the following settings: 200 kV, 50 milliamps, and 0.5 mm Tin filter. Images were collected from 1600 pixels horizontal, 2024 pixels vertical, 0.5 s detector exposure, averaging of 4 images per rotation position with a one-exposure skip and a total of 2200 rotational positions. The average thickness per layer was ~15.12 µm and the uncertainty for each layer was Three, 0.914 m (36 in) long, 0.051 cm wall thickness, 1.27 cm outer diameter SS304  and instrumentation, a Labview Virtual Instrument software and National Instrument (NI) CompactDAQ hardware was used to collect all sensor data to be displayed in real-time on a laptop. The sampling rate of all the sensor measurements was set to 16 Hz. Two NI-9214 TC modules read the signals from all the T-type TCs. NI 9205, an analog input module, read all the voltage signals from pressure transducers. The Labview VI controlled the opening and closing of the solenoidal valves (SVs), through a combination of NI-USB 6009 and Solid-State relay. In the case of continuous flow, the relay energized the solenoid valve after receiving a constant voltage signal. For pulse flow, the relay energized and de-energized SV according to a rectangular waveform voltage signal generated by the Labview VI. Signals of the two mass flow meters (Alicat M3000 - SLPM) downstream of the vaporizers were read by the program directly without the NI DAQ system.o  at each station as shown in Fig. Fifteen TCs were soldered to the outside of each tested tube. Five stations were spaced out axially in Fig. 29; uncertainties for test section dimensions, vacuum chamber dimensions, and thermal properties were similar as in29. Standard error propagation rules were applied to compute uncertainties in chilldown time (2.1%), propellant mass consumed at steady state (2.5%), mass flux (2.8%), and Re number (3.3%). The median relative uncertainties were 8–10% in Burggraf heat flux, total heat flux, and HTC, and 25% in parasitics across all the bare tube cases. The number of outliers in relative uncertainties were on the order of 101 or fewer in each case and occurred post-chilldown. Therefore, the 95% quantile accurately represents the maximum relative uncertainties in Burgraff heat flux, parasitics, total heat flux, and HTC which are reported in Table Root-sum-square uncertainty analysis was conducted in a similar fashion as in refs. n time 2.%, propelThe experimental methodology to conduct a test was as follows: At the start, the needle valve was set to the target position to set test section pressure, Vap 1, Vap2, and Vap3 were heated up to 550 K, and the vacuum pump system was turned on. The total time from engaging the pump until reaching 1 Pa inside the vacuum chamber was ~15 min. Concurrently, the inner tube inside the subcooler was chilled by pressurizing the dewar, opening GV2, and directing the flow through 3V3 and 3V2 to the fill port of the subcooler. The subcooler took ~10 min to completely chill and fill. Then, 3V2 was shut off to stop the flow from 3V3, and the supply dewar was pressurized by opening the pressure regulator to the desired gauge pressure for the dewar. Pressurization was done as quickly as possible before the liquid inside the dewar could re-saturate at the new dewar pressure, and also before the liquid inside the plumbing upstream of the test section could gain enough heat to start boiling. Shortly after, 3V3 was turned to start the flow into the test section to begin a chilldown test. Once the TC readings dropped below the saturation temperature and maintained a steady temperature, GV2 and SV was closed. This marked the end of the test. In preparation for the next test, Heater 1 was turned on, and both 3V2 and 3V3 were rearranged so that warm gas could enter the test section to carry out the reheat process. After reheating was finished, NV1 was set at the new position. Vap2 and Vap3 were allowed to heat up to above 550 K, and the subcooling process was repeated to account for the lost LN2. At this time, the system is ready for another run.Further information on research design is available in the 33, at somewhat similar inlet pressure, period, and duty cycle, the mass savings in going from continuous flow with a bare tube to pulse flow with a coated tube is slightly higher in microgravity (~75%) versus in 1-g (67%) at similar high Re. The lower mass savings in 1-g can easily be attributed to the fact that the duty cycle of the 1-g coated tube pulse flow test is 20% compared to 10% of 0-g coated tube pulse flow test in the current work. Lower duty cycle is predicted to increase mass savings33 which means that a 1-g coated tube pulse flow test performed at 10% duty cycle would have >67% propellant mass savings. At high Re, the mass savings would be roughly equal in microgravity and 1-g because of forced convection dominating over buoyancy effects. However, at low Re, 1-g results would be expected to yield higher mass savings due to the aforementioned lack of buoyancy-assisted cooling in microgravity at low Re, whether comparing 1-g pulse flow to 0-g pulse flow or 1-g coated tube flow to 1-g coated tube flow. Therefore, with optimization of coating thickness and pulse characteristics performed a priori, coated tube and pulse flow can be used for transfer line chilldown to significantly save chilldown time and mass for all future in-space cryogenic transfers.Figure Reporting Summary


Many biomedical applications, such as classification of binding specificities or bioengineering, depend on the accurate definition of protein binding interfaces. Depending on the choice of method used, substantially different sets of residues can be classified as belonging to the interface of a protein. A typical approach used to verify these definitions is to mutate residues and measure the impact of these changes on binding. Besides the lack of exhaustive data this approach generates, it also suffers from the fundamental problem that a mutation introduces an unknown amount of alteration into an interface, which potentially alters the binding characteristics of the interface. In this study we explore the impact of alternative binding site definitions on the ability of a protein to recognize its cognate ligand using a pharmacophore approach, which does not affect the interface. The study also provides guidance on the minimum expected accuracy of interface definition that is required to capture the biological function of a protein. The residue level description or prediction of protein interfaces is a critical input for protein engineering and classification of function. However, different parametrizations of the same methods and especially alternative methods used to define the interface of a protein can return substantially different sets of residues. Typical experimental or computational methods employ mutational studies to verify interface definitions, but all these approaches inherently suffer from the problem that in order to probe the importance of any one position of an interface, an unknown amount of alteration is introduced into the very interface being studied. In this work, we employ a pharmacophore-based approach to computationally explore the consequences of defining alternative binding sites. The pharmacophore generates a hypothesis for the complementary protein binding interface, which then can be used in a search to identify the corresponding ligand from a library of candidates. The accurate ranking of cognate ligands can inform us about the biological accuracy of the interface definition. This study also provides a guideline about the minimum required accuracy of protein interface definitions that still provides a statistically significant recognition of cognate ligands above random expectation, which in turn sets a minimum expectation for interface prediction methods. Protein-protein interactions are key players in many biological processes including metabolism, development, and regulation. Residue level descriptions of protein binding interfaces are essential for explaining, classifying, and modulating the formation of specific protein complexes. The biological function of a protein binding interface critically relies on its specificity, the ability to selectively recognize its cognate binding partners. However, the question of what exactly comprises a biological interface does not have a clear answer. Many interface prediction approaches exist. Some of these employ a radial distance cutoff-based approach , 2, whicA major strategy, both computationally –25 and eIn this paper, we computationally explore various protein interface definitions by probing the minimum interface necessary to successfully recognize its cognate ligand. Through this analysis we quantitatively investigate how much overlap alternative receptor interface patches must share with their true biological interface to still recognize their cognate partner reliably. Similarly, we investigate how many true interface residues can be lost without diminishing a proteins ability to accurately recognize its cognate partners.The analysis presented here does not perturb the integrity of the protein structure with mutations. Instead, we utilize a computational approach, ProtLID , to obtaIn this work, we study three protein interfaces: PD1:PD-L1, CTLA4:CD80, and CTLA4:CD86. These interactions are critical in regulating cellular functions. PD1 and PD-L1 form a co-inhibitory complex that can limit the development of T-cell response. PD1-PD-L1 interaction helps ensure that the immune system is activated only at the appropriate time to minimize autoimmune inflammation . CTLA4, Our results show that, on average, one can misdefine a protein interface by about 20–30% while still preserving its ability to recognize its cognate partner with statistically significant results. These results suggest that methods that predict protein interfaces should achieve above 70% accuracy to be useful. Our results also show that receptors with higher binding affinity (CTLA4:CD80) compared to receptors with lower binding affinity (CTLA4:CD86) are harder to destroy by removing true interface residues and adding false interface residues.The goal of this work is to assess the impact of alternative interface definitions for a protein receptor by monitoring how well each alternatively defined interface can recognize its cognate binding partner(s) from an ensemble of candidates. We consider the receptor-ligand interactions in three protein-protein complexes: PD1:PD-L1, where the interface on PD1 comprises 14 residues, and CTLA4:CD86/CD80, with a smaller interface on CTLA4 of 9 and 10 residues, respectively . CTLA4 wth percentile for the PD1 receptor when we consider interface patches with gradually reduced degree of overlap with the original interface. The original interface . nterface ), was abnterface . If we cIt is interesting to consider protein receptor interfaces with a smaller number of known cognate ligands as well. We consider two CTLA4 interfaces in complex with CD80 and CD86. We collected all the docked poses of CTLA4:CD80 and CTLA4:CD86 generated by ZDOCK that have the same interface size as CTLA4 in complex with its respective cognate binding partners . BecauseThe large degree of overlap between the two CTLA4 interfaces , defined using the CTLA4:CD80 complex or the CTLA4:CD86 complex, enables us to assess the ability of the CTLA4:CD80 interface to recognize CD86, and the CTLA4:CD86 interface to recognize CD80. As shown in Fig. S1, the ability of the CTLA4:CD80 interface to recognize CD86 as a cognate ligand is poorer than the average ability of it to recognize its own cognate ligands. In contrast, as shown in Fig. S2, the ability of the CTLA4:CD86 interface to recognize CD80 as a cognate ligand is better than the average ability of it to recognize its own cognate ligands. This suggests that CD80 has greater specificity than CD86 for recognizing the CTLA4 interface.In general, we find that the performance of the CTLA4:CD80 interface is better on average than that of the CTLA4:CD86 interface in recognizing cognate partners. This observation possibly implies that the binding affinity of CTLA4 for CD80 is markedly higher than the affinity for CD86 –49. The In an alternative approach, we explored what happens when we gradually eliminate residues in a combinatorial way from the original interface, without replacing them with non-interface residues (thereby reducing the size of the interface). We systematically removed residues in cohorts  from the residues comprising the rs-pharmacophore to assess the ability of the reduced rs-pharmacophore to recognize its cognate ligands. Protein interfaces can be defined using various approaches, which include using radial cutoffs, monitoring change in solvent accessible surface areas, or employing Voronoi polyhedra calculations. The disagreement among the results of these methods can be traced back to the fact that these approaches aim at identifying interfaces from a physical point of view using alternative criteria. In contrast, in this work we tried to assess the impact of alternative interface definitions from a biological point of view, from the perspective of a protein to maintain its ability to selectively recognize its cognate binding partner. The central question we ask in this work is how imperfect can the interface definition be whilst retaining the ability to recognize the cognate ligand(s). We assessed this by pursuing alternative interface patches that had decreasing overlap with the original biological interface. In this work, none of the alternative interfaces sampled produced higher accuracy results than the original definition obtained from CSU. This does not validate CSU as a uniformly accurate approach, but simply reflects the fact that we picked well performing cases, where the pharmacophore reliably identified the cognate ligand partners from a set of decoys. This means that the original definition of the interface had to be reasonably good to start with.ab-initio methods , CTLA4:CD80 (PDB code: 1I8L), and CTLA4:CD86 (PDB code: 1I85). A rs-pharmacophore is calculated with ProtLID treating PD1 , CTLA4 , and CTLA4  as receptor proteins. Each receptor rs-pharmacophore is screened against a library of 103 structures of the immunoglobulin superfamily (Suppl. Table 1). There are 24 available structures of the cognate ligand of receptor PD1   was usedProtein Ligand Interface Design,  is a comskewness = (Pm3/ Psd3) where Pm3 is the third moment of a ProtLID score distribution, and Psd is the standard deviation of ProtLID scores. Once a pharmacophore for a given protein interface is generated, we enumerate all possible 5-mer combinations of the calculated residue preferences to screen a ligand structure database. This results in a certain number of matches for each potential ligand out of all combinatorial possibilities. For instance, for a 15-residue large rs-pharmacophore, the number of 5-mer enumerations is 3003 (15C5). The ProtLID score is the number of 5-mer hits for a particular ligand. Skewness is calculated over the distribution or scores obtained for all possible ligands. Only interface patches for which the skewness is above 2.5 are deemed reliable (Mathematical skewness of ProtLID scores assesses the reliability of the rs-pharmacophore generated by ProtLID . Skewnesreliable , while o


Ustilago maydis, we tried to address the effects of the ectopic activation of TOR on virulence. We obtained gain-of-function mutations in the Rheb GTPase, one of the conserved TOR kinase regulators. We have found that unscheduled activation of Rheb resulted in the alteration of the proper localization of the pheromone receptor, Pra1, and thereby pheromone insensitivity. Since pheromone signaling triggers virulence in Ustilaginales, we believe that the Rheb-induced pheromone blindness was responsible for the associated lack of virulence. Strikingly, although these effects required the concourse of the Rsp5 ubiquitin ligase and the Art3 α-arrestin, the TOR kinase was not involved. Several eukaryotic organisms have shown that Rheb transmits environmental information through TOR-dependent and -independent pathways. Therefore, our results expand the range of signaling manners at which environmental conditions could impinge on the virulence of phytopathogenic fungi.The target of the rapamycin (TOR) signaling pathway plays a negative role in controlling virulence in phytopathogenic fungi. However, the actual targets involved in virulence are currently unknown. Using the corn smut fungus  Fungal pathogens often use the adverse conditions found on plant surfaces (nutrient limitation and presence of some stresses) as triggers of the virulence program. Previous reports have shown that conditions or mutations resulting in unscheduled activation of the TORC1 pathway  disable the virulence program in phytopathogenic fungi. Using mutations that activate Rheb1, the GTPase responsible for TORC1 activation, we have addressed whether unscheduled TORC1 activity impairs the virulence of Ustilago maydis, the agent responsible for the corn smut disease. Strikingly, we have found that overactivation of Rheb1 resulted in an inhibition of virulence, but this effect was TORC1-independent. Several reports in other eukaryotic systems demonstrated non-canonical roles of the Rheb regulatory axis. In this work, we added the control of virulence in U. maydis to this increasing list. We also tried to determine at which steps Rheb is controlling the virulence in U. maydis, and we have found that the ability to sense the presence of pheromone, the trigger of the virulence program in this fungus was affected. For many phytopathogenic fungi, the activation of the virulence program is a consequence of the response to environmental cues found on plant surfaces, mainly chemical- and surface-dependent stimuli. This extracellular information interacts with the internal cell status  at various levels in a sort of "fungal brain," which integrates exogenous and endogenous signals to determine the coordinated cellular response. One of the more essential elements for appropriated signal integration in a eukaryotic cell is the TORC1 (Target Of Rapamycin Complex 1) pathway, widely conserved in fungi . IntegraFusarium oxysporum We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.Yours sincerely,Eva H. Stukenbrock, PhDAssociate EditorPLOS GeneticsGregory P. CopenhaverEditor-in-ChiefPLOS GeneticsThree reviewers provide below their detailed comments to the manuscript. These should all be considered in a revised and re-submitted version of the manuscript. This includes improved clarity in the presentation of the results. A summary figure with a schematic illustration of signaling branches would be helpful. Moreover, reviewer 1 points to sections in the results that clearly can be shortened while other details related to Rhb1KR mediated avirulence could be more extended.The authors should also carefully take into account comments regarding missing controls. Although the reviewers acknowledge the use of different controls to support findings from the genetic analyses, some basic controls are missing in some of the experimental assays .Several gene strains were generated, but the authors have not mentioned if the mutants were impaired in growth or in other ways showed a distinct phenotype. Reviewer 2 and 3 both highlight the necessity of providing information to mutant phenotypes as well as considering the possibility that deletion mutants could have accumulated suppressor mutations that complement gene loss.Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: In the manuscript by de la Torre and Perez-Martin, the authors investigate the relationship between TOR pathway activity and the virulence program in the corn smut fungus Ustilago maydis. The authors observed that Tor1 inhibition inhibits growth and components of the TORC1 but not TORC2 complexes are essential for growth in U. maydis. The further established Maf1 and Aga1 phosphorylation as potential read-out for TORC1 activity, respectively. To investigate the effects of increased TORC1 activity, the authors express a constitutive active alle of the Rheb GTPase Rhb1 or delete the Rhb1 GAP Tsc2. Rhb1KR expression results in aberrant localization and, most likely degradation of the nitrate transporter Nrt1, which is rescued upon rapamycin treatment, or deletion of the Rsp5 ubiquitin-ligase encoding gene (function downstream of Rhb1). Furthermore, in Rhb1KR expressing strains the virulence program is non-functional and filamentous growth during pheromone perception and formation of the infectious filament in haploid pathogenic background is blocked. Importantly, this phenotype appears to be independent of TORC1 activity as rapamycin treatment did not rescue the pheromone response. As a possible explanation the authors find that localization of the pheromone receptor Pra1 is disturbed upon Rhb1KR expression, which is rescued upon deletion of rsp5 or art3. In case of art3 deletion also rescues, in part the virulence defect of Rhb1KR expressing strains.In conclusion, this manuscript provides first insight into TOR signaling in U. maydis, but focusses in its main part on the Tor-independent effect of Rhb1 overactivation on the virulence program. The underlying mechanism of Rhb1KR-dependent avirulence might relate to delocalized Pra1 but is not fully explored, understood or experimentally tested. This might also relate to various other reasons, as increased Rhb1 activity results in pleiotropic events.Overall, the manuscript reports on several interesting observations, but needs to be overworked and restructured to improve coherence and focus. Several findings, appear to be unrelated to the main part of the paper. And based on the pleiotropic nature of many introduced mutations, conclusions are in some cases speculative.Specific points:1. To allow the reader appreciate the central and important findings of the manuscript some parts relating to Figures 1 + 2 + 4 should be more concise and the Rhb1KR mediated avirulence more expanded. Most of the observations in the first sections (page 6-10) conclude with knowledge that is expected and conserved and thus should be clearly shortened. 2. Please be more concise when introducing the relevant components of the TOR pathway in the individual sections and/or include, when necessary, in the introduction.3. It would be important to find out if Pra1 delocalization is causal for the loss of virulence in Rhb1KR expressing strains. This hypothesis could be challenged by expression of prf1-con in the haploid pathogenic SG200 or HA103 background (together with Rhb1RK), as this de-necessitates the pheromone response pathway for virulence.4. Please restructure and reword for clarity, grammar, conciseness and correct use of tenses. This is formally a minor point, but based on the numerous occasions these corrections are required to follow the logical flow of experiments.5. To me, the title of the paper is somewhat misleading and not fully supported by the experiments. Please rephrase. Based on the data one can conclude that constitutive Rhb1 activity interferes with mating, the pheromone response and formation of infectious filaments. As SG200 is as well affected in virulence and filamentous growth, the Pra1 localization effect is unlikely to be the (only) cause for reduced virulence.The conclusion that if filament formation is reduced in SG200, this shows that there is no defect in cell fusion in compatible matings is not justified (page 15). Rather it appears that both morphological transitions are not functioning.Minor points:1. Please clarify why the effect of Rhb1KR expression on Nrt1 functionality is relevant for the manuscript. In Fig 4E it is referred to RHB1KR-depndent degradation of Nrt1-GFP, although, overall abundance is clearly reduced. As this might as well result from altered translation, the degradation hypothesis is speculative and/or should be tested by further assays (e.g. cycloheximide treatment) or rephrased.2. The authors should comment on why their protocols deviate from established U. maydis assays. For instance, precultures for virulence assays are normally done in YEPS medium, pheromone response is normally tested in CM and mating assay on PD charcoal plates. Here the authors use minimal media with ammonium as nitrogen source. It would be important to understand the underlying logic.3. Formation of conjugation tubes and Fuz7DD filaments should be quantified.4. Page 16: The main factor determining Prf1 activity is the differential phosphorylation by Kpp2 and PKA and not the mRNA level, which rather serves as an additional boost for signaling. Please clarify.5. Page 17 : The suppression of Rhb1KR effects by Fuz7DD expression is not really convincing, as the morphological alterations upon Fuz7DD expression in WT and Rhb1KR cells are clearly different and do not resemble conjugation tubes. It is hard to see filamentous growth at all.6. I was wondering if the authors could comment on the possibility whether Rhb1KR expression might interfere with cell cycle arrest, as this would explain the majority of the observed phenotypes .7. In some instances rsp5 is misspelled rps5 in the manuscript.8. In the discussion it is supposed that TORC1 activity is required to form infective filaments, although no data is presented in this respect. Rather the data indicates that high TORC1 activity might be detrimental for virulence.Reviewer #2: SummaryIn frame with previous discoveries, emphasizing the requirement of an obligate inhibition of Tor-signalling for efficient virulence in fungal pathogens, the work conducted by Antonio de la Torre and José Pérez-Martín aimed to further unravel Tor-signalling networks during virulence but also during vegetative growth in the phytopathogen, Ustilago maydis. Because very little is known about Tor-signalling in the vast majority of fungal pathogens, the authors had as prerequisite to assess the existence of TORC components and Tor-signalling, which they remarkably conducted. Besides putting impressive efforts that lead them i) to assess the essentiality of the Tor kinase, ii) to identify experimentally critical components of both TORC1 and TORC2 subunits as well as iii) TORC1 downstream effectors (Sch9 &Aga1 as direct effectors), they also remarkably identified Tsc2 and Rheb as TORC1 upstream regulators and used several genetic approaches, including deletant strains (for Tsc2), conditional alleles and hyperactive mutants, to assess their function as upstream regulators of TORC1. This contrasts with previous studies that mainly addressed Tor-regulation in pathogenic fungi through indirect approaches . As a major achievement, the authors have elegantly demonstrated that overactivation of Rheb1 hinders poor nitrogen source uptake through internalization and Rsp5-mediated degradation of the nitrate permease Nrt1 in a TORC1-dependent manner. With this in hand, Antonio de la Torre and José Pérez-Martín then focused on their initial problematic (impact of TORC1 overactivation in the pathogenic context) and showed that overactivity of Rheb1 abolishes the ability of U. maydis to form infective structures and thus their ability to infect plants. This was then shown to be due to the pheromone insensitivity, caused by misdistribution of the receptor Pra1, thereby hindering U. maydis to trigger the virulence program. Interestingly, the authors have shown that the inappropriate misdistribution of Pra1 in the virulence context also involves Rsp5 ubiquitin-ligase, as for the TORC1-dependent Ntr1 mis-signalisation previously demonstrated in this work. Strikingly, the Rheb1-mediated pheromone blindness during virulence was Tor-independent which was unexpected. It is noteworthy that the authors have managed to describe two Rheb1 and Rps5-dependent pathways both regulating external cues modulators of which only one is TORC1-dependent while the other is not.Significance/originalityThe proposed work will be of interest for a broad readership, including the fungi community but also people from the Tor-signalling field, notably because the present work uncovered Tsc-Rheb(-TORC1) dependent pathways that are not or poorly understood even in well-studied organisms. One out of the many strengths of the proposed work is the impressive effort the authors have put to evidence TOR-signalling in Ustilago maydis. It is highly appreciable that the authors dug into the TOR field in filamentous fungi, a field that is dramatically lacking scientific knowledge. It is easily expectable that lessons from this work are transposable to other fungi and will provide important perspectives for future research topics in fungal biology. I would like to point out the fact that the authors have unexpectedly but successfully deleted several genes that were expected to be essential . The significance of this research obviously also arises from the description of a critical regulation axis for virulence, and thereby evidences potential specific targets for the development of chemicals.Quality of the manuscriptThe manuscript is dense with information and data with very well thought out and carefully planned experiments and controls. The authors have carefully considered alternatives and addressed them with control experiments in nearly all cases. Their conclusions are clearly supported by the data presented. The manuscript is well-written, clear, easy to follow, and the conclusions are well justified. They provide a clear and complete introduction that will be appreciable for readers that are out of the higher fungi biology field. I also appreciated that all along the manuscript, the authors lead the readers by clearly establishing their aims, their issues, how they circumvent these issues and technical details about fungal biology or about the strategies they have employed. This is also appreciable in the figures within the main manuscript and in the Supplementary Material.As a general observation, besides the below-mentioned remarks (mainly concerning blots), the figures are of high quality, designed in a sober way which greatly facilitates the reader to catch the author’s message. I also appreciated the way the authors have organized the figures between the main manuscript and the supplementary materials.Remarks• A main strategy used by the authors was to introduce an ectopic version of Rheb-K127R under nar1 promoter (overexpressed in the presence of nitrate (MMD) but not YPD). My understanding is that the endogenous Rheb might be inhibited on MMD media leading to TORC1 inhibition due to the poor nitrogen source. At the same time, the ectopic Rheb-K127R might be overexpressed leading to active TORC1 which is expected to induce Ntr1 degradation. Consequently, the cell should have defects in nitrate uptake which would lead to a negative feedback loop on the nar1 promoter. Nevertheless, the authors have shown that in spite of this potential negative feedback, the ectopic Rheb-K127R is overexpressed. This is surprising but can be explained due to the presence of alternative nitrate permeases.My concern here is about the appropriateness of using a nitrate dependent promoter when aiming to unravel mechanistic insight involving nitrate uptake (i.e. via the Ntr1 permease). This point is according to me highly significant, and the authors should provide strong arguments defending why this strategy is still appropriate .• The authors have conducted several immunoprecipitation (IP) experiments and evidenced changes in the phosphorylation status as readouts for TORC1-dependent and -independent pathways. They applied this approach in several cases by taking advantage from band shifts using SDS-PAGE conducted with phostag-gels. Although their results are convincing per se, I was somewhat surprised by the systematic lack of evident controls that must be done when conducting IP experiments. These include a blot showing the inputs that were submitted for IPs.• Several blots are overexposed which hinders the evaluation of potential changes in protein amounts or potential double-bands . Nevertheless, the authors could in this case cut the blot or display two expositions).• Fig 4C: Loading control is present but no quantification. Because the loading is not homogenous, it is difficult to fully exploit this blot without any quantification. This is even more true because of that the loading control signal is somewhat saturated. Especially, I am pointing this out because the protein amount of Rhb1 wt + rps5 deletant strain on MMD (control lane 4) is clearly lower than the 3 other control conditions (when comparing Tubulin amounts). Thus, it is expectable that Nrt1-GFP level is comparable in the Rhb1 wt + rps5 deletant strain (if not higher) to the control strain on MMD. This would even further support the authors’ expectations. Quantifications would also facilitate the confrontation between protein (4C) and mRNA (4D) levels.• The authors have successfully deleted several genes, including some of them that were expected to be essential. One might question whether these deletant strains have not accumulated suppressor mutations that complement the loss of the targeted gene? This point is not clearly discussed. How would the authors argue to this?https://doi.org/10.1093/genetics/iyab152). I wondered why they didn’t take advantage from this system to assess the essentiality of the targeted genes.• In their work the authors attempted to demonstrate the essential character of several genes. To assess this, they took advantage of inducible/repressible promoters and convincingly reached their aims. However, the authors have recently successfully developed an Auxin-degron based approach in Ustilago maydis to create conditional null-mutants  contains lower amount of Atg8-GFP than YPD torNAR. Is this due to differences in sample loading or to a physiological effect? This enables one to speculate that free GFP would also be present in the control strain on MMD if an equal amount was loaded. To avoid such issues, a loading control would be necessary. Also, the signals are saturated, and quantification is missing, even if I totally understand that the aim here is on the qualitative level rather than quantitative.• Fig 8C: The blot is of low quality but sufficient to understand the authors’ message. For more clarity, I would suggest lowering “Pra1-GFP” mention in front of the expected band . Do the authors have an idea to what correspond the upper band at 250 kDa? Here I would also suggest to kindly remind the readers that defect in virulence due to variations in Pra1 levels was excluded as shown in figure 7B.• The authors showed that while rapamycin has a mild effect on growth in U. maydis  it was sufficient to restore all phenotypes observed under Tsc-Rheb-TORC1 overactivation , ii) restoring the PM-localisation of Nrt1 in a rhb1-K127R  as well as iii) restoring its protein level ). The authors concluded that these pathways are TORC1 dependent. Because of the slight inhibitory effect of Rapamycin on growth, could the authors envisage to delete the proline isomerase FKBP12 homolog in U. maydis? In this context, Rapamycin treatment might not induce the above-mentioned events which would further demonstrate their TORC1-dependencies.• The authors reported Aga1 as being the closest homolog of YPKs (from S. cerevisiae) and Gad8 (from S. pombe). In contrast to YPK1/2 homologs, which are direct TORC2 targets, S. cerevisiae also encodes an additional YPK homolog (YPK3) which is a substrate of TORC1. Of note, once activated by TORC1, YPK3 phosphorylates Rps6. In their work, the authors showed that Aga1 deletion had only a mild impact on growth, then they showed it to be regulated by TORC1 and concluded it to be a substrate of TORC1. However, they didn’t discuss, nor have they tested, the possibility of Aga1 being a functional homolog of YPK3 rather than of YPK1/2. This arises the possibility that the TORC2 substrate  in U. maydis was not identified.• I would also suggest changing the term S6K by AGC kinases to designate YPK and Sch9 homologs. In facts, S6K kinases are a sub-group of AGC kinases (doi:10.1038/nrm2822 for AGC classification) to which YPK do not belong (they belong to AGC/Akt kinases). Whether Aga1 and other kinases mentioned in the manuscript indeed belong to the Akt or to the S6k is not clear.• The authors have obtained viable deletant strains of genes expected to encode specific components of TORC2. In spite those genes were not essential in U. maydis, I wondered whether the authors tested the impact of those deletions (i. e. rct and sin1 deletant) for virulence ? Conversely have they tested the impact of torin1 (inhibiting TORC1 & TORC2 in other systems) treatment during virulence?• Page 16: There is a little error of inattention while choosing the terms: The authors reported “Elegant previous work had described that Prf1 is submitted to posttranscriptional control by protein phosphorylation… ”. The herein cited references have indeed evidenced an posttranscriptional regulation of Prf1 but also a posttranslational regulation. I think that the authors meant “posttranslational control” instead of posttranscriptional since they refer to Prf1 phosphorylation.• Primers table used in the work is missing and should be included• Suggestion: If I might, although very clearly presented, readers might appreciate a schematic recapitulating both signalling branches . This schematic could even include the TORC1/(2) upstream and downstream components evidenced here.Reviewer #3: The authors (de la Torre and Pérez-Martin) present an investigation of the role of TORC1 in the virulence of Ustilago maydis using a variety of genetic, pharmacological and biochemical approaches. They made the interesting discovery that a TORC1-independent pathway influences the virulence of the fungus by interfering with the response to pheromone. The main approach was to use a gain-of-function version of the Rheb GTPase, an upstream regulator of TOR, and to assess the roles of TOR-related functions, degradative functions for permeases and receptors, and components of the pheromone signaling pathway. The study is impressive because of the methodical and detailed evaluation of functions related to TORC1 activity, the pursuit of relevant side avenues , the use of regulated promoters to bypass essentiality, and the linkage with virulence via infection assays in maize. Overall, the data are clearly presented and interpreted. I have a few minor suggestions for improvements and clarification.1.There are a number of situations where incorrect words are used. For example, the following sentence appears in the Abstract: “Strikingly, this negative control does not imply the TOR pathway.” The word imply is not used correctly. Other example words are: concourse, acuation, appropriated, subdued, coherent, seek, etc. I recommend that the authors proof read the entire manuscript to check work usage.2.Page 7. Lst8 is briefly mentioned as a candidate gene to be deleted but the authors don’t follow up with information about whether the gene was successfully deleted and, if so, whether there was a relevant phenotype.3. Fig. 3F and page 11. More explanation is needed for the statement: “This effect was more apparent in the rhb1K127R allele expression than upon tsc2 removal.”4. It wasn’t clear why YPD was used as the restrictive condition for the nar1 promoter – the comparison with MMD as the permissive condition could potentially be confounded by other differences in media composition.5. Figure 6B,C. Were different concentrations of rapamycin tested to ensure that an appropriate level was employed?6. Figure 8C is quite messy and the GFP bands are highly overexposed. Perhaps the authors can comment on the reliability of their interpretations given the presentation.7. Given the complexity of the study in terms of the number of genes/mutants/regulated strains employed, the authors might consider adding a summary diagram.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). 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The initial version of the manuscript submitted by the authors was significantly improved and I have no other issues to address.Reviewer #3: The authors adequately addressed my comments.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). 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Background: Inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, is characterized by chronic relapsing intestinal inflammation. There are few data on the efficacy and safety in clinical practice of infliximab (CT-P13) in subcutaneous formulation (SC) for the treatment of patients with IBD. Methods: Multicenter, prospective study of patients with IBD in clinical remission, who had their treatment changed from intravenous (IV) infliximab to SC. Two groups of patients were evaluated according to whether they were on IV infliximab treatment at standard or intensified doses before the switch. Results: A total of 30 patients were on standard dosing and another 30 in intensified therapy. Treatment persistence in both groups at 6 months was greater than 95%. In both groups after the change, neither the biomarkers of inflammation nor the activity indices underwent significant changes at 3 and 6 months compared to the baseline value. Similarly, in both groups, infliximab trough levels showed a significant increase 3 and 6 months after the change to SC. No serious adverse events were registered. Conclusions: The CT-P13 SC brings a new anti-TNF era. Achieving much higher drug levels that are constant over time opens new paths to explore the management of patients with IBD: less immunogenicity, better perianal disease control and higher achievement of mucosal healing. Inflammatory bowel disease (IBD) is a heterogeneous group of chronic inflammatory disorders: the main phenotypes comprise ulcerative colitis (UC) and Crohn’s disease (CD). IBD is characterized by chronic relapsing intestinal inflammation. UC is restricted to the colon, whereas CD is characterized by involvement of the gastrointestinal tract from the mouth to anus in a discontinuous fashion, with the development of strictures, abscesses or fistulas that penetrate neighboring organs or the perianal skin. It has been a worldwide healthcare problem with a continually increasing incidence. It is thought that IBD results from an aberrant and continuing immune response to the microbes in the gut, catalyzed by the genetic susceptibility of the individual. Although the etiology of IBD remains largely unknown, it involves a complex interaction between the genetic, environmental or microbial factors and the immune responses ,2,3.® to the biosimilar CT-P13 is safe and effective , it is an ELISA and its assay range is 0.035–14.4 μg/mL. In recent months two centers have changed the measurement system to i-TrackerInfliximab (Theradiag) which is an automated chemiluminescence immunoassay on human serum or plasma samples. Their assay ranges are 0.3–24 µg/mL.The aim of our study was to evaluate the persistence of SC CT-P13 in patients with IBD who were switched from intravenous infliximab to subcutaneous administration. The secondary aims were the following: to determine the levels of infliximab before and in each of the three-monthly evaluations, to assess the possible clinical changes (using the HBI for CD and the PMI for UC), to evaluate the possible changes in the inflammatory biomarkers (CRP and FCP) and determine the occurrence of possible side effects attributable to the drug.p values < 0.05.We used basic descriptive statistics, which included the mean and standard deviation for continuous variables and absolute frequency and percentages for discrete variables. To determine de differences between clinical indices, serum and fecal biomarkers and serum infliximab concentrations; the Friedman test was used. The Statistical Package for the Social Sciences (SPSS) 22 was used to describe and analyze the data, considering The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Consorcio Hospital General Universitario de Valencia on 8 April 2021, and the reference number is 142/2021. Informed consent was obtained from all subjects involved in the study.® or Inflectra®) and 11 from original infliximab (Remicade®). A total of 30 patients were on standard dosing and another 30 in intensified therapy. In Of the 77 patients to whom the change was proposed, 60 [78%] accepted the change to SC IFX. Of these 32 [53.3%] were male and had Crohn’s disease 33 [55%]. A total of 49 patients switched from CT-P13 , all patients maintained treatment. At the time of the analysis, 21 patients had completed 6 months of follow-up, in which the persistence of treatment was 95.2% .The clinical indices remained at remission values (HBI < 5 and PMI < 2) except for one patient who presented a mild flare-up at 3 months and was treated with budesonide.In the short term (3 months), all patients persisted with treatment and also remained in remission based on clinical indices. Of the initial 30 patients, at the time of the analysis, 17 had already completed the 6-month follow-up and all of them were still on treatment and in clinical remission. One patient had an HBI of 6 that was attributed to previous surgery for his CD.Anti-IFX antibodies were detected in only one patient with very low IFX levels, that were already present before the switch. At three months, one patient had withdrawn azathioprine without observing clinical changes, and this withdrawal was maintained at 6 months of follow-up.No patient had anti-drug antibodies. At three months of follow-up, three patients had withdrawn azathioprine and the patient on methotrexate was also able to remove it, without observing clinical changes. These withdrawals were maintained at 6-month follow-up without observing any clinical worsening. No patient required an increase in the standard dosage of 120 mg/every 2 weeks nor did any patient return to intravenous dosing either due to adverse events or clinical decision.A total of nine patients had perianal disease (five in the standard dosage group and four in the intensified group). The months of treatment with IV infliximab prior to the change were a median of 30 months (interquartile range (IQR) 73). No clinical worsening of perianal disease was observed in any of them after switching to SC. On Regarding the adverse events described, only one patient had pain and erythema at the injection site that did not require withdrawal or change. Another patient presented mild elevation of transaminases (<5×) that she had already presented on other occasions and that was not considered to be related to the drug.No patient required neither hospital admission nor surgery for their IBD.Our study shows that SC CT-P13 maintains high rates of drug persistence beyond 6 months. Both the HBI and the PMI remained at inactive values throughout the follow-up, as did the biomarkers (CRP and FCP). Infliximab levels increased significantly with the switch to subcutaneous CT-P13.After more than 20 years of existence of infliximab in its intravenous formulation, the appearance of the subcutaneous formulation represents a revolution in the management of the drug and places it as a true “infliximab 2.0”. We think that this is due to its pharmacokinetic advantages that have been evaluated both in clinical trials and in real clinical practice data, such as those shown in the present study. We think that this is due to its pharmacokinetic advantages that have been evaluated both in clinical trials and in real clinical practice data, such as those shown in the present study.In our study, the rate of drug persistence was high both at 3 and 6 months, in agreement with what was previously published by Smith et al.  in whichAs in other studies published to date ,15,16,17It has been suggested that patients with CD and perianal involvement would require higher drug levels to achieve improvement of the fistulous disease ,21. In oThe rate of acceptance of the change was 78%. These are results similar to those reported by Buisson et al. , in whicDrug tolerance was excellent. Regarding the adverse events described, they were mild and did not require drug withdrawal, in accordance with what has been published to date ,11.The association of immunosuppressive and anti-TNF drugs has been associated with an increased risk of infections and other adverse events ,29. On tOnce again, in view of the results in drug levels, we consider that another field to explore is the spacing of treatment beyond 2 weeks in patients with very high levels and in clinical-biological remission. In our group, and after 9 months of follow-up, this possibility has been explored in 2 patients in whom we spaced treatment for 3 weeks, having observed maintenance of clinical remission 3 months after spacing.Several methodologies have been developed to measure serum concentrations of anti-TNFα drugs, including different types of ELISA, radioimmunoassay, liquid chromatography-based homogeneous mobility shift assay, liquid chromatography coupled to mass spectrometry, etc. . Among tIn conclusion, the subcutaneous infliximab formulation, CT-P13, brings a new anti-TNF era. Achieving much higher drug levels that are constant over time opens new paths to explore the management of patients with IBD: less immunogenicity with the possibility of not combining immunosuppressant drugs, better perianal disease control, higher achievement of mucosal healing, the possibility of using it in patients with severe ulcerative colitis with high inflammatory load and even spacing treatments beyond 2 weeks.


The assessment consisted of perceptual voice analysis, objective voice evaluation and video-laryngo-stroboscopic exam. In addition, patients rated their voices and completed the Voice related Quality of Life (VR-QOL) questionnaire. The results were compared with those obtained in a matched control group.15 patients with glottic Tis and T1 squamous cell carcinoma treated with CO2 laser, functional results are acceptable, with minimal repercussion in their quality of life. Avaliação da voz em pacientes submetidos à cordectomia com laser de CO2.Most of the patients presented some degree of hoarseness on perceptual voice analysis, mainly rough and breathy voices. Their acoustic evaluation compared with the control group showed a small increase in fundamental frequency, but with no statistically significant difference, and the values of jitter, shimmer and noise to harmonic ratio were worse and statistically significant. As regards to video-laryngo-stroboscopic findings, better results were achieved in the less extensive resection group. Patients have had minimal repercussion in their life quality in respect to voice. Conclusions: In spite of voice alterations in patients submitted to cordectomy by CO Cancer of the larynx is the second most frequent tumor of the upper airway and upper digestive tract, second only to oral cavity tumors. Brazil occupies the second place in the world in the incidence of cancer of the larynx. This disease affects mostly males in the sixth and seventh decades of life2 laser. These approaches provide satisfactory local control and similar survival ratesInitial glottic carcinomas, classified as in situ carcinomas (Tis) and T1, may be treated by open surgery , radiotherapy and laryngeal microsurgery with conventional instruments or by COCordectomy is the surgical procedure used for early glottic tumors, where the affected vocal fold is resected at varying depths.2 laser in laryngeal surgery in 1972Since Strong & Jako introduced CO2 laser treated patients with early glottic tumors vary from 89% to 100%Cure rates for CO2 laser compared to other treatment strategies include lower morbidity compared to radiotherapy side effects , lower cost as patients do not require traqueostomy, improved homeostasis, and the fact that CO2 laser does not exclude other forms of treatment if the disease recurs locally, regionally or at a distanceThe advantages of COWhen different treatment strategies offer similar cure rates, resulting voice quality becomes paramount in the therapeutic decision. There are few published papers in medical literature about voice following laser cordectomy for the treatment of laryngeal tumors.2 laser cordectomy is similar to that in radiotherapy patientsMost of the studies compare cordectomy with radiotherapy, and results are controversial. Some authors have reported worse outcomes in patients undergoing laser surgery, which may limit its use2 laser cordectomy provides improved results in the voice quality of patients, compared to conventional cordectomy2 laser cordectomy have been published to date.CO2 laser cordectomy for the treatment of laryngeal tumors.The factors that stimulated this study where the importance of preserving the phonatory function when treating early glottic tumors and the lack of guidelines on voice quality in patients undergoing CO2 laser cordectomy.Therefore, the aim of this study was to assess voice quality in patients with stages Tis and T1 glottic carcinoma undergoing CO2 laser cordectomy for the treatment of early vocal fold SCC. Early glottic SCC was defined in this study as patients with vocal fold Tis and T1 tumors. Patients were referred to us from the Head and Neck Surgery Unit at Sao Paulo University - Paulista School of Medicine.Between January and May 2004 we assessed fifteen patients  who had undergone COAges varied from 43 to 82 years, average 63.1 years. Average follow-up time was 14.6 months, varying from 3 to 38 months.Patients were staged, based on data in their charts, using the clinical classification of the International Union Against Cancer (UICC) and the American Joint Committee on Cancer (AJCC) 2002.Three patients were staged as TisN0 and twelve as T1aN0. Types of cordectomy were classified into five types according to the European Laryngological Society2 laser unit. A 5 to 7 Watts superpulse mode 0.5 mm spot was used. Safety margins were 1 to 2mm. Following tumor resection upper, lower, anterior, posterior and deep margins were sent to frozen section examination, to assure surgical safety margins.Surgery was done under general anesthesia. A 400mm lens microscope was coupled to a Visograf® COAll patients included in this study had been operated at least three months before enrollment, and were referred to post-operative speech therapy. No patient showed signs of local and/or regional recurrence and none had a second primary tumor.Patients underwent subjective and objective voice assessment and videolaryngostroboscopic evaluation between the 3rd and 38th post-operative month. Subjective testing was done by perceptive-auditory voice analysis according to the GRBASI scaleGRBASI's scale assesses the global impression of dysphonia (G) considering hoarseness (R), soprosity (B), asteny (A), tension (S) and instability (I), which are scored 0 to 3, 0 (no change), 1 , 2  and 3 . V-RQOL is a protocol whereby patients relate their voice to quality of life in a 0 to 100 score where 100 is improved quality of lifeComputer analysis was made based on open and isolated phonation of the sustained vowel /e/ picked up by an AKG model C410 professional microphone at a standard 5 cm distance from the patient. Voice recording was done by the Computerized Speech Lab - CSL, model 4300B, and analyzed by the Kay Elemetrics Corp.'s MDVP  voice analysis program.Each patient was asked to emit an isolated and sustained vowel /e/. A voice sample was considered as the most stable 3 second period, whenever possible eliminating the beginning and the end of sound emission or a maximum period of voice stability. The following acoustic measurement parameters were used in this study: fundamental frequency, the perturbation quotients Jitter (variations of frequency) and Shimmer (variations of amplitude), and noise measurements (harmonics-to-noise ratio).Computer analysis compared fundamental frequency, jitter, shimmer, and harmonics-to-noise ratio with a patient control group (average age 57.6 years) that had normal larynxes, and no complaints about voice.Videolaryngostroboscopy was used to analyze the following parameters:1.Glottic coaptation: complete or incomplete2.Laryngeal vestibule constriction: normal or with hyperconstriction.3.Presence of vibration: absent, glottic, supraglottic or combined. Vibration was considered as absent if there was any area of akinesia in the resected glottic region, with no supraglottic compensation.The Stata 7.0 program was used for statistical analysis. Data had a normal distribution allowing us to use Student's t-test for non-related samples to compare numerical data in the study and the control groups. The significance level was 0.10.Student's t-test was used to compare the average fundamental frequency of males in the study group and the control group (139.3Hz). The descriptive value was 0.2008, which suggests no differences between both groups. There were not sufficient females in the study group to apply statistical tests. Descriptively, however, the 226.24 Hz average value may be compared to the average control group value .Student's t-test for independent samples demonstrated that average jitter in patients was higher than in the control group (p = 0.001), that average shimmer in patients was higher than in the control group (p = 0.0007), and that patients had a higher average harmonics-to-noise ratio compared to the control group (p = 0.053).There is no agreement about voice quality after different treatment strategies for glottic tumors, particularly Tis and T1 tumors. Important factors influencing the decision for a specific treatment procedure, except for voice quality, include oncological results, cost and morbidity.2 laser cordectomy, and only rarely is there a comparison with a control group of normal individuals; there are no such papers published in Brazil to date. The importance of voice is evident as we measure voice changes in patients undergoing CO2 laser cordectomy compared to normal individuals and to how much this affects their lives. Furthermore, future comparisons may be made both within groups undergoing similar treatment and between groups subjected to other strategies such as radiotherapy and conventional surgery.There are few papers in literature assessing voice following COIn our patients age ranged from 43 to 82 years (average 63.1 years), with 80% males. This is similar to other published studies, evidence that malignant laryngeal tumors are more frequent in men in the sixth and seventh decades of lifeAlthough all patients were staged as Tis and T1a, the type of cordectomy varied in the group. Less extensive cordectomies, classified as type I and II, were done in 53% of patients. Type III and IV cordectomies were done in the remaining 47%.Subjective analysis demonstrated that the majority of patients had some degree of dysphonia, usually hoarseness and soprosity. These results are not surprising, as hoarseness (R) is related to vocal crepitation and bitonality due to irregular vibration of the vocal folds, and soprosity (B) is related to air escape during phonation. One naturally expects a scar and local irregularity when cordectomy is done, as part of the vocal fold is removed.Patient opinions about their voice following treatment were also of interest in this study. Using Sittel et al's (1998)2 laser cordectomy had some degree of dysphonia, the effect on activities of daily life was minor. It is important to underline that no patient in this study used his or her voice professionally, which reduces the impact of voice changes on the quality of life. In fact, the V-RQOL is not a sensitive method to assess professional voice usersThe impact of voice on the patient's quality of life should be a constant concern in the treatment of laryngeal tumors. This concept, however, is recent; Zeitels (1995)2 laser surgery. Results confirm good quality of life in both treatment strategies. Specific head & neck questionnaire questions about swallowing solid food, xerostomy and dental problems scored worse for patients undergoing radiotherapy, although there was no difference in voice quality. Schneider, Guidicelli & Stöckli in 2000Stoeckli et al (2001)2 laser cordectomy (p< 0.1) comparing results of the surgery group to controls.Computer voice acoustic analysis using the Kay Elemetrics Corp. Computerized Speech Lab-CSL model 4300 MDVP  voice analysis program showed that, except for the fundamental frequency, all voice acoustic analysis parameters in this study - percentage of jitter and shimmer, and harmonics-to-noise ratio - are significantly affected by COThe average fundamental frequency for operated male patients was higher compared to controls, although this difference was not statistically significant. There were not sufficient females in the study group to apply statistical tests, but a minor increase of 24.01 Hz in the fundamental frequency was seen compared to controls (202.23Hz). Similar results have also been reported by McGuirt et al. (1992)Both jitter and shimmer were significantly higher in the study group compared to controls. These results reflect increased aperiodicity in the glottic cycle and sound wave variability. The harmonics-to-noise ratio is directly related to voice quality2 laser cordectomy is compromised compared to individuals with no voice-related problems. A further issue is that when these patients seek medical help, they already have some degree of dysphonia by having a glottic tumor. Pre-operative voice assessment was not evaluated in this study, but is suggested for future studies.Results of objective voice analysis clearly suggest that voice quality in patients undergoing COTheoretically, regardless of the treatment, voice quality of patients with glottic tumors will never be equal to their voice before the disease. Even the smallest cordectomy will involve resection of part of the mucosa, reducing the ability for vibration at that point.Although considered a more conservative strategy, radiotherapy also has negative effects on voice quality compared to controlsVideolaryngostroboscopy found that 75% (n=6) of patients undergoing type I and II cordectomy presented complete glottic closure. In this same group glottic mucosal vibration was always present; only 1 patient  had supraglottic vibration. This same patient was the only one in this group (cordectomy types I and II) that had laryngeal vestibule hyperconstriction. All subjective and objective parameters in this patient, except for the fundamental frequency, were worse compared to other patients in the same group. A further interesting point is that even in type II cordectomy, glottic closure was complete in 4 of the 5 operated patients. This is relevant because voice quality is intimately related to glottic closure. Frequently in situ carcinoma is treated with type I cordectomy, and the surgeon may find himself surprised with the pathological examination showing compromised surgical resection margins. The next step would be to widen the surgical margins, which means the patient has to undergo a second operation or be referred to radiotherapy. Knowing that the post-operative functional result is similar in type I and II cordectomies, a safer and wider margin procedure for Tis could be advised.Videolaryngostroboscopy of type III and IV cordectomies showed that 57.1% (n=4) of patients presented incomplete glottic closure, an increased trend to have laryngeal hyperconstriction as a compensation mechanism and no mucosal vibration. Stroboscopic changes correlated with voice quality data in study group patients, probably due to the amount of tissue removed, decreased mucosal vibration and compensation mechanisms.2 laser cordectomy have abnormal voice parameters, the possibility of preserving communication is itself reason for satisfaction. Frequently patients lose motivation when receiving a diagnosis of cancer of the larynx, believing that treatment is necessarily related to voice loss, which is so important for social interactions. Expectations of voice professionals may not be reached, but this is not what happens to most patients; they frequently surprise us by being satisfied with their resulting voice quality.Although results suggest that patients undergoing CO2 laser cordectomy presented some degree of dysphonia, usually hoarseness and soprosity.Voice perceptive-auditory analysis indicates that most patients undergoing CO2 laser cordectomy produces acceptable functional results, with minor repercussion on the patient's lives.Both the general voice assessment made by patients and the quality of life protocol scores suggest that CO2 laser cordectomy.Acoustic voice analysis showed a minor increase in the fundamental frequency of operated patients compared to controls. This difference was not statistically significant. Worse jitter, shimmer and the harmonics-to-noise ratio results were seen in patients undergoing CO